U.S. patent application number 15/164798 was filed with the patent office on 2016-10-06 for slip chip device and methods.
The applicant listed for this patent is University of Chicago. Invention is credited to Delai Chen, Wenbin Du, Rustem F. Ismagilov, Jason Eugene Kreutz, Liang Li, Kevin Paul Flood Nichols, Feng Shen.
Application Number | 20160288121 15/164798 |
Document ID | / |
Family ID | 42781435 |
Filed Date | 2016-10-06 |
United States Patent
Application |
20160288121 |
Kind Code |
A1 |
Ismagilov; Rustem F. ; et
al. |
October 6, 2016 |
Slip Chip Device and Methods
Abstract
A device is described having a first surface having a plurality
of first areas and a second surface having a plurality of second
areas. The first surface and the second surface are opposed to one
another and can move relative to each other from at least a first
position where none of the plurality of first areas, having a first
substance, are exposed to plurality of second areas, having a
second substance, to a second position. When in the second
position, the plurality of first and second areas, and therefore
the first and second substances, are exposed to one another. The
device may further include a series of ducts in communication with
a plurality of first second areas to allow for a substance to be
disposed in, or upon, the plurality of second areas when in the
first position.
Inventors: |
Ismagilov; Rustem F.;
(Chicago, IL) ; Du; Wenbin; (Chicago, IL) ;
Li; Liang; (Chicago, IL) ; Shen; Feng;
(Chicago, IL) ; Nichols; Kevin Paul Flood;
(Chicago, IL) ; Chen; Delai; (Cambridge, MA)
; Kreutz; Jason Eugene; (Chicago, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Chicago |
Chicago |
IL |
US |
|
|
Family ID: |
42781435 |
Appl. No.: |
15/164798 |
Filed: |
May 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13257811 |
Sep 20, 2011 |
9415392 |
|
|
PCT/US2010/028316 |
Mar 23, 2010 |
|
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15164798 |
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61340872 |
Mar 22, 2010 |
|
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61262375 |
Nov 18, 2009 |
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61162922 |
Mar 24, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0861 20130101;
B01L 3/502761 20130101; B01L 2200/025 20130101; B01F 13/0094
20130101; B01L 2300/0809 20130101; H01L 2924/0002 20130101; B01L
2300/0864 20130101; C12Q 1/703 20130101; B01L 2400/065 20130101;
B01L 7/52 20130101; G01N 33/54386 20130101; B01L 2300/0609
20130101; B01L 2300/12 20130101; B01L 3/502738 20130101; B01L
2300/0816 20130101; B01L 2300/168 20130101; B01L 3/5025 20130101;
B01L 2200/027 20130101; G01N 21/78 20130101; B01L 2300/0867
20130101; B01L 2300/0887 20130101; B01L 2300/0893 20130101; C12Q
1/025 20130101; B01L 3/502715 20130101; H01L 2924/0002 20130101;
H01L 2924/00 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; B01F 13/00 20060101 B01F013/00 |
Goverment Interests
[0002] This invention was made with governmente support under grant
numbers EB012946, GM074961 and DP1OD003584 awarded by the National
Institutes of Health (NIH) and CHE-0526693 awarded by the National
Science Foundation. The government has certain rights in the
invention.
Claims
1. An apparatus for determining the quantity of an analyte present
in a sample, the apparatus comprising: a top plate comprising a
plurality of areas arranged to form a plurality of rows extending
parallel to one another; and a bottom plate comprising a plurality
of areas arranged to form a plurality of rows extending parallel to
one another, and a plurality of channels extending perpendicularly
to the plurality of rows of the bottom plate; wherein the top plate
and the bottom plate are assembled together so that the top plate
is on top of the bottom plate and the areas of the top plate
communicate with the areas of the bottom plate so as to form a
plurality of rows; wherein at least one of the top plate and the
bottom plate is configured to slide relative to the other of the
top plate and the bottom plate in order to form a plurality of
columns, with each of the plurality of columns in communication
with each of the plurality of channels; and wherein the areas in
the top plate and the areas in the bottom plate extend at a 45
degree angle relative to the axis of a row.
2. The apparatus of claim 1, wherein the top plate is
transparent.
3. The apparatus of claim 1, wherein at least one of the plurality
of rows formed in the top plate comprises an inlet and an
outlet.
4. The apparatus of claim 1, wherein each of the plurality of
channels is connected to an area formed in the bottom plate.
5. The apparatus of claim 1, wherein the top plate is preloaded
with one or more capture agents which absorb, adsorb or react with
the analyte present in the sample.
6. The apparatus of claim 5, wherein the top plate is preloaded
with at least two different capture agents in separate areas.
7. The apparatus of claim 5, wherein the capture agent is a nucleic
acid, a peptide, a protein, an antibody, an aptamer, a bead, a
particle or a cell.
8. The apparatus of claim 1, further comprising ink positioned in
an area in a row adjacent to the plurality of channels.
9. The apparatus of claim 1, wherein the top plate and the bottom
plate are made from a material selected from the group consisting
of glass, silicon, plastics, ceramics and metal oxide.
10. The apparatus of claim 1, wherein the analyte is a nucleic
acid, a peptide, a protein, an antibody, a cell, an organism, an
allergen, a drug or its metabolites, a toxin, or an environmental
pollutant.
11. An apparatus for determining the quantity f an analyte present
in a sample, the apparatus comprising: a top plate comprising a
plurality of areas arranged to form a plurality of rows extending
parallel to one another; and a bottom plate comprising a plurality
of areas arranged to form a plurality of rows extending parallel to
one another, and a plurality of channels extending perpendicularly
to the plurality of rows of the bottom plate; wherein the top plate
and the bottom plate are assembled together so that the top plate
is on top of the bottom plate and the areas of the top plate
communicate with the areas of the bottom plate so as to forma
plurality of rows; wherein at least one of the top plate and the
bottom plate is configured to slide relative to the other of the
top plate and the bottom plate in order to form a plurality of
columns, with each of the plurality of columns in communication
with each of the plurality of channels; and wherein each of the
plurality of channels is connected to an area formed in the bottom
plate.
12. The apparatus of claim 11, wherein top plate is
transparent.
13. The apparatus of claim 11, wherein at least one of the
plurality of rows formed in the top plate comprises an inlet and an
outlet.
14. The apparatus of claim 11, wherein the areas in the top plate
and the areas in the bottom plate extend at a 45 degree angle
relative to the axis of a row.
15. The apparatus of claim 11, wherein the top plate is preloaded
with one or more capture agents which absorb, adsorb or react with
the analyte present in the sample.
16. The apparatus of claim 15, wherein the top plate is preloaded
with at least two different capture agents in separate areas.
17. The apparatus of claim 15, wherein the capture agent is a
nucleic acid, a peptide, a protein, an antibody, an aptamer, a
bead, a particle or a cell.
18. The apparatus of claim 11, further comprising ink positioned in
an area in a row adjacent to the plurality of channels.
19. The apparatus of claim 11, wherein the top plate and the bottom
plate are made from a material selected from the group consisting
of glass, silicon, plastics, ceramics and metal oxide.
20. The apparatus of claim 11, wherein the analyte is a nucleic
acid, a peptide, a protein, an antibody, a cell, an organism, an
allergen, a drug or its metabolites, a toxin, or an environmental
pollutant.
21. A method for determining the quantity of an analyte present in
a sample, the method comprising: providing an apparatus comprising:
atop plate comprising a plurality of areas arranged to form a
plurality of rows extending parallel to one another; and a bottom
plate comprising a plurality of areas arranged to form a plurality
of rows extending parallel to one another, and a plurality of
channels extending perpendicularly to the plurality of rows of the
bottom plate; wherein the top plate and the bottom plate are
assembled together so that the top plate is on top of the bottom
plate and the areas of the top plate communicate with the areas of
the bottom plate so as to form a plurality of rows; wherein at
least one of the top plate and the bottom plate is configured to
slide relative to the other of the top plate and the bottom plate
in order to form a plurality of columns, with each of the plurality
of columns in communication with each of the plurality of channels;
binding a capture agent in at least one area forming one of the
plurality of rows of the top plate; introducing a sample into the
at least one area so that an analyte contained in the sample is
bound to the capture agent, and binding a probe to the bound
analyte; and positioning a reagent in an area adjacent to the row
containing the capture agent, bound analyte and bound probe; and
positioning ink in an area in a row adjacent to the plurality of
channels; sliding one of the top plate and the bottom plate
relative to the other of the top plate and the bottom plate so as
to form the plurality of columns, with each column being in
communication with one of the plurality of channels; and
determining the quantity of the analyte present in the sample by
detecting the longitudinal position of the ink contained in the
plurality of channels.
22. The method of claim 21, wherein the top plate is
transparent.
23. The method of claim 21, wherein at least one of the plurality
of rows formed in the top plate comprises an inlet and an
outlet.
24. The method of claim 21, wherein the areas in the top plate and
the areas in the bottom plate extend at a 45 degree angle relative
to the axis of a row.
25. The method of claim 21, wherein each of the plurality of
Channels is connected to an area formed in the bottom plate.
26. The method of claim 21, wherein the top plate is preloaded with
one or more capture agents which absorb, adsorb or react with the
analyte present in the sample.
27. The method of claim 26, wherein the top plate is preloaded with
at least two different capture agents in separate areas.
28. The method of claim 26, wherein the capture agent is a nucleic
acid, a peptide, a protein, an antibody, an aptamer, a bead, a
particle or a cell.
29. The method of claim 21, wherein the top plate and the bottom
plate are made from a material selected from the group consisting
of glass, silicon, plastics, ceramics and metal oxide.
30. The method of claim 21, wherein the analyte is a nucleic acid,
a peptide, a protein, an antibody, a cell, an organism, an
allergen, a drug or its metabolites, a toxin, or an environmental
pollutant.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 13/257,811, filed Sep. 20, 2011, which claims priority to
PCT/US2010/28316 filed Mar. 23, 2010, which claims priority to U.S.
Provisional Patent Application Ser. No. 61/162,922 filed on Mar.
24, 2009, and entitled "Slip Chip Device And Methods"; U.S.
Provisional Patent Application Ser. No. 61/262,375, filed on Nov.
18, 2009, and entitled "Slip Chip Device And Methods"; and U.S.
Provisional Patent Application Ser. No. 61/340,872, filed on Mar.
22, 2010, and entitled "Slip Chip Device And Methods", the
entireties of all of which are incorporated herein by
reference.
BACKGROUND
[0003] Known devices and methods for carrying out a reaction are
limited in the way two or more substances may be exposed to one
another. Such devices employ a series of chambers configured for
subjecting a substance to a specific processing step, but require
each chamber to be individually filled and/or exposed to another
chamber for carrying out a reaction in that chamber. These devices
are not designed to minimize the possibility of cross-contamination
or contamination from external sources. Moreover, to perform
multiple reactions with multiple substances, these devices must be
re-loaded with additional substances, thus taking additional time
and increased chance of contamination. Accordingly, it is a
time-consuming process to perform each combination of reactions for
a specific substance.
BRIEF SUMMARY
[0004] The present invention includes a device and method for
carrying out a reaction. In one embodiment the device includes a
base having a first surface, at least one first area located along
a portion of the first surface where the at least one first area is
configured to maintain at least one first substance. A plate having
a second surface is opposed to the first surface and at least one
second area is located along a portion of the second surface, where
the at least one second area is configured to maintain at least one
second substance, where at least one of the first surface of the
base and the second surface of the plate is configured to move
relative to the other between a first position, where one of the at
least one first area is only exposed to one of the at least one
second areas and form a closed system.
[0005] In another embodiment, the device for carrying out a
reaction includes a base having a first surface and a plurality of
areas formed along a portion of the first surface, where each of
the plurality of first areas is configured to maintain at least one
first substance. A plate having a second surface is opposed to the
first surface and a plurality of second areas is formed along a
portion of the second surface. Each of the plurality of second
areas is configured to maintain at least one second substance
wherein at least one of the first surface of the base and the
second surface of the plate is configured to slide relative to the
other between a first position, where at least some of the
plurality of the first areas are not exposed to any of the
plurality of the second areas, and a second position in a direction
substantially perpendicular to the normal of the first surface,
wherein in the second position at least one of the plurality of the
first areas and at least one of the plurality of the second areas
are only exposed to one another.
[0006] In another embodiment of the present invention, the device
includes a base having a first surface and a first area located
along a portion of the first surface where the first area is
configured to maintain at least one first substance. A first duct
is formed along a portion of the first surface and is not exposed
to the first area. A plate having a second surface is opposed to
the first surface and a second area is located along a portion of
the second surface, where the second area is configured to maintain
at least one second substance wherein the first surface and the
second surface are configured to slide relative to one another
between a first position and a second position, wherein in the
first position the first duct is exposed to the second area and the
first area and the second area are not exposed to one another, and
wherein in the second position the first area and the second area
are only exposed to one another.
[0007] In another embodiment of the present invention the device
includes a base having a first surface and a first plurality of
first areas located along a portion of the first surface, where the
first plurality of first areas have a first pattern and are
configured to maintain at least one first substance. A first set of
ducts is formed along a portion of the first surface and are not
exposed to the first plurality of first areas. A plate having a
second surface is opposed to the first surface and a plurality of
second areas are located along a portion of the second surface, the
plurality of second areas having a pattern substantially similar to
the pattern of the first plurality of first areas where the
plurality of second areas are configured to maintain at least one
second substance, wherein the first surface and the second surface
are configured to slide relative to one another between a first
position, where the first set of ducts is exposed to the plurality
of second areas, and a second position, where at least one of the
first plurality of first areas and at least one of the plurality of
second areas are only exposed to one another.
[0008] In another embodiment of the present invention the device
includes a base having a first surface, a first area located along
a portion of the first surface, and the first area configured to
maintain at least one first substance. An upper plate has a second
surface facing the first surface and has a second area located
along a portion of the second surface and is configured to maintain
at least one second substance. An intermediate plate is disposed
between the first surface of the base and the second surface of the
upper plate and the intermediate plate has an opening formed
therethrough, wherein the base, the upper plate and the
intermediate plate are configured to slide relative to one another
from a first position where the first area is not exposed to the
second area via the opening to a second position where the first
area is exposed to the second area via the opening.
[0009] Yet another embodiment of the present invention includes a
kit for carrying out a reaction including a base having a first
surface and a first area located along a portion of the first
surface where the first area is configured to maintain at least one
first substance, and a plate having a second surface and a second
area located along a portion of the second surface, where the
second area is configured to maintain at least one second
substance, and at least one of a first substance in the first area,
and a second substance in the second area, and a substrate disposed
between the first surface and the second surface, wherein the first
surface of the base and the second surface of the plate are
configured such that when fitted together, they are opposed to each
other and move relative to the other between a first position,
where the first area and the second area are not exposed to one
another, and a second position where the first area and the second
area are only exposed to one another.
[0010] Yet another embodiment of the present invention includes a
kit for carrying out a reaction including a base having a first
surface, a first area located along a portion of the first surface
and configured to maintain at least one first substance and a first
duct formed along a portion of the first surface and not exposed to
the first area, a plate having a second surface and a second area
located along a portion of the second surface and configured to
maintain at least one second substance, and at least one of a first
substance in the first area, a second substance in the second area,
and a substrate disposed between the first surface and the second
surface, wherein the first surface and the second surface are
configured such that when fitted together they slide relative to
one another between a first position and a second position, wherein
in the first position, the first duct is exposed to the second
area, and the first area and the second area are not exposed to one
another, and wherein in the second position, the first area and the
second area are only exposed to one another.
[0011] Yet another embodiment of the present invention includes a
kit for carrying out a reaction including a base having a first
surface, a first plurality of first areas located along a portion
of the first surface, where the plurality of first areas have a
first pattern and are configured to maintain at least one first
substance, and a first set of ducts formed along a portion of the
first surface and not exposed to the first plurality of first
areas. The embodiment further includes a plate having a second
surface and a plurality of second areas located along a portion of
the second surface where the plurality of second areas have a
pattern substantially similar to the pattern of the first plurality
of first areas and the plurality of second areas are configured to
maintain at least one second substance, and at least one of a first
substance in the first area, a second substance in the second area,
and a substrate disposed between the first surface and the second
surface, where the first surface and the second surface are
configured such that when fitted together they slide relative to
one another between a first position, where the first set of ducts
is exposed to the plurality of second areas, and a second position,
where at least one of the first plurality of first areas and at
least one of the plurality of second areas are only exposed to one
another.
[0012] Yet another embodiment of the present invention includes a
kit for carrying out a reaction including a base having a first
surface and a first area located along a portion of the first
surface, where the first area is configured to maintain at least
one first substance, an upper plate having a second surface and a
second area located along a portion of the second surface
configured to maintain at least one second substance, an
intermediate plate disposed between the first surface of the base
and the second surface of the upper plate having an opening formed
therethrough, and at least one of a first substance in the first
area, a second substance in the second area, and a substrate
disposed between the first surface and the second surface, wherein
the base, the upper plate and the intermediate plate are configured
such that the intermediate plate can be disposed between the first
and second surfaces and can slide relative to the base and upper
plate from a first position, where the first area is not exposed to
the second area via the opening, to a second position, where the
first area is exposed to the second area via the opening.
[0013] Yet another embodiment of the present invention includes a
method for carrying out a reaction, the method includes the steps
of providing a device in a first position where the device
comprises a base having a first surface, a first area located along
a portion of the first surface where the first area is configured
to maintain at least one first substance, a first substance in the
first area, a plate having a second surface opposed to the first
surface, a second area located along a portion of the second
surface, where the second area is configured to maintain at least
one second substance, and a second substance in the second area,
wherein the first surface of the base and the second surface of the
plate are configured to move relative to one another, and wherein
the first area and the second area are not exposed to one another
when in the first position, and moving the device from the first
position into a second position by moving the first surface of the
base and the second surface of the plate relative to one another,
and wherein in the second position, the first area and the second
area are only exposed to one another, thereby reacting the first
and second substances.
[0014] Yet another embodiment of the present invention includes a
method for carrying out a reaction, the method includes the steps
of providing a device in a first position, the device including a
base having a first surface, a plurality of first areas formed
along a portion of the first surface, where each of the plurality
of first areas is configured to maintain at least one first
substance, at least one first substance in at least one of the
plurality of first areas, a plate having a second surface opposed
to the first surface, wherein the first surface of the base and the
second surface of the plate are configured to move relative to one
another in a direction substantially perpendicular to the normal of
the first surface, a plurality of second areas formed along a
portion of the second surface, where each of the plurality of
second areas is configured to maintain at least one second
substance, at least one second substance in at least one of the
plurality of second areas, and where at least some of the plurality
of first areas are not exposed to any of the plurality of second
areas in the first position, and moving the device from the first
position to a second position, wherein in the second position at
least one of the plurality of the first areas and at least one of
the plurality of the second areas are only exposed to one another,
thereby reacting the at least one first and second substances.
[0015] Yet another embodiment of the present invention includes a
method for carrying out a reaction, the method includes the steps
of providing a device in a first position, with the device
including a base having a first surface, a first area located along
a portion of the first surface, where the first area is configured
to maintain at least one first substance, at least one first
substance maintained in the first area, a first duct formed along a
portion of the first surface and not exposed to the first area, a
plate having a second surface opposed to the first surface, wherein
the first surface and the second surface are configured to slide
relative to one another from the first position to a second
position, a second area located along a portion of the second
surface, where the second area is configured to maintain at least
one second substance, and at least one second substance maintained
in the second area, wherein when in the first position, the first
duct is exposed to the second area, and the first area and the
second area are not exposed to one another, and moving the device
from the first position into the second position, wherein in the
second position, the first area and the second area are only
exposed to one another, thereby reacting the at least one first and
second substances.
[0016] Yet another embodiment of the present invention includes a
method for carrying out a reaction, the method including the steps
of providing a device in a first position, wherein the device
includes a base having a first surface, a first plurality of first
areas located along a portion of the first surface, where the first
plurality of first areas have a first pattern and are configured to
maintain at least one first substance, at least one first substance
maintained in at least one first area, a first set of ducts formed
along a portion of the first surface and not exposed to the first
plurality of first areas, a plate having a second surface opposed
to the first surface, wherein the first surface and the second
surface are configured to slide relative to one another, a
plurality of second areas located along a portion of the second
surface, the plurality of second areas having a pattern
substantially similar to the pattern of the first plurality of
first areas, the plurality of second areas configured to maintain
at least one second substance, and at least one second substance
maintained in at least one of the second areas, wherein in the
first position the first set of ducts is exposed to the plurality
of second areas, and moving the device from the first position into
a second position, wherein in the second position, at least one of
the first plurality of first areas and at least one of the
plurality of second areas are only exposed to one another, thereby
reacting the at least one first and second substances.
[0017] Yet another embodiment of the present invention includes a
method for carrying out a reaction, the method including the steps
of providing a device in a first position, wherein the device
includes a base having a first surface, a first area located along
a portion of the first surface, where the first area is configured
to maintain at least one first substance, at least one first
substance in the first area, an upper plate having a second surface
facing the first surface, a second area located along a portion of
the second surface configured to maintain at least one second
substance, at least one second substance in the second area, and an
intermediate plate disposed between the first surface of the base
and the second surface of the upper plate having a opening formed
therethrough, wherein the base, the upper plate and the
intermediate plate are configured to slide relative to one another,
and wherein in the first position the first area is not exposed to
the second area via the opening, and moving the device from the
first position into a second position, wherein in the second
position, the first area is exposed to the second area via the
opening, thereby reacting the at least one first and second
substances.
[0018] Yet another embodiment of the present invention includes a
kit for offering an inventory of reagents, receiving from the
customer a desired subset of reagents, and delivering a kit to the
customer, wherein the kit includes a base having a first surface
and a first area located along a portion of the first surface,
where the first area is configured to maintain at least one first
substance, and a plate having a second surface and an second area
located along a portion of the second surface, where the second
area is configured to maintain at least one second substance, and
either a first substance in the first area or a second substance in
the second area, wherein the first surface of the base and the
second surface of the plate are configured such that when fitted
together they are opposed to each other and move relative to the
other between a first position, where the first area and the second
area are not exposed to one another, and a second position, where
the first area and the second area are exposed to one another, and
wherein at least one of the first substance and the second
substance is an element of the desired subset of reagents.
[0019] Yet another embodiment of the present invention includes a
kit for offering an inventory of reagents, receiving from the
customer a desired subset of reagents, and delivering a kit to the
customer, wherein the kit includes a base having a first surface, a
first plurality of first areas located along a portion of the first
surface, where the first plurality of first areas have a first
pattern and are configured to maintain at least one first
substance, and a first set of ducts formed along a portion of the
first surface that are not exposed to the first plurality of first
areas, a plate having a second surface and a plurality of second
areas located along a portion of the second surface where the
plurality of second areas have a pattern substantially similar to
the pattern of the first plurality of first areas, and where the
plurality of second areas configured to maintain at least one
second substance, and at least one of a first substance in the
first area and a second substance in the second area, wherein the
first surface and the second surface are configured such that when
fitted together they slide relative to one another between a first
position, where the first set of ducts is exposed to the plurality
of second areas, and a second position, where at least one of the
first plurality of first areas and at least one of the plurality of
second areas are only exposed to one another.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1A is a side view of a slip chip device according to
one embodiment of the invention in a first position.
[0021] FIG. 1B is a side view of the slip chip device of the
embodiment shown in FIG. 1A in a second position.
[0022] FIG. 2 is a partial view of a slip chip device according to
another embodiment of the invention.
[0023] FIG. 3A is a perspective view of a slip chip device
according to another embodiment of the invention in a first
position.
[0024] FIG. 3B is a side view of the slip chip device shown in FIG.
3A in a second position.
[0025] FIG. 3C is a side view of the slip chip device shown in FIG.
3A in a third position.
[0026] FIG. 3D is a side view of the slip chip device shown in FIG.
3A in a fourth position.
[0027] FIG. 4A is a side view of a slip chip device according to
another embodiment of the invention in a first position.
[0028] FIG. 4B is a side view of the slip chip device shown in FIG.
4A in a second position.
[0029] FIG. 4C is a side view of the slip chip device shown in FIG.
4A in a third position.
[0030] FIG. 5A is a side view of a slip chip device according to
another embodiment of the invention in a first position.
[0031] FIG. 5B is a side view of the slip chip device shown in FIG.
5A in a second position.
[0032] FIG. 6A is a top view and a cross-sectional view of a slip
chip device according to another embodiment of the invention in a
first position.
[0033] FIG. 6B is a top view and a cross-sectional view of the slip
chip device of the embodiment shown in FIG. 6A in a second
position.
[0034] FIG. 7A is a partial view of a slip chip device according to
another embodiment of the invention in a first position.
[0035] FIG. 7B is a partial view of the slip chip device shown in
FIG. 7A in a second position.
[0036] FIG. 8A is a partial top view of a slip chip device
according to another embodiment of the invention in a first
position.
[0037] FIG. 8B is a partial view of a slip chip device shown in
FIG. 8A in a second position.
[0038] FIG. 8C is a partial view of a slip chip device shown in
FIG. 8A in a third position.
[0039] FIG. 8D is a partial view of a slip chip device shown in
FIG. 8A in a fourth position.
[0040] FIG. 9A is a top view of a slip chip device according to
another embodiment of the invention in a first position.
[0041] FIG. 9B is a top view of the slip chip device shown in FIG.
9A in a second position.
[0042] FIG. 10A is a partial top view of a slip chip device
according to another embodiment of the invention in a first
position.
[0043] FIG. 10B is a partial top view of the slip chip device shown
in FIG. 10B in a second position.
[0044] FIG. 11A is a partial top view of a slip chip device
according to another embodiment of the invention in a first
position.
[0045] FIG. 11B is a partial top view of the slip chip device shown
in FIG. 11A in a second position.
[0046] FIG. 11C is a partial top view of a slip chip device
according to another embodiment of the invention in a first
position.
[0047] FIG. 11D is a partial top view of the slip chip shown in
FIG. 11C in a second position.
[0048] FIG. 12A is a top view of a slip chip device according to
another embodiment of the invention in a first position.
[0049] FIG. 12B is a top view of the slip chip device shown in FIG.
12A in a second position.
[0050] FIG. 13A is a perspective view of a slip chip device
according to another embodiment of the invention in a first
position.
[0051] FIG. 13B is a perspective view of the slip chip device shown
in FIG. 13A in a second position.
[0052] FIG. 14A is a partial top view of a slip chip device
according to another embodiment of the present invention in a first
position.
[0053] FIG. 14B is a partial top view of the slip chip device shown
in FIG. 14A in a second position.
[0054] FIG. 15 is a partial side view of a slip chip device
accordingly to another embodiment of the invention.
DETAILED DESCRIPTION OF THE DRAWINGS AND THE PRESENTLY PREFERRED
EMBODIMENTS
[0055] The invention is described with reference to the drawings in
which like elements are referred to by like numerals. The
relationship and functioning of the various elements of this
invention are better understood by the following detailed
description. However, the embodiments of this invention as
described below are by way of example only, and the invention is
not limited to the embodiments illustrated in the drawings. While
not intending to be bound by theory, in several of the examples
below the inventors propose theories by which the invention is
believed to operate. Any statements which propose a scientific
theory by which an invention is believed to operate are not
intended as, and should not be treated as, a limitation on the
claimed invention.
[0056] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" indicate plural references
unless the context clearly dictates otherwise. Thus, for example,
reference to "a substance" includes a single substance as well as a
plurality of substances, reference to "an area" includes a single
area as well as a plurality of areas, "a duct" includes a single
duct as well as a plurality of ducts, and so forth.
[0057] The term "area" as used herein refers to a site where two or
more substances are exposed to one another. The "area" may also
refer to a portion along a surface that is capable of maintaining a
substance therein or therealong. The "area" may take on a physical
structure such as a hole, a well, cavity, or indentation, and have
any cross-sectional shape along its length, width or depth, such as
rectangular, circular, or triangular.
[0058] The term "between" when used in the context of moving
between "a first position" and a "second position" may mean to move
only from a first position to a second position, move only from a
second position to a first position, or move from a first position
to a second position and from the second position to the first
position.
[0059] The term "closed system" may refer to a system that can
exchange heat and energy but not matter, with its surroundings. For
certain embodiments, the closed system can be one in which liquid
cannot be exchanged with its surroundings, but gases, such as water
vapor or oxygen, can be. For certain embodiments, the closed system
can be one in which liquid water cannot be exchanged with its
surroundings, but gases, such as water vapor or oxygen, or
substances that can permeate a lubricating layer or substrate, can
be. also be non-Newtonian fluids, for example shear-thickening
fluids. May also be gels, including hydrogels. May also be
carbohydrate-rich or lipid-rich phases, including lipidic cubic
phase and other lipid mesophases. In some embodiments, permeability
to gases may be desirable, for example in some applications that
use live cells and tissues inside the SlipChip.
[0060] The term "duct" may refer to a three-dimensional enclosure
through which a substance may be transported. Alternatively, it can
also refer to an open groove or a trench in a surface through which
a substance may also be transported. A duct can assume any form or
shape such as tubular or cylindrical, have a uniform or variable
(e.g., tapered) diameter along its length, and have one or more
cross-sectional shapes along its length such as rectangular,
circular, or triangular. As used herein, the term "duct" includes
microducts that are of dimensions suitable for use in devices. A
duct may be connected to at least one other duct through another
duct, area, or any other type of conduit.
[0061] In certain embodiments areas may also be ducts, and in
certain embodiments ducts may also be areas.
[0062] As mentioned above, the duct can have any cross-sectional
shape (circular, oval, triangular, irregular, square or
rectangular, or the like) and can be covered or uncovered. In
embodiments where it is completely covered, at least one portion of
the duct can have a cross-section that is completely closed, or the
entire duct may be completely enclosed along its entire length with
the exception of inlet(s) and outlet(s). A duct generally will
include characteristics that facilitate control over substance
transport, e.g., structural characteristics and/or physical or
chemical characteristics (hydrophobicity vs. hydrophilicity) or
other characteristics that can exert a force on a fluid. The
substance within the duct may partially or completely fill the
duct. In some cases where an open duct is used, the substance, such
as a fluid, may be held within the duct, for example, using surface
tension (i.e., a concave or convex meniscus).
[0063] The duct may be of any size, for example, having a largest
dimension perpendicular to the direction of flow of a substance,
for example a fluid, of less than about 50 mm, less than about 5
mm, less than about 2 mm, less than about 1 mm, less than about 500
microns, less than about 200 microns, less than about 60 microns,
less than about 50 microns, less than about 40 microns, less than
about 30 microns, less than about 15 microns, less than about 10
microns, less than about 3 microns, less than about 1 micron, less
than about 300 nm, less than about 100 nm, less than about 30 nm,
or less than about 10 nm. In some cases the dimensions of the duct
may be chosen such that a substance is able to freely flow through,
or into, an area or other ducts. The dimensions of the duct may
also be chosen, for example, to allow a certain volumetric or
linear flow rate of fluid in the duct. Of course, the number of
ducts and the shape of the ducts can be varied by any method known
to those of ordinary skill in the art.
[0064] The term "exposed" as used herein is a form of communication
between two or more elements. These elements may include a
substance, an area, a duct, a passage, a channel, a lumen, or any
combination thereof. In some instances, "exposed" may mean that two
or more substances are in fluidic communication with each other, or
alternatively, it may mean that two or more substances react with
one another.
[0065] The term "fluidic communication," as used herein, refers to
any duct, channel, tube, pipe, or pathway through which a
substance, such as a liquid, gas, or solid may pass substantially
unrestricted when the pathway is open. When the pathway is closed,
the substance is substantially restricted from passing through. In
embodiments where a substrate is present, a substance may pass from
one reaction area to another through the substrate when the device
is in the closed position, if the reaction areas are spatially
positioned to allow diffusion via the substrate versus passage via
a pathway. Typically, limited diffusion of a substance through the
material of a plate, base, and/or a substrate, which may or may not
occur depending on the compositions of the substance and materials,
does not constitute fluidic communication.
[0066] The terms "react" or "reaction" refer to a physical,
chemical, biochemical, or biological transformation that involves
at least one substance, e.g., reactant, reagent, phase,
carrier-fluid, or plug-fluid and that generally involves (in the
case of chemical, biochemical, and biological transformations) the
breaking or formation of one or more bonds such as covalent,
noncovalent, van der Waals, hydrogen, or ionic bonds. The term
includes typical photochemical and electrochemical reactions,
typical chemical reactions such as synthetic reactions,
neutralization reactions, decomposition reactions, displacement
reactions, reduction-oxidation reactions, precipitation,
crystallization, combustion reactions, and polymerization
reactions, as well as covalent and noncovalent binding, phase
change, color change, phase formation, dissolution, light emission,
changes of light absorption or emissive properties, temperature
change or heat absorption or emission, conformational change, and
folding or unfolding of a macromolecule such as a protein.
[0067] The term "substance" as used herein refers to any chemical,
compound, mixture, solution, emulsion, dispersion, suspension,
molecule, ion, dimer, macromolecule such as a polymer or protein,
biomolecule, precipitate, crystal, chemical moiety or group,
particle, nanoparticle, reagent, reaction product, solvent, or
fluid, and any one of which may exist in the solid, liquid, or
gaseous state, and which is typically the subject of an
analysis.
[0068] A for carrying out a reaction is shown in FIGS. 1A and 1B.
FIGS. 1A and 1B are a cross-sectional view of the device 10 taken
along a longitudinal axis. The device 10 includes a base 12 and a
plate 14. A first surface 16 is formed along a portion of the base
12. A first area 18 is located along a portion of the first surface
16. A second surface 20 is formed along a portion of the plate 14
and has a second area 22 located along a portion of the second
surface 20. The first and second surfaces 16, 20 may be fixedly
opposed to one another and may be substantially planar, or
alternatively, may have complimentary surface characteristics to
permit relative movement between the first and second surfaces 16,
20. Moreover, the second surface 20 may be complex, non-planar,
and/or nonparallel to the first surface 16. The first and second
surfaces 16, 20 are capable of interfacing closely with one
another, and in some embodiments, pressure sealing techniques may
be employed, e.g., by using external means to urge the pieces
together (such as clips, springs, pneumatic or hydraulic means, or
clamping apparatuses). Moreover, to ensure that uniform pressure is
applied over the first and second surfaces 16, 20, the shape of the
surfaces may vary to ensure when pressure is applied in discrete
locations along the device 10, a uniform pressure across the
surfaces 16, 20 results. For example, when the two surfaces are
conical, pressure may be applied to bring two surfaces into close
contact. One or more of the plates may be designed to deform as the
pressure is applied, to re-distribute local pressure into uniform
pressure over entire surface.
[0069] In some embodiments, areas are filled with reagents that,
when exposed to one another, consume a gas, or cause a decrease in
pressure, and are configured such that they form a closed system.
For example, at least one first area may contain sodium hydroxide
and at least one second area may be filled with carbon dioxide.
Once the parts of the device 10 are moved to expose the at least
one first area to the second area, the reaction of the sodium
hydroxide with the carbon dioxide can form a partial vacuum. This
partial vacuum produces a force acting to hold the base 12 and the
plate 14 of the device 10 together.
[0070] The first and second surfaces 16, 20 may be planar or
nonplanar. For example, the surfaces can be cylindrical. The
relative motion in a cylindrical device 10 of the base 12 and plate
14 will be rotational. If the relative motion of base 12 and plate
14 is to be carried out manually, a handle could be fixed to either
base 12, plate 14 or both. It will be apparent to one skilled in
the art that the surfaces 16, 20 can be other closely interfacing
shapes. The first and second surfaces may be concentric
spheres.
[0071] The first and second surfaces 16, 20 may be made out of the
same material as the base 12 and plate 14, respectively.
Alternatively, the surfaces 16, 20 may be made out of any other
suitable material having a low coefficient of friction and may have
hydrophobic or hydrophilic properties. Moreover, the first and
second areas 18, 22 may also be made out of a different material,
or have different properties, than the first and second surfaces
16, 20 or the base 12 and plate 14, respectively.
[0072] Both the first area 18 and the second area 22, as shown in
the FIGS. 1A and 1B embodiment, are areas 23 configured to maintain
a substance therein. However, the first area 18 and the second area
22 may also be a surface pattern 25 of a substance, as shown in
FIG. 2, or a through hole, as shown in FIG. 4. As shown in FIG. 2,
it is not necessary that the first area 18 and the second area 22
have the same structural configuration, or maintain the same
substance, as the other.
[0073] Areas 18, 22 may also contain porous materials, for example
porous glass, aluminum oxide, or cellulose matrix found in paper.
Such areas may be made by deposition of the matrix into the area.
Alternatively, they may be made by patterning a porous layer and
filling the porous layer around the areas. For example, paper may
be patterned by methods described in Martinez, A. W., Phillips, S.
T., Carrilho, E., Thomas III, S. W., Sindi, H., Whitesides, G. M.,
Simple telemedicine for developing regions: Camera phones and
paper-based microfluidic devices for real-time, off-site diagnosis
(2008) Analytical Chemistry, 80 (10), pp. 3699-3707, Martinez, A.
W., Phillips, S. T., Butte, M. J., Whitesides, G. M. Patterned
paper as a platform for inexpensive, low-volume, portable bioassays
(2007) Angewandte Chemie--International Edition, 46 (8), pp.
1318-1320, Martinez, A. W. FLASH: A rapid method for prototyping
paper-based microfluidic devices (2008) Lab Chip, and Macek, K.,
Be{hacek over (c)}va{hacek over (r)}ova, H. Papers, ready-for-use
plates, and flexible sheets for chromatography (1971)
Chromatographic Reviews, 15 (1), pp. 1-28, and other materials may
be patterned by methods described in Vozzi, G., Flaim, C.,
Ahluwalia, A., Bhatia, S. Fabrication of PLGA scaffolds using soft
lithography and microsyringe deposition (2003) Biomaterials, 24
(14), pp. 2533-2540, Desai, T. A., Hansford, D. J., Leoni, L.,
Essenpreis, M., Ferrari, M. Nanoporous anti-fouling silicon
membranes for biosensor applications (2000) Biosensors and
Bioelectronics, 15 (9-10), pp. 453-462, Pichonat, T.,
Gauthier-Manuel, B. Development of porous silicon-based miniature
fuel cells (2005) Journal of Micromechanics and Microengineering,
15 (9), pp. S179-S184, Cohen, M. H., Melnik, K., Boiarski, A. A.,
Ferrari, M., Martin, F. J. Microfabrication of silicon-based
nanoporous particulates for medical applications (2003) Biomedical
Microdevices, 5 (3), pp. 253-259, De Jong, J., Ankone, B.,
Lammertink, R. G. H., Wessling, M. New replication technique for
the fabrication of thin polymeric microfluidic devices with tunable
porosity (2005) Lab on a Chip--Miniaturisation for Chemistry and
Biology, 5 (11), pp. 1240-1247, Ohji, H., Lahteenmaki, S., French,
P. J. Macro porous silicon formation for micromachining (1997)
Proceedings of SPIE--The International Society for Optical
Engineering, 3223, pp. 189-197, Chu, K.-L., Gold, S., Subramanian,
V., Lu, C., Shannon, M. A., Masel, R. I. A nanoporous silicon
membrane electrode assembly for on-chip micro fuel cell
applications (2006) Journal of Microelectromechanical Systems, 15
(3), pp. 671-677, Petronis, S., Gretzer, C., Kasemo, B., Gold, J.
Model porous surfaces for systematic studies of material-cell
interactions (2003) Journal of Biomedical Materials Research--Part
A, 66 (3), pp. 707-721, Wang, M., Feng, Y. Palladium-silver thin
film for hydrogen sensing (2007) Sensors and Actuators, B:
Chemical, 123 (1), pp. 101-106, to fill and/or coat the regions
around the areas, all of which are incorporated herein by
reference.
[0074] Referring back to the embodiment shown in FIGS. 1A and 1B,
the first and second surfaces 16, 20 are substantially opposed to
one another. A substrate 24 may be disposed between the first and
second surfaces 16, 20 to help maintain a substance within each
area 18, 22, or may operate to protect each area 18, 22 from
cross-contamination. The substrate 24 is typically comprised of a
material that is substantially inert with respect to the substances
that will be in contact with and/or transported through the device
10. The substrate 24 is also typically comprised of a material that
is substantially immiscible with the substances that will be in
contact with and/or transported through the device 10.
[0075] The substrate 24 may be a hydrocarbon or a fluorinated
substance, Fluorinated substances that can be used in the invention
include but are not limited to fluorocarbons, perfluorocarbons,
alkyl and aryl fluorocarbons, halofluorocarbons, fluorinated
alcohols, fluorinated oils, and liquid fluoropolymers including
perfluoropolyethers). Examples include, but are not limited to,
perfluorooctyl bromide, perfluorooctylethane,
octadecafluorodecahydronaphthalene,
1-(1,2,2,3,3,4,4,5,5,6,6-undeca-fluorocyclohexyl)ethanol,
C.sub.6F.sub.11C.sub.2H.sub.4OH, Flourinert (3M), Krytox oils,
Fomblin oils, and Demnum oils. Hydrocarbon substances include but
are not limited to, alkanes or mixtures of alkanes (e.g. paraffin
oils such as hexane, hexadecane, and mineral oil), other organic
materials and polymers. Other fluid material includes silicon oils
and various greases (e.g. Dow Corning high vacuum grease, Fomblin
vacuum grease, Krytox greases), and ionic fluids. Fluids can also
be non-Newtonian fluids, for example shear-thickening fluids, gels,
including hydrogels, and carbohydrate-rich or lipid-rich phases,
including lipidic cubic phase and other lipid mesophases. In some
embodiments, permeability to gases may be desirable, for example in
some applications that use live cells and tissues inside the
SlipChip. Surfactants may be added to the substrate, for example to
cause or prevent surface aggregation and/or to influence the
stability of substances. Lubricating powders or bead could also be
used. Variations or versions of some of the above materials may
apply here and include but are not limited to various Teflon beads
or powders which could be composed of PTFE, PFA or FEP Teflon
materials. Other dry lubricants include graphite, molybdenum
disulfide and tungsten disulfide. The substrate may also be a solid
membrane. For example, if bead-based reagents are used in an area,
the membrane may be capable of preventing motion of the beads from
an area 18 to an area 22 while still allowing diffusion of other
substances from area 18 to area 22. Such a membrane could be, for
example, a Teflon membrane or a polycarbonate membrane or a
cellulose membrane or any other membranes. In certain embodiments,
typically when the substrate 24 is a liquid, it may partially fill
areas and/or ducts of the device. In particular, in certain
embodiments, surface tension may cause substrate 24 to divide a
sample fluid present in a volume into separate plugs or droplets
separated by substrate 24. If the volume varies in cross-section
along its length, the substrate 24 may, for example, be mostly
present in the portions of the volume with a larger cross-sectional
area, for example in ducts, and the sample may be mostly present in
the portions of the volume with a larger cross-sectional area.
[0076] FIG. 1A further illustrates the device 10 in a first
position, referred to as "Position A," and FIG. 1B illustrates the
device in a second position, referred to as "Position B". The
device 10, when in the first position, is in an orientation where
the first surface 16 is opposed to the second surface 20 and is
configured to move in a direction substantially perpendicular to
the normal of the second surface 20 such that the vertical distance
(as defined when the device is oriented as shown in FIG. 1A)
between the first surface 16 and the second surface 20 remains at a
substantially constant value. The distance, or gap, between the
first surface 16 and the second surface 20 may vary depending on
the existence of a substrate and the type of substrate. In certain
embodiments, the distance may vary in different device positions,
for example due to design or due to surface roughness. Generally
speaking, the gap may range anywhere from 0.2 nanometers to 20
micrometers.
[0077] When in the first position, the first area 18 and the second
area 22 each contain a substance, but the first and second areas
18, 22 and therefore the substances, are not exposed to one
another. When in the second position, at least one of the base 12
or the plate 14 moves relative to the other in a direction
perpendicular to the normal of the base 12 thereby exposing the
first and second areas 18, 22 to each other. In this embodiment, as
depicted in FIGS. 1A and 1B, the first and second areas 18, 22 are
only exposed to one another when one overlaps with the other.
However, the level of exposure and overlap may vary, and as shown
in FIG. 2, the second position may be reached when only a portion
of the first and the second areas 18, 22 overlap. It is also
contemplated that other configurations will allow two or more areas
to be exposed to each other without any of the areas overlapping,
as will be discussed later with respect to other embodiments of the
present invention. Independent of how the first and second areas
18, 22 are exposed to one another, the exposure allows the
substances in the first and second areas 18, 22 to react with each
other.
[0078] However, in each of the embodiments discussed herein it is
contemplated that when the device 10 is in the second position
there may be at least one first area 18 and corresponding second
area 22 overlapping such that no other substance will be exposed
to, or in communication with, that first and second areas 18, 22.
Accordingly, respective first and second areas 18, 22 will not be
exposed to, or in communication with, any channel, duct, inlet,
outlet, or any other structure that is configured to provide a
substance therein.
[0079] At least one of the base 12 and plate 14 may further move
with respect to the other to separate the first and second areas
18, 22 such that they are no longer exposed to each other. The base
12 and/or plate 14 may move back to the first position, or move to
a third position that is different from the first position to
separate the first and second areas 18, 22. The relative movement
between the base 12 and plate 14 may be guided by a guide/track
(not shown) configuration, or a ball bearing configured to
slidingly engage the base 12 and the plate 14 in order to limit the
direction and amount of relative movement between the base 12 and
the plate 14. In addition, the relative movement between the base
12 and the plate 14 may be automated. In any of the embodiments
discussed herein, the device 10 may also include a detector, such
as an imaging or sensor components to record and/or measure
reactions within the device 10. Examples of such detectors and
imaging devices can be found in U.S. Publication No. 2009/0010804
and WO 2008/002267, both of which are incorporated herein by
reference. The detector may be any detector suitable to detect the
may be selected from the group consisting of: a web camera, a
digital camera, a digital camera in a mobile phone and a video
camera, as described in published patent application WO
2008/002267, incorporated by reference herein in its entirety.
Alternatively, the detector can be a camera or imaging device which
has adequate lighting and resolution for spatially resolving
individual signals produced by the device, as described in US
2009/0010804, incorporated by reference in its entirety. In this
regard, an imaging device of the present invention can be any known
in the art that is compatible with the various designs and
configurations of the instant device. For example, the camera can
employ any common solid state image sensor including a charged
coupled device (CCD), charge injection device (CID), photo diode
array (PDA), or complementary metal oxide semiconductor (CMOS). The
device may incorporate markers, such as lines, dots or visible
substances in ducts and/or areas to enable registration and/or
analysis. Registration marks may be included on the device to allow
for automatic correction of optical aberrations, or adjustment of
the image for the angle and orientation at which the picture was
taken. For detecting fluorescent output, chirped excitation/readout
can be used. For example blue excitation light may be shined on the
device for, for example, nanoseconds, then turned off, and
fluorescence may be detected a, for example, nanosecond later.
Then, ten nanoseconds later, for example, another image is
collected (without an initial excitation flash) to produce a
background intensity image for subtraction. In this manner,
fluorescence can be analyzed even in daylight. For safety, the
detector could be designed to automatically recognize the device,
for example if the device comprised a recognizable pattern, such
that the detector would only produce the excitation light when
pointed at the device. Sia, et al., Angewandte Chemie International
Edition, (43), 4, 498-502, incorporated by reference herein,
describes additional means for detecting signals in multifluidic
devices, including using pulse modulation to reduce noise.
Detection can also be improved by using the polarization of
excited/emitted light, as is known to those skilled in the art.
[0080] It can be appreciated that the number, configuration, or
orientation of first and/or second areas 18, 22 is application
dependent and may vary from application to application and can
include an infinite number of configurations. Accordingly, by way
of example, FIGS. 3A-D illustrates another embodiment of the device
10. In this and other figures where appropriate, solid lines
indicate features associated with the plate 14 and the second
surface 20 and dashed lines indicate features associated with the
base 12 and first surface 16. In this embodiment, the device 10
includes the base 12 having one first area 18 and the plate 14 now
having two second areas 22. The two second areas 22 are located
along a portion of the second surface 20, but are separate and not
directly exposed to each other.
[0081] Depending on the relative movement between the base 12 and
the plate 14, the first area 18 may be exposed to only one of the
second areas 22, the other second area, or simultaneously to both
of the second areas 22. For instance, as shown in Position A of
FIG. 3A, the first area 18 is not exposed to the two second areas
22 and the two second areas 22, in this position, are also not
exposed to each other.
[0082] The base 12 and/or plate 14 may move relative to the other
from Position A, of FIG. 3A, to another position such that the
first area 18 is now only exposed to one of the second areas 22, as
shown in Position B in FIG. 3B, or is only exposed to the other of
the second areas 22, as shown in Position C in FIG. 3C, or is
simultaneously exposed to both the second areas 22 as shown in
Position D in FIG. 3D. The base 12 and/or plate 14 may further move
from the second position to additional positions that will allow
for different configurations and reactions. Of course, the sequence
in which the first area 18 and at least one of the two second areas
22 are exposed to the other will govern the substances to be
reacted and the reaction itself.
[0083] The embodiment of FIGS. 3A-D only contains one plate 14,
however, there are other embodiments that contemplate using more
than one plate 14. For example, in the embodiment shown in FIGS.
4A-C, between the plate 14 and the base 12 is an intermediate plate
46. Similar to the plate 14 and base 12, the intermediate plate 46
is configured to slide relative to either element and further
defines an opening 48 therein.
[0084] As shown in FIGS. 4A-C, the device 10 in this embodiment is
configured to have three different substances disposed therein,
with one substance being disposed within, or along, the first area
18, a second substance being disposed within, or along, the second
area 22 and a third substance being disposed within, or along, the
opening 48. The first surface 16, the second surface 20, and the
intermediate plate 46 are all configured to move relative to one
another. In this embodiment, the device 10 is configured to move
from a first position, Position A in FIG. 4A, where the first area
18, the second area 22, and the opening 48 are not exposed to one
another, to a second position, Position B in FIG. 4B, where the
second area 22 is exposed to the opening 48, and to a third
position, Position C in FIG. 4C, where the first area 18, second
area 22, and opening 48 are all exposed to one another to allow for
the three substances to react. It can be appreciated that the order
in which the areas 18, 22 and the opening 48 are exposed to one
another, if at all, can vary and the number of intermediate plates
48 may vary depending on the application.
[0085] The embodiment of the device 10 in FIGS. 5A and B, is one
example having more than one intermediate plate 46 in a stacked
configuration. This embodiment includes a plurality of intermediate
plates 46, with each of the plurality of intermediate plates 46
having an opening 48 therethrough to form, when aligned with the
other openings 48, a continuous column 50. A substance then may be
disposed within the column 50 through one of the openings 48 or via
an inlet port (not shown). The stack of intermediate plates 46 can
be used for multiple substance testing, or be used to fill and
store a plurality of intermediate plates 46 for future tests. A
holder (not shown) may also be included to provide stability,
control of relative movement of plates 46, and control of
evaporation of a substance contained by the plates 46.
[0086] As mentioned above, this embodiment of the device 10 may be
used for multiple substance testing. For example, one intermediate
plate 46 can be moved relative to the other intermediate plates 46,
or partially "slipped" out, in at least a first direction from a
first position, Position A of FIG. 5A, to a second position,
Position B of FIG. 5B, such that the opening 48 of that
intermediate plate 46 can be exposed to the first area 18 along the
first surface 16 of the base 12 which is in the form of a receiving
structure 52 configured to receive the intermediate plate 46.
[0087] The stack of intermediate plates 46 may have a biasing
mechanism or system to apply a biasing force when one of the
intermediate plates 46 is removed such that the column 50 is kept
intact. For example, the stack of intermediate plates 46 may be
bounded by an upper plate 54 and a lower plate 56, such that when
the top intermediate plate 46 is removed the biasing mechanism will
push the remaining stack of plates upwardly such that the next
intermediate plate 46 is now adjacent to the upper plate 54.
[0088] Alternatively, the intermediate plate 46 that is exposed to
the first surface 16 may be slipped in a direction substantially
perpendicular to the first direction and in a direction
substantially opposite from the first direction such that that
intermediate plate 46 is placed within the stack of intermediate
plates 46 but the opening 46 is no longer in communication of the
column 50, and the column 50 is no longer intact. The remaining
openings 48 in the intermediate plates 46 may then be subsequently
slipped out and caused to be exposed to another, or the same, first
area 18 of the receiving structure 52. It can be appreciated that a
plurality of intermediate plates 46 may be used for a single device
10, and the embodiment described above is exemplary of the multiple
contemplated configurations. Moreover, the features discussed
herein with respect to the embodiments having only the base 12 and
the plate 14 without the intermediate plate 46 may also be
accomplished in embodiments having one or more intermediate plates
46.
[0089] In certain embodiments, any of the base or plates may be
replaced with another new base or plate containing, where the new
base or plate has a different configuration of areas and/or a
different substance in its areas. For example, a device may be used
to conduct a solid-phase reaction, such as an on-bead synthetic
reaction, in an area on a base. The reagents for this reaction
would be added in one or more steps using any of the techniques
described herein. After the reaction is complete, the plate may be
removed and replaced by a new plate, containing ducts and/or areas
suitable for assaying the products of the reaction that are located
on the beads within the area of the base. Optionally, the plate may
have reagents preloaded to conduct the assay. In another
embodiment, the reaction product may be cleaved off the beads in
the base and allowed to diffuse into a reaction area on the plate,
and then the base is removed and a new base is added containing
ducts and/or areas and/or reagents for further reactions and/or
assays.
[0090] Moving on to the two plate embodiment shown in FIGS. 6A and
6B, the number and configuration of first areas 18 may also be
greater than one and coincide with the number of second areas 22.
FIGS. 6A and B are top fragmentary views of one embodiment of the
device 10, having the base 12 illustrated in dashed lines and the
plate 14 illustrated in solid lines, with a plurality of first and
second areas 18, 22. A series of discrete ducts 26 are formed along
a portion of the first surface 16. The series of discrete ducts 26
are independent from one another and do not independently form a
continuous fluidic path. The number of discrete ducts 26 may range
from one to more than one. The physical characteristics may vary
between each duct 26 of the series of ducts 26 and are application
dependent.
[0091] The series of discrete ducts 26 are spaced apart from, and
not in communication with, the plurality of first areas 18. One or
more of the discrete ducts 26 may include an inlet duct 28 and
another may be an outlet duct 30. The inlet duct 28 and the outlet
duct 30 may be formed along the first 16 or second 20 surface, and
it is not required that the inlet duct 28 and the outlet duct 30 be
formed along the same surface 16, 20. In the embodiment shown in
FIGS. 6A-B, the inlet duct 28 and outlet duct 30 are formed along
the second surface 18. In some embodiments having a plurality of
first and second areas 18, 22, the number of inlet ducts 28 will be
less than half the total number of areas 18, 22 for that particular
embodiment. In other embodiments, the number of outlet ducts 30
will be less than half the total number of areas 18, 22.
[0092] When in the first position, as shown as Position A in FIG.
6A, the first and second surfaces 16, 20 are fixedly opposed to one
another and the plurality of second areas 22 are exposed to the
series of discrete ducts 26, for example, to allow fluid
communication between the series of ducts 26 and the second areas
22 to dispose a first substance along, or within, the second areas
22. In this embodiment, the first substance is provided to the
series of discrete ducts 26 and the second areas 22 via the inlet
duct 28. Any excess substance is exited via the outlet duct 30.
[0093] Once the substance is disposed within or along the second
areas 22, the base 12 and/or plate 14 may move relative to one
another towards the second position, shown as Position B of FIG.
6B. When in Position B, the fluidic communication between the
series of discrete ducts 26 and the second areas 22 is broken, and
no additional substance provided by the inlet duct 28 may be
disposed within or along the second areas 22. The second position,
referred to as Position B, is user defined, and in this embodiment
is attained when each second area 22 is exposed with the respective
first area 18. The exposure of each second area 22 to the
respective first area 18 allows the first substance to communicate,
and possibly react, with any other substance that is disposed
within or along the first areas 18. The base 12 and/or plate 14 may
then be moved to another position, if necessary.
[0094] A device may be configured with an inlet duct or area in a
base capable of being dipped into a sample. The inlet duct or area
may be concave, in order to capture a sample, or may contain a
wicking material. An inlet duct or area designed for capturing
sample may be exposed to the environment, that is, not covered by
an opposing plate, in a first, loading position, but covered by an
opposing plate in a second position, after motion of the base and
opposing plate relative to one another.
[0095] In an alternative embodiment, as shown in FIGS. 7A and 7B,
the first and second areas 18, 22 may be formed along, or within,
the same surface. For example, in this embodiment, the first and
second areas 18, 22 are formed along the first surface 16 of the
base 12. The ducts 26 in this embodiment are formed along the
second surface 20 of the plate 14.
[0096] In this embodiment, the device 10 is configured to move from
a first position, Position A of FIG. 7A, where two or more second
areas 22 are in fluidic communication with, or exposed to, the
ducts 26, but where none of the first and second areas 18, 22 are
exposed to one another, to a second position, Position B of FIG.
7B. When in the second position, corresponding first and second
areas 18, 22 are exposed to one another via one of the ducts 26.
Specifically, the relative movement between the first and second
positions caused the ducts 26 to move with respect to the first 18
and second 22 area from the first position, Position A, where each
duct 26 was exposed two adjacent second areas 22 and allowed for
fluidic communication therebetween to the second position, Position
B, shown in FIG. 7B, where each duct 26 is now exposed to one first
area 18 and one corresponding second area 22. Accordingly, as shown
in this embodiment, the first and second areas 18, 22 may be
exposed to one another via the duct 26 and it is not required that
the first and second areas 18, 22 physically overlap. However, the
number and orientation of the areas 18, 22 configured to be exposed
to each other via the duct is application dependent.
[0097] In any embodiment discussed above, the relative movement
between the base 12 and the plate 14 of the device 10, and any
intermediate plates 46, may vary in direction and in distance. For
example, unlike the single direction of movement disclosed in FIGS.
4A-C, the embodiment of the device 10 shown in FIGS. 8A-D
illustrates a plurality of first and second areas 18, 22, with each
set of areas having its own set of discrete ducts 26 in a matrix
configuration. Specifically, the device 10 of this embodiment
includes a plurality of first areas 18 on the first surface 16 of
the base 12, and has a series of first ducts 40 formed within the
first surface 16 that are not in direct fluid communication with
the first areas 18. The second surface 20 of the plate 14 includes
a plurality of second areas 22 and a series of second ducts 42
formed therein that are not in direct fluidic communication with
the second areas 22.
[0098] When in the first position, as shown as Position A in FIG.
8A, the first surface 16 is fixedly opposed to the second surface
20 in an orientation such that the first areas 18 are in fluidic
communication, or exposed, to the second set of ducts 42, but the
second areas 22 are not in fluidic communication, or exposed to,
the first areas 18 or the first set of ducts 40. When in this
position, the first areas 18 can be filled with a substance, or
each row of first areas 18 can be filled with a different
substance. The relative movement in the first direction between the
first surface 16 and the second surface 20 to the second position,
Position B in FIG. 8B, isolates each one of the first area 18 and a
corresponding one of the second set of ducts 42 from other first
areas 18 and second set of ducts 42. Further movement in a second
direction, to Position C in FIG. 8C, which is substantially
perpendicular to the first direction, causes the second areas 22 to
be in fluidic communication, or exposed to, the first set of ducts
40 and allows the second areas 22 to be filled with another
substance, or each column of second areas 22 can be filled with a
different substance. Further movement to Position D in FIG. 8D, in
a direction opposite from the first direction, causes the first
areas 18 to be at least partially exposed to the second areas 22.
It can be appreciated that devices 10 may have a greater or lesser
number of rows and columns and the relative movement between the
first surface 16 and the second surface 20 may vary depending on
the particular application.
[0099] As mentioned above, the device 10 when moving between any
two positions moves in a direction substantially perpendicular to
the normal of the first surface 16. Accordingly, the direction may
be linear, rotational, or a combination of both. In some instances,
two-dimensional motion (e.g., X-Y motion) may be accomplished
through a combination of linear and/or rotational movements. For
example, sliding and rotating means may be employed to effect
linear and rotational sliding motion. In addition, such means for
producing relative sliding motion may be constructed from, for
example, motors, levers, pulleys, gears, hydraulics, pneumatics, a
combination thereof, or other electromechanical or mechanical means
known to one of ordinary skill in the art. Other examples of
methods of controlling the motion of one part relative to another
include, but are not limited to, sliding guides, rack and pinion
systems (U.S. Pat. No. 7,136,688), rotational plates (U.S. Pat. No.
7,003,104), slider assemblies (US 2007/015545 and US 2008/0058039),
guide grooves (U.S. Pat. Nos. 5,805,947 and 5,026,113),
piezoelectric actuators (US 2005/0009582), ball bearings and
notches (U.S. Pat. No. 2,541,413) and drive cables (U.S. Pat. No.
5,114,208). These patents and patent applications are incorporated
herein by reference in their entireties.
[0100] Moreover, motion of the base 12 and plate 14 or plates
relative to one another may be constrained by notches, retainers,
and/or a system of holes and mating pins, for example, as are
typically used alone or in combination in electrical connectors.
Also, the motion of the base 12 and plate 14 or plates relative to
one another may be constrained by a case, posts, grooves and
ridges, gears, or, for example in the case of rotational motion, a
central axle. In certain embodiments, the device 10 is configured
to be manipulated by a robot.
[0101] For example, in the embodiment shown in FIGS. 9A and 9B, the
relative movement between the first and second surfaces 16, 20 is
rotational in nature. Specifically, the device shown in FIG. 9A
moves from the first position, Position A, where the second areas
22 are in fluidic communication with the series of ducts 26 and an
inlet duct 28. It can be appreciated that in this embodiment, there
may be no outlet duct 30. The way in which the first substance is
disposed in, or along, the second areas 22 may vary. For example,
an external pump may create a line pressure to help dispose the
first substance 32 in, or along, the second areas 22.
Alternatively, and as shown in the embodiment in FIGS. 9A-B, the
rotation of the entire device 10 creates a centrifugal force that
helps the first substance 22 to travel from the inlet duct 28 to
the second areas 22.
[0102] The base 12 and plate 14 are then moved from the first
position, Position A, to the second position, Position B shown in
FIG. 9B, by relative rotational movement. In this position, at
least one first area 18 is exposed to at least one second area 22.
The relative rotational movement may be caused, in part, by, for
example, an automated gear assembly 36 or by manual movement.
[0103] The pattern and shape of each embodiment of the device 10
may also vary and is application dependent. For example, in an
alternate embodiment, shown in FIGS. 10A and 10B, the first area 18
is a continuous channel 21, configured to maintain a substance,
formed within the first surface 16. A series of post members 38 are
formed along the second surface 20, adjacent to the second areas 22
that do not impede the continuality of the first area 18 when in
the first position, Position A in FIG. 10A. When in Position A, the
first area 18 is not exposed to the second areas 22. However, when
moved into the second position, Position B, the series of post
members 38 engage with a portion of the first area 18, such as to
compartmentalize the previously continuous channel 21 into a
plurality of discrete first areas 18 that are not in fluid
communication with the other discrete first areas 18. Each of the
discrete first areas 18, when in Position B shown in FIG. 10B, is
exposed to the second areas 22.
[0104] Moreover, a combination of a post member 38 along the second
surface 18 and the first area 18 along the first surface 16 may be
used to generate pressure as the surfaces 16, 18 move relative to
one another. For example, positive pressure may be generated in
front of the direction of the post member 38, and negative pressure
may be generated behind. It may be used to load a substance into
the device 10 or dispose substance out of the device 10, or move a
substance within the device 10, or to introduce separations as
discussed infra, including filtrations. Flow may also be generated
by such movement.
[0105] In addition to the variance of the shape of the first and
second areas 18, 22 between embodiments, the amount of exposure and
the relative exposure between each respective set of first and
second areas 18, 22, may also vary and is application dependent.
For example, the embodiment of the device 10 shown in FIGS. 11A and
11B varies the amount of exposure between each respective set of
first and second areas 18, 22 when in the second position, Position
B as shown in FIG. 10B. The varied, or graduated, amount of
exposure between each set of first and second areas 18, 22 can be
achieved by, for example configuring the pattern of the first set
of areas 18 and the second set of areas 22.
[0106] For example, the amount of exposure, or diffusion, between
the first and second areas 18, 22 may be attained in a number of
ways. For example, as shown in FIGS. 11A and 11B, the first and
second areas 18, 22 are substantially square shaped with the amount
of overlap between each set of the first and second areas 18, 22
varied by the graduated diagonal pattern of the first areas 18 when
in the second position as shown in FIG. 11B. Alternatively, in the
embodiment shown in FIGS. 11C and 11D, the amount of exposure, or
diffusion, is controlled by varying the shape and/or diameter of an
inlet portion 34 of the second area 22 that is exposed to the first
area 18 when in the second position as shown in FIG. 11D. As
discussed herein, gradients may be generated by controlling
diffusion of substances between areas of the device. The level of
diffusion in each step of the method of gradient generation of the
present invention may be controlled according to the slip position
of the device. Gradients of the present invention are useful in
studying biological phenomena that depend on gradient
concentration, such as cell-surface interactions, high-throughput
screening using arrays of cells, and in cell-based biosensors. In
particular, studies involving chemotaxis, haptotaxis and migration
take advantage of the relatively compact and stable gradients
achievable by the present invention. As chemotactic cells may be
sensitive to concentration differences as small as 2% between the
front and back of the cell, gradients with a resolution on the
order of a single cell (10-100 .mu.m, 2-20% per 100 .mu.m) can be
useful. The invention provides the ability to generate gradients of
proteins, surface properties, and fluid streams containing growth
factors, toxins, enzymes, drugs, and other types of biologically
relevant molecules. In addition, gradients of diffusible substances
having chemoattractant or chemorepellent properties can play an
important role in biological pattern formation, and angiogenesis
and axon pathfinding provide examples of processes that can make
use of gradients. The invention also provides the superimposition
of gradients (similar or dissimilar) of different substances in
studying higher organisms. The sawtooth gradients of the present
invention can also be used in investigating biological processes.
The gradients of the present invention may be used for additional
applications as described in US 2004/0258571, U.S. Pat. No.
6,705,357, U.S. Pat. No. 7,314,070 and U.S. Pat. No. 6,883,559, the
entireties of all of which are herein incorporated by
reference.
[0107] Other embodiments exist of the device 10 where the first and
second areas 18, 22 form a continuous channel to expose two or more
substances to each other. For example, embodiment of the device 10
as shown in FIGS. 12A and B, includes an inlet duct 28 in a
branch-like formation formed along the second surface 20. Also
formed within, or along, the second surface 20 are multiple series
of the second areas 22. The multiple series of second areas 22 and
the inlet duct 28 however, are not directly in fluidic
communication with, or exposed to, each other when in the first
position, Position A as shown in FIG. 12A. A multiple series of
first areas 18 are formed within, or along, the first surface 16
along with multiple outlet ducts 30, each of which is aligned with
each series of first areas 18, but are not in direct fluid
communication with, or exposed to, each other when in Position
A.
[0108] In this embodiment, a substance or a series of substances
may be placed within or along each of the first areas 18. When the
first and second surfaces 16, 20 move relative to each other from
the first position to the second position, Position B as shown in
FIG. 12B, a first area 18 and a second area 22 for each series of
areas 18, 22 overlap, or are exposed to one another, to form a
continuous series of first and second areas 22, 22, as shown in
FIG. 12B. Additionally, when in the second position, Position B as
shown in FIG. 12B, at least one of the first areas 18 is exposed
to, or in fluidic communication with, one branch of the inlet duct
28. Moreover, one of the second areas 22 is exposed to, or in
fluidic communication with, one of the outlet ducts 30 forming a
continuous path between the inlet duct 28 and the outlet duct 30 to
allow a substance to be exposed to the series of first areas 18.
The orientation and number of branches of the inlet duct 28 and
outlet ducts 30 may vary and is application dependant. However, as
can be seen in this embodiment, a plurality of substances may be
placed within or formed along each of the first areas 18 and can
react with the substance provided by the inlet duct 28 when in the
second position, Position B.
[0109] Continuous channels may also be used to preload other
reactions areas. For example in an alternative embodiment of the
device 10, as shown in FIGS. 13A and 13B, the device 10 may be
configured to fill, or preload, a number of second areas 22 with a
substance. In this embodiment, the base 12 has a continuous channel
44 configured to carry a first substance. The second area 22 of the
plate 14 and continuous channel 44 of the base 12 are configured to
move from a first position, Position A shown in FIG. 13A, where the
second area 22 or areas 22 are not in fluidic communication, or
exposed, to the continuous channel 44, to a second position,
Position B as shown in FIG. 13B, where at least a portion of the
second area 22, or areas 22, are exposed, or in fluidic
communication, with the channel 44 and thereby filling, or
disposing, the substance within, or along, the second area 22. The
base 12, and/or plate 14, are then configured to move relative to
one another to a third position (not shown) such that the second
area 22, or areas 22, are now filled with the substance. The plate
14 preloaded with the filled substance may then be used for
subsequent uses, some of which are described herein. It can be
appreciated that the base 12 may instead have the discrete first
areas 18 and the continuous channel 44 be formed within the plate
14 (not shown in this figure). Moreover, instead of preloading the
areas, this embodiment may also be used for exposing a second
substance within the second area 22 to continuous channel 44 filled
with the first substance within, or along, the first surface 16, or
vice versa.
[0110] In another embodiment a fragmentary view of which is shown
in FIGS. 14A and 14B the relative movement between the first and
second surfaces 16, 20 is rotational in nature. The first and
second etc. areas 18, 22 may be formed along, or within, the same
surface. The inlet duct 28 and series of ducts 26 in this
embodiment are formed along the second surface 20. In this
embodiment, the device is configured to rotate from a first
position, Position A as shown in FIG. 14A, where a set of two or
more first areas 18, a set of two or more second areas 22, etc. are
each in fluidic communication with, or exposed to, a corresponding
set of ducts 26, but where none of the first and second etc. areas
18, 22 are exposed to one another. For example, as shown in FIGS.
14A and 14B, when in Position A, as shown in FIG. 14A, seven first
areas 18 are exposed to one another via a series of radially
connected ducts 26 but are not exposed to any of second areas
22.
[0111] In a second position, Position B, as shown in FIG. 14B, the
first and second surfaces 16, 20 are then moved from the first
position, Position A, to the second position, Position B, by
relative rotational movement. In this position, at least one first
area 18 is exposed to at least one second area 22, etc. When in
Position B, corresponding first and second, etc. areas 18, 22 are
exposed to one another via a series of spirally connected ducts 26.
For example, as shown in FIGS. 14A and 14B, the relative movement
between the first and second positions caused the ducts to move
with respect to the first and second areas, etc. 18, 22 from the
first position, Position A, where each duct was exposed to adjacent
first areas 18 and allowed for fluidic communication between the
row of first areas 18 to Position B, where each duct is now exposed
to one first area 18, one corresponding second area 22, etc. via a
series of spirally connected ducts 26. The first and second
surfaces 16, 20 may then be moved from the second position,
Position B, to a third position, Position C (not shown in FIG. 14A
or 14B), by relative rotational movement in the same direction as
the motion from Position A to Position B. In this position as in
Position A, two or more first areas 18, two or more second areas
22, etc. are in fluidic communication with, or exposed to, the
ducts 26, but where none of the first and second etc. areas 18, 22
are exposed to one another. In this third position, Position C, two
or more first areas 18 are exposed to one another via a series of
ducts 26 connected radially but are not exposed to second areas 22
etc.
[0112] The first and second surfaces 16, 20 may then be moved from
the third position, Position C, to a fourth position, Position D
(not shown in FIG. 14A or 14B), by further relative rotational
movement. In this position as in Position B, at least one first
area 18 is exposed to at least one second area etc. 22 via a series
of spiraling ducts 26.
[0113] In each sequential slip position each duct originating from
an inlet duct 28 slips over to the next adjacent first or second
etc. area 18, 22. For each sequential slip position the ducts 26
alternate between connecting rows of first areas 18, rows of second
areas 22, etc. and connecting a first area 18 to a second area 22
etc. via a spiraling series of ducts 26.
[0114] In certain embodiments of the invention the areas retain an
amount of the substances they are exposed to. This can be done by
functionalization of the surface of an area, deposition of a
material on an area, attaching a monomer in a polymerization
reaction (such as peptide or DNA synthesis) to an area, etc. Prior
to assembling this device the areas could be loaded with beads or
gels that are trapped, thus whatever absorbs, adsorbs, or reacts
with these beads or gels is also trapped. This device also
comprises an outlet duct or alternative outlet such as a
gas-permeable element. Although the above description pertains to a
device with one base and one plate, alternative embodiments may
include a plurality of intermediate plates as described for FIGS.
5A-B.
[0115] Potential uses for this device include running assays of
enzyme activity, cell viability, cell adhesion, cell binding etc.,
screening for catalytic activity or selectivity, screening for
storage ability or sequestration (such as absorption of gas or
trapping of toxic compounds, etc.), and testing various properties
such as electrical, magnetic, optical, etc.
[0116] The invention described herein may also be used for the
synthesis of radioisotopes. Typical methods for making
radioisotopes are disclosed in U.S. Pat. Nos. 7,235,216; 6,567,492;
5,264,570; and 5,169,942, the entireties of all of which are
incorporated herein by reference. These multistep methods may be
performed by controlling conditions at each subsequent slip
position of the device.
[0117] The materials used to form the substrates and the devices 10
of the invention as described above are selected with regard to
physical and chemical characteristics that are desirable for proper
functioning of the device 10. In microfluidic applications, the
first and second surfaces 16, 20, first and second areas, 18, 22,
and ducts 26, 28, 30, are typically fabricated from a material that
enables formation of high definition (or high "resolution")
features, e.g., microchannels, chambers, mixing features, and the
like, that are of millimeter, micron or submicron dimensions. That
is, the material should be capable of microfabrication using, e.g.,
dry etching, wet etching, laser etching, laser ablation, molding,
embossing, or the like, so as to have desired miniaturized surface
features; preferably, the substrate is capable of being
microfabricated in such a manner as to form features in, on and/or
through the surface of the substrate. Microstructures can also be
formed on the surface of a substrate by adding material thereto,
for example, polymer channels can be formed on the surface of a
glass substrate using photo-imagable polyimide. Also, all device
materials used are preferably chemically inert and physically
stable with respect to any substance with which they come into
contact when used to introduce a fluid (e.g., with respect to pH,
electric fields, etc.). Suitable materials for forming the present
devices include, but are not limited to, polymeric materials,
ceramics (including aluminum oxide, silicon oxide, zirconium oxide,
and the like), semiconductors (including silicon, gallium arsenide,
and the like) glass, metals, composites, and laminates thereof.
Glass Etching Fabrication of Slip Chip:
[0118] The device 10 may be composed of two pieces of glass slides
with complementary patterns were made with using standard
photolithographic and wet chemical etching techniques. (See He, et
al., Sens Actuators B Chem. 2008 Feb. 22; 129(2): 811-817, for
example.) Soda-lime glass plates with chromium and photoresist
coating were obtained from Telic Company (Valencia, Calif.). The
glass plate with photoresist coating was aligned with a photomask
containing the design of the microducts and areas using a Karl
Suss, MJBB3 contact alighner. The photomask may also contain marks
to align the mask with the plate. The glass plate and photomask
were then exposed to UV light for 1 min. The photomask was removed,
and the glass plate was developed by immersing it in 0.1 mol/L NaOH
solution for 2 min. Only the areas of the photoresist that were
exposed to the UV light dissolved in the solution. The exposed
underlying chromium layer was removed using a chromium etchant (a
solution of 0.6:0.365 M
HClO.sub.4/(NH.sub.4).sub.2Ce(NO.sub.3).sub.6). The plate was
rinsed with Millipore water and dried with nitrogen gas, and the
back of the glass plate was taped with PVC sealing tape
(McMaster-Carr) to protect the back side of glass. The taped glass
plate was then carefully immersed in a plastic container with a
buffered etching agent composed of 1:0.5:0.75 mol/L
HF/NH.sub.4F/HNO.sub.3 to etch the soda-lime glass at the
temperature of 40.degree. C. The etching speed was controlled by
the etching temperature, and the area and duct depth was controlled
by the etching time. After etching, the tape was removed from the
plates. The plate was then thoroughly rinsed with Millipore water
and dried with nitrogen gas. The remaining photoresist was removed
by rinsing with ethanol, and the remaining chromium coating was
removed by immersing the plate in the chromium etchant. The surface
of the glass plate were rendered hydrophobic by silanization with
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United
Chemical Technologies, Inc.). Access holes were drilled with a 0.76
mm diameter diamond drill bit.
[0119] One method to establish fluidic communication between two or
more areas of the SlipChip includes the use of a channel with at
least one cross-sectional dimension in the nanometer range, a
nanochannel, which can be embedded in the SlipChip. The
nanochannels could be embedded into multilayer SlipChip. The height
of nanochannel can be varied with nanometer scale resolution, for
instance this would prohibit transfer of micron sized cells between
the wells, but enable transfer of proteins, vesicles, micelles,
genetic material, small molecules, ions, and other molecules and
macromolecules, including cell culture media and secreted products.
The width, length, and tortuosity of the nanochannels can also be
manipulated in order to control transport dynamics between wells.
Nanochannels can be fabricated as described in Bacterial
metapopulations in nanofabricated landscapes, Juan E. Keymer, Peter
Galajda, Cecilia Muldoon, Sungsu Park, and Robert H. Austin, PNAS
Nov. 14, 2006 vol. 103 no. 46 17290-17295, or by etching
nanochannels in the first glass piece and bringing it in contact
with the second glass piece, optionally followed by a bonding step.
Applications include filtration, capturing of cells and particles,
long term cell culture, and controlling interactions among cells
and cellular colonies and tissues.
[0120] Devices 10 of the PDMS/Glass type may also be made using
soft lithography (McDonald, J. C.; Whitesides, G. M. Accounts Chem.
Res. 2002, 35, 491-499.) similarly as described previously (Angew.
Chem. Int. Ed. 2004, 43, 2508-2511). The device used contains two
layers, each layer was composed of a thin membrane of PDMS with
ducts and areas, and a 1 mm thick microscope glass slides with size
of 75 mm.times.25 mm. To make the device, the glass slides were
cleaned and subjected to an oxygen plasma treatment. Dow-Corning
Sylgard 184 A and B components were mixed at a mass ratio of 5:1,
and poured onto the mold of the SlipChip. A glass slide was placed
onto the PDMS before cure. A glass bottom with iron beads were
place onto the glass slides to make the PDMS membrane thinner. The
device were pre-cured for 7 hour at room temperature, then move to
60.degree. C. oven and cured overnight. After cure, the device were
peeled off the mold and silanized with
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane. Access
holes were drilled with a 0.76 mm diameter diamond drill bit.
[0121] Polymeric materials suitable for use with the invention may
be organic polymers. Such polymers may be homopolymers or
copolymers, naturally occurring or synthetic, crosslinked or
uncrosslinked. Specific polymers of interest include, but are not
limited to, polyimides, polycarbonates, polyesters, polyamides,
polyethers, polyurethanes, polyfluorocarbons, polystyrenes,
poly(acrylonitrile-butadiene-styrene)(ABS), acrylate and acrylic
acid polymers such as polymethyl methacrylate, and other
substituted and unsubstituted polyolefins, and copolymers thereof.
Generally, at least one of the substrate or a portion of the device
10 comprises a biofouling-resistant polymer when the microdevice is
employed to transport biological fluids. Polyimide is of particular
interest and has proven to be a highly desirable substrate material
in a number of contexts. Polyimides are commercially available,
e.g., under the tradename Kapton.RTM., (DuPont, Wilmington, Del.)
and Upilex.RTM. (Ube Industries, Ltd., Japan).
Polyetheretherketones (PEEK) also exhibit desirable biofouling
resistant properties. Polymeric materials suitable for use with the
invention include silicone polymers, such as polydimethylsiloxane,
and epoxy polymers.
[0122] The devices 10 of the invention may also be fabricated from
a "composite," i.e., a composition comprised of unlike materials.
The composite may be a block composite, e.g., an A-B-A block
composite, an A-B-C block composite, or the like. Alternatively,
the composite may be a heterogeneous combination of materials,
i.e., in which the materials are distinct from separate phases, or
a homogeneous combination of unlike materials. As used herein, the
term "composite" is used to include a "laminate" composite. A
"laminate" refers to a composite material formed from several
different bonded layers of identical or different materials. Other
preferred composite substrates include polymer laminates,
polymer-metal laminates, e.g., polymer coated with copper, a
ceramic-in-metal or a polymer-in-metal composite. One preferred
composite material is a polyimide laminate formed from a first
layer of polyimide such as Kapton.RTM., that has been co-extruded
with a second, thin layer of a thermal adhesive form of polyimide
known as KJ.RTM., also available from DuPont (Wilmington,
Del.).
[0123] The device can be fabricated using techniques such as
compression molding, injection molding or vacuum molding, alone or
in combination. Sufficiently hydrophobic material can be directly
utilized after molding. Hydrophilic material can also be utilized,
but may require additional surface modification. Further, the
device can also be directly milled using CNC machining from a
variety of materials, including, but not limited to, plastics,
metals, and glass. Microfabrication techniques can be employed to
produce the device with sub-micrometer feature sizes. These
include, but are not limited to, deep reactive ion etching of
silicon, KOH etching of silicon, and HF etching of glass.
Polydimethylsiloxane devices can also be fabricated using a
machined, negative image stamp. In addition to rigid substrates,
flexible, stretchable, compressible and other types of substrates
that may change shape or dimensions may be used as materials for
certain embodiments of the SlipChip. In certain embodiments, these
properties may be used to, for example, control or induce
slipping.
[0124] In some instances, the base 12 and plate 14 and substrate
may be made from the same material. Alternatively, different
materials may be employed. For example, in some embodiments the
base 12 and plate 14 may be comprised of a ceramic material and the
substrate may be comprised of a polymeric material.
[0125] The device may contain electrically conductive material on
either surface 16, 20. The material may be formed into at least one
area or patch of any shape to form an electrode. The at least one
electrode may be positioned on one surface 16 such that in a first
position, the at least one electrode is not exposed to at least one
first area on the opposing surface 20, but when the two parts of
the device 12, 14 are moved relative to one another to a second
position, the at least one electrode overlaps the at least one area
18. The at least one electrode may be electrically connected to an
external circuit. The at least one electrode may be used to carry
out electrochemical reactions for detection and/or synthesis. If a
voltage is applied to at least two electrodes that are exposed to a
substance in an area or a plurality of areas in fluidic
communication or a combination of areas and ducts in fluidic
communication, the resulting system may be used to carry out
electrophoretic separations, and/or electrochemical reactions
and/or transport. Optionally, at least one duct and/or at least one
area may be present on the same surface as the at least one
electrode and may be positioned so that in a first position, none
of the at least one duct and the at least one electrode are exposed
to an area 18 on the opposing surface, but when the two parts of
the device 12, 14 are moved relative to one another to a second
position, the at least one duct and/or at least one area and the at
least one electrode overlaps the at least one area 18.
[0126] Several embodiments of the current invention require
movement of a substance through, into, and/or across at least one
duct and/or area. For example movement of a substance can be used
for washing steps in immunoassays, removal of products or
byproducts, introduction of reagents, or dilutions.
[0127] Loading of a substance may be performed by a number of
methods, as described herein. Loading may be performed either to
fill the ducts and areas of the device, for example by designing
the outlets to increase flow resistance when the substance reaches
the outlets. This approach is valuable for volume-limited samples
or to flow the excess volume through the outlets, while optionally
capturing analyte from the substance. Analytes can be essentially
any discrete material which can be flowed through a microscale
system. Analyte capture may be accomplished for example by
preloading the areas of the device with capture elements that are
trapped in the areas (such as particles, beads or gels, retained
within areas via magnetic forces or by geometry or with relative
sizes of beads and ducts or with a membrane), thus whatever
absorbs, adsorbs, or reacts with these beads or gels is also
trapped. These areas will then retain an amount or component or
analyte of the substances they are exposed to. This can also be
done by functionalization of the surface of an area, deposition of
a material on an area, attaching a monomer in a polymerization
reaction (such as peptide or DNA synthesis) to an area, etc.
[0128] Other examples of capture elements include antibodies,
affinity-proteins, aptamers, beads, particles and biological cells.
Beads may be for example, polymer beads, silica beads, ceramic
beads, clay beads, glass beads, magnetic beads, metallic beads,
inorganic beads, and organic beads can be used. The beads or
particles can have essentially any shape, e.g., spherical, helical,
irregular, spheroid, rod-shaped, cone-shaped, disk shaped, cubic,
polyhedral or a combination thereof. Capture elements are
optionally coupled to reagents, affinity matrix materials, or the
like, e.g., nucleic acid synthesis reagents, peptide synthesis
reagents, polymer synthesis reagents, nucleic acids, nucleotides,
nucleobases, nucleosides, peptides, amino acids, monomers, cells,
biological samples, synthetic molecules, or combinations thereof.
Capture elements optionally serve many purposes within the device,
including acting as blank particles, dummy particles, calibration
particles, sample particles, reagent particles, test particles, and
molecular capture particles, e.g., to capture a sample at low
concentration. Additionally the capture elements may be used to
provide particle retention elements. Capture elements are sized to
pass or not pass through selected ducts or membranes (or other
microscale elements). Accordingly, particles or beads will range in
size depending on the application.
[0129] A substance may be introduced to fill the majority of
reaction areas and ducts. Filling may be continued further to
provide excess sample, larger than the volume of areas and ducts.
Introducing a volume of substance which is greater than the volume
of areas and ducts will increase the amount of analyte which may be
captured within the capture. Introducing a wash fluid after the
introduction of a substance may be performed to wash the capture
elements and analytes which are bound to the capture elements.
Subsequent further slipping may be performed to conduct reactions
and analysis of the analytes.
[0130] The approach described above is beneficial when analyzing
samples with low concentrations of analytes, for example rare
nucleic acids or proteins, markers and biomarkers of genetic or
infectious disease, environmental pollutants, etc. (See e.g., U.S.
Ser. No. 10/823,503, incorporated herein by reference). Another
example includes the analysis of rare cells, such as circulating
cancer cells or fetal cells in maternal blood for prenatal
diagnostics. This approach may be beneficial for rapid early
diagnostics of infections by capturing and further analyzing
microbial cells in blood, sputum, bone marrow aspirates and other
bodily fluids such as urine and cerebral spinal fluid. Analysis of
both beads and cells may benefit from stochastic confinement (See
e.g., PCT/US08/71374, incorporated herein by reference).
[0131] In certain embodiments, the device 10 may be used for rapid
detection and drug susceptibility screening of bacteria in samples,
including complex biological matrices, without pre-incubation.
Unlike conventional bacterial culture and detection methods, which
rely on incubation of a sample to increase the concentration of
bacteria to detectable levels, this method may be used to confine
individual bacteria into areas nanoliters in volume. When single
cells are confined into areas of small volume such that the loading
is less than one bacterium per area, the detection time is
proportional to area volume. Confinement increases cell density and
allows released molecules to accumulate around the cell,
eliminating the pre-incubation step and reducing the time required
to detect the bacteria. This approach may be called `stochastic
confinement`. The device may, for example, be used to determine an
antibiogram--or chart of antibiotic sensitivity--of bacteria, such
as methicillin-resistant Staphylococcus aureus (MRSA) to many
antibiotics in a single experiment and to measure the minimal
inhibitory concentration (MIC) of the drugs against such strains.
In addition, this device may be used to distinguish between
sensitive and resistant strains of S. aureus in samples of human
blood plasma. The device also enables multiple tests to be
performed simultaneously on a single sample containing bacteria.
The device provides a method of rapid and effective
patient-specific treatment of bacterial infections and could be
extended to a variety of applications that require multiple
functional tests of bacterial samples on reduced timescales.
[0132] Stochastic confinement has been used in other systems. See
for example, "Detecting bacteria and determining their
susceptibility to antibiotics by stochastic confinement in
nanoliter droplets using plug-based microfluidics", Boedicker J.
Q., Li L., Kline T. R., Ismagilov R. F. Lab on a chip 8(8):1265,
2008 August, published U.S. patent application 60/962,426, M. Y.
He, J. S. Edgar, G. D. M. Jeffries, R. M. Lorenz, J. P. Shelby and
D. T. Chiu, Anal. Chem., 2005, 77, 1539-1544; Y. Marcy, T. Ishoey,
R. S. Lasken, T. B. Stockwell, B. P. Walenz, A. L. Halpern, K. Y.
Beeson, S. M. D. Goldberg and S. R. Quake, PLoS Genet., 2007, 3,
1702-1708; A. Huebner, M. Srisa-Art, D. Holt, C. Abell, F.
Hollfelder, A. J. Demello and J. B. Edel, Chem. Commun., 2007,
1218-1220; S. Takeuchi, W. R. DiLuzio, D. B. Weibel and G. M.
Whitesides, Nano Lett., 2005, 5, 1819-1823; P. Boccazzi, A.
Zanzotto, N. Szita, S. Bhattacharya, K. F. Jensen and A. J.
Sinskey, App. Microbio. Biotech., 2005, 68, 518-532; V. V.
Abhyankar and D. J. Beebe, Anal. Chem., 2007, 79, 4066-4073.
Similar techniques have been used for single molecule and single
enzyme work. (H. H. Gorris, D. M. Rissin and D. R. Walt, Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 17680-17685; A. Aharoni, G.
Amitai, K. Bernath, S. Magdassi and D. S. Tawfik, Chem. Biol.,
2005, 12, 1281-1289; 0. J. Miller, K. Bernath, J. J. Agresti, G.
Amitai, B. T. Kelly, E. Mastrobattista, V. Taly, S. Magdassi, D. S.
Tawfik and A. D. Griffiths, Nat. Methods, 2006, 3, 561-570; J.
Huang and S. L. Schreiber, Proc. Natl. Acad. Sci. U.S.A, 1997, 94,
13396-13401; D. T. Chiu, C. F. Wilson, F. Ryttsen, A. Stromberg, C.
Farre, A. Karlsson, S. Nordholm, A. Gaggar, B. P. Modi, A. Moscho,
R. A. Garza-Lopez, O. Orwar and R. N. Zare, Science, 1999, 283,
1892-1895; J. Yu, J. Xiao, X. J. Ren, K. Q. Lao and X. S. Xie,
Science, 2006, 311, 1600-1603), the entireties of all of which are
incorporated by reference. The device also enables simultaneous
execution of numerous assays of bacterial function from a single
bacterial sample in the same experiment, which is especially useful
for rapid antibiotic susceptibility screening. Previously, gel
microdroplets had been utilized for susceptibility screening. (Y.
Akselband, C. Cabral, D. S. Shapiro and P. McGrath, J. Microbiol.
Methods, 2005, 62, 181-197; C. Ryan, B. T. Nguyen and S. J.
Sullivan, J. Clin. Microbiol., 1995, 33, 1720-1726.)
[0133] The device may be used to detect organisms. The term
"organism" refers to any organisms or microorganism, including
bacteria, yeast, fungi, viruses, protists (protozoan, micro-algae),
archaebacteria, and eukaryotes. The term "organism" refers to
living matter and viruses comprising nucleic acid that can be
detected and identified by the methods of the invention. Organisms
include, but are not limited to, bacteria, archaea, prokaryotes,
eukaryotes, viruses, protozoa, mycoplasma, fungi, and nematodes.
Different organisms can be different strains, different varieties,
different species, different genera, different families, different
orders, different classes, different phyla, and/or different
kingdoms. Organisms may be isolated from environmental sources
including soil extracts, marine sediments, freshwater sediments,
hot springs, ice shelves, extraterrestrial samples, crevices of
rocks, clouds, attached to particulates from aqueous environments,
and may be involved in symbiotic relationships with multicellular
organisms. Examples of such organisms include, but are not limited
to Streptomyces species and uncharacterized/unknown species from
natural sources.
[0134] Organisms included genetically engineered organisms. Further
examples of organisms include bacterial pathogens such as:
Aeromonas hydrophila and other species (spp.); Bacillus anthracis;
Bacillus cereus; Botulinum neurotoxin producing species of
Clostridium; Brucella abortus; Brucella melitensis; Brucella suis;
Burkholderia mallei (formally Pseudomonas mallei); Burkholderia
pseudomallei (formerly Pseudomonas pseudomallei); Campylobacter
jejuni; Chlamydia psittaci; Clostridium botulinum; Clostridium
botulinum; Clostridium perfringens; Coccidioides immitis;
Coccidioides posadasii; Cowdria ruminantium (Heartwater); Coxiella
burnetii; Enterovirulent Escherichia co//group (EEC Group) such as
Escherichia coli--enterotoxigenic (ETEC), Escherichia
coli--enteropathogenic (EPEC), Escherichia coli--O157:H7
enterohemorrhagic (EHEC), and Escherichia coli--enteroinvasive
(EIEC); Ehrlichia spp. such as Ehrlichia chaffeensis; Francisella
tularensis; Legionella pneumophilia; Liberobacter africanus;
Liberobacter asiaticus; Listeria monocytogenes; miscellaneous
enterics such as Klebsiella, Enterobacter, Proteus, Citrobacter,
Aerobacter, Providencia, and Serratia; Mycobacterium bovis;
Mycobacterium tuberculosis; Mycoplasma capricolum; Mycoplasma
mycoides ssp mycoides; Peronosclerospora philippinensis; Phakopsora
pachyrhizi; Plesiomonas shigelloides; Ralstonia solanacearum race
3, biovar 2; Rickettsia prowazekii; Rickettsia rickettsii;
Salmonella spp.; Schlerophthora rayssiae varzeae; Shigella spp.;
Staphylococcus aureus; Streptococcus; Synchytrium endobioticum;
Vibrio cholerae non-O1; Vibrio cholerae O1; Vibrio parahaemolyticus
and other Vibrios; Vibrio vulnificus; Xanthomonas oryzae; Xylella
fastidiosa (citrus variegated chlorosis strain); Yersinia
enterocolitica and Yersinia pseudotuberculosis; and Yersinia
pestis. Further examples of organisms include viruses such as:
African horse sickness virus; African swine fever virus; Akabane
virus; Avian influenza virus (highly pathogenic); Bhanja virus;
Blue tongue virus (Exotic); Camel pox virus; Cercopithecine
herpesvirus 1; Chikungunya virus; Classical swine fever virus;
Coronavirus (SARS); Crimean-Congo hemorrhagic fever virus; Dengue
viruses; Dugbe virus; Ebola viruses; Encephalitic viruses such as
Eastern equine encephalitis virus, Japanese encephalitis virus,
Murray Valley encephalitis, and Venezuelan equine encephalitis
virus; Equine morbillivirus; Flexal virus; Foot and mouth disease
virus; Germiston virus; Goat pox virus; Hantaan or other Hanta
viruses; Hendra virus; Issyk-kul virus; Koutango virus; Lassa fever
virus; Louping ill virus; Lumpy skin disease virus; Lymphocytic
choriomeningitis virus; Malignant catarrhal fever virus (Exotic);
Marburg virus; Mayaro virus; Menangle virus; Monkeypox virus;
Mucambo virus; Newcastle disease virus (WND); Nipah Virus; Norwalk
virus group; Oropouche virus; Orungo virus; Peste Des Petits
Ruminants virus; Piry virus; Plum Pox Potyvirus; Poliovirus; Potato
virus; Powassan virus; Rift Valley fever virus; Rinderpest virus;
Rotavirus; Semliki Forest virus; Sheep pox virus; South American
hemorrhagic fever viruses such as Flexal, Guanarito, Junin,
Machupo, and Sabia; Spondweni virus; Swine vesicular disease virus;
Tickborne encephalitis complex (flavi) viruses such as Central
European tickborne encephalitis, Far Eastern tick-borne
encephalitis, Russian spring and summer encephalitis, Kyasanur
forest disease, and Omsk hemorrhagic fever; Variola major virus
(Smallpox virus); Variola minor virus (Alastrim); Vesicular
stomatitis virus (Exotic); Wesselbron virus; West Nile virus;
Yellow fever virus; and South American hemorrhagic fever viruses
such as Junin, Machupo, Sabia, Flexal, and Guanarito.
[0135] Further examples of organisms include parasitic protozoa and
worms, such as: Acanthamoeba and other free-living amoebae;
Anisakis sp. and other related worms Ascaris lumbricoides and
Trichuris trichiura; Cryptosporidium parvum; Cyclospora
cayetanensis; Diphyllobothrium spp.; Entamoeba histolytica;
Eustrongylides sp.; Giardia lamblia; Nanophyetus spp.; Shistosoma
spp.; Toxoplasma gondii; Filarial nematodes and Trichinella.
Further examples of analytes include allergens such as plant pollen
and wheat gluten.
[0136] Further examples of organisms include fungi such as:
Aspergillus spp.; Blastomyces dermatitidis; Candida; Coccidioides
immitis; Coccidioides posadasii; Cryptococcus neoformans;
Histoplasma capsulatum; Maize rust; Rice blast; Rice brown spot
disease; Rye blast; Sporothrix schenckii; and wheat fungus. Further
examples of organisms include worms such as C. Elegans and
pathogenic worms or nematodes.
[0137] Sample may obtained from a patient or person and includes
blood, feces, urine, saliva or other bodily fluid. Food samples may
also be analyzed. Samples may be any sample potentially comprising
an organism. Environments for finding organisms include, but are
not limited to, geothermal and hydrothermal fields, acidic soils,
sulfotara and boiling mud pots, pools, hot-springs and geysers
where the enzymes are neutral to alkaline, marine actinomycetes,
metazoan, endo and ectosymbionts, tropical soil, temperate soil,
arid soil, compost piles, manure piles, marine sediments,
freshwater sediments, water concentrates, hypersaline and
super-cooled sea ice, arctic tundra, Sargasso sea, open ocean
pelagic, marine snow, microbial mats (such as whale falls, springs
and hydrothermal vents), insect and nematode gut microbial
communities, polar bear nostrils, plant endophytes, epiphytic water
samples, industrial sites and ex situ enrichments. Additionally, a
sample may be isolated from eukaryotes, prokaryotes, myxobacteria
(epothilone), air, water, sediment, soil or rock, a plant sample, a
food sample, a gut sample, a salivary sample, a blood sample, a
sweat sample, a urine sample, a spinal fluid sample, a tissue
sample, a vaginal swab, a stool sample, an amniotic fluid sample, a
fingerprint, aerosols, including aerosols produced by coughing,
skin samples, tissues, including tissue from biopsies, and/or a
buccal mouthwash sample.
[0138] To monitor the presence and metabolically active bacteria in
the device, a fluorescent viability indicator alamarBlue.RTM. may
be added to the cultures. The active ingredient of alamarBlue is
the fluorescent redox indicator resazurin. (J. O'Brien and F.
Pognan, Toxicology, 2001, 164, 132-132.) Resazurin is reduced by
electron receptors used in cellular metabolic activity, such as
NADH and FADH, to produce the fluorescent molecule resofurin.
Therefore, fluorescence intensity in an area is correlated with the
presence and metabolic activity of a cell, in this case, a
bacterium. Because resazurin indicates cell viability,
resazurin-based assays have been used previously in antibiotic
testing. (S. G. Franzblau, R. S. Witzig, J. C. McLaughlin, P.
Torres, G. Madico, A. Hernandez, M. T. Degnan, M. B. Cook, V. K.
Quenzer, R. M. Ferguson and R. H. Gilman, J. Clin. Microbiol.,
1998, 36, 362-366; A. Martin, M. Camacho, F. Portaels and J. C.
Palomino, Antimicrob. Agents Chemother., 2003, 47, 3616-3619; K. T.
Mountzouros and A. P. Howell, J. Clin. Microbiol., 2000, 38,
2878-2884; C. N. Baker and F. C. Tenover, J. Clin. Microbiol.,
1996, 34, 2654-2659.) Resazurin may be used to detect both the
presence of a live bacterium and the response of bacteria to drugs,
such as antibiotics. Stochastic confinement decreases detection
time because in an area that has the bacterium, the bacterium is at
an effectively higher concentration than in the starting solution,
and the signal-to-noise required for detection is reached sooner
since the product of reduction of resazurin accumulates in the area
more rapidly.
[0139] Detecting low concentrations of species (down to single
molecules and single bacteria) is a challenge in food, medical, and
security industries. The device may allow one to concentrate such
samples and perform analysis. For example, a sample containing
small amounts of DNA of interest in the presence of an excess of
other DNA may be amplified. Amplification may be detected if areas
are made small enough that some areas contain single DNA molecules
of interest, and other areas contain no DNA molecules of interest.
This separation into areas effectively creates areas with higher
DNA of interest concentration than in the original sample.
Amplification of DNA in those areas, for example by PCR, may lead
to higher signal than amplification of the original sample. In
addition, localization of bacteria in areas by a similar method may
create a high local concentration of bacteria (1 per very small
area), making them easier to detect. For some bacteria that use
quorum sensing, this may be a method to activate and detect them.
Such bacteria may be inactive/non-pathogenic and difficult to
detect at low concentrations due to lack of activity, but at a high
concentration of bacteria, the concentration of a signaling
molecule increases, activating the bacteria. If a single bacterium
is localized in an area, the signaling molecule produced by a
bacterium cannot diffuse away and its concentration will rapidly
increase, triggering activation of the bacterium, making it
possible for detection. In addition, the device may be used to
localize cells and bacteria by creating gels or matrixes inside
areas. Bacteria and other species (particles and molecules) may be
collected and concentrated into plugs by flowing air through a
fluid such as water, and then using that fluid to fill a plurality
of areas. This results in concentrated sample-containing areas
because some of the areas do not contain any of the analyte.
[0140] PCR techniques are disclosed in the following published US
patent applications and International patent applications: US
2008/0166793, WO 08/069884, US 2005/0019792, WO 07/081386, WO
07/081387, WO 07/133710, WO 07/081385, WO 08/063227, US
2007/0195127, WO 07/089541, WO 07030501, US 2007/0052781, WO
06096571, US 2006/0078893, US 2006/0078888, US 2007/0184489, US
2007/0092914, US 2005/0221339, US 2007/0003442, US 2006/0163385, US
2005/0172476, US 2008/0003142, and US 2008/0014589, all of which
are incorporated by reference herein in their entirety.
[0141] The following articles, describing methods for concentrating
cells and/or chemicals by making small volume areas with low
numbers of items to no items being incorporated into the areas,
with specific applications involving PCR, are incorporated by
reference herein: Anal Chem. 2003 Sep. 1; 75(17):4591-8.
Integrating polymerase chain reaction, valving, and electrophoresis
in a plastic device for bacterial detection. Koh C G, Tan W, Zhao M
Q, Ricco A J, Fan Z H; Lab Chip. 2005 April; 5(4):416-20. Epub 2005
Jan. 28. Parallel nanoliter detection of cancer markers using
polymer microchips. Gulliksen A, Solli L A, Drese K S, Sorensen O,
Karlsen F, Rogne H, Hovig E, Sirevag R.; Ann N Y Acad Sci. 2007
March; 1098:375-88. Development of a microfluidic device for
detection of pathogens in oral samples using upconverting phosphor
technology (UPT). Abrams W R, Barber C A, McCann K, Tong G, Chen Z,
Mauk M G, Wang J, Volkov A, Bourdelle P, Corstjens P L, Zuiderwijk
M, Kardos K, Li S, Tanke H J, Sam Niedbala R, Malamud D, Bau H;
Sensors, 2004. Proceedings of IEEE 24-27 Oct. 2004
Page(s):1191-1194 vol. 3. A microchip-based DNA purification and
real-time PCR biosensor for bacterial detection. Cady, N. C.;
Stelick, S.; Kunnavakkam, M. V.; Yuxin Liu; Batt, C. A.; Science.
2006 Dec. 1; 314(5804):1464-7. Microfluidic Digital PCR Enables
Multigene Analysis of Individual Environmental Bacteria. Elizabeth
A. Ottesen, Jong Wook Hong, Stephen R. Quake, Jared R. Leadbetter;
Electrophoresis 2006, 27, 3753-3763. Automated screening using
microfluidic chip-based PCR and product detection to assess risk of
BK virus associated nephropathy in renal transplant recipients.
Govind V. Kaigala, I, Ryan J. Huskins, Jutta Preiksaitis, Xiao-Li
Pang, Linda M. Pilarski, Christopher J. Backhouse; Journal of
Microbiological Methods 62 (2005) 317-326. An insulator-based
(electrodeless) dielectrophoretic concentrator for microbes in
water. Blanca H. Lapizco-Encinas, Rafael V. Davalos, Blake A.
Simmons, Eric B. Cummings, Yolanda Fintschenko; Anal. Chem. 2004,
76, 6908-6914. Electrokinetic Bioprocessor for Concentrating Cells
and Molecules. Pak Kin Wong, Che-Yang Chen, Tza-Huei Wang, and
Chih-Ming Ho; Lab Chip, 2002, 2, 179-187. High sensitivity PCR
assay in plastic micro reactors. Jianing Yang, Yingjie Liu, Cory B.
Rauch, Randall L. Stevens, Robin H. Liu, Ralf Lenigk and Piotr
Grodzinski; Anal. Chem. 2005, 77, 1330-1337. High-Throughput
Nanoliter Sample Introduction Microfluidic Chip-Based Flow
Injection Analysis System with Gravity-Driven Flows. Wen-Bin Du,
Qun Fang, Qiao-Hong He, and Zhao-Lun Fang; Science Vol 315 5 Jan.
2007, 81-84. Counting Low-Copy Number Proteins in a Single Cell. Bo
Huang, Hongkai Wu, Devaki Bhaya, Arthur Grossman, Sebastien
Granier, Brian K. Kobilka, I, Richard N. Zare; Nature Biotechnology
Vol 22 (4), April 2004. A nanoliterscale nucleic acid processor
with parallel architecture. Hong J W, Studer V, Hang G, Anderson W
F, and Quake S R; Electrophoresis 2002, 23, 1531-1536. A nanoliter
rotary device for polymerase chain reaction. Jian Liu, Markus
Enzelberger, and Stephen Quake; Biosensors and Bioelectronics 20
(2005) 1482-1490. Microchamber array based DNA quantification and
specific sequence detection from a single copy via PCR in nanoliter
volumes. Yasutaka Matsubara, Kagan Kerman, Masaaki Kobayashi,
Shouhei Yamamura, Yasutaka Morita, Eiichi Tamiya; US Patent
Application 2005/0019792, "Microfluidic device and methods of using
same"; and Nature Methods 3, 541-543 (2006) "Overview: methods and
applications for droplet compartmentalization of biology" John H
Leamon, Darren R Link, Michael Egholm & Jonathan M
Rothberg.
[0142] Flourogenic media, which change color in the presence of
specific bacteria, can also be used to detect cells. Chromogenic
media include, for example, Difco mEI agar, Merck/EMD
Chromocult.TM. Coliform Agars, Chromocult.TM. Enterococci
Agar/Broth, or Fluorocult.RTM. LMX Broth, BL agar, IDEXX Colilert,
CPI ColiTag and Merck/EMD ReadyCult.RTM.. Typical enzyme substrates
linked to chromogens or fluorogens include ONPG, CPRG, and MUG.
These are also available in ready-to-use format, e.g. BBL ml agar
and `convenience` packs, e.g. IDEXX Colilert, CPI ColiTag and
Merck/EMD ReadyCult.RTM..
[0143] To perform an antibiotic screen, areas may contain
antibiotics, and the areas filled with the sample may be allowed to
incubate to permit growth of microorganisms. Antibiotics are
recognized and are substances which inhibit the growth of or kill
microorganisms. Examples of antibiotics include, but are not
limited to, chlorotetracycline, bacitracin, nystatin, streptomycin,
polymicin, gramicidin, oxytetracyclin, chloramphenicol, rifampicin,
cefsulodin, cefotiam, mefoxin, penicillin, tetracycline,
minocycline, doxycycline, vancomycin, kanamycin, neomycin,
gentamycin, erythromycin, cephalosporins, geldanamycin, and analogs
thereof. Examples of cephalosporins include cephalothin,
cephapirin, cefazolin, cephalexin, cephradine, cefadroxil,
cefamandole, cefoxitin, cefaclor, cefuroxime, cefonicid,
ceforanide, cefotaxime, moxalactam, ceftizoxime, ceftriaxone, and
cefoperazone. Additional examples of antibiotics that may be used
are in US 2007/0093894 A1, hereby incorporated by reference in its
entirety. Detection of differences in growth and microbial
populations in the absence and presence of each antibiotic would
provide information on antibiotic susceptibility. First the
bacteria in the sample are counted. Then, the bacteria sample is
exposed to areas containing different growth media and different
antibodies along with some as "blank" media and "blank" antibiotics
areas, and areas are assayed for bacterial growth.
[0144] Other applications include detecting bacteria for
applications in homeland security and safety of the food chain and
water. It is also possible to apply these methods of detection to
the areas of sepsis, bioenergy, proteins, enzyme engineering, blood
clotting, biodefense, food safety, safety of water supply, and
environmental remediation. The following patents and patent
applications are hereby incorporated by reference in their
entireties: WO 2005-010169 A2, U.S. Pat. No. 6,500,617, WO
2007-009082 A1.
[0145] Examples of means to cause movement of a substance include,
but are not limited to, centrifugal force, for example when the
device contains an array of areas in fluidic communication is used,
gradients of surface tension, osmotic pressure, capillary pressure,
positive or negative pressure, generated externally, for example
using pumps or syringes, slipping, for example by compressing or
expanding an area containing fluid, electric forces, electroosmotic
forces, magnetic forces, and chemical reactions or processes, which
may be initiated externally or initiated by slipping.
[0146] For example a plurality of liquid or solid substances may be
brought into that together produce a gaseous product, thereby
generating pressure. For example a solution of sulfuric acid and a
carbonate salt may be used. Alternatively, a catalyst may be added
to an area containing substances that do not react or only react
slowly in the absence of the catalyst but which react more rapidly
in the presence of the catalyst. One example is a mixture of sodium
bicarbonate with a solid acid, for example tartaric acid, activated
by addition of water, acting as a catalyst. A number of such
mixtures capable of being activated by catalysts are used as baking
powders. Alternatively, substances may be brought together such
that a gaseous substance is consumed, thereby generating negative
pressure and inducing motion of a substance in a device. For
example, sodium hydroxide and carbon dioxide will react in such a
manner. Phase transitions may also be used to induce motion of a
substance in a device. In addition, wicking may be used. For
example a first area may contain, or be composed of a material that
absorbs a substance in order to induce motion. In another example
of inducing movement of a substance, differential pressures due to
surface tension and flow resistance can be used to drive flow after
slipping, even without applying external pressure. In one instance,
a device may contain one or more main channels through which flow
is desired as well as an array of one or more capillary channels,
which are smaller than the main channel and therefore have a higher
capillary pressure than the main channel. The device can be slipped
to bring the main channel(s) into fluidic communication with the
array of capillary channels, thus creating a fluidic path that has
higher pressure in the capillary channels than in the main channel,
which drives flow into the main channel. The device and the
slipping motion can be tuned to provide control over the rate and
duration of flow. For example, reservoirs of fluid that are open to
the atmosphere can be located at controlled distances the capillary
and/or main channel, to control the pressure due to flow
resistance. These reservoirs can optionally be connected via a duct
to the capillary and/or main channel, to further decrease flow
resistance and thus increase the flow rate. This could for example
be used to drive flow through a washing channel, to wash during an
immunoassay, or to drive slow flow over a perfusion culture of
cells or a suspension of beads.
[0147] The device of the present invention can be used to load
multiple areas with the same substance easily and economically. For
example, with respect to FIGS. 12A and 12B, the device can be
manufactured to include multiple, areas 22 and areas 18. In the
open position, each area is connect to each other and to an inlet
28, allowing easy loading. In the closed position, each of the
areas 18 and 22 are isolated from each other, allowing, for
example, detection of small amount of substances in individual
areas (e.g., through stochastic confinement of single molecules,
beads, cells and bacteria). Methods for detecting small amounts of
substances in individual areas are described in, for example,
PCT/US08/071374, PCT/US07/02532, and PCT/US08/71370, all
incorporated by reference herein.
[0148] The device of the present invention can also be used to
easily load a first substance into multiple areas preloaded with
various second substances. For example, with respect to FIGS. 12A
and 12B, each area 18 and 22 may contain a different first
substance, which may be attached to the surface of the areas (e.g.,
different antibiotics). When a second substance (e.g., a sample
containing a bacteria) is loaded into the device in the open
position through inlet 28, it will load into each area. After the
device is slipped into the closed position, individual areas can be
monitored for the affect of the first substance on the second
substance. Methods for measuring susceptibility of bacteria to
antibiotics are described in PCT/US08/71374, incorporated by
reference herein.
[0149] Embodiments of the invention described herein may be used
for microbial culturing. For example, anaerobic microbes may be
cultured in devices made of glass in which the microbes have been
loaded anaerobically. The anaerobes could then be manipulated,
grown, analyzed etc. without exposing the organism to oxygen. Such
devices may be used in applications such as analyzing aerobic or
anaerobic microbes, analyzing intestinal biota, diagnostics,
determining antibiotic susceptibility of anaerobic infections.
Applications of these microbial culturing devices are disclosed in
patent applications PCT/US08/71374 and PCT/US08/71370. After
microbial species have been confined to areas of the device they
may be manipulated via multistep processes such that conditions
(i.e., anaerobic, chemical, etc.) are controlled at each subsequent
slip position. For example, a microbe may be confined in the
initial slip position, then stimulated to produce a virulence
factor in the following slip position and then in a final slip
position the virulence factor may be contacted with a detection
reagent.
[0150] Additionally, embodiments of the invention described herein
may be used for culturing and manipulating prokaryotic and
eukaryotic cells including multicellular organisms such as
nematodes. For example organisms may be cultured in devices
designed to supply cells and organisms with nutrients in the first
slip position, supply stimuli in the second slip position and
remove waste products in the third slip position. Optional
additional slip positions may be used to capture products secreted
by the organisms within the device as disclosed in patent
applications PCT/US08/71374 and PCT/US08/71370. The device may be
designed to be compatible with high resolution imaging of the
confined organisms.
[0151] Similarly, the device of the present invention can be
designed to load multiple areas with different substances easily
and economically. For example, in FIGS. 8A-D, the device is
manufactured to include multiple areas 18 on one surface and
multiple areas 22 on the opposing surface. In position A, parallel
rows of areas 22 can be loaded with different, first substances.
After slipping into position C, parallel columns of areas 18 can be
loaded with different, second substances. In position D, the
various first and second substances can combine, forming an array
of different reactions. In this embodiment, for example, a device
containing 10 areas in each of 10 rows and 10 areas in each of 10
columns can be used to set up 100 reactions. In other embodiments,
the device could contain areas configured in the same locations as
standard multiwell plates which may contain, for example, 6, 24,
96, 384, 1536, 3456, or 9600 sample wells. In other embodiments,
the device could contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 20, 24, 30, 40, 48, 50, 60, 70, 80, 90, 96, 100, 200, 300, 384,
400, 500, 512, 1000, 1500, 1536, 2000, 2500, 3000, 3456, 3500,
4000, 4500, 5000, 6000, 7000, 8000, 9000, 9600, 10000, 1500, 2000,
2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,
30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000,
200000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000 or
more areas. Standard multiwell configurations are described in US
20070015289, incorporated by reference herein in its entirety. For
example, a device can contain an array of 100,000 areas, wherein
each area is a cube approximately 200 micrometers on a side,
enabling 1 milliliter of sample to be divided into 100,000 volumes
of 10 nL each. Such a device can be used, for example to detect
analytes present at very low concentrations.
[0152] In some embodiments the device of the present invention can
be preloaded with substances and stored prior to use. For example,
if one or more substances are dried into the areas, a solution
could be added to the device in the open position to
rehydrate/dissolve the substances. Methods of drying substances for
storage are described in US 2008/0213215, US2009/0057149 and U.S.
Pat. No. 7,135,180, incorporated by reference herein in their
entireties.
[0153] The present invention can be used with plug technology, such
as disclosed in U.S. Pat. No. 7,129,091 and patent publications US
2007/0172954, US 2006/0003439, US 2005/0087122, PCT/US08/71374,
PCT/US08/71370, and PCT/US07/26028 to the same inventor, all
incorporated by reference herein in their entireties. For example,
an area can comprise a channel on a base capable of being filled
with an array of plugs. The device can comprise an opposing plate
containing a set of at least one areas that, in a first position,
each overlap at least one plug in the array of plugs, and in a
second position, do not overlap any plugs of the array of
plugs.
[0154] One embodiment of such device 10 is shown in FIGS. 12A-B.
One way to manufacture the device 10 shown in FIGS. 12A-B is out of
glass. The glass slide with areas or channels were made by etching
as described above. The area size is approximately 130.times.50
.mu.m and depth is about 15 .mu.m. There are 2048 areas in each
layer of the device, which were composed of 32 rows of 64 areas.
All 32 row areas connected to a single inlet by a Y shape tree
distribution style. After slipping, the device generated 4096
individual compartments. The size of the device was 1 cm.times.2
cm.
[0155] Two pieces of glass slides with complementary patterns were
aligned under microscope to make through-channels and clamped with
paper clips. When the areas are aligned, they formed a continuous
channel connected with inlet. And the other end of the channel
connected with a bigger channel which went all way down to the edge
of the device.
[0156] FC-40 was first injected via the inlet to fill all the
channels. Since the glass was silanized and FC-40 wet the glass,
the oil not only filled all the channels, but also all contact area
between two glass layers. Air was pushed in through the inlet to
replace FC-40, while keep the contacted area of two layer still wet
by FC-40. And also, there are small amount of FC-40 residue in
channel or a FC-40 thin layer still covers the surface of the
channel. A solution of 0.5 .mu.M fluoroscein in 10 mM Tris pH 7.8
was injected into the channels through the inlet.
[0157] In some embodiments, the sample loaded into the device may
have beads, such as those capable of immobilizing a substance or
magnetic beads. Beads can be confined into different areas in the
device by sizing the ducts that connect the areas in the open
position to be smaller than the beads. Magnetic beads can
additionally be directed or trapped in specific areas by applying a
focused magnetic field to the area. In some embodiments, the areas
are loaded with beads containing a first substance (e.g., a first
amino acid). By slipping the device between an open and closed
position (or through several different open and closed positions),
the beads can be washed, deprotected, reacted with a second
substance (e.g. a second amino acid), washed, etc. In this manner,
arrays of new molecules (e.g. polypeptides) can be formed.
Ultimately, the new molecule could be released from the bead and
either analyzed or even collected. Examples of the types of beads
that may be used in the present invention are listed in US
2009/0035847, WO 2009/018348, WO 2009/013683, WO 2009/002849 and WO
2009/012420, all incorporated by reference herein in their
entireties.
[0158] In some embodiments, the speed of mixing of first and second
substances can be increased by slipping the device between the open
and closed positions multiple times.
[0159] In some embodiments, multiple areas are aligned to allow
consecutive addition of substances (and possibly further reactions)
by slipping more than one time into further closed positions. In
this embodiment, the slipping can be in the same or different
directions as described with respect to FIGS. 14A and 14B,
discussed supra.
[0160] In some embodiments, the volume of the areas is controlled
such that mixing of two areas is quantitative allowing the
concentration of the substances to be monitored. In some
embodiments, multiple areas are aligned allowing for serial
dilution of substance when the device is slipped into further
closed positions. For example, a first set of at least one first
areas on a base can be filled with a substance, for example via
ducts, in a first position, and then sequentially the area can be
moved into different positions, where, in each position, the at
least one first area is exposed to one of a second set of
pre-filled areas, for example on an opposing plate, that contains a
diluent, for example a buffer. The exposure at each position is
maintained for enough time for the substance to be fully diluted
with the diluents. At each successive position, the substance is
diluted by volume of diluents. If, for example, a first area
contains 1 nanoliter of substance and each of a set of five second
areas are 9 nanoliters in volume, after the first area is exposed
to each of the second areas in turn, the second areas will be
filled with substances diluted approximately 10-fold, 100-fold,
1,000-fold, 10,000-fold and 100,000-fold. The second set of areas
may then be exposed to further areas and substances to conduct
further reactions.
[0161] In an alternative embodiment, a row containing a plurality
of first areas on a base can be filled with a substance, for
example via ducts, in a first position, and then sequentially the
plurality of areas can be moved into different positions, where, in
each position, each one of the plurality of first areas is exposed
to a corresponding second set of pre-filled areas, for example on
an opposing plate, that each contain a diluent, and where each one
of the plurality of first areas is exposed to a different number of
areas in the second set of pre-filled areas. For example, four
first areas can be filled in a first position, and then in a second
position, a first first area is exposed to a diluting area, but the
other three first areas are not. In a third position, the first and
second areas are exposed to diluting areas, but the remaining two
are not. In a fourth position, the first, second and third areas
are exposed to diluting areas, but the remaining one is not. The
result of these actions is to fill a series of four first areas
differing in concentration that may then be moved to at least one
further positions at which they are exposed to reagents, for, for
example, assaying protein binding or inhibition activity. It will
be apparent to one skilled in the art that the number of first
areas and second areas in this example could be readily varied to
any desired value, subject to the available area on the device, and
the amount of substance available.
[0162] Using these techniques, solutions for, for example, protein
activity assays, and/or protein-binding assays, in which a large
range of protein and/or inhibitor concentrations are needed to get
accurate data, can quickly be prepared using small amounts of
material.
[0163] In some embodiments, areas on a first surface are aligned
with those on the opposing second surface so that the area on the
second surface bridges two or more areas on the first surface in
the closed position. In this embodiment, the formed bridge allows
for controlled diffusion from one area on the first surface to
another area on the first surface via the bridging area on the
second surface. This embodiment is especially useful for protein
crystallization.
[0164] A few exemplarily experiments were conducted to illustrate
the usefulness of this device for protein crystallization. One
experiment conducted, referred to as "Crystallization of RC on
SlipChip (L16L025-26)," incorporated the use of the device 10
illustrated in FIGS. 6A-B. Specifically, the experiment occurred on
an aligned PDMS/glass SlipChip (patterned as FIGS. 6A-B, 25
mm.times.75 mm size). The gap between the two layers was filled by
FC-40 before use. The device contains 160 areas for protein and 160
areas for precipitants on two layers, which are complementary for
sliding. All of the areas have a depth of 100 .mu.m and width of
300 .mu.m, and a changing length was used to control the volume in
the range of 8.8 to 14.2 nL. 16 precipitants and reaction center
sample were loaded onto the SlipChip by pipetting. Each precipitant
filled an array of 10 areas with volumes from 8.8 nL to 14.2 nL
(with a steady increment of 0.6 nL between neighboring areas), the
protein fills all 160 areas opposite to the precipitant areas, with
a volume of 14.2 to 8.8 nL. 16 precipitants included No. 1 to No.
14 of CrystalScreen kit (Hampton Research) and two identical
control solutions: 4 M (NH.sub.4).sub.2SO.sub.4 in 50 mM
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 pH 6.0. When loading each
precipitant, a 100 .mu.L pipetter was used. 40 .mu.L of solution
was loaded into the pipetter. To load the solution into the
SlipChip, the end of the pipetter tip was pushed against the
corresponding inlet hole. The solution was then pushed out and
pipetter tip was released when the whole channel was filled. Once
all precipitants were loaded into the chip, the reaction center
sample (.about.24 mg/mL in 4.5% TEAP, 7% 1,2,3-heptanetriol, 0.08%
LDAO and 20 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 pH 6.0. A 10
.mu.L pipetter was used and .about.6 .mu.L of RC sample was loaded
onto the Chip. Sliding was achieved by hand and RC was brought into
contact with the correlated precipitants. After one day's
incubation, only the control precipitant generated crystals. The
other 14 conditions did not yield crystals even after one week.
[0165] Another experiment, referred to as "Crystallization of
lysozyme in hybrid device (notebook page L16L032)" occurred on an
aligned PDMS/glass SlipChip embodiment shown in FIGS. 6A-B and
7A-B. On an aligned PDMS/glass SlipChip (25 mm.times.75 mm size),
which consisted of both FID (FIGS. 7A-B) and microbatch styles
(FIGS. 6A-B), one precipitant (30% PEG 5000 MME, 1 M NaCl in 0.1 M
NaOAc pH 4.8) was loaded into 16 different ports through 16 inlets.
Precipitant filled 12 areas connected with each inlet. These 12
areas were composed of 6 areas for microbatch optimization of
mixing ratio and 6 areas for optimization of free interface
diffusion. For the microbatch experiment, the mixing volume of
protein to precipitants were: 7.8 nL: 15.8 nL; 9.4 nL:14.2 nL; 11.0
nL: 12.6 nL; 12.6 nL:11.0 nL; 14.2 nL:9.4 nL; 15.8 nL:7.8 nL. For
FID, the protein volume and precipitants used were both 16 nL.
There was a bridging duct with cross section of 50 .mu.m.times.50
.mu.m which connected the protein and precipitant areas, with a
distance of 160 .mu.m, 220 .mu.m, 280 .mu.m, 320 .mu.m, 360 .mu.m,
400 .mu.m. The same pipetting procedure for pipetting precipitants
was performed as described above. Lysozyme sample (.about.120 mg/mL
in 0.1 M NaOAc pH 4.8) was loaded into the chip using the same
procedure of loading protein as described above. Within 30 minutes'
incubation, crystals started to appear, first in microbatch style
and then FID style.
[0166] Yet another experiment, referred to as "Crystallization of
lysozyme, thaumatin in FID (L16L24, L16L095)" was conducted on the
embodiment shown in FIGS. 7A-B, referred to as the PDMS/glass
SlipChip with FID style. First, the bottom layer containing areas
of protein sample and precipitants was immersed in FC-40 contained
in a Petri-dish. 7.5 nL solutions of precipitants from Crystal
Screen (Hampton research) and double concentrated wizard I (Emerald
BioSystems) were deposited into the precipitant areas. To deposit
7.5 nL of solution, the solution was first aspirated into a piece
of Teflon tubing (100 .mu.m I.D. and 250 .mu.m O.D.) which was
connected to a 10 .mu.L syringe by another piece of Teflon tubing
(.about.360 .mu.m I.D.). The two pieces of Teflon tubing were
sealed by wax. The syringe was driving by a syringe pump. The pump
was set to use 10 mL syringe at an infusion rate of 300 .mu.L/min.
It was set at volume mode and 7.5 .mu.L was designated to be
dispensed every time. Considering the offset of syringe size, the
actually dispensed volume is 7.5 nL.
[0167] It can also be appreciated that the device 10 of the present
invention can be combined with other microfluidic crystallization
techniques, including those described in U.S. Pat. Nos. 6,409,832;
6,994,749; 7,306,672; 7,015,041; and 6,797,056, all of which are
incorporated by reference herein.
[0168] Moreover, the device of the present invention can be used to
carry out vapor diffusion crystallization experiments. Vapor
diffusion experiments are described in patent applications
WO/2006/101851 and U.S. Publication No. 2005/0087122 and U.S. Pat.
Nos. 6,808,934 and 4,755,363, all incorporated by reference herein.
In some embodiments useful for vapor diffusion crystallization
experiments, at least one first area can be connected to at least
one second area via at least one duct or third area wherein the
duct or third area contains a first substance. In some embodiments,
the at least one first area contains a second substance to be
crystallized dissolved in a solvent, and the at least one second
area contains at least one third substance dissolved in the same
solvent such that the osmotic pressures of the solution in the at
least one first area and at least one second area differ, for
example, by differing in the concentration of a salt. Typically,
the solution in the second area contains a higher salt
concentration than the solution in the first area. The first
substance may be a gas such as air or an oil, but may be any
substance through which the solvent can equilibrate between the
first and second areas. Typically, some portion of the solvent, for
example, water, will diffuse towards equilibrium, moving from the
solution of lower salt concentration, which contains the second
substance to be crystallized, to the solution of higher salt
concentration. This diffusion will concentrate the second substance
to be crystallized, thereby making it more likely to crystallize.
It will be apparent that all of the techniques described herein,
for example, moving a suitable base and a suitable plate that
contain the appropriate areas and/or ducts relative to one another,
can be used to prepare the solutions necessary for such
experiments.
[0169] After deposition, the top layer containing the connecting
"necks" was aligned on top of the bottom layer to connect the
correlated areas for protein samples. After alignment, the two
layers were clamped using four paper clips. Thaumatin solution
(.about.80 mg/mL in water) and lysozyme solution (22 mg/mL) were
injected into the areas through the inlets, respectively. After all
the sample areas were all filled by one of the two samples, sliding
was performed manually. The previous deposition was performed in
such a way that the precipitants from Crystal Screen would be
connected to the thaumatin sample by "necks" while those from
double concentrated Wizard I would be connected to the lysozyme
sample. Within five days, thaumatin was crystallized with condition
29 (0.8 M Sodium potassium tartrate in 0.1 M HEPES pH 7.5) of
Crystal Screen and lysozyme was crystallized with condition 16
(3.75 M NaCl in 0.1 M sodium potassium phosphate buffer pH 6.2) of
double concentrated Wizard I.
[0170] In some embodiments, a substance is immobilized in an area.
For example, catalyst, analyte, and biomolecules (i.e.,
carbohydrates, peptides, proteins, DNA, antibodies, etc.) can be
immobilized using known methods, such as those described in U.S.
Pat. Nos. 4,071,409, 5,478,893, 7,319,003, 6,203,989, 5,744,305 and
6,855,490, all of which are incorporated by reference herein.
[0171] The devices of the present invention can be analyzed using a
variety of known detection methods (optical, x-ray, MALDI, FP/FCS,
FCS, fluorometric, colorimetric, chemiluminescence,
bioluminescence, scattering, Surface Plasmon Resonance,
electrochemical, electrophoresis, lasers, mass spectrometry, Raman
spectrometry, FLIPR.TM. (Molecular Devices), etc.). The device can
be analyzed directly when suitable materials are used (i.e.,
optically transparent materials used for optical detection
methods). For those detection methods, such as optical absorption,
in which the signal is a function of pathlength, multiple areas can
be formed on the device such that they contain identical contents,
but differ only in pathlength. In this way, the chances are
increased that the signal obtained from at least one of the areas
will be within the dynamic range of the detector. A computer system
configured to account for the differing pathlengths could be used
to obtain the final desired result, for example an analyte
concentration. The device alternatively can be opened and
individual areas analyzed or designed to allow slippage into a
further position that allows for access to individual areas (e.g.,
through access holes). In some embodiments, amplification of the
reaction areas may be conducted (e.g. silver-based amplification,
microphage amplification, etc.).
[0172] In some embodiments, once loaded into a duct, an electric
field can be used to separate constituents of a sample
(electrophoresis).
[0173] The device of the present invention can be used to study and
perform coagulation/clotting, protein aggregation, protein
crystallization (including the use of lipidic cubic phase),
crystallization and analysis of small molecules, macromolecules,
and particles, crystallization and analysis of polymorphs,
crystallization of pharmaceuticals, drugs and drug candidates,
biomineralization, nanoparticle formation, the environment (via
aqueous and air sampling), culturing conditions (e.g., stochastic
confinement, lysis of cells, etc.), drug susceptibility, drug
interactions, etc. Techniques for crystallization are described in
US patent and publications U.S. Pat. No. 7,129,091, US
2007/0172954, US 2006/0003439, and US 2005/0087122, incorporated by
reference in their entireties. Methods for assaying blood
coagulation/clotting are described in PCT/US07/02532, incorporated
by reference in its entireties, and are further discussed infra.
These methods, as individual tests or their combinations, include
PT, aPTT, ACT, INR, assays for individual coagulation factors,
measurement of fibrinogen concentration, measurement of platelet
function, thrombelastography and various modifications of this
method, and viscosimetric methods. These methods can be deployed on
slipchip, and can be enhanced by taking advantage of the movement
of the layers of the SlipChip. Protein aggregation assays are
described in U.S. Pat. Nos. 6,949,575; 5,688,651; 7,329,485; and
7,375,190 and US publication 2003/0022243, incorporated by
reference in their entirety. The study of culturing conditions is
described in PCT/US08/71370, incorporated by reference in its
entirety. The device of the present invention can be used in
various assays, including high throughput screening (e.g. one first
substance with many, different second substances; many, different
first substances with many, different second substances), multiplex
assays (e.g. PCR, Taqman, immunoassays (e.g. ELISA, etc.)),
sandwich immunoassays, chemotaxis, ramification amplification
(RAM), etc. The device of the present invention can be used for
various syntheses, including catalysis, multistep reactions,
immobilized multistep synthesis (e.g., small molecule, peptide and
nucleic acid syntheses), solid state synthesis, radioisotope
synthesis, etc. Finally, the device of the present invention can be
used for purification and enrichment of samples.
[0174] As discussed above, embodiments of the invention described
herein may be used for assaying coagulation and platelet function
of blood samples. For example the invention provides a device and
method that may be used to assay blood clotting. The method
includes contacting blood fluid from a subject with at least two
patches, where each of the patches includes stimulus material which
is capable of initiating a clotting pathway when contacted with a
blood fluid from a subject. The stimulus material in one patch
differs from the stimulus material in the other patch; or the
concentration of stimulus material in the one patch differs from
the second patch; or one patch has a surface area different from
the other patch; or one patch has a shape different from the other
patch; or one patch has a size different from the other patch. The
method includes determining which patch initiates clotting of the
blood fluid from the subject. The invention may be used for all
standard coagulation and platelet function assays. Techniques for
assaying coagulation and platelet function in a microfluidic device
are described in the following patent application and herein
incorporated by reference: PCT/US07/02532 (publication number WO
2007/089777).
[0175] In some embodiments, the device can contain areas that are
used as positive or negative controls. To make positive controls,
the analyte that is being tested for in other areas on the device
can be preloaded in the control areas, such that when the device
parts are moved as described herein, the pre-loaded analyte is
exposed to reactions and detected using the same method as the
sample to be measured. When a positive control does not give the
expected result, it can be sign of improper storage or usage of the
device. Similarly, negative control areas can be prepared that
contain no analyte, which would be expected to give no signal when
exposed to the reagents for analysis. Additive verification
controls can also be used to determine integrity of the assay.
Using the techniques of the present invention, a known amount, X,
of analyte can be added to the sample containing the unknown amount
of analyte, and then both the sample containing additional material
and the original sample containing the unknown amount are assayed
for analyte concentration using the same method, preferably on the
same device to give results Y, for the unknown sample, and Z, for
the unknown sample with added amounts of analyte. The difference
between Z and Y should be X, and any deviation from X indicates a
problem with the assay, such as degradation of the assay
reagents.
[0176] Optionally, a detectable, such as a colored, substance, for
example, black ink, or a dye, can be placed in specific control
areas of the device and located such that, movement of the parts of
the device in the manner needed to carry out the desired reactions
in other regions of the device exposes the colored substance to
other areas on either the same or different part of the device such
that a specific known detectable pattern is created. If the
expected pattern is not created, it can be a sign of improper
storage of the device, leakage of the device, or incomplete motion
of the parts of the device through the desired sequence of motions.
In some embodiments, the expected pattern is a barcode. The pattern
may be read by a human or a machine.
[0177] In other embodiments, a user adds sample to a device, slips
through one or more steps, and a readout is obtained as a pattern
of areas that convey information about the presence of analytes and
their concentrations.
[0178] One method of measuring concentration is to take advantage
of multiplexing many assays with different response
characteristics, and then using statistics to calculate the
expected value and the confidence interval. This is analogous to
approaches used in the computer industry such as is done with RAID
for disks and the HP approach to constructing supercomputers using
many potentially faulty chips.
[0179] Alternatively, reactions can be set up in different areas
such that each area displays a different threshold response. That
is, each area has a different sensitivity to the analyte. For
example, for a given analyte, sets of areas can be set up to only
give a response if the concentration exceeds, for example an array
of, for example, 16 areas divided into sets of, for example, four
areas can be formed, where each set only gives a response if, for
example, 20, 25, 30 or 35 concentration units are present. After a
sample is introduced to the 16 areas, if, for example, the sample
really contains the substance at 27 concentration units, then the
concentration can be reported as between 25 and 30 concentration
units with high confidence if all the areas with thresholds of 20
and 25 concentration units respond and none with thresholds of 30
and 35 concentration units respond. Successively greater deviations
from this response pattern will result in successively lower
degrees of confidence in the reported result.
[0180] Mechanisms for generating a threshold response are reported
in PCT US2008/071374, PCT/US07/02532, and PCT/US08/71370, all
incorporated by reference herein in its entirety. In one embodiment
of the device, a first area on the plate of a device comprises the
sample to be analyzed. A second area on the base of a device
comprises a capture area. The capture area contains a substance
capable of capturing an amount of the analyte just below the
threshold level. The threshold for detection is set by the amount
of the substance capable of capturing analyte in the capture area.
For example, the capturing substance could be surface- or
bead-bound antibodies, aptamers or other molecules selective for
the analyte. The device is slipped in order to expose the sample to
be analyzed to the capture area. If beads are used, a membrane
could be disposed between the base and plate to prevent their
movement outside the capture area. After a time sufficient to allow
exchange has occurred, the device is slipped again to expose the
sample to be analyzed to an exchange area placed on the base. The
exchange area contains bound catalyst capable of being displaced by
the analyte. The catalyst may be, for example, functionalized gold
nanoparticles capable of being bound by bead- or surface-bound
antibodies or aptamers. Catalyst will only be displaced in the
exchange area if the capacity of the capture area is exceeded,
leading to analyte being carried over to the exchange area. The
device is then slipped again to exposed displaced catalyst in the
first area to a detection area located on the base. The detection
area contains substances that react in the presence of the catalyst
to produce a detectable signal. For example, if the catalyst is a
functionalized gold nanoparticle, the detection area may be
comprised of two areas one of which contains, for example,
silver(I) and the other of which contains a reducing agent, such as
hydroquinone. They two areas may be located so that they are not
exposed to one another until the first area containing catalyst is
slid over them. Once they are both exposed to catalyst, the gold
nanoparticle catalyzes reduction of silver to form detectable
silver metal. It will be apparent to one skilled in the art that,
at each step of the process, the device should be left in position
for a time sufficient for the reaction to occur, and that the
dimensions and other characteristics of the device could be
optimized, taking into account diffusion, for example, to make this
time longer or shorter.
[0181] In some embodiments, measuring concentration can be done by
measuring intensity or time to reach intensity. Time resolution can
be automatic or manual. For visual or photometric detection, the
device may include a computer with a timer to control or signal at
what time or times an image should be acquired or a test area
observed.
[0182] The device may optionally contain a timer region. The timer
region could contain a standard reaction that indicates when the
device should be moved from one position to the next. A reaction
that undergoes an abrupt visible transition could be used.
Preferably the timer region reaction or reactions are carried out
in separate areas and are initiated by the same movements that
initiate the reactions to be timed.
[0183] Alternatively, a concentration can be determined
geometrically by filling a volume with capture sites, introducing
the analyte at one end, side or edge of the volume and choosing the
conditions such that the analyte binds quickly relative to the rate
of diffusion of the molecule and the rate at which the substance
carrying the analyte flows through the volume so that the analyte
saturates the capture sites as it diffuses and/or flows across the
volume. If a color change or other detectable difference occurs
when the analyte is bound to capture sites, measuring the length or
size of the capture zone directly gives a measure of the amount of
analyte. Alternatively, a competitive strategy in which a complex
of a capture molecule and a labeled analyte is pre-formed in the
volume, then added analyte displaces the labeled analyte, and
finally the labeled analyte is detected as described elsewhere
herein and as will be apparent to one skilled in the art.
[0184] The present invention could be used to for determining copy
number variation of a target polynucleotide in a genome of a
subject including amplification based techniques such as is
described in US 2009/0069194, PCR reactions, such as is described
in US 2008/0129736 and WO 2008/063227, assays of nucleic acid and
protein targets, such as are described in US 2008/0108063, US
2007/0134739, WO 2008/063227, WO 2008/043041 and U.S. Pat. No.
7,413,712, noninvasive fetal gene screening, such as is described
in US 2007/0202525, polynucleotide sequencing, such as is described
in U.S. Pat. No. 7,501,245 and WO 06/088876, cell-based assays such
as are described in US 2008/0107565, US 2007/0077547, U.S. Pat. No.
7,122,301, US 2009/0062134 and WO 2008/063227, biosensors, such as
are described in US 2009/0068760, and high throughput screening,
such as is described in WO 2007/081387, all of which are
incorporated by reference herein. SlipChip may be used to analyze a
few cells obtained from a mammalian embryo, including human, mouse,
rat, bovine and other embryos. Tests may include genetic tests,
including those to establish the presence or absence of certain
genes or mutations in genes, detection of chromosomal abnormalities
including inversions and deletions. PCR, FISH, whole genome
amplification and comparative genomic hybridization and other
technologies may be used on SlipChip. Tests may be applied for
embryo selection, embryo screening, preimplantation genetic
diagnosis, to enable gene therapy, to enable in-vitro
fertilization, and other applications. Conditions for which tests
may be performed include cystic fibrosis, Beta-thalassemia, sickle
cell disease and spinal muscular atrophy type 1, myotonic
dystrophy, Huntington's disease and Charcot-Marie-Tooth disease;
fragile X syndrome, haemophilia A and Duchenne muscular dystrophy.
PCR, FISH and other techniques for analysis and amplification of
nucleic acids may be used, as described in this application.
SlipChip may be used to analyze bilirubin or bilirubin-albumin
complex in blood of neonates.
[0185] Embodiments of the invention described herein may be used
for PCR-based single nucleotide polymorphism (SNP) genotyping or
quantitative measurement of gene expression by real-time PCR in
applications such as plant and animal diagnostics, food and water
safety testing, ecology, agricultural genetics and human disease
research. For example the pathogen E. coli O157:H7 which has been
found in ground beef, unpasteurized milk, bottled juices and sewage
contaminated water, and individual virulence genes of the pathogen
can be rapidly screened for and identified by performing parallel
PCR in the device described herein.
[0186] In addition, the present invention can be used to assay
enzyme concentration and/or activity of enzymes, including but not
limited to glycosidases, peptidases, esterases, phosphatases,
peroxidases, sulfatases, phospholipases, luciferases, Cytochrome
P450, kinases, lipases, phospholipases, oxidases, secretases,
proteases, and peptidases, and to carry out immunoassays, using for
example reagents sold by Life Technologies, Carlsbad, Calif. and/or
Biosynth, Switzerland.
[0187] The device can be used to perform a heterogeneous
immunoassay without a washing step. For example, in one embodiment,
a partial view of which is shown in FIG. 15, a plate 14 of the
device 10 contains an area A, optionally B, C, D, and E, all of
which are preloaded with appropriate reagents or beads. In a first
position, the sample containing the analytes is loaded into the at
least one area A. Anti-analyte capture antibodies are loaded into
an area F on the opposing base. The capture antibody may be
immobilized, for example on beads or on the surface of the area F.
When the base and plate are moved relative to one another to a
second position, the area F is exposed to area A, and analyte
molecules bind to the capture antibody. In a third, optional
position, area F is exposed to area B, which contains buffer and/or
other reagents that help remove potential interfering molecules. In
a fourth position area F is exposed to area C, which contains
detection antibody. The detection antibody is chosen to bind
strongly to the analyte. The detection antibody may be labeled with
an enzyme. Alternatively, it may also be labeled with a fluorescent
tag or other tags, or may be unlabeled, depending on the specific
immunoassay configuration. In a fifth position, area F is exposed
to area D. Area D contains an antibody which binds to the detection
antibody, but with an affinity that is weaker than the detection
antibody-antigen interaction. The antibody in D may be immobilized
on either beads or the surface of area D. The antibody in area D
removes excessive detection antibody from the solution. In a sixth
position, area F is exposed to area E. Area E contains a substrate
solution, which may be converted to a product in the presence of
the enzyme that is linked to the detection antibody. This step is
optional for some immunoassay configurations. Typically, in each
position area F is only exposed to one of areas A, B, C, D, and E.
The device can be configured to perform a single such immunoassay
on a single sample or a plurality of samples, or many different
such immunoassays on a single sample or a plurality of samples.
[0188] The device may be used to perform sample preparation and for
sample storage. For example, the device may be used to remove cells
from blood using filtration and for adding reagents to preserve a
blood sample. Plasma may be filtered from blood using the device by
first introducing the blood into an input volume in a device
comprised of at least one first area and/or ducts. The input volume
is exposed to at least one second area separated from the input
volume by a membrane, such that some or all of the plasma passes
through the membrane into the at least one second area. Excess
plasma may be collected in at least one third area exposed to the
at least one second area but not directly to the input volume.
Optionally, in the same device, the at least one second area may be
filled with plasma as described above, and other at least one
fourth area may be filled with whole blood by exposure through, for
example, a disrupted membrane, or no membrane.
[0189] After filling areas with plasma, they can be used for a
variety of reactions and manipulations. For example, by using the
relative motion of the parts of the device to expose the at least
one second area to additional areas, plasma can be preserved by
addition of citrate or EDTA to prevent coagulation. Other
preservatives or reagents can be added similarly. The whole device
may be then stored and transported for analysis. For analysis, all
or some of the plasma can be removed from areas and used in other
assays outside of the device. In addition, the at least one area
containing plasma may be moved into additional positions to perform
additional analysis. This analysis could be done using reagents
preloaded in additional areas on the device. This analysis could
also be performed using user-added reagents; this method is
attractive for assays that involve those reagents that are
difficult to preload and that are easier to add immediately prior
to the assay. Optionally, assays can be performed on the device at
the time of sample collection, or at a later time, for example, in
a setting in which external temperature is more readily controlled,
or external detectors are available.
[0190] The device can be used with, and/or incorporate, a
chemistrode for sampling (See: Chen, et al., PNAS, Nov. 4, 2008,
vol. 105, no. 44 16843-16848; Keats, J., "Jargon Watch," Wired
Magazine 17.03, 2/23/09; Armstrong, G., Nature Chemistry (14 Nov.
2008), doi: 10.1038/nchem.89, Research Highlights.).
[0191] A single device could be used to store and/or perform a
single assay or a plurality of assays on samples from a single
patient, or to store and/or perform a single assay or a plurality
of assays on samples from a plurality of patients. Other types of
sample preparation and storage can also be performed, for example
for preparing and storing other bodily fluids, or environmental
samples. Additionally, the areas 18, 22, the ducts 26, or
combinations of areas 18, 22 and ducts 26 of one embodiment of the
device 10, may constitute a separation path or a separation area.
Separation may be carried out by the methods known in the art,
using chromatography, electrical potentials including gel and
capillary electrophoresis, hydrodynamic separations, filtration,
separations by centrifugations, separations based on magnetic and
optical forces. A variety of species may be separated including
molecules including proteins and nucleic acids, macromolecules,
particles and cells. Patents and published applications discussing
the separation path or area include U.S. Pat. Nos. 5,707,850;
5,772,889; 5,948,624; 5,993,631; 6,013,166; 6,274,726; 6,436,292;
6,638,408; 6,716,642; 6,858,439; 6,949,355; and U.S. Publication
No. 2002/0076825. These patents and patent applications are
incorporated herein by reference in their entirety.
[0192] Membranes can be incorporated into the SlipChip. For
example, a dialysis membrane may be used to concentrate
macromolecules on chip, for example for macromolecular and protein
crystallization. Membranes can be used to perform other
separations, for example separate cells, including blood cells, and
to separate components of blood and other biological fluids.
[0193] Slipping the two plates relative to one another may be used
to carry out a transformation for example: reconfiguring separation
path or area, capturing a separated product, bringing reagents to
the separation path or area to detect, visualize or analyze.
[0194] In some embodiments, the slip chip can be used for two stage
reactions. For example, a slip chip capable of moving between a
first, second and third position can be configured with areas such
that at least one first area overlaps at least one second area in
the second position, and the second areas are smaller (for example
one-tenth or one-twentieth the size) than the at least one third
area that the first area overlaps in the third position. Such a
device may be used for a two-stage protein crystallization
experiment. The at least one first area is filled with protein to
be crystallized. The at least one second area is pre-filled or
user-filled with a substance expected to induce nucleation, for
example a higher concentration of precipitant, or a solution of
methyl-.beta.-cyclodextrin or a solution of another substance
capable of removing detergent. The at least one third area may
contain, for example, a lower concentration of precipitant. To use
the chip, first, areas would be filled. Then, the device would be
moved to the first position to nucleation, and either held there
for a time sufficient to induce nucleation or moved continuously
across the first position such that the at least one first area and
at least one second area are in contact for a time sufficient to
induce nucleation. The time could be, for example, 1 second, 30
seconds, or 5 minutes. The device would then be moved to the third
position. The small size of the at least one second area prevents
significant dilution of the sample.
[0195] In some embodiments, a user-loaded SlipChip can be used to
perform multiplexed nanoliter-scale experiments by combining a
sample with multiple different reagents, each at multiple mixing
ratios. The mixing ratios, characterized, for example, by diluting
a fluorescent dye, can be controlled by the volume of each of the
combined areas. Such a SlipChip design was used to screen the
conditions for crystallization of a soluble protein, glutaryl-CoA
dehydrogenase from Burkholderia pseudomallei, against 48 different
reagents; each reagent was tested at 11 different mixing ratios,
for a total of 528 crystallization trials, each on the scale of
.about.12 nL. This experiment was conducted using 3 identical
SlipChip devices, each screening 16 different reagents. The total
consumption of the protein sample was .about.10 .mu.L. Conditions
for crystallization were successfully identified. The
crystallization experiments were successfully scaled up in plates
using the conditions identified in the SlipChip. Crystals were
characterized by X-ray diffraction and provided a protein structure
in a different space group and at a higher resolution than the
structure obtained by conventional methods. The user-loaded
SlipChip reliably handles fluids of diverse physicochemical
properties, such as viscosities and surface tensions. Quantitative
measurements of fluorescence intensities and high-resolution
imaging were straightforward to perform in these glass SlipChips.
Surface chemistry was controlled using fluorinated lubricating
fluid, analogous to the fluorinated carrier fluid used in
plug-based crystallization. This approach can be used in a number
of areas beyond protein crystallization, especially those areas
where droplet-based microfluidic systems have demonstrated
successes, including, for example, measurements of enzyme kinetics
and blood coagulation, cell-based assays, and chemical
reactions.
[0196] In certain embodiments, the SlipChip can be used to combine
a sample with many different reagents, each at many different
mixing ratios, to perform multiplexed nanoliter-scale experiments
in a user-loaded fashion. In certain embodiments, this can be done
without the need for equipment external to the SlipChip, such as
extra fluid-handling equipment. Multiplexed experiments are common
in the areas of biological assays, chemical synthesis,
crystallization of proteins and any area where chemical space is
widely explored. U.S. Patent Application 61/162,922, incorporated
by reference in its entirety herein, describes additional features
and embodiments of the SlipChip. Wide exploration of chemical space
benefits from technologies for faster experiments and lower
consumption of samples, both to make these processes more
productive and to reduce the amount of chemical waste. Microfluidic
technology has both the capacity for high throughput screening and
the ability to manipulate fluids on nanoliter and smaller scales.
Although various microfluidic systems have been developed for such
applications, these systems often require pumps, valves, or
centrifuges. Certain embodiments of the SlipChip can be used to
perform multiplexed microfluidic reactions without pumps or valves
and its operation, in certain embodiments, requires only pipetting
of a sample into the chip followed by slipping one part of the chip
relative to another to combine the sample with pre-loaded reagents
and initiate the reactions. (Additional exemplary means of
configuring the SlipChip for slipping are described in Chung, et
al., Lab Chip, 2009, 9, 2845-2850, incorporated by reference herein
in its entirety.) In certain embodiments of the SlipChip the sample
is combined with pre-loaded reagents. For certain embodiments,
pre-loading the reagents onto the chips in a centralized facility
and distributing chips to researchers is attractive to dramatically
simplify the experiment for the user. In certain embodiments, a
SlipChip does not have to be pre-loaded with reagents. The
inventors have demonstrated that the SlipChip can be used to
perform multiplexed nanoscale experiments with many different
reagents, each at multiple different mixing ratios, allowing
exploration of chemical space on the regional scale.
[0197] The inventors used this approach to screen conditions for
crystallization of a soluble protein. Obtaining crystals of
proteins remains one of the bottlenecks to solving their structures
and elucidating their functions at the molecular level. Getting
"diffraction-quality" crystals requires high throughput screening
of multiple precipitants at various concentrations, i.e.,
performing, for example hundreds or thousands of crystallization
trials. Microfluidic technology using either valves or droplets to
accurately handle nanoliter and even picoliter volumes has been
described, and has also been applied to crystallization of
proteins. Although these two approaches can successfully
crystallize proteins, most individual laboratories are still
setting up crystallization trials by pipetting microliters of
solutions into 96-well plates, suggesting that there is still a
need for a system for crystallizing proteins that is simple,
inexpensive, fast, and controllable. Here we describe embodiments
of a user-loaded SlipChip that satisfies these criteria.
[0198] In some embodiments of a user-loaded SlipChip, the two
plates of the SlipChip can be aligned such that the sample areas
and sample ducts are aligned to form a continuous fluidic path, and
the reagent areas and reagent ducts are offset. The sample can be
loaded through a continuous fluidic path formed by overlapping
sample areas (top plate) with sample ducts (bottom plate). The
device can be slipped such that the reagent areas (bottom plate)
and reagent ducts (top plate) are now aligned. Reagents can be
loaded into the individual fluidic paths formed by overlapping
reagent areas and sample areas. The device can be slipped a second
time, and the sample areas from the top plate are exposed to the
reagent areas of the bottom plate. The order of loading reagents
and sample can be determined by the user.
[0199] In one embodiment of the invention, the SlipChip was used to
screen a protein sample against 16 different precipitants, at 11
mixing ratios each, for a total of 176 experiments, each on the
scale of .about.12 nL, and requiring only 3.5 .mu.L of the protein
sample for all of the experiments. The SlipChip contained 16
separate fluidic paths for the reagents, each path with 11 areas,
and a single, continuous fluidic path for the protein sample with
176 areas. In some embodiments of the SlipChip, the inlets for
fluidic paths of reagents were spaced in a way to match the spacing
of areas in a 96-well plate and spacing of tips in a multichannel
pipettor. This SlipChip consisted of two plates. The top plate
contained separate inlets for the reagent and the sample, ducts for
the sample, and areas for the reagent. The bottom plate contained
ducts for the reagent which were connected to an inlet on the top
plate, areas for the samples, and an outlet. The two plates were
separated by a layer of lubricating fluid, for which the inventors
used fluorocarbon, a mixture of perfluoro-tri-n-butylamine and
perfluoro-di-n-butylmethylamine (FC-40). When the two plates were
first assembled, the inlet and areas for the reagent in the top
plate were aligned on top of the ducts for the reagent in the
bottom plate. In this orientation, each reagent was pipetted into
the inlet, flowed through the ducts, and filled the areas. After
loading the reagents, the top plate of the chip was "slipped" to a
new orientation, where the ducts for the sample in the top plate
were aligned on top of the areas for the sample in the bottom
plate. In this orientation, the sample was pipetted into the inlet,
flowed through the ducts, and filled the areas. After loading both
sample and reagents, the top plate of the chip was slipped again to
position the areas for the reagent on top of the areas for the
sample and initializing the interaction between the reagent and the
sample by diffusion.
[0200] In one embodiment of a user-loaded SlipChip the top plate
consisted of an outlet duct, a reagent inlet, a sample inlet
aligned to sample ducts, and reagent areas. The bottom plate
consisted of an outlet aligned with reagent ducts and sample areas.
The top plate and bottom plate were assembled and filled with
fluorocarbon to generate a SlipChip ready for use. In this
orientation, a continuous fluidic path was formed by the reagent
inlet, the reagent areas, and the outlet. A reagent was introduced
by pipetting. The reagent flowed through the continuous fluidic
path and filled the reagent areas. The chip could be slipped into a
second position. In this second position, a continuous fluidic path
was formed by the sample inlet, the sample ducts, and the sample
areas. The sample may be introduced by pipetting. The sample flowed
through the continuous fluidic path and filled the sample areas.
The chip could be slipped again into the third position, where the
reagent areas were aligned on top of the sample areas, and the
sample and reagent in the aligned areas combined by diffusion.
[0201] In certain embodiments, during the slipping steps an
undesired thin film of reagent solution can form between the two
plates of the SlipChip. This thin film can, in certain embodiments,
connect the duct for the reagent to the area for the reagent
instead of keeping them separated. Cross-contamination after the
slipping steps can be minimized by controlling the contact angle
between the solutions (sample or reagents) and the plates of the
SlipChip, measured under the lubricating fluid. The inventors
measured the contact angle under the lubricating fluid used for
certain embodiments, fluorocarbon (FC), and determined that, for
certain embodiments, a contact angle above .about.130.degree. is
preferred to minimize cross-contamination. To confirm this, when
the inventors loaded a solution of reagents containing no
surfactants and having a contact angle of 139.degree., reagents did
not get trapped between the plates of the SlipChip after the first
slipping step. The contact angle preference was found to be the
same for the second slipping step; when the inventors added
surfactant to the sample solution, the contact angle dropped to
110.degree., and a thin film of the surfactant solution was trapped
between the two plates of the SlipChip. To minimize this problem
for certain embodiments, the inventors spin-coated the plates with
thin layers of fluorinated ethylene propylene (FEP) increasing the
contact angle to 154.degree.. After spin coating, the slipping
steps were performed without cross-contamination.
[0202] Using this embodiment of the SlipChip, the inventors
controlled the volumes, and thus the mixing ratio, of both the
sample and reagents that were combined into each trial. The
inventors designed this SlipChip with areas for reagent and samples
such that the total volume of a trial, created by slipping to
combine the two areas, was always .about.12 nL, and the mixing
ratio of reagent and sample in each trial varied from 0.67:0.33 to
0.33:0.76 by volume, with nine evenly spaced ratios in between.
[0203] Experimental results using a fluorescent dye solution as the
sample and a buffer solution as the reagent confirmed that this
design did lead to a controlled mixing ratio in each of the 11
areas. The relationship between the relative concentrations of the
sample from the experiment and the predicted concentrations based
on the design showed good agreement: the disparity between the
experimental and predicted concentrations was lower than 10% for
all except one of the areas.
[0204] In one embodiment of the present invention, the SlipChip had
areas for the sample in the bottom plate containing a fluorescent
dye solution and areas for the reagent in the top plate containing
a buffer solution. Each area was a different size and held a
different volume of fluid. Areas ranged in volume from 8 nL
(relative volume of 0.67) to 4 nL (relative volume of 0.33). Once
the chip was slipped to combine the reagents and the sample, the
total volume of a trial was always 12 nL. A graph of the relative
concentrations of the diluted sample from the experiment plotted
against the relative concentrations that were predicted based on
the designed volume showed good agreement between the experimental
and predicted concentrations (slope=0.98; R.sup.2=0.9938). The
concentration was inferred from the measurements of fluorescent
intensities. A histogram of the number of areas with different
disparity values was generated. The disparity was calculated as the
percentage difference in concentration between the experiment
results and the predicted concentration, and tooks into account
errors and deviations in fabrication of the areas, filling of the
areas, slipping, and measurements of intensity.
[0205] The inventors identified the variability in reagent
concentrations using this approach with crystallization of a model
membrane protein, the photosynthetic reaction center (RC) from
Blastochloris viridis. Seven replicate trials, each with 11
different mixing ratios of a precipitant (3.2 M
(NH.sub.4).sub.2SO.sub.4 in 40 mM
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 6.0) and RC, were performed
on the SlipChip and were reproducible. Different mixing ratios were
randomly arranged across the rows of the SlipChip. That is, instead
of beginning at a mixing ratio of 0.33 precipitant to 0.67 protein
and ending at a mixing ratio of 0.67 precipitant to 0.33 protein
with evenly spaced mixing ratios in between, the areas were
arranged from left to right in the following order with regard to
the relative precipitant concentration: 0.33, 0.63, 0.4, 0.57,
0.47, 0.5, 0.53, 0.43, 0.6, 0.37, and 0.67. This arrangement was
chosen so that any artifacts of manufacturing or evaporation that
might systematically skew the results from one side to another
could be easily differentiated from the effects of mixing ratios.
This arrangement also kept the distance between two adjacent areas
similar, keeping the duct length similar to the area size, making
fabrication of the SlipChip simpler. The results obtained were the
same as when the different mixing ratios were arranged sequentially
across the rows of the SlipChip, indicating that any effects due to
manufacturing or evaporation are minimal.
[0206] To help understand the behavior of crystallization, the
inventors digitally re-arranged the microphotographs of the areas
in order of increasing concentration of the precipitant. At mixing
ratios of precipitant to protein from 0.33:0.67 to 0.43:0.57, none
of the seven trials formed protein crystals. At a mixing ratio of
0.47:0.53, one trial formed protein crystals, and at 1:1 four
trials formed protein crystals. At mixing ratios of 0.53:0.47,
0.57:0.43 and 0.6:0.4, all seven trials formed protein crystals. At
0.63:0.37, all seven trials formed precipitate. At 0.67:0.33, two
trials formed protein crystals while the remaining five formed
precipitate. Crystallization of RC was found to be sensitive to
precipitant concentration. As the inventors increased the relative
concentration of precipitant, the inventors observed a transition
from the protein remaining in solution to crystallizing to
precipitating. Decreasing protein concentration was observed to
reduce nucleation to a certain extent. Crystallization outcome was
not monotonic with mixing ratio, with regions of larger single
crystals separated by regions of microcrystals. In addition to the
seven rows used for the seven experiments described here, on this
chip two rows were intentionally left blank and the additional
seven trials were performed at a higher concentration of
precipitant.
[0207] The inventors also screened the conditions for
crystallization of protein samples using many different reagents,
each at many different mixing ratios, on a single user-loaded
SlipChip. The inventors chose a soluble protein as the target:
glutaryl-CoA dehydrogenase from Burkholderia pseudomallei. The
protein sample was obtained from the Seattle Structural Genomics
Center for Infectious Disease (SSGCID). It was screened in parallel
without the use of a SlipChip to yield crystals under vapor
diffusion conditions in conditions using 20% (w/v) PEG-3000, 0.1 M
HEPES pH 7.5, 0.2M NaCl (PDBid 3D6B). These crystals yielded a
structure of 2.2 .ANG. resolution and space group
P2.sub.12.sub.12.sub.1 (PDBid 3D6B). Without any knowledge of those
crystallization conditions, the protein was screened on an
embodiment of the SlipChip against 48 different reagents from a
home-made screening kit based on the Wizard screen. For each
reagent, 11 different mixing ratios of protein sample and reagent
were screened, ranging from 0.33:0.67 to 0.67:0.33 as described
above. The screen successfully identified two conditions for
crystallization of the protein. From these results, optimal
conditions were chosen: a 0.57:0.43 mixing ratio with 45% (w/v)
PEG-400, 0.2 M MgCl.sub.2 and 0.1 M Tris, pH 7.8 and a 0.67:0.33
mixing ratio with 30% (w/v) PEG-8000 and 0.1 M Hepes, pH 7.8. The
latter condition is similar, but not identical, to the one
identified by using traditional technologies at SSGCID. Each of
these conditions was reproduced in area plates, and crystals were
obtained in both cases. The crystals from the area plates
diffracted X-rays at resolutions of 1.6 .ANG., space group P21 and
2.9 .ANG., space group P212121 respectively. Consequently, the
inventors determined the structure of the protein at the resolution
of 1.73 .ANG., with the data set collected from the crystal that
diffracted X-rays to the higher resolution, 1.6 .ANG., and the
inventors could assign the loops missing in the 2.2 .ANG.
P2.sub.12.sub.12.sub.1 structure.
[0208] In some embodiments the SlipChip does not require external
equipment for operation. For example, in certain embodiments, the
sliding can be done manually. In certain embodiments internal
guides can be used to constrain the motion of the plates relative
to one another. In some embodiments, the results of a reaction or
reactions carried out on the device can be read out without
specialized equipment, for example, using widely available
equipment e.g., a camera on a cell phone, or by eye, or using a
barcode scanner. In certain embodiments, readout is facilitated by
having each area of the device function as a pixel in a digital
display, wherein different results produce different overall
patterns that can be perceived and/or interpreted by a human and/or
a machine.
[0209] In certain embodiments of the present invention, a
user-loaded, SlipChip can be used to perform multiplexed reactions
by screening many different reagents against a substrate at
different mixing ratios and accurately meter nanoliter volumes.
Certain embodiments of the SlipChip can be delivered to researchers
preloaded with reagents at multiple mixing ratios or user-loaded at
the site of use, depending on the requirements of a given
application. The fluid paths can be designed to include extra ducts
to increase fluidic resistance and to provide adequate filling of
all areas. This method is functionally akin to the droplet-based
hybrid method where many different conditions are screened in a
droplet-based array. The inventors have demonstrated the use of the
SlipChip in screening conditions for crystallization for a soluble
protein. X-ray diffraction data for the protein were obtained by
replicating crystallization conditions in well plates,
demonstrating that crystallization conditions identified in a
SlipChip can be reliably scaled up outside of the SlipChip.
Crystallization by free interface diffusion on a different
embodiment of a SlipChip can be performed and, in yet another
embodiment, a composite SlipChip can be used to perform both
microbatch and free interface diffusion crystallizations in
parallel.
[0210] In addition to crystallization, user-loaded SlipChip
embodiments are applicable to a number of other multiplexed
reactions and assays where testing both different reagents and
their concentrations is desirable. A fluorinated lubricating fluid,
for example, can be used to directly transfer established
approaches for control of surface chemistry into certain
embodiments of the SlipChip. Assays similar to those performed in
plug-based systems, such as those using enzymes, and cells can be
performed in certain embodiments of the SlipChip. The inventors
found imaging certain embodiments of the SlipChip to be readily
accomplished, as positions of all areas are defined. Certain
embodiments of user-loaded SlipChips can be used for those
applications where droplet-based approaches, especially the hybrid
approach, have been demonstrated. In general, attractive
applications of user-loaded SlipChips include diagnostics, drug
discovery, combinatorial chemistry, biochemistry, molecular biology
and materials science.
Example
[0211] Chip Design and Fabrication
[0212] Slipchip was fabricated using glass etching fabrication of
SlipChip as described elsewhere in this application, except for the
following changes: In this example, .about.45 minutes of etching
was used to yield a depth of .about.60 .mu.m. Access holes were
drilled with a diamond drill bit 0.030 inches in diameter. The
surfaces of the etched glass plates were cleaned with Millipore
water, followed by ethanol and subjected to an oxygen plasma
treatment before silanization or Fluorinated Ethylene Propylene
(FEP) coating.
[0213] Spin Coating FEP.
[0214] An aqueous emulsion of FEP (TE-9568, Dupont) was first
diluted 4 times with Millipore water before use. Following plasma
cleaning the SlipChip device, the solution was evenly spread onto
the device by using a plastic pipette. For spin coating, the spin
speed was set at 1500 rpm and the process was executed for 30
seconds, or the spin speed was set at 2000 rpm and the process was
executed for 30 seconds. Once the coating was finished, the
SlipChip was transferred to a 120.degree. C. oven and incubated for
10 minutes. After incubation, the SlipChip was baked at 250.degree.
C. on a hot plate for 10 minutes, followed by baking while
increasing the temperature to 265.degree. C. for another 10
minutes. After baking, the SlipChip was sintered at 340.degree. C.
on a hot plate for 1 minute. The sintered Chip was then cooled to
room temperature.
[0215] Assembling the SlipChip.
[0216] The SlipChip was assembled under FC-40. The bottom plate was
first immersed into FC-40 in a Petri dish, with the patterns facing
up. The top plate was then laid on top of the bottom plate, with
the patterns facing down. The two plates were aligned into the
position, by moving them relative to each other and then fixed by
using four micro binder clips. The SlipChip was ready for use after
the extra FC-40 on the surface was removed.
[0217] Measuring Contact Angles.
[0218] The plate of the SlipChip was first immersed into
fluorocarbon in a tank. The plate, facing down, was clamped by two
micro binderclips on each end to create a gap between the plate and
the bottom of the tank. 5 .mu.L of the measured aqueous solution
was pipetted into the gap, and the aqueous droplet contacted the
plate due to its buoyancy in the surrounding fluorocarbon. The
contact angle of the droplet on the substrate was then measured by
using an optical contact angle meter (Rame-Hart Instrument Co.,
Model 500).
[0219] Food Dye Assays.
[0220] All the solutions used for food dye assays were filtered
with a 0.45 .mu.m PVDF syringe filter before use. Four food dyes
(brown, pink, red, and blue, Ateco, Glen Cove, N.Y.) were diluted
.about.10 times from their stock solutions and were pipette-loaded
into 16 reagent ducts. To load each duct, 4 .mu.L of dye was first
pushed through the inlet using a pipette until the dye solution
emerged from the outlet. After loading reagents, the SlipChip was
slipped to form a continuous fluidic path for the sample. A green
dye was diluted 20 times and then loaded through the sample inlet.
Using a pipette 4 .mu.L of dye was loaded into the Chip until all
the sample ducts were fully filled. Once the sample was loaded, the
SlipChip was slipped again to mix the solutions by diffusion.
[0221] Quantifying Mixing Ratio.
[0222] The loading procedure was similar to that for the food dye
assays. Two solutions, the fluorescent solution (44.8 .mu.M
Alexa-488 in 10 mM Tris, pH 7.8) and the buffer (10 mM Tris, pH
7.8), were used. The outermost four fluidic paths, each path
containing 11 areas, were loaded with the fluorescent solution, and
the remaining 12 fluidic paths were loaded with the buffer. The
fluorescent solution was also used as the sample. After the areas
for the reagent and areas for the sample were combined, the
SlipChip was incubated for one hour in the dark to allow complete
mixing. The SlipChip was then slipped a second time to separate the
areas for the reagent from those for the sample. The outermost four
fluidic paths containing the fluorescent solution were not diluted,
providing a control for calibrating intensity measurements.
[0223] Quantifying Mixing Ratio:
[0224] Measuring fluorescence. To confirm that the fluorescence
intensity of Alexa-488 is linearly correlated with the
concentration in the working range of the fluorescent microscope,
the inventors made a dilution curve on a SlipChip. First, four
solutions, including one buffer (10 mM Tris, pH 7.8) and three
solutions at concentrations of 1/4, 1/2, and 1 times the
concentrations of the original Alexa-488 solution (44.8 micromolar
in 10 mM Tris pH 7.8), were loaded into four separated fluidic
paths in a pre-assembled user-loaded SlipChip. The top plate was
slipped relative to the bottom plate so that all the areas were
separated. The fluorescence intensity of the loaded areas on the
bottom plate was then measured by using a Leica DMI6000 microscope
(Leica Microsystems) with a 10.times.0.4 NA Leica objective and a
Hamamatsu ORCAER camera. A GFP filter was used to collect Alexa-488
fluorescence. An exposure time of 4 ms was used. Images were
acquired and analyzed by using Metamorph imaging system version
6.3r1 (Universal Imaging). To extract the intensity of the
fluorescent signal, a region of 100 pixels by 100 pixels was
selected in the middle of every area of interest. The average
integrated intensity of the regions belonging to areas with the
same Alexa-488 concentration (five areas for each concentration)
was plotted against the corresponding concentration to obtain a
calibration curve.
[0225] The fluorescent measurement was then performed by using the
sample areas. The inventors measured the fluorescence intensity of
the areas in the bottom plate. This ensured that the working
parameters for measuring fluorescence intensity were consistent.
The same setup for the fluorescent microscope was used in this
experiment as was used in making the dilution curve. The intensity
from the measurements was then converted to concentration based on
the dilution curve. To calibrate the microscope, the fluorescence
intensity of a fluorescence reference slide for GFP was recorded
and used for background correction. Images were acquired and
analyzed by using Metamorph imaging system version 6.3r1 (Universal
Imaging).
[0226] Quantifying Mixing Ratio:
[0227] Characterization of area sizes. The wet etching of glass is
assumed to be isotropic, and the speed of etching is the same in
all directions. The size of the areas after etching was measured by
using a Leica MZ 16 Stereoscope calibrated by a micro-ruler and the
volume of the areas were calculated accordingly.
[0228] Quantifying Mixing Ratio: Data Analysis.
[0229] To calibrate the intensity measurements, the background
intensity was first subtracted from all the fluorescent images. The
intensity of each area was then extracted from the integrated
intensity of a 100 pixel by 100 pixel region located at the center
of each area. The dilution ratio for each area was obtained by
dividing the intensity of that area by the intensity of a area of
the same size that did not get diluted.
[0230] RC Crystallization.
[0231] A sample of the photosynthetic reaction center (RC) from
Blastochloris viridis was obtained. The loading procedure was
similar to that for the food dye assays. The precipitant (3.2 M
(NH.sub.4).sub.2SO.sub.4 in 40 mM
NaH.sub.2PO.sub.4/Na.sub.2HPO.sub.4, pH 6.0) was loaded into seven
reagent ducts and the protein sample (36 mg/mL RC in 0.07% (w/v)
LDAO, 7% (w/v) 1,2,3-heptanetriol, 4.5% (w/v) triethylamine
phosphate (TEAP), 17 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH
6.0) was loaded into the sample duct. The SlipChip containing the
trials was then stored in FC-70 in a Petri dish at room temperature
in the dark. The trials were monitored over 10 days to check for
the formation of crystals.
[0232] Crystallization of Glutaryl-CoA Dehydrogenase from
Burkholderia pseudomallei in SlipChip.
[0233] The protein sample was obtained from the Seattle Structural
Genomics Center for Infectious Disease (SSGCID). 48 precipitants
from a home-made screening kit based on the Wizard screen were
loaded into three SlipChips, 16 precipitants in each Chip; the same
loading procedure was the same as in the food dye experiments. Each
SlipChip was then immersed into FC-70 in separate Petri dishes. The
Petri dishes were incubated at room temperature and the results
were monitored for two weeks. Images of areas containing crystals
were taken by a SPOT Insight camera (Diagnostic Instruments, Inc.,
Sterling Heights, Mich.) coupled to a Leica MZ 16 Stereoscope.
[0234] Crystallization of Glutaryl-CoA Dehydrogenase from
Burkholderia pseudomallei in Well Plates, not Using a SlipChip.
[0235] Once a crystallization condition for glutaryl-CoA
dehydrogenase was identified, the experiment was scaled up in a
sitting-drop well plate (Hampton research) using the microbatch
method. At the same mixing ratio identified by the screening
experiments on the SlipChip, the protein sample was mixed with the
precipitant to obtain a final volume of 3 .mu.L in the well. In the
reservoir, Millipore water was mixed with the precipitant to give
the same precipitant concentration as in the well; the final volume
was 600 .mu.L. Each condition had one duplicate. The plate was then
sealed with sealing tape (Hampton research) and incubated at room
temperature. Images of crystals were taken by a SPOT Insight camera
(Diagnostic Instruments, Inc., Sterling Heights, Mich.) coupled to
a Leica MZ 16 Stereoscope.
[0236] X-Ray Diffraction and Data Processing.
[0237] Crystals for x-ray diffraction were obtained from the well
plate experiments. For precipitants that contained PEG-400, the
mother liquor was used as a cryo-protectant, and the concentration
of PEG-400 was changed to be 25% (w/v). For other precipitants, the
mother liquor plus 20% (v/v) glycerol was used as a
cryo-protectant. A crystal was first transferred from the original
well to the well containing the cryo-protectant by using a nylon
loop. Then the crystal was frozen in liquid nitrogen. The X-ray
diffraction assays were performed at GM/CA Cat station 23 ID-D of
the Advanced Photon Source (Argonne National Laboratory). X-ray
data were collected at 100 K using a wavelength of 1.0332
.ANG..
[0238] The data were processed and analyzed using HKL-2000.
[0239] X-Ray Structure Determination of Glutaryl-CoA
Dehydrogenase.
[0240] The structure of glutaryl-CoA dehydrogenase was solved by
molecular replacement using the PDBid 3D6B structure as a starting
model and the MOLREP program in CCP4 suite. The data collected from
crystals grown in the condition containing PEG-400 were used. The
rigid-body, positional, and temperature factor refinement was
performed using maximum likelihood target with the program REFMAC5.
The SigmaA-weighted 2Fobs-Fcalc and Fobs-Fcalc Fourier maps were
calculated using CCP4. The Fourier maps were displayed and examined
in COOT. The search for new solvent molecules was performed with
help of COOT. The coordinates and structure factors have been
deposited in the Protein Data Bank with entry code 3119
(pending).
[0241] In certain embodiments of the SlipChip, multi-parameter
screening can be performed for nanoliter protein crystallization
combining free interface diffusion and microbatch methods. In
certain embodiments of the present invention, a SlipChip-based free
interface diffusion (FID) method and a SlipChip-based composite
method that simultaneously performs microbatch and FID
crystallization methods in a single device can be performed.
[0242] In one embodiment, the FID SlipChip was designed to screen
multiple reagents, each at multiple diffusion equilibration times,
and was used to screen conditions for crystallization of two
proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and
dihydrofolate reductase/thymidylate synthase from Babesia bovis
against 48 different reagents at 5 different equilibration times
each, consuming 12 .mu.L of each protein for a total of 480
experiments using three SlipChips. The composite SlipChip was
designed to screen multiple reagents, each at multiple mixing
ratios and multiple equilibration times, and was used to screen
conditions for crystallization of two proteins, enoyl-CoA hydratase
from Mycobacterium tuberculosis and dihydrofolate
reductase/thymidylate synthase from Babesia bovis. To prevent
cross-contamination while keeping the solution in the neck ducts
for FID stable, the plates of the SlipChip were etched with a
pattern of nano-scale areas. This nanopattern was used to increase
the contact angle of aqueous solutions on the surface of the
silanized glass. Nanopatterning is generally described in Z. Burton
and B. Bhushan, Nano letters, 2005, vol. 5, no8, pp. 1607-1613,
incorporated by reference in its entirety. The composite SlipChip
increased the number of successful crystallization conditions and
identified more conditions for crystallization than separate FID
and microbatch screenings. Crystallization experiments were scaled
up in well plates using conditions identified during the SlipChip
screenings, and X-ray diffraction data were obtained to yield the
protein structure of dihydrofolate reductase/thymidylate synthase
at 1.95 .ANG. resolution. This free-interface diffusion approach
provides a convenient and high-throughput method of setting up
gradients in microfluidic devices, and can also be used for
cell-based assays.
[0243] A SlipChip-based approach can be used to simultaneously
perform two methods for protein crystallization, microbatch and
free interface diffusion (FID), in a single microfluidic device.
Currently, there are challenges to protein crystallization. To
crystallize proteins, a large chemical space must be searched to
determine the conditions required. The search for the right
precipitants and the right concentrations of protein and
precipitant is expedited by faster assays and smaller sample sizes,
and a simple, fast, and controllable system advances the discovery
of new protein structures. A particularly attractive method to
crystallize proteins is nanoliter-scale FID because it explores the
phase diagram for crystallization as both the concentration of
protein and the concentration of precipitant are gradually changed
by diffusion, provides a higher transient supersaturation level for
crystal nucleation, and eliminates precipitation induced by fast
mixing. Nanoliter-scale FID is consequently efficient for
crystallization, but currently it is only implemented with
valve-based systems. FID is mechanistically very similar to the
well-established counter diffusion methods that are typically
implemented on microliter scales, including chip-based and gel
acupuncture-based approaches. The use of valves in FID requires
external control equipment, and valves are often composed of PDMS.
PDMS devices have the additional complication of requiring control
of the atmosphere and evaporation. Valve-free approaches to
implement FID simplify the method and make it more widely
available. Different methods of crystallization explore different
paths towards the equilibrated condition where crystals of protein
form, and therefore yield different crystallization results. These
methods can be modified to alter the kinetics of crystallization
and thus explore different routes to form crystals of proteins;
however, different methods require different techniques to combine
the protein solution and precipitant solution. While it is
desirable to use more than one method of crystallization, it is
technologically challenging to use two techniques in one
experiment.
[0244] The SlipChip technology described herein addresses these
challenges. It has been demonstrated in both pre-loaded and
user-loaded formats. In some embodiments, the user-loaded format
can be used to demonstrate an FID technique based on a SlipChip and
also combined FID and microbatch techniques in one "composite"
SlipChip.
[0245] The inventors designed an embodiment of the SlipChip to
incorporate the FID method. This SlipChip was designed to screen a
sample against 16 different precipitants at five different
equilibration times. Each equilibration time was investigated in
duplicate, for a total of 160 assays in a single SlipChip. The
SlipChip can be configured to form 16 separate fluidic paths for
the precipitants, each containing 10 areas, and a single fluidic
path for the protein sample containing 160 areas. To incorporate
the FID method, when the SlipChip was "slipped" to connect the
protein areas and the precipitant areas, the microducts (ducts 21
.mu.m in depth) that had formed the continuous fluidic path for the
protein sample became the neck duct connecting the protein area to
the precipitant area. By gradually increasing the distance between
the protein areas and the precipitant areas, the length of the neck
was increased from 91 .mu.m to 491 .mu.m; by decreasing the width
of the ducts, the width of the neck was decreased from 104 .mu.m to
58 .mu.m. The geometry of the necks, defined as the length of the
neck duct divided by the cross-sectional area of the duct, was
consequently altered.
[0246] A SlipChip was designed to screen a protein against 16
different precipitants using the FID method of crystallization.
Multiple precipitants, as well as multiple equilibration times for
mixing the protein with each precipitant, can be screened on the
same SlipChip. The top plate contains ducts for the protein and
ducts for the precipitant. The ducts for the protein will become
the neck ducts that connect the protein areas and the precipitant
areas, and these ducts gradually decrease in width from left to
right, gradually changing the equilibration time. The bottom plate
has areas for the protein and areas for the precipitant. The
distance between the areas for the protein and areas for the
precipitant is gradually increased from left to right, gradually
changing the equilibration time. When the two plates are assembled,
the fluidic path for the protein and the fluidic path for the
precipitants are formed. After "slipping", protein and precipitant
areas from the bottom plate are bridged by narrow ducts in the top
plate.
[0247] The geometry of the neck controlled the equilibration time,
and the inventors found that the equilibration time increased
linearly with the neck geometry, consistent with numerical
simulations. Equilibration time occurring in the steady state with
fully developed diffusion profiles is different than the time to
establish these profiles; the latter time scales with the square of
distance. The FID assays were set up easily in the SlipChip,
requiring no valves and only involving pipetting and slipping. In
this approach, the ducts for the protein sample were used to set up
the FID assays, so little sample was wasted. Because the necks were
designed to be thin compared to the areas containing precipitant or
protein, the change in volume caused by changing the neck geometry
was negligible compared to the total volume of the crystallization
assay. The volume of the neck constituted only 4-8% of the total
volume of the crystallization trial. The inventors have
demonstrated how changing the equilibration time affects protein
crystallization.
[0248] Changing the geometry of the duct changes the equilibration
time in the SlipChip. Each condition represents a different
equilibration time, and was done in duplicate. Diffusion profiles
were obtained for various neck geometries by using a model
fluorescent dye, DTPA. Average intensities in the area for protein
were measured by linescan through the areas. The diffusion profiles
depended on the neck geometry. The 50% equilibration time and neck
geometry are linearly related. 50% equilibration time was defined
as the time it took for the average intensity in the protein areas
to reach half of the maximum equilibrated intensity; neck geometry
was defined by the length of the neck divided by the
cross-sectional area of the neck. At the shortest equilibration
time, only precipitates were obtained. As equilibration time
increased, fewer, larger crystals were obtained.
[0249] The inventors first demonstrated the effect of equilibration
time on the kinetics of crystallization by crystallizing the
photosynthetic reaction center from Blastochloris viridis using the
FID SlipChip. The inventors demonstrated that as the equilibration
time increased, the protein progressed from precipitate to many
small crystals to fewer larger crystals. The inventors then used
the FID SlipChip to screen crystallization conditions for two
proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and
dihydrofolate reductase/thymidylate synthase from Babesia bovis.
Approximately 12 .mu.L of each protein was consumed to screen
against a screening kit containing 48 precipitants for a total of
480 experiments. This was performed on three SlipChips, each
SlipChip with 16 precipitants and five conditions in duplicate per
precipitant, for a total of 160 experiments per chip and consuming
4 .mu.L of protein per chip. The inventors also screened both
proteins using certain embodiments of the user-loaded SlipChip
using the microbatch method against the same precipitants, and
compared the microbatch results to the FID results.
[0250] The two proteins assayed represent different kinetics of
nucleation: enoyl-CoA hydratase nucleates quickly while
dihydrofolate reductase/thymidylate synthase nucleates slowly. For
enoyl-CoA hydratase, FID minimizes nucleation and yields crystals
in conditions where only precipitation is observed in microbatch.
Using the FID SlipChip, the inventors obtained crystals of
enoyl-CoA hydratase under several conditions. Under conditions that
yield crystals in both methods, such as for the photosynthetic
reaction center from Blastochloris viridis, FID yields fewer large
crystals while microbatch yields many small crystals. For
dihydrofolate reductase/thymidylate synthase assays where crystals
formed, few crystals were obtained in each trial, indicating that
the crystallization of dihydrofolate reductase/thymidylate synthase
is nucleation-limited. Only one precipitant condition produced
crystals using the FID method, but three precipitant conditions
produced crystals in the microbatch method. This implies that
proteins with different nucleation kinetics will require different
crystallization techniques, and using multiple techniques in
parallel increases the likelihood of identifying suitable
conditions to produce protein crystals.
[0251] In another embodiment of the SlipChip the two methods (FID
and microbatch) were screened simultaneously in addition to
identifying a precipitant and its concentration for
crystallization. In certain embodiments a continuous fluidic path
for the protein sample and 16 separate fluidic paths for different
precipitants can be configured. In this embodiment, areas designed
for microbatch experiments and areas designed for FID experiments
were in each fluidic path, allowing a single protein to be screened
against 16 precipitants each at multiple mixing ratios and
equilibration times. In this embodiment FID areas have multiple
mixing ratios (1:2, 1:1, and 2:1) for a total of 176 experiments
per chip, five microbatch experiments and six FID experiments for
each of 16 precipitants.
[0252] In the composite SlipChip, multiple precipitants and
multiple volumes and equilibration times for mixing the protein can
be screened on the same SlipChip using both microbatch and FID
methods. The top plate contains areas for the protein and ducts for
the precipitant (microbatch) and ducts for both the protein and
precipitant (FID). The bottom plate has ducts for the protein and
areas for the precipitant (microbatch) and areas for both the
protein and precipitant (FID). When the two plates are assembled,
the fluidic path for the protein and the fluidic paths for the
precipitants are formed to fill areas for both microbatch and FID
methods. In microbatch the two areas are aligned with one another,
in FID the two areas are connected by a narrow duct.
[0253] In certain embodiments, unwanted cross-contamination could
potentially occur during the slipping step: a thin film of solution
can form between the two plates of the SlipChip, connecting the
ducts and areas that should be separated. To minimize unwanted
cross-contamination, a contact angle between the solutions and the
plates of the SlipChip in the lubricant fluorocarbon of greater
than .about.130.degree. is preferred, and in other experiments, it
is preferred to spin-coat the plates with thin layers of
fluorinated ethylene propylene. In certain embodiments of the FID
method, the solution in the neck duct is not stable at such high
contact angles and tends to break up to minimize the surface
energy. The inventors solved this problem by patterning the surface
of the SlipChip to make it more hydrophobic than the surface inside
the areas and neck ducts. To do so, the inventors introduced an
extra step of fine etching before washing off the coating left from
the previous etching steps. This generated patterns of 10 .mu.m
diameter areas that were 250 nm deep. Without nanopatterning, the
average contact angle of a 0.1% N,N-Dimethyldodecylamine N-oxide
(LDAO) sample solution was only 112.2.degree., with nanopatterning,
the average contact angle of the same LDAO sample solution was
134.2.degree.. In addition, nanopatterning decreased the surface
area of glass that was directly exposed to the solution edge during
the slipping step. The small areas trapped lubricating fluid and
created a barrier to prevent solution leakage.
[0254] The performance of the nanopatterning was affected by the
geometry of the nanopattern, including the nano-scale area size,
spacing, and etched depth. These parameters can be varied, and the
contact angle of each nanopatterning can be measured. Both the
depth and the surface area of the nano-scale areas should affect
the contact angle. Silanized glass with nanopatterning typically
had a contact angle higher than glass without nanopatterning, and
the contact angle increased with the depth of etching. The contact
angle was above 130.degree. for those glass plates where the
nanopatterning depth was in the range of 196 nm.about.3.81 .mu.m.
For nanopatterns with a depth of 3.81 .mu.m, the maximum contact
angle was 153.62.degree. (RSD=1.01%, n=5, measured after 5 min of
droplet setup). The contact angle decreased with time, as observed
by measuring the contact angle 5 min later. The amount of the
decrease was affected by the nanopattern depth. Nanopatterns with
less than 200 nm depth had a faster decrease in contact angle than
those nanopatterns that were deeper than 200 nm. The composite
SlipChip was also used to screen conditions for crystallization of
the same two proteins studied using separate FID and microbatch
experiments, enoyl-CoA hydratase from Mycobacterium tuberculosis
and dihydrofolate reductase/thymidylate synthase from Babesia
bovis. The composite approach made the search for relevant
crystallization conditions more efficient, as two routes to
nucleation and crystal growth were investigated simultaneously,
while the same small amount of protein (.about.12 .mu.L) was
consumed to screen each protein against the same screening kit.
Both microbatch and free-interface diffusion components of the
composite SlipChip functioned, and identified crystallization
conditions for both proteins. In the composite SlipChip, the
majority of conditions identified by separate microbatch and FID
screenings were also identified. For enoyl-CoA hydratase, two new
conditions not identified in either of the individual screens were
picked up by the hybrid screen.
[0255] Screening crystallization conditions for proteins using the
composite SlipChip matched results from microbatch and FID methods.
All areas contained reagent 41 (45% (W/V) PEG-3000, 0.1 M CHES, pH
9.5). Using the microbatch method, crystals formed at a mixing
ratio of 2:1 (protein:precipitate). Using the FID method, crystals
formed at a mixing ratio of 1:2. The composite method produced as
many or more crystallization hits than either microbatch or FID
alone for both enoyl-CoA hydratase and dihydrofolate
reductase/thymidylate synthase.
[0256] The inventors scaled up one of the three conditions for
crystallization of dihydrofolate reductase/thymidylate synthase
identified in the microbatch SlipChip. The condition chosen was the
protein sample at a mixing ratio of 0.33:0.57 with 20% (w/v)
PEG-8000, 0.2 M NaCl and 0.1 M CHES, pH 9.5. The inventors scaled
up dihydrofolate reductase/thymidylate synthase instead of
enoyl-CoA hydratase because dihydrofolate reductase/thymidylate
synthase is more difficult to crystallize, as indicated by fewer
recognized hits. The precipitant, 20% (w/v) PEG-8000, 0.2 M NaCl
and 0.1 M CHES, pH 9.5, produced crystals with the best-defined
shape at the chosen mixing ratio. It is straightforward to
translate the microbatch method crystallization trial from
SlipChips to well plates, and the inventors successfully obtained
crystals from the scale up approach. The inventors collected a full
X-ray diffraction data set and determined the structure at a
resolution of 1.95 .ANG., space group P212121. The structure has
been deposited in the Protein Data Bank, PBDid: 3KJR. The same
protein was screened in parallel using Seattle Structural Genomics
Center for Infectious Disease (SSGCID) and Accelerated Technologies
Center for Gene to 3D Structure (ATCG3D) facilities to yield
crystals using microfluidic microbatch in a crystal card in
conditions using 20% (w/v) PEG-8000, 0.1 M CHES pH 9.5. These
crystals yielded a 2.35 .ANG. structure, space group P1 (PDBid
3D6B). Screens were conducted double-blind, without any information
about crystallization conditions shared until after the screens
were completed and crystals were obtained--the screening of
crystallization of dihydrofolate reductase/thymidylate synthase on
the SlipChip and the concomitant scale up assays were performed
without any knowledge of conditions obtained by the screening in
facilities SSGCID and ATCG3D. Similar conditions, sharing the same
PEG and buffer and differing only by the presence of NaCl in the
SlipChip screen, were independently discovered to yield structures.
The inventors obtained a higher resolution structure, with a
different space group.
[0257] The inventors have demonstrated a SlipChip-based FID
approach to crystallize proteins and a composite SlipChip-based
approach to use microbatch and FID crystallization techniques
simultaneously. Certain embodiments of the SlipChip provide a
simple and easy-to-use method to set up over 160 experiments in
free interface diffusion and 176 experiments in both microbatch and
free interface diffusion, and all assays can be setup
simultaneously with a single slip. For applications such as protein
crystallization, where each trial does not necessarily need to be
controlled individually, the absence of valves dramatically
simplifies both the execution of assays and fabrication of devices.
Fabrication of devices is further simplified by using a SlipChip
platform, because the SlipChip is compatible with inexpensive
molding technologies and common plastics. More advanced techniques
already demonstrated in plug-based crystallization techniques are
compatible with the SlipChip design. In addition to screening
multiple precipitants, mixing ratios, and equilibration times, the
composite SlipChip enables the comparison of two different protein
crystallization techniques on the nanoliter scale in the same
device. By using a single device, the surface chemistries and
solutions used are the same, and any advantage of one method over
the other can be identified and realized. Microbatch corresponds to
rapid mixing through a larger interface, leading to more rapid
nucleation. Free interface diffusion corresponds to slower mixing
through a smaller interface, corresponding to slower nucleation.
Control of the neck geometry enables the continuum of methods
bridging microbatch and FID methods. Crystallization based on
counter diffusion approaches is mechanistically similar to FID
methods. Counter diffusion for crystallization can be implemented
on the SlipChip on smaller scale and in more multiplexed format
than in traditional methods. The composite SlipChip provides a
platform on which to assay many proteins and the opportunity to
learn more about important characteristics of protein
crystallization.
[0258] After crystallization conditions are identified,
high-quality crystals suitable for X-ray diffraction are preferred
for characterizing the crystals and determining protein structures.
To produce crystals large enough for X-ray diffraction, typically a
minimum trial volume of .about.10 nl is required, and even much
smaller crystals can be analyzed using, for example, recent
advances in synchrotron x-ray science, so the crystals obtained in
the SlipChip can be large enough for structural characterization.
There are several options for obtaining X-ray diffraction data from
crystals grown in a SlipChip including extraction of the crystals
or in situ diffraction. In certain embodiments, the SlipChip is not
sealed, therefore, the two plates can be separated and crystals
extracted as has been done for a well-based chip. Diffraction in
situ can prevent damage to the crystals during post-crystallization
manipulations and can increase throughput. X-ray diffraction in
situ can be performed in the SlipChip since the SlipChip can be
constructed of material that is compatible with in situ
diffraction, such as PDMS, PMMA, and cyclo-olefin-copolymers, or,
if necessary, the glass can be etched to create areas with
sufficiently thin walls.
[0259] If certain crystals grown in a SlipChip don't yield
high-quality X-ray diffraction data, the crystallization
experiments can be scaled up using the conditions identified by the
SlipChip screenings. Microbatch experiments are easily scaled-up in
well plates. Another success has been achieved using the same
strategy with ribose-phosphate pyrophosphokinase from Burkholderia
pseudomallei. A condition (20% (w/v) PEG-3350, 0.2M magnesium
formate, pH 5.9) found by conventional vapor diffusion method
yielded crystals in space group of 1222. The crystal structure was
determined at 2.3 .ANG. resolution (PDBid: 3DAH). In parallel using
an embodiment of the SlipChip, the inventors found a different
condition (11% (w/v) PEG-8000, 37 mM sodium citrate, pH 5.5)
yielding crystals in space group of P43212. The inventors obtained
a data set at 1.83 .ANG. with crystals produced by scaling up.
Using other techniques, the FID approach can be less trivial to
scale up because the diffusion profiles and kinetics need to be
replicated and thoughtfully controlled on a larger scale. The
predictable diffusion profile the inventors determined for FID
SlipChip enables the rational design of scaled up scalable
SlipChips both down to, for example, picoliter-scales and up to,
for example, microliter-scales.
[0260] The technology described here has many applications beyond
protein crystallization. For example, the nanometer-scale etching
used to create a superhydrophobic surface will impact surface
patterning technologies. In addition, the techniques used for the
FID method can be expanded to control equilibration times when
combining solutions in other experiments. This control of
equilibration can be useful for setting up concentration gradients
in a range of applications, e.g. when studying chemotaxis and in
other cell-based assays.
Example
[0261] Fabrication of SlipChip with Nanopatterning
[0262] The inventors followed the glass etching fabrication
procedure described elsewhere in the application with the following
modifications. A blank glass plate (Soda-lime glass, thickness: 0.7
mm; chromium coating: 1025 .ANG.; AZ photoresist: 1 .mu.m) was
first cut to be 3 in.times.1 in. Step 1: The glass etching
fabrication procedure was followed until the point where the
backside of the glass plate was sealed with PVC tape. Next, the
inventors placed cross marks for aligning the second photomask on
the edge of the glass plate; these marks were also taped to prevent
etching. In this example, the etching time was .about.30 min to
etch areas that were 40 .mu.m deep into the glass plate. The plate
was thoroughly rinsed with Millipore water and dried with nitrogen
gas. Step 2: Using another photomask containing the design for the
ducts and an etching time of .about.15 min, 20 .mu.m deep ducts
were etched on to the glass plate using the same procedure as in
Step 1. Care was taken to align the glass plate with the photomask.
During this step, the 40 .mu.m deep areas were further etched to be
60 .mu.m deep. The plate was thoroughly rinsed with Millipore water
and dried with nitrogen gas. Step 3: After ducts and areas were
etched into the plate, the plate was aligned with a nanopatterning
photomask and the same procedure was followed as in Step 1. After
removing the chromium coating, the glass plate was immersed in
50:25:37.5 mmol/L HF/NH4F/HNO3 etching solution, and etched for 20
min at room temperature (.about.23.degree. C.) to produce
.about.250 nm deep patterns over the surface. Finally, the glass
plate was rinsed with ethanol to strip the undeveloped photoresist,
and immersed in the chromium etchant to remove the chromium
coating. The glass was then rinsed with ethanol and Millipore water
and dried with nitrogen gas. The method described here integrates
nanometer-deep designs and various micrometer-deep designs on one
glass substrate. It can also be used to create nanometer/micrometer
hybrid ducts for other nanofluidic/microfluidic applications. The
etched patterns were measured with a Veeco Dektak 150 profilometer
(Figure S2). The glass plates were cleaned and subjected to an
oxygen plasma treatment, and then the surfaces were rendered
hydrophobic by silanization in a vacuum desiccator for 3 hours with
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane as
previously described. After silanization, the glass plates were
baked in a 120.degree. C. oven for 30 min, rinsed by immersing in a
tank of FC-3283, and dried in a 60.degree. C. oven overnight.
FEP Spin Coating
[0263] Spin coating FEP was performed as described elsewhere in
this application.
Measuring Contact Angles of Nanopatterning
[0264] Glass plates were etched with nanopatterns by using the
nanopatterning photomask described in Step 3 of Fabrication of
SlipChip with nanopatterning, and different nano-scale area depths
were obtained by controlling the etching time. All glass was
silanized and cleaned before measuring contact angles. The glass
plate was immersed into fluorocarbon in a glass tank. The plate,
with patterned surface facing down, was clamped by two micro
binderclips on each end to create a gap between the plate and the
bottom of the tank. 5 .mu.L of the measured aqueous solution was
pipetted into the gap, and the aqueous droplet with 0.1% LDAO
contacted the plate due to its buoyancy in the surrounding
fluorocarbon (FC-40). The contact angle of the droplet on the
substrate was then measured by using an optical contact angle meter
(Rame-Hart Instrument Co., Model 500). The contact angle was
measured immediately after the droplet contacted the glass plate
and again 5 min after contact.
Food Dye Assay in a FID Device
[0265] A FID device was made with the method described above
without nanopatterning or FEP coating. The two plates of the device
were assembled under FC-40. In the resulting orientation, fluidic
ducts for all 16 reagents and one sample were formed. All the
solutions used for food dye experiments were filtered with a 0.45
.mu.m PVDF syringe filter before use. Four food dyes (yellow, pink,
red, and blue) were diluted .about.10 times from their stock
solutions and were pipette-loaded into 16 reagent ducts. To load
each duct, 4 .mu.L of dye was first pushed through the inlet using
a pipette until the dye solution emerged from the outlet. A green
dye was diluted 20 times and was mixed with 0.04% (w/v) LDAO to
mimic a protein sample. The green dye was then loaded through the
sample inlet. Using a pipette, 10 .mu.L of the dye was loaded into
the Chip until all the sample ducts were fully filled. Once the
sample was loaded, the SlipChip was slipped such that the
connections between adjacent areas were disconnected and the
vertical ducts formed a bridging diffusion duct for the sample
areas and relative reagents areas under it. Sequential images (time
interval of 3 min) were taken with a Leica MZ 16 Stereoscope with a
Plan APO 0.63.times. objective.
Fluorescent Dye Diffusion Assay in a FID Device
[0266] A FID device was made with the method described above with
nanopatterning. The SlipChip was assembled and solutions were
loaded as described for the food dye experiment. 250 .mu.M MPTS in
PBS buffer (1.times., pH 7.4) was loaded by pipetting into two
reagent ducts. 0.01% (w/v) LDAO solution was loaded into the sample
duct to fill all sample areas. The SlipChip were slipped under a
Leica MZ 16 Stereoscope to form 20 free interface diffusion
experiments with 5 different duct geometries. The starting time
point of FID was recorded with a timer. The device was quickly
transferred to a Leica DMI6000 microscope (Leica Microsystems) with
a 5.times.0.4 Leica objective and a Hamamatsu ORCAER camera. A DAPI
filter with an exposure time of 20 ms was used to collect MPTS
fluorescence. To calibrate the microscope, the fluorescence
intensity of a fluorescence reference slide for the DAPI filter was
recorded and used for background correction. Images were acquired
and analyzed by using Metamorph imaging system version 6.3r1
(Universal Imaging) with multi-dimension acquisition function.
Images were taken every 10 minutes. To obtain the average intensity
in the sample area, the inventors obtained linescans on each sample
area. The intensity along the linescan was averaged, and the
average intensity was plotted over time. The time was corrected by
accounting for the delay between setting up the FID experiments and
the start of imaging.
Food Dye Assay in a Hybrid Device
[0267] A hybrid SlipChip was made by using the nanopatterning
method described above. It was assembled under FC-40. In the
resulting orientation, fluidic ducts for both 16 reagents and one
sample were formed. All the solutions used for food dye experiments
were filtered with a 0.45 .mu.m PVDF syringe filter before use.
Four food dyes (yellow, pink, red, and blue, Ateco, Glen Cove,
N.Y.) were diluted .about.10 times from their stock solutions and
were pipette-loaded into 16 reagent ducts. To load each duct, 4
.mu.L of dye was first pushed through the inlet using a pipette
until the dye solution emerged from the outlet. A green dye was
diluted 20 times and was mixed with 0.04% (w/v) LDAO to mimic a
protein sample. The green dye was then loaded through the sample
inlet. Using a pipette, 10 .mu.L of the dye was loaded into the
Chip until all the sample ducts were fully filled. Once the sample
was loaded, the SlipChip was slipped such that the reagent areas
overlapped with the sample areas in the microbatch sections, and
the reagent areas were connected to the sample areas by the necks
(ducts connecting the fluidic path of the sample before slipping)
in the FID sections.
Crystallization of Enoyl-CoA Hydratase from Mycobacterium
tuberculosis with Microbatch SlipChip.
[0268] The protein sample was obtained from the Seattle Structural
Genomics Center for Infectious Disease (SSGCID). The microbatch
SlipChips were made by glass etching, surface-coated by fluorinated
ethylene propylene (FEP), and assembled under lubricant
fluorocarbon, a mixture of perfluoro-tri-n-butylamine and
perfluoro-di-n-butylmethylamine (FC-40). 48 precipitants from a
home-made screening kit were loaded into three assembled SlipChips,
16 precipitants in each Chip. Precipitants were combined with the
protein sample by slipping. Each SlipChip was then immersed into
FC-70 in separate Petri dishes. The Petri dishes were stored in a
23.degree. C. incubator and the results were monitored for two
weeks. Images of areas containing the crystallization trials were
taken over the two weeks by using a SPOT Insight camera (Diagnostic
Instruments, Inc., Sterling Heights, Mich.) coupled to a Leica MZ
16 Stereoscope.
Crystallization of Enoyl-CoA Hydratase with FID Chip
[0269] The FID SlipChip for protein crystallization was made by
using the nanopatterning method described above. 48 precipitants
from a home-made screening kit were loaded into three SlipChips, 16
precipitants in each Chip; the loading procedure was the same as in
the food dye experiments of FID Chip. After slipping, the
precipitant areas and protein areas were connected in pairs by the
protein neck to initiate FID experiments. Each SlipChip was then
immersed in FC-70 in separate Petri dishes. The Petri dishes were
stored in a 23.degree. C. incubator and the results were monitored
for two weeks. Images of areas containing crystals were taken over
the two weeks.
Crystallization of Enoyl-CoA Hydratase with Hybrid SlipChip.
[0270] The hybrid SlipChip for protein crystallization was made by
using the nanopatterning method described above. 48 precipitants
from a home-made screening kit were loaded into three hybrid
SlipChips, 16 precipitants in each Chip; the loading procedure was
the same as in the food dye experiments of the hybrid Chip. After
one step of slipping, both microbatch and FID experiments were set
up. Each SlipChip was then immersed in FC-70 in separate Petri
dishes. The Petri dishes were stored in a 23.degree. C. incubator
and the results were monitored for two weeks. Images of areas
containing crystals were taken over the two weeks.
Crystallization of Dihydrofolate Reductase/Thymidylate Synthase
from Babesia bovis with Microbatch SlipChip.
[0271] The protein sample was obtained from SSGCID. The screening
assays using microbatch SlipChips were performed in the same way as
described for enoyl-CoA hydratase.
Crystallization of Dihydrofolate Reductase/Thymidylate Synthase
with FID Chip
[0272] The protein sample was obtained from SSGCID. The screening
assays using FID SlipChips were performed in the same way as
described for enoyl-CoA hydratase.
Crystallization of Dihydrofolate Reductase/Thymidylate Synthase
with Hybrid SlipChip
[0273] The protein sample was obtained from SSGCID. The screening
assays using hybrid SlipChips were performed in the same way as
described for enoyl-CoA hydratase.
Visualization of Protein Crystals Using a UV-Microscope
[0274] To confirm the crystals obtained in all of the
crystallization assays on SlipChips were indeed protein crystals,
the inventors used a UV-microscope (PRS-1000, Korima Inc., Carson,
Calif.). Both brightfield images and images under UV-light were
taken. The crystals were confirmed as protein crystals when UV
signals from the crystals were detected, and the corresponding
crystallization conditions were identified as hits.
Crystallization of Dihydrofolate Reductase/Thymidylate Synthase in
Well Plates.
[0275] Crystallization of dihydrofolate reductase/thymidylate
synthase was performed in well plates as described for glutaryl-CoA
dehydrogenase from Burkholderia pseudomallei.
X-Ray Diffraction and Data Processing
[0276] X-ray diffraction and data processing were performed as
described elsewhere in this application.
X-Ray Structure Determination of Dihydrofolate
Reductase/Thymidylate Synthase.
[0277] The structure of dihydrofolate reductase/thymidylate
synthase was solved by molecular replacement using the PDBid 3I3R
structure as a starting model and the MOLREP program in CCP4 suite.
The data collected from crystals grown in the condition containing
PEG-400 was used. Rigid-body, positional, and temperature factor
refinements were performed using a maximum likelihood target with
the program REFMAC5. The SigmaA-weighted 2Fobs-Fcalc and Fobs-Fcalc
Fourier maps were calculated using CCP4. The Fourier maps were
displayed and examined in COOT. The search for new solvent
molecules was performed with help of COOT. The structure has been
deposited in the Protein Data Bank, PBDid: 3KJR.
Quantifying Mixing Ratio: Characterization of Area Sizes
[0278] The original (before etching) area is a hexagon with two
opposing right angles between the first and second sides and the
fourth and fifth sides. The volume of the area is expressed in
Equation 1, where W1 is the original width of the area (the
distance between the third and sixth sides), L is the original
length of the area (the length of the third and sixth sides), r is
the expanding distance, and d is the depth of the area after
etching.
Volume=W.sub.1Ld+0.5W.sub.1.sup.2d+0.707.pi.rdW.sub.1+0.666.pi.dr.sup.2+-
0.5.pi.rdL Eq. 1
[0279] The size of the areas after etching was measured by using a
Leica MZ 16 Stereoscope calibrated by a micro-ruler. The expanding
distance r was then calculated using Equation 2, where W2 is the
width (along the same axis as W.quadrature.) of the area after
etching.
r=0.5(W.sub.2-W.sub.1) Eq. 2
[0280] The inventors assumed that the etching speed was the same in
all directions, so the original pattern of the area expanded the
same distance in all directions. The expanding distance, r, was
assumed to be the same as the depth, d. Therefore, the volume of
the areas can be calculated by combining Equations 1 and 2.
Volume = W 1 L W 2 - W 1 2 + 0.5 W 1 2 W 2 - W 1 2 + 0.707 .pi. ( W
2 - W 1 ) 2 4 W 1 + 0.666 .pi. ( W 2 - W 1 ) 3 8 + 0.5 .pi. ( W 2 -
W 1 ) 2 4 L Eq . 3 ##EQU00001##
The areas of the SlipChip can be designed such that W1 was always
236 .mu.m and L was varied to be 0, 20, 40, 60, 80, 100, 120, 140,
160, 180 and 200 .mu.m. The angles of the hexagon are 90 or 135
degrees. By etching the areas to be 60 .mu.m deep, the areas can be
designed with volume of 4.0, 4.4, 4.8, 5.2, 5.6, 6.0, 6.4, 6.8,
7.2, 7.6 and 8.0 nL, respectively.
[0281] In certain embodiments of the present invention, the
SlipChip can be used to perform bead-based assays such as bead
based immunoassays. In certain embodiments, bead-based SlipChip
methods can involve multi-step slipping, loading beads into the
chip, handling beads in areas, transfer of beads from one layer to
another, and then from one area to another area, by slipping.
Washing beads can be performed by many mechanisms including back
and forth sliding, forward sliding and serial dilution. Hydrophilic
areas can be used to maintain thin layers of fluid in an area, can
be used for effective serial dilution by creating small volumes
that can be washed with large volumes and can be used to speed up
diffusion in and out of the thin layer. Nano-scale areas which are
very thin (between, for example, about 100 nm, 1 um, 10 um) can
contain immobilized antibodies for very rapid immunoassays. Such
immunoassays can be valuable for rapid analysis, for example to
detect Parathyroid Hormone. In addition, removal of excess material
by slipping over such an area can be used to evaluate weaker
binding, for example in applications described in Maerkl S J, Quake
S R. "A Systems Approach to Measuring the Binding Energy Landscapes
of Transcription Factors" Science, 2007, 315:233-237. For SlipChip
immunoassays, when beads are held down, or capture antibody is
immobilized on the surface, washing can be performed directly by
running fluid through aligned areas and ducts (to reduce
cross-contamination, it is preferred to wash all areas in parallel,
not sequentially).
[0282] Cell cultures can be grown, maintained, or assayed in areas.
There may be at least one cell in an area, and analyzing can be
performed by, for example, immunoassay. This may involve a
secretion of the cell, a lysed cell, stimulating cells and then
analyzing the result by any method including, for example, by
immunoassay, or stimulating by slipping to add a reagent, and
analyzing by any method including immunoassay.
[0283] The SlipChip can be used to analyze many samples which may
be obtained from other devices including, for example, the
chemistrode.
[0284] In some embodiments, many small-volume samples can be
analyzed in parallel using bead-based ELISA assays in the SlipChip.
Situations in which analyzing small-volume samples are important
include, but are not limited to, analyzing samples from the
chemistrode. Understanding biological systems can involve tools to
deliver, capture, and interpret molecular signals with high
temporal resolution. The newly developed chemistrode addresses this
unmet need by recording molecular signals in an array of hundreds
of nanoliter-volume plugs, which are subsequently analyzed by
multiple independent techniques in parallel. The chemistrode can
benefit from methods to analyze the nanoliter-volume recording
plugs with high sensitivity, specificity, and throughput.
Immunoassays are one of the most frequently used techniques for
detecting molecular markers with high specificity and sensitivity
in biological research. Developing immunoassays for these nL-plugs
enhances the analyzing abilities of the chemistrode. Other
situations in which analyzing small-volume samples are important
include, but are not limited to, diagnostics and clinical research.
For example, serially monitoring a tumor over time requires
repeated sampling of small volumes and analyzing them. Also, to
avoid unnecessary depletion of blood samples deposited in blood
banks, testing requires analysis of small volumes. Other situations
in which analyzing small-volume samples are important include, but
are not limited to, single-cell analysis, nano-flow sampling from
live tissue, e.g., the retina (Lu, Miao-Jen, et al. Exp Diabetes
Res. 2007; 2007: 39765), small samples (e.g., material from an
embryo). The biggest bottleneck in certain situations is processing
(such as combining samples, separating samples with beads, and
adding reagents) many small volumes in parallel. Typical methods
for manipulating nanoliter droplets serially process plugs
one-by-one. For certain embodiments, this is less preferred when
indexing of plugs is important, because errors can accumulate. Many
examples of current devices for arranging nanoliter droplets in
arrays do not allow manipulations (adding reagents, handling beads)
of droplets. Digital microfluidics works with microliter volumes.
Many microfluidic devices rely on laminar flow to introduce the
sample: these can have large dead volumes and/or adsorption
problems. Certain embodiments of the SlipChip are capable of
robustly handling many multi-step reactions in parallel without
using complex instruments. The inventors developed a simple
approach that uses a SlipChip to perform bead-based ELISA to
analyze many small-volume samples in parallel. The inventors have
designed certain embodiments of the SlipChip to incorporate
multi-step slipping, and performed experiments to demonstrate
loading and washing of beads. Multi-step slipping allows us to
transfer beads from one layer to another, and then from one area to
another area. In these embodiments, beads can be washed by two
mechanisms: forward sliding with serial dilutions, and back and
forth sliding. Hydrophilic areas maintain thin layers of fluid in
the areas of this SlipChip. The hydrophilic areas also allow for
effective serial dilution by creating small volumes that can be
washed with larger volumes. The detection limit the inventors
achieved with one embodiment of the SlipChip was down to the pM
range, which is in the physiological concentration of many
molecular markers.
[0285] An embodiment of the SlipChip has been designed to be able
to perform 48 immunoassays in parallel. It contains two sections,
section A and section B. Section A is used for loading many small
volume samples: the design of this section is variable to
accommodate the different requirements of different sources of
samples. To demonstrate performing bead-based ELISA, a device was
built with six groups of seven areas each (1 nL, 10 .mu.m deep).
When the areas for the sample (bottom plate) and ducts for the
sample (top plate) are aligned, six separate fluidic paths are
formed, and each fluidic path is filled by pipetting into an
individual inlet. Each fluidic path also contains a separate outlet
for the solutions. In these experiments, six standard calibrators
were loaded into the six fluidic paths for the sample. Section B is
used for performing the bead-based ELISA: this is the core section
of the device. It contains six rows of 48 areas (9 nL, 80 .mu.m
deep). Areas in the first row are used to load the mixed solution
containing magnetic beads coupled with the capture antibody and the
enzyme-labeled detection antibody. Areas in the second, third,
fourth, and fifth rows are used to load the washing buffer. Areas
in the sixth row are used to load the solution containing the
substrate. The top layer of an embodiment of the SlipChip may
contain inlets, outlets, and ducts to load the sample, and inlets,
outlets, and areas for the various reagents. The bottom layer of an
embodiment of the SlipChip may contain the areas for the sample,
and ducts to load the reagents.
[0286] In certain embodiments, a SlipChip may be composed of two
layers of microfabricated glass. The top layer may contain all the
inlets, outlets and ducts for the sample and areas for the
reagents. The bottom layer may contain areas for the sample ducts
for the reagents. For improved filling of the areas, the surfaces
of the device can be silanized to be hydrophobic while keeping the
areas hydrophilic. The areas can be protected during silanization
to maintain a hydrophilic surface. A potential source of
cross-contamination is the formation of a thin film of solution
between the two plates of certain embodiments of the SlipChip that
connect areas that should be separated after slipping. This is
caused when the solutions wet the surface of the SlipChip. To
minimize the wetting of the BSA-containing solutions on the surface
of certain embodiments of the SlipChip except inside the areas and
the ducts, a nanopattern can be fabricated on the surface outside
the areas and the ducts. The nanopatterning increases the contact
angle between the solution and the surface, preventing wetting of
the surface.
[0287] In certain embodiments, using a SlipChip to perform
immunoassays involves three general steps (a) preload reagents, (b)
load samples, and (c) perform the assay. In certain embodiments
reagents may be preloaded in eight steps: (1) A SlipChip is
assembled so that the areas of row 1 are connected by reagent
ducts. (2) The reagent solution containing, for example,
capture-antibody coated superparamagnetic beads and enzyme-labeled
detection antibody is injected into the SlipChip and the areas in
row 1 are filled. (3) The chip is slipped to connect the areas of
row 2 by ducts. (4) Fluorocarbon is injected through the ducts to
remove any remaining solution in the ducts. (5) Washing buffer is
injected to fill the areas in that row in the SlipChip. (6) The
chip is slipped to connect the areas of the next row by ducts. (7)
Steps (4), (5), and (6) are repeated three times to fill rows 3 and
4 with buffer. (8) Fluorocarbon is injected through the ducts to
remove any remaining solution, and the enzymatic substrate is
injected to fill row 6.
[0288] In one embodiment, samples are loaded in two steps: (1) The
SlipChip is slipped to connect areas by ducts (this is the
ready-to-use state for the users), (2) Solutions of the analyte are
injected by pipetting through the inlets.
[0289] In certain embodiments, assays may be performed in five
steps: (1) The SlipChip is slipped to combine the analyte and
reagent solution of, for example, antibodies and beads, and the
solution is incubated to allow an antibody sandwich to form, (2) A
magnet is brought up against the back of the bottom layer to pull
the beads down into the area of the bottom plate, and the assay
solutions and the washing buffer are combined by slowly slipping
the SlipChip so that the beads remain in the areas of the bottom
plate, though the magnet is moved away, (3) Step (2) is repeated
three times, (4) A magnet is used to pull the beads down into the
area of the bottom plate, and the SlipChip is slipped to combine
the antibody-sandwich and the substrate, (5) The increase of
fluorescence is monitored using a fluorescence microscope. The
fluorescence is correlated with the concentration of analyte using
techniques known to those skilled in the art.
[0290] In one embodiment containing two sections, A and B, eight
steps may be used to pre-load reagents into the SlipChip. The areas
of row 1 of Section B can be connected by the reagent ducts. The
reagent solution containing the capture-antibody coated
superparamagnetic beads and enzyme-labeled detection antibody can
be injected into the SlipChip to fill the areas in row 1 of Section
B. The SlipChip can be slipped to connect the areas of row 2 of
Section B by ducts. Fluorocarbon can be injected through the ducts
to remove any remaining solution in the ducts. Washing buffer can
be injected to fill the areas in that row in the SlipChip. The
SlipChip can be slipped to connect the areas of the next row by
ducts. These steps can be repeated three times to fill rows 3, 4,
and 5 of Section B with buffer. Fluorocarbon can be injected
through the ducts to remove any remaining solution, and the
enzymatic substrate can be injected to fill row 6 of Section B. Two
steps can be used to load the sample into this embodiment of the
SlipChip: The SlipChip can be slipped to connect the areas in
section A by the ducts for the sample. Solutions of the analyte can
be injected by pipetting through the inlets. Five steps can be used
to perform the immunoassay: The SlipChip can be slipped to combine
the analyte and reagent solution of antibodies and beads, and
incubate the solution to allow the antibody sandwich to form. A
magnet can be used to pull the beads down into the area of the
bottom plate, and the SlipChip can be slipped to combine the assay
solutions and the washing buffer. Steps can be repeated as
necessary. A magnet can be used to pull the beads down into the
area of the bottom plate, and the SlipChip can be slipped to
combine the antibody-sandwich and the substrate. The increase of
fluorescence can be monitored using a fluorescence microscope.
[0291] It will be apparent to one skilled in the art that
embodiments similar to those described above that contain, for
example, rows 1 through 6, can be made in which a plurality of sets
of, for example, six row sections can be built onto a single
SlipChip, such that a plurality of assays can be performed in
parallel.
[0292] In other embodiments, analyzing many, for example, nanoliter
samples simultaneously using the SlipChip may be performed by using
an insulin bead-based ELISA. To demonstrate this, the inventors
injected a solution containing superparamagnetic beads coated with
the capture-antibody, alkaline phosphatase-labeled anti-insulin
monoclonal antibody, and blocking buffer in areas of a first row in
a first section to form a sandwich complex. To detect the
enzyme-labeled detection antibody, the inventors used a fluorescent
substrate for the enzyme, fluorescein diphosphate (FDP), which
becomes fluorescent upon hydrolysis by the enzyme alkaline
phosphatase (ALP). The inventors injected six standard calibrator
solutions of insulin (0 pM, 7 pM, 70 pM, 350 pM, 1050 pM, and 2100
pM) in the areas in a section of the same chip. Fluorescence
intensity in each area was measured over time. The inventors found
that the limit of detection, defined as three times the deviation
of the background signal, was about 9 pM.
[0293] An insulin immunoassay may be performed with multiple small
samples in parallel on a SlipChip. Fluorescence intensity of
multiple, for example, nanoliter samples on the same SlipChip in
different areas of the insulin immunoassay may be measured over
time.
[0294] In certain embodiments of the SlipChip, superparamagnetic
bead-based assays may be performed. The inventors have demonstrated
these beads stayed in areas during slipping: the beads did not get
trapped between the two plates, and there was <3% loss. For
certain embodiments, retention of beads is preferred to improve the
accuracy of the results. The beads can be moved using a moving
magnet to facilitate mixing of solutions. For certain embodiments,
this is preferred for both washing and transferring the beads from
area to area. Moving beads with a magnet will increase mixing,
increasing the efficiency of washing. A magnet can also be used to
pull beads into a bottom area prior to slipping, increasing the
number of beads that were transferred from row to row in the
SlipChip. Residual enzyme-labeled detection antibody will diffuse
into the washing buffer, and can be exponentially diluted to
eventually reached a negligible level, washing the beads. In
certain embodiments, after four cycles of washing, the residual
reagents are diluted by a factor of 104, assuming complete mixing
in every washing cycle. The inventors demonstrated, in certain
embodiments, that the level of enzyme-labeled detection antibody
was below the detection limit after washing in the SlipChip.
[0295] Fabrication of SlipChip with hydrophilic areas. The
inventors used the glass etching fabrication of SlipChip procedure
described elsewhere in this application with the following
modifications. A blank glass plate (Soda-lime glass, thickness: 0.7
mm; chromium coating: 1025 .ANG.; AZ photoresist: 1 .mu.m) was
first cut to be 2 in.times.1 in. After the photomask was removed
from the glass plate, the glass plate was developed by immersing it
in 0.5% NaOH solution for 1 min. In this example, certain areas on
the front of the glass plate were also taped with PVC tape to form
thinner areas. After the glass plate was taped with PVC tape, it
was immersed in the etching solution and a 25.degree. C.
constant-temperature water bath shaker was used to control the
etching speed. By controlling the etching time (.about.50 min),
areas and ducts that were 70 .mu.m deep were etched into the glass
plate. The plate was thoroughly rinsed with Millipore water and
dried with nitrogen gas.
[0296] Next, the tape protecting the thinner areas was removed and
the plate was immersed in the etching solution for .about.7 min. 10
.mu.m deep areas were etched on to the glass plate where the tape
was removed. During this step, the 70 .mu.m deep areas and ducts
were further etched to be 80 .mu.m deep. The plate was thoroughly
rinsed with Millipore water and dried with nitrogen gas.
[0297] After the ducts and areas were etched into the plate, the
glass plate was rinsed with ethanol to strip the undeveloped
photoresist. Then, the plate was coated with OmniCoat and baked at
200.degree. C. for 1 min. Next, the plate was coated with a 10
.mu.m thick layer of SU8 2010, and the plate was covered with a
photomask that protected the areas on the plate that were to be
hydrophobic. UV light was shined from the back of the glass plate.
In the area exposed by the photomask, UV light only passed through
the plate where the chromium coating was removed, so only the SU8
in the areas remained after developing. The SU8 in the areas
protected the areas and prevented them from being made hydrophobic.
OmniCoat on the exposed surface was developed by immersion in CD-26
for 4 min.
[0298] Next, a layer of S1813 positive photoresist was coated on
top of the plate and baked at 95.degree. C. for 1 min. The plate
then was aligned with a nanopatterning photomask and the same
procedure was followed as described for etching the areas and
ducts. After removing the chromium coating, the glass plate was
immersed in the glass etching solution described above that was
diluted 10 times, and etched for 10 min at room temperature
(.about.20.degree. C.) to produce .about.300 nm deep patterns over
the surface. Finally, the glass plate was rinsed with ethanol to
strip the undeveloped photoresist, and immersed in the chromium
etchant to remove the chromium coating. The glass was then rinsed
with ethanol and Millipore water and dried with nitrogen gas.
[0299] The etched patterns were measured with a Veeco Dektak 150
profilometer. The glass plates were cleaned and subjected to an
oxygen plasma treatment, and then the surfaces were rendered
hydrophobic by silanization in a vacuum desiccator for 3 hours with
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane as
previously described. After silanization, the glass plates were
baked in a 120.degree. C. oven for 30 min, rinsed by immersion into
a tank of FC-3283, and dried in a 60.degree. C. oven overnight.
Finally, the SU8 in the areas was stripped by immersing the glass
plates in Remover PG at 80.degree. C. for 30 min.
[0300] Assembling a SlipChip. The SlipChip was assembled under
FC-40. The bottom plate was first immersed into FC-40 in a Petri
dish, with the patterns facing up. The top plate was then laid on
top of the bottom plate, with the patterns facing down. The two
plates were aligned into the positions shown in FIG. 3a, by moving
them relative to each other and then fixed by using two micro
binder clips. The SlipChip was ready for use after the extra FC-40
on the surface was removed.
[0301] Food Dye Illustration. All the food dye solutions were
filtered with a 0.45 .mu.m PVDF syringe filter before use. A
solution of mouse monoclonal anti-insulin coupled to paramagnetic
particles was concentrated six times by centrifuging. The resulting
bead suspension and two food dyes (orange, and blue, Ateco, Glen
Cove, N.Y., diluted .about.10 times from their stock solutions)
were pipette-loaded into the reagent ducts. To load each duct, 2.5
.mu.L of dye was first pushed through the inlet using a pipette
until the dye solution emerged from the outlet. After loading
reagents, the SlipChip was slipped to form a continuous fluidic
path for the sample. A red dye was diluted 10 times and then loaded
through the sample inlet. Using a pipette, 2.5 .mu.L of dye was
loaded into each of the six fluidic sample path of the Chip.
[0302] Insulin bead-based ELISA. The loading procedure was similar
to that for the food dye illustration. The reagent areas were
loaded and the six sample paths were loaded with the six standard
insulin solutions. After the areas for the antibodies and areas for
the samples were combined the SlipChip was incubated for half an
hour at 37.degree. C. to allow complete reaction. The SlipChip was
then slipped to perform the assay.
[0303] Images of areas were taken by a SPOT Insight camera
(Diagnostic Instruments, Inc., Sterling Heights, Mich.) coupled to
a Leica MZ 16 Stereoscope. The fluorescence intensity of the areas
was measured by using a Leica DMI6000 microscope (Leica
Microsystems) with a 20.times.0.4 NA Leica objective and a
Hamamatsu ORCAER camera. A GFP filter was used to collect
fluorescein fluorescence. Images were acquired and analyzed by
using Metamorph imaging system version 6.3r1 (Universal Imaging).
The maximum intensity of the images was first plotted against time,
and then the initial increasing rates were extracted and subtracted
by the rate of the negative control (the assay was the same except
that no detection antibody was added), and the initial rates were
plotted against the corresponding concentration to obtain a
calibration curve.
[0304] In certain embodiments of the SlipChip an immunoassay can be
performed in seven steps: (A) Nanoliter-volumes of analyte solution
are deposited on the areas in the bottom layer of SlipChip immersed
under fluorocarbon. (B) The SlipChip is assembled and the reagent
solution containing the capture-antibody coated superparamagnetic
beads and enzyme-labeled detection antibody is injected into the
fluidic path formed by the ducts of the bottom plate and the areas
of the top plate. (C) The SlipChip is slipped to combine the
analyte and reagent solution, and a magnet is used to settle the
beads down into the areas of the bottom plate. The solutions are
incubated to allow antibody sandwiches to form. (D) The SlipChip is
slipped back into the configuration in (B) and washing buffer is
injected into the fluidic path formed by the ducts of the bottom
plate and the areas of the top plate. (E) The SlipChip is slipped
to combine the washing buffer and the assay solutions. Steps (D)
and (E) are repeated to remove loosely bound enzyme-labeled
detection antibody. (F) The SlipChip is slipped and the enzymatic
substrate is injected into the fluidic path formed by the ducts of
the bottom plate and the areas of the top plate. (G) The SlipChip
is slipped a final time to combine the substrate and
antibody-sandwich. The concentration of analyte is monitored by
measuring the increase of fluorescence. The increase in
fluorescence is correlated with the concentration of analyte. In
one example of beads being loaded, transferred, and washed in an
embodiment of the SlipChip, beads are uniformly loaded into the
areas of the SlipChip by pipetting, beads are transferred from one
layer to another by using magnets and slipping. The beads will stay
in the areas during slipping. Beads can be moved using a moving
magnet to facilitate mixing of solutions. In certain embodiments,
this is preferred for efficient washing. In certain embodiments,
such as certain enzymatic reactions, mixing is preferred to improve
homogeneity of the reaction mixture.
[0305] In one example, manipulating superparamagnetic beads in a
SlipChip involved the following: nanoliter-volume solutions were
deposited in the bottom plate, and the SlipChip was assembled,
beads suspended in a solution were injected into the SlipChip,
slipping and magnetic force were used to settle the beads down into
the areas of the bottom plate, the SlipChip was slipped back to the
original configuration and buffer was injected into the SlipChip to
remove any residual solution in the fluidic path.
[0306] Next, an example of washing superparamagnetic beads to
remove substantially all loosely bound detection antibody in the
SlipChip is described. The inventors first deposited solutions of
enzyme-labeled detection antibody (alkaline phosphatase labeled
anti-insulin monoclonal antibody) in areas 13-24 and 37-48 of the
bottom plate. As a control, the inventors also deposited buffer
solutions in wells 1-12 and 25-36. Then, the inventors injected the
capture-antibody coated superparamagnetic beads suspended in the
blocking buffer into the SlipChip. The inventors slipped the device
and combined the beads with detection antibody. To introduce the
washing buffer, the inventors settled the beads into the areas of
the bottom plate using magnetic force and slipped the device.
Washing buffer was injected. Next, the inventors slipped the device
to combine washing buffer with the beads. Loosely bound detection
antibody will diffuse into the washing buffer while the beads
remain in the areas of the bottom plate. By repeating the wash
steps, residual enzyme-labeled detection antibody was exponentially
diluted and eventually reached a negligible level; at this point
beads were considered to be washed. In one case, wash steps were
repeated 12 times, and the amount of residual detection antibody
was .about.0.2% of the starting concentration assuming complete
mixing in every washing cycle. To detect residual enzyme-labeled
detection antibody, the inventors used a fluorescent substrate for
the enzyme, fluorescein diphosphate (FDP), which becomes
fluorescent upon hydrolysis by the enzyme alkaline phosphatase
(ALP). The inventors slipped the device and injected FDP. Finally,
the inventors slipped the device to combine the substrate and any
residual enzyme-labeled detection antibody in the area.
Fluorescence intensity in each area was measured. The inventors
found that the fluorescence intensity was very weak and the same
for areas deposited with ALP-antibody and areas deposited with
buffer. The fluorescence intensities were also the same as the
fluorescence of substrate solution mixed with buffer. This result
indicated that the level of enzyme-labeled detection antibody was
below the detection limit after washing. As a positive control, the
inventors added FDP to the areas without washing by leaving out the
washing steps. The areas deposited with ALP-antibody showed strong
fluorescence, indicating that the reagents and the method were
effective for detecting residual ALP-antibody. Together, these
experiments show that residual detection antibody can be
substantially removed from the beads by washing with back-and-forth
slipping in the SlipChip.
[0307] The forward-slipping method in the SlipChip can be modified
to incorporate analysis of single cells on-chip and to analyze
samples collected in plugs. The inventors used the forward-slipping
method to measure insulin secretion from single .beta.-cells loaded
on-chip (insulin secretion from mouse islets sampled by
chemistrode). First, the inventors modified the design of section A
to allow analysis of single cells loaded on-chip. In this design,
Section A has two rows of areas (one in the bottom plate and one in
the top plate) and Section B is the same as previously described.
The inventors loaded and cultured single .beta.-cells in the first
row of areas on the top layer--this is the second row of areas in
Section A. The inventors loaded glucose solutions in the row of
areas on the bottom layer--the first row of areas in Section A.
This design involved one additional slipping step to combine the
.beta.-cells and the glucose solutions. After the .beta.-cells and
the glucose solutions were combined, the inventors slipped the
samples through section B to perform the insulin bead-based ELISA
as described above. This design can be used to grow pure cultures
of cells in the areas starting from a single cell. This design can
also be used to stimulate and analyze single cells. The cell can be
stimulated by slipping to bring it into contact with a particular
reagent, and either the secretions of the cell or the cell lysates
can be analyzed by immunoassay (as described previously) or by
other methods.
[0308] The inventors also modified the design of section A to allow
analysis of insulin secretion from single islets sampled by a
chemistrode. In this design, Section A has two rows of areas in the
top plate. The first row is loaded with the plugs captured using
the chemistrode, the second row is preloaded with buffer. The six
rows of section B are preloaded as described previously. The
inventors stimulated a single islet by glucose and sampled the
insulin release in plugs using the chemistrode. In this case, the
chemistrode generated an array of plugs representing temporal
resolution of insulin release. The SlipChip was assembled under
fluorocarbon the inventors first directly deposited the sample
plugs in the first row of areas on the top layer before assembling
the two layers, then carefully aligned the two layers such that the
first row of areas on the top layer was lined up with the row of
wells in the bottom layer. The top row of this SlipChip design
contained no inlets or outlets because the plugs were directly
deposited onto the areas of the SlipChip. The inventors first
slipped the sample to dilute it by slipping into buffer. The
inventors then slipped the diluted sample through section B to
perform the insulin bead-based ELISA as described above.
[0309] The SlipChip can also be designed with very thin areas (for
example, about 100 nm, 1 .mu.m, or 10 .mu.m) that contain
immobilized antibodies for very rapid immunoassays. Washing can
also be done by an active method: if the beads are immobilized by a
magnetic field or if the capture antibody is immobilized on the
surface of the areas, the beads can be washed directly by running
fluid through the aligned areas and ducts. To avoid
cross-contamination in active washing, the areas are washed in
parallel instead of sequentially. In certain embodiments, it is
preferred to design the device so the pressure drop along the inlet
duct and the outlet duct is smaller (10 fold for example) than the
pressure drop along the individual fluid paths that are being
washed. When nano-scale areas are washed, the flow resistance is
likely to be high and this condition is likely to be satisfied. The
inlet duct and the outlet duct for the washing fluid can be
dead-ending, with narrow ducts pointing towards the other duct.
When the areas containing immobilized antibodies are slipped and
aligned to connect the inlet duct and the outlet duct, the washing
fluid can pass through and wash the areas.
[0310] Certain embodiments of the SlipChip can be used to carry out
sample preparation using beads: by transferring beads from area to
area in the SlipChip and exposing them to different reagents,
sample purification and preparation can be accomplished, for
example as done in the Kingfisher system. Washing and concentrating
can also be enhanced by a number of fields and effects, for
example, electrical concentration uses electrical fields to
concentrate molecules near nanopores or ducts.
[0311] Certain embodiments of the SlipChip are compatible with
magnetic immunoassays, including, for example, those developed by
the Philips Corporation. Certain embodiments of the SlipChip may be
used to obtain epigenetic information. For example, acetylation,
methylation, ubiquitylation, phosphorylation and sumoylation of
histones can be analyzed, and certain embodiments of the SlipChip
may be used to perform and analyze chromatin immuno-precipitation
(ChIP), down to the single-cell level.
[0312] Certain embodiments of the SlipChip can be used to perform
PCR experiments. At least three different SlipChip-based PCR
examples follow: a preloaded SlipChip to perform multiplexed PCR
experiments, a SlipChip designed for digital PCR experiments, and a
procedure to trap bacteria onto beads and load the beads into a
SlipChip for PCR.
[0313] Certain embodiments of the SlipChip may include slipping on
top of an oil area, the use of non-fluorinated oil/mineral oil;
and/or non-fluorinated silanization on glass, the use of dried
reagents (for example, primers) with oil on top, and areas that are
shallower than the area with the PCR mix so the PCR mix drop
touches the primer. When slipping an area containing an aqueous
solution over an area containing oil that optionally has reagents,
the contents of the top area displaced the oil and then can react
with the reagents deposited in the bottom. Some oil remains in the
area to provide control of thermal expansion, and in some instances
the total volume of oil may be greater than the volume of aqueous
solution. Certain embodiments of the multiplexed PCR device may
also include overlapping a larger square and a smaller circle. This
geometry achieves two goals: it reduces errors due to thermal
expansion so some oil is trapped in the larger square and it
reduces errors due to touching the dried primer in the bottom
area.
[0314] In certain embodiments of the SlipChip there are oval areas
that overlap. In certain embodiments the oval areas (areas extended
in the direction of filling) provide strong overlap, and low
pressure drop for loading, and can be slipped a small distance to
break up overlap among them and create overlap with oil areas. In
certain embodiments the oval areas can be used to center droplets
for better imaging of the droplets.
[0315] Certain embodiments of the SlipChip may be used to trap
bacteria using magnetic beads. Bacteria from plasma may be trapped
on beads and loaded into certain embodiments of the SlipChip and
then analyzed using, for example, PCR reactions.
[0316] The devices and methods described here can be used for a
number of applications. In particular, applications that require
changes in temperature can be performed using these devices.
Applications include analysis of DNA by PCR and RNA by RT-PCR,
including analysis of mRNA. Other applications include processes
that require thermal denaturation of enzymes and other molecules,
processes that require thermal activation or inactivation of
components and reactions, and processes that require non-ambient
temperature (e.g., many catalysis reactions).
[0317] Certain embodiments of the SlipChip can be used for a number
of applications involving human, animal and environmental samples
that include, but are not limited to, samples from blood, urine,
CSF, stool, eye, ear, genital tracts, lower respiratory tracts,
nose, and throat. These applications include measurement of viral
loads for viral infections such as HIV and hepatitis, analysis of
mutations and drug resistance of viruses and bacteria and fungi,
panels for identification of viruses and bacteria, analysis of
cancer cells and their mutations, genetic variability, clonal
evolution, and drug resistance. Microbes of interest include, but
are not limited to, Staphylococcus aureus, Beta-hemolytic
streptococci, Streptococcus pneumonia, Enterococcus,
Erysipelothrix, Listeria monocytogenes, Haemophilus influenza,
Pseudomonas aeruginosa, Mold, Actinomyces sp., lecithinase or
lipase positive anaerobic Gram-positive organisms, and the
Bacteroides fragilis group.
[0318] Viral detection can be performed on the SlipChip using many
different assays including but not limited to nucleic acid testing
(NAT) technology to amplify and detect viral target RNA or DNA
sequences. In some embodiments, HIV detection can be performed on
the slipchip using NAT technology to amplify and detect HIV target
sequences.
[0319] Capturing cells on beads or area surfaces of certain
embodiments of the SlipChip is attractive for analysis and
manipulation of cells, e.g., multiplexed PCR analysis, relevant for
applications, including but not limited to, cancer diagnostics,
prenatal diagnostics and infectious disease.
[0320] In certain embodiments, SlipChip devices were fabricated by
using glass etching fabrication of SlipChip as described elsewhere
in this application, except for the following changes: In this
example, .about.45 minutes of etching yielded a depth of .about.60
microns. Access holes were drilled with a diamond drill bit 0.030
inches in diameter. The surfaces of the etched glass plates were
cleaned with Millipore water, followed by ethanol and subjected to
an oxygen plasma treatment before silanization. The glass was
silanized by using dichlorodimethylsilane (a non-fluorinated
silane) in vapor phase for one hour. Then the glass slides were
rinsed with chloroform, acetone, and ethanol, and finally dried
with nitrogen gas.
[0321] The following describes one embodiment of a preloaded
multiplexed PCR SlipChip. The top plate of the PCR SlipChip
contained square sample areas of 640 .mu.m in length, 70 .mu.m in
depth and the bottom plate contained ducts for the samples and
preloaded circular areas containing different PCR primer sets. The
circular areas were 560 .mu.m in diameter and 30 .mu.m in depth.
The areas in the bottom plate were first loaded with 0.5 .mu.L of
primer solution (1 .mu.M), and dried at room temperature. Then, the
bottom plate was placed in a Petri dish containing mineral oil.
Fluorinated or non-fluorinated mineral oils may be used in PCR
SlipChip experiments. By placing the bottom plate in a Petri dish
containing oil, a layer of oil formed on top of the preloaded dry
primer. The areas containing primer were designed to be smaller in
both depth and width than the top areas containing the PCR master
mix. This allowed the droplet containing the PCR master mix loaded
in the top area to efficiently reach the primer in the bottom area
through the layer of oil on top of the primer. Next, the top plate
of the PCR SlipChip was aligned on top of the bottom plate such
that the sample areas and sample ducts lined up to form a
continuous fluidic path. The PCR mixture containing EvaGreen
supermix (Bio-rad), 1 mg/mL BSA (Roche) and either DNA template or
water (for the control set) was flowed through the fluidic path to
load the sample areas. The PCR SlipChip was slipped to align the
square sample areas with the circular primer areas. Because there
was a layer of oil between the two areas, the aqueous PCR mixture
formed a droplet within the areas to reduce surface tension. When
the PCR mixture touched the primer on the bottom of the primer
area, the PCR primer dissolved in the reaction mixture. After the
SlipChip was slipped, thermocycling was performed using an
Eppendorf mastercycler with an in-situ adapter. PCR readout was
performed by using fluorescence measurements and gel
electrophoresis of the sample areas.
[0322] During thermocycling, the aqueous solution in the areas
expanded in volume due to the increase in temperature. In certain
embodiments, when using a SlipChip with only square areas, the
aqueous solution can fill the square area, risking, after an
increase in temperature, the aqueous solution leaking out of the
areas, resulting in a loss of material and unmonitorable changes in
concentration. When a smaller, circular area containing oil was
brought into contact with a square area containing aqueous
solution, the aqueous solution forms a droplet within the area,
providing room for expansion during thermocycling. Certain shapes
and sizes of the bottom area are preferable for forming a single
droplet of consistent size in the center of the two areas.
Consistently sized droplets minimize variations in the
concentration of reagents within the droplets.
[0323] The inventors set up the experiments in this embodiment of
the PCR SlipChip to have two rows of control areas with no template
and two rows of areas with 5 pg/.mu.L of S. aureus gDNA. The
inventors found that no contamination occurred in the SlipChip, as
only areas containing template showed amplification. All areas
containing template showed amplification, verifying the robustness
of the PCR SlipChip. Fluorescence intensity measurements and gel
electrophoresis showed that areas without template had no DNA
present after thermocycling and areas with template only contained
one DNA sample.
[0324] Quantitative data analysis confirmed no contamination in the
PCR SlipChip. To further verify that there was no contamination or
cross-contamination in the SlipChip, we preloaded the bottom chips
with two different primer sets, alternating primer sets for the nuc
gene (from S. aureus) and the mecA gene (from MRSA). 5 pg/.mu.L S.
aureus genomic DNA was injected into chips as described above.
Since the nuc gene is present only in S. aureus genomic DNA, while
the mecA gene is present only in MRSA, only the areas loaded with
primers for the nuc gene showed an increase in fluorescence, and
other areas containing mecA gene did not show fluorescence. A
linescan of the fluorescence intensity quantitatively showed that
areas without template did not show significant fluorescence.
[0325] In certain embodiments, thermocycling is performed by
placing the entire PCR SlipChip into a thermocycler that will raise
and lower the ambient temperature surrounding the device. In
different embodiments of the PCR SlipChip, thermocycling takes
place within the device. Here, the thermocycler is replaced by a
steady temperature distribution within the device, and the areas
are physically moved from one temperature to the next. Aqueous
droplets are first formed by slipping to combine areas containing
aqueous solution with areas containing oil as previously described.
Certain embodiments of the SlipChip are designed such that the
aqueous droplets that are formed can be moved by slipping without
loss of solution. These droplets are then slipped to regions of the
SlipChip that are maintained at a specific temperature for a
specified period of time. The temperature distribution within
certain embodiments of the SlipChip can be generated by using, for
example, IR heaters or a thermoelectric device under a P2i coating.
The size of these "hotspots" and "coldspots" can be small enough to
accommodate individual areas, or large enough to accommodate rows
or arrays of areas. For example, a rotary device can move areas
from the cold half of the device to the hot half of the device. The
presence of multiple temperature spots within the device can be
used to add another dimension to certain embodiments of the PCR
SlipChip device: annealing temperature. As different primers have
different annealing temperatures, a wider range of primers can be
screened on this device.
[0326] The following describes one embodiment of a digital PCR
SlipChip. One embodiment of the SlipChip contained 1,280 areas, and
each area was about 5 nL in volume, and was fabricated using the
photolithographic and wet chemical etching techniques described
above. This embodiment contained oval-shaped ducts or areas; the
two plates were patterned with overlapping oval areas of dimensions
400 .mu.m.times.200 .mu.m and 50 .mu.m in depth. The two plates
were also patterned with circular areas of dimensions 200 .mu.m in
diameter and 50 .mu.m in depth. By using overlapping oval areas,
the pressure drop in the device was small, allowing for filling by
simple pipetting. By slipping the SlipChip a short distance, the
oval areas were separated and were overlaid on top of circular
areas containing a layer of oil. For digital PCR, a primer was
added to the PCR mixture instead of being preloaded into the
circular areas. The oval areas were designed so that the width of
the oval areas was the same as the diameter of the circular areas.
The design enabled the droplets to be centered in the areas,
allowing for better imaging. The design also produced droplets of
consistent size, therefore producing droplets with consistent
concentrations of reagents. The design created an aqueous droplet
surrounded by oil within the area, as in the previously described
PCR SlipChip, allowing room for thermal expansion during
thermocycling.
[0327] Certain embodiments of the digital PCR SlipChip were able to
detect template DNA at concentrations as low as 100 fg/10
.mu.L.
[0328] The dynamic range of digital PCR can be increased by, for
example, using a combination of large and small areas. For example,
in a device containing 2,000 areas, one would get a larger dynamic
range and higher confidence in the statistics if 1,000 areas
contained 1 nL of solution and 1,000 areas contained 10 nL of
solution. The distribution of area sizes that gives the best
dynamic range and highest confidence interval can be predicted.
[0329] In certain embodiments of the digital PCR SlipChip, multiple
area sizes can be designed by using a rotational design. The large
areas can be placed on the outside at a lower density, and the
small areas can be placed on the inside at a higher density. As
this embodiment of the SlipChip is rotated to slip, the large areas
will move more than small areas and all the areas will each contact
their corresponding areas on the bottom plate simultaneously.
[0330] In certain embodiments of the SlipChip, trapping bacteria
can be performed using magnetic beads. The inventors used magnetic
beads (Bug Trap version C) to capture MRSA from human pooled plasma
(HPP). HPP was spiked with MRSA for a final concentration of
1.times.107 cfu/mL of MRSA. Then, 100 .mu.L of this solution was
incubated with Bug Trap beads for 20 minutes at room temperature.
The beads were pulled down with magnets, and washed with
1.times.PBS buffer five times. Then, the beads were mixed with
EvaGreen PCR supermix, 1 mg/mL BSA, primers and injected into a
SlipChip for thermal cycling. The SlipChip design used here was the
same as for the multiplex PCR experiments.
[0331] The techniques described herein may be used for parallel
analysis of many individual cells, viruses, particles, molecules,
and other objects. For example, certain embodiments of the SlipChip
may be used to perform such measurements on populations of cancer
cells to determine variability and heterogeneity of genetic makeup,
phenotype, dynamics of responses, including responses to potential
treatments and combination of treatments. SlipChip can be used to
evaluate cells and tissues, for example blood cells, for markers of
radiation damage, resulting, for example, from radiation therapy,
industrial accidents or acts of war or acts of terrorism. This
analysis may be used to estimate the radiation dose received by a
person, and such knowledge may be used to take appropriate
countermeasures, e.g. adjusting the dose of radiotherapy,
administration of chelation therapy or ingestion of non-radioactive
isotopes or additional methods. These markers can be, for example,
markers of double-stranded DNA breaks. Proteins, mRNA, miRNA
markers and small molecules may be used, both general markers and
organ-specific markers. One example of such marker is
phosphorylation of Histone H2AX. The markers can be analyzed on
SlipChip, for example, via enzyme assays, via immunoassays,
electrophoresis, western blotting, via nucleic acid amplification
techniques, including analysis of RNA levels, and combinations of
methods. Measurements performed at single-cell level would provide
further valuable information to distinguish a dose of radiation
received globally from a dose received locally, even from
circulating cells. For example, global damage could lead to similar
levels of damage shown by the damaged cells or a single-peak
distribution of famage, while local damage could lead to a
variation of levels of damage shown by cells, or a bimodal or a
more complex distribution of damage. Amplification of genetic
material from individual viruses followed by genotyping the viruses
to determine their resistance patterns enables early detection of
resistant phenotypes, preferred for treatment in, for example, HIV
and Hepatitis infections.
[0332] The techniques described herein may be integrated with
multiphase flow techniques including plug-based and/or
droplet-based microfluidic systems and other techniques. Certain
embodiments of the SlipChip are suitable for the analysis of arrays
of droplets, plugs and other fluid volumes surrounded by an
immiscible fluid, including volumes generated on a SlipChip
directly or generated externally and introduced into the SlipChip,
such as plugs generated by a chemistrode or elsewhere.
[0333] This application describes a SlipChip device used for
separation that can be integrated with a number of different
separation techniques and sample types. This is a more detailed
description of capabilities already described in U.S. provisional
application 61/162,922 (see, for example, sections 00102, 00104,
00122, and 00188). The SlipChip was constructed as described for
the SlipChip used for FID protein crystallization above.
[0334] In certain embodiments, the SlipChip may be used for
diffusion-based separation. Many medical diagnostics rely on
isolated clear bodily fluids such as blood plasma to diagnose
diseases, but generating clear body fluids often requires expensive
centrifuges, time, and labor. A SlipChip can be designed to let
small molecular weight proteins, nuclear acids, and viruses in
whole blood diffuse into an area containing buffer while red blood
cells are retained in the original areas. This separation is based
on a difference in diffusion coefficients. For example, in 5
minutes, the Hepatitis B virus can diffuse 600 .mu.m but a red
blood cell can only diffuse 4 .mu.m. For example, the inventors
designed a SlipChip in which whole blood is mixed with 5 .mu.mol/L
8-methoxypyrene-1,3,6 trisulfonic acid (MPTS) and the mixture is
loaded into a left area by pipetting a 10 .mu.L blood sample.
1.times.PBS buffer was loaded into right areas. The device was
slipped to connect the blood areas with the buffer areas. The MPTS
diffused into the buffer areas in 30 min, while the blood cells did
not move.
[0335] This SlipChip design can utilize a separation medium in the
ducts or areas to induce a separation. At least one area/duct can
contain the separation medium. Examples of separation media that
can be integrated into the areas include, but are not limited to,
gels (e.g., silica gel or polyacrylamide gels), buffers, polymer
filters and membranes, binding agents, chromatography media,
surfaces of living cells, biological membranes (i.e. lipid
bilayers) with and without proteins, arrays of particles, and
nanoparticles. Alternatively, the separation medium can be on the
surface of the device. For example, thin layer chromatography
(TLC), gel electrophoresis, and isoelectric focusing can be
implemented on a SlipChip. Separation can also be driven by
diffusion and external fields and environments. Examples of fields
and environments to induce separation include magnetic fields,
electric fields, optical fields, gravitational fields, a chemical
gradient, a temperature gradient, active transport, and shear
forces. Fields may be produced by elements that have been
integrated on-chip or externally. For example, electrodes can be
incorporated into the areas and/or ducts or other areas of the
SlipChip, or can be applied externally via the inlets and outlets
of the SlipChip. With the integration of electrodes into the
SlipChip, one can use electrophoresis to do separation without
pretreatment of samples by placing a gel in the ducts for
electrophoresis. Fields can be switched on/off or modulated in
strength by slipping the SlipChip from one position to another.
Separations can also enabled by tags that modify objects'
properties with respect to an applied field. For example, magnetic
susceptibility, electrophoretic mobility, and diffusion
coefficients can be modified by binding the object of interest to
another object. Surface modified magnetic beads can be utilized to
bind specific bacteria, followed by separation by magnetic fields.
The SlipChip can be used to separate and detect small molecules
such as drugs and their metabolites and complexes, hormones,
environmental pollutants, antibiotics, nicotine and its
metabolites, drugs of abuse, stress hormones, other molecules
associated with chronic and acute stress. These separation methods
can also be used for separation of cells and isolation of cells
from biological fluids. Such cells of interest include circulating
tumor cells, fetal cells in blood, stem cells, bacterial and fungal
cells, T-cells and B-cells, and other subpopulations of cells
expressing specific markers. These cells can be isolated from
blood, urine, cerebral spinal fluid, interstitial fluid, tear
fluid, amniotic fluid, bone marrow, and tissue biopsies. For
example, a separation may be useful to determine the aggregation
states and post-translational modifications of proteins and
peptides involved in neurodegenerative diseases.
[0336] This SlipChip can also be used to study objects that can
move independently, such as cells and organisms. Chemotaxis (active
transport), thermotaxis, and magnetotaxis can be studied by setting
up chemical, thermal, and magnetic gradients within the SlipChip.
For example, chemotaxis can be used to isolate bacteria or
leukocytes in blood.
[0337] Separations can be integrated with all the other
capabilities of the SlipChip. For example, after slipping to
separate a mixture into various fractions, the SlipChip can be
slipped a second time to introduce reagents to visualize detection,
such as in delivering antibodies for Western blotting. Also,
detection of phosphorylation and glycosylation levels in cells is
important for diagnostics and drug discovery. Combining separation
with immunostaining is attractive for detection of phosphorylation
and glycosylation, and the SlipChip may be used to implement such
measurements of phosphorylation and glycosylation down to
single-cell levels. A series of slips can be used to isolate a
single cell, lyse it, perform a separation, stain the separated
fraction with antibody, and perform a detection assay. After an
initial separation, multiple fields can be combined in a single
step or multiple steps to perform one-dimensional, two-dimensional,
or higher dimensional separations. For example, separations may be
combined with protein crystallization. By continuing separation
during crystallization, various aggregation states of proteins
during crystallization can be separated. This separation can yield
crystals of high quality and purity.
[0338] In certain embodiments of the SlipChip strong intrinsic
mixing can be generated by vortex magnetic fields. See Martin,
Shea-Rohwer, Phys Rev E Stat Nonlin Soft Matter Phys. 2009 July;
80(1 Pt 2):016312 incorporated by reference in its entirety.
"Vortex" magnetic fields can be applied to a suspension of
spherical magnetic particles, which create strong, homogeneous
mixing throughout the fluid volume. Stirring a laminar flow within
a microchannel can be done by applying an alternating magnetic
field to ferrimagnetic beads inside a channel. See Rida and Gijs
Anal Chem. 2004 Nov. 1; 76(21):6239-46 incorporated by reference in
its entirety. Stir-bar strategies using microscale magnetic bars
exposed to a spatially uniform rotating magnetic field can be used.
Permanent structures that can be used include fabricated magnetic
rods driven by a standard magnetic stirrer. Beads bound to
microchains can also be used for mixing. Mixing can be achieved by
exposing beads to a simple rotating field. Beads can provide a
modest level of mixing within a fluid. Permanent magnets (and
magnetic stirrers) to create vortex magnetic fields are preferred
for certain embodiments because of the simplicity of the setup.
Other methods of mixing commonly used in microfluidic devices,
including ultrasonic mixing, "bubble mixers", and mixing and flow
driven by electrical fields, including alternating current
dielectrophoresis, can be used on SlipChip.
[0339] An example of using a magnetic stirrer and strong permanent
magnet.
[0340] A microfluidic device containing 1 micron magnetic beads was
placed 1-1.5 cm away from a rotating strong magnet and 4 strong
magnets were added on top at approx. the same distance. Strong
mixing did occur inside the .about.6 nL areas. Without the top
magnets, or rotating magnet below, the strong mixing stops. Without
being bound by theory, it is thought that a vortex magnetic field
was generated.
[0341] Buffering chambers can be used in any of the devices
described herein. These are preferably closer to the inlet,
upstream of the set of areas and ducts. They are capable of
trapping some of a sample and, for example, can prevent
overshooting when a small amount of sample is pushed too far into a
SlipChip. Buffering chambers are preferred when loading with
positive displacement devices (pipettes, etc).
[0342] In SlipChips used to perform FID crystallization,
differently-sized ducts that connect areas (for example, differing
in at least one of length, width and depth) on the same device can
be used to create a plurality of diffusion profiles across
different areas.
[0343] For certain embodiments of the SlipChip, it is preferable to
have varying degrees of overlap between areas and/or ducts within a
set of overlapping areas and/or ducts.
[0344] In SlipChips used to perform certain reactions, including
FID crystallization, a plurality of ducts can connect to a single
area to create multiple concentration gradients connected to the
same area.
[0345] A SlipChip can be designed to perform more than one type of
reaction. For example, a device can be configured to carry out both
FID crystallization and microbatch crystallization on the same
device. In some embodiments, a branching supply duct is used to the
sample between the regions of the device used for the different
reactions.
[0346] SlipChips similar to the devices described above for
carrying out FID crystallization can be used for other types of
experiments. For example, a cell-migration or cell polarization
assay can be carried out in such a device. One slips the device to
connect at least two areas, creating a gradient along which cells
can migrate up or down, or in response to which cells may polarize.
One can connect multiple areas to establish complex gradients and
countergradients. In addition, such devices can be used to
co-culture and monitor cell-cell interactions.
[0347] Many fields and forces can be used to transfer a volume from
one area to another area. Examples of fields that can enable
transfer of volumes between areas include surface tension, magnetic
fields, electric fields, gravitational fields, temperature
gradients, and shear forces. In certain embodiments, the SlipChip
can be used for metering and transferring multiple volumes of
liquid into a single volume. The SlipChip can be designed with
areas of varying volumes, to transfer and mix samples of different
volumes into a single volume. The ability to transfer and mix
samples of different volumes into a single volume can be used as a
general method for rehydrating dry reagents and can be followed by
relevant assays. It can be used for unidirectional transfer of
reagents in assays, for PCR and other applications that require
thermal expansion, in protein crystallization experiments, blood
coagulation, assays and reactions, for adding reagents to arrays of
trapped droplets, as on "drop spot arrays" and in other arrays as
described, for example in the following publications: Schmitz, C.
H. J.; Rowat, A. C.; Koster, S.; Weitz, D. A., Lab Chip 2009, 9,
44-49; Shim, J. U.; Olguin, L. F.; Whyte, G.; Scott, D.; Babtie,
A.; Abell, C.; Huck, W. T. S.; Hollfelder, F., J. Am. Chem. Soc.
2009, 131, 15251-15256, which are herein incorporated by reference.
Different geometries, sizes, and surface modifications of the areas
and plates of the SlipChip can be utilized to transfer and
compartmentalize droplets in the areas. As the droplet shape and
volume is restricted by the area shape and area volume, areas can
be filled to differing extents, including areas that are fully
filled, small droplets trapped within bigger areas, and droplets
that are contained in areas that are only slightly bigger than the
droplet. Areas of different sizes and different extents of filling
can be used to transfer and combine volumes in certain embodiments
of a SlipChip.
[0348] One example of how to use these combinations of area and
droplet sizes to transfer volumes in the SlipChip involves the
following: One can slip and overlap one area fully filled with one
substance with a larger area containing a droplet composed of a
second substance. When these two areas come into contact, the
liquid in the first area merges with the droplet in the larger and
remains in the larger area to minimize the surface tension.
[0349] Different geometries can be used to trap droplets that are
smaller than the area. For example, sloped areas can be used to
confine the droplet or a three-layer SlipChip can be used to
confine a droplet in a middle layer. These designs can be used to
precisely position the droplets and can be used to avoid the escape
of the droplet while slipping. In addition, they are also very
useful for devices that need to be opened to extract droplets for
off-chip analysis.
[0350] The SlipChip can also be used to induce mixing in droplets.
In certain embodiments, if an area is not completely filled, there
is an additional layer of lubricating fluid between the solution in
the area and the lubricating fluid between the two plates of the
SlipChip. As the SlipChip is slipped, the motion of the two plates
can induce mixing in the droplet, transmitted by the motion of the
lubricating fluid. A nonlinear or irreversible slipping pattern can
be used to enhance mixing.
[0351] In certain embodiments, in areas that are not completely
filled, the presence of an additional layer of lubricating fluid
between the solution and the surface of the other plate of the
SlipChip can prevent cross-contamination. The additional barrier
between the solution and the facing plate of the SlipChip will
reduce the possibility of residue being left on the surface of the
SlipChip, in addition to, or as an alternative to adjusting the
contact angle of the solution by surface modifications.
[0352] Surface modification of an area can be used to control
positioning and mass transfer in the areas. For example, one can
create an area with a hydrophilic bottom surface that will trap the
droplet in the bottom, as the bottom of the area will be
preferentially wetted by the aqueous solution. In another example,
the entire area can be made hydrophilic, so that an aqueous
solution will wet the area. Different solutions can have different
shapes and surface curvatures (surface energy) in the same size
area. Surface modification can also be used to transfer solutions
from one area to another. For example, two areas can be connected
with a hydrophilic bridge, connecting one area that is not fully
filled to another that is full. Using surface tension and
diffusion, substances can be transported from one area to
another.
[0353] One mechanism of transferring a volume of fluid from a first
area (e.g., a metering area) to a second area (e.g., a reactor
area) is when the first area and the second area have geometries
such that the volume of fluid inside the first area has a higher
surface tension than the volume of fluid inside the second area.
For example, this condition can be satisfied when the first area is
shallower and smaller than the second area. The user can load the
first area using a duct and then slip such that the first area and
second area overlap. The droplet in the first area prefers to go
into the bigger area because of surface tension. This approach can
be applied for example to rehydrate a preloaded dry reagent. This
approach can also be used to combine multiple reagents within the
same volume; for example two, three, four, five or more reagents
can be added sequentially to the same volume without loss of the
reagents already added.
[0354] In another example of transferring droplets using surface
tension, a surface of a reactor area can be modified to be
hydrophilic, while the remaining surfaces of the SlipChip device
are hydrophobic. When using hydrophilic reactor areas and
hydrophobic metering areas, the relative size of the areas is
unimportant, as an aqueous solution being transferred by the
metering area will preferentially wet the hydrophilic surface of
the reactor area. For example, large and small fully filled
hydrophobic areas, as well as partially filled hydrophobic areas,
can be used to fill the reactor area.
[0355] A multiplexed SlipChip for high efficiency screening of many
combinations was developed based on the multi-step transferring
strategy. The device can be used to set up a reaction matrix, each
area with a different combination of solutions. More steps can be
carried out to introduce third and fourth reagents in both vertical
and horizontal directions. For example, such an N.times.N design
can be used to rehydrate a dry reagent, add a sample, add reagents
(in, for example, the vertical direction), and then add another set
of reagents (in, in this example, the horizontal direction). The
device can also be designed with areas of different volumes, adding
an additional dimension to the multiplexed screening. The device
can utilize the mechanisms of volume transfer based on surface
tension described above.
[0356] Other mechanisms that can be used to transfer solutions from
a first area to a second area are described. The density
differences of the liquids can be used to float a droplet or
deposit a droplet into a larger area. If magnetic beads are added
to a droplet in a first area, a magnet can be used to move the
droplet into a second area. One can also integrate electrodes onto
certain embodiments of the SlipChip to move droplets containing
charged solutions or particles. After filling a second area with a
first solution, one can slip back and fill the second area with
another solution, then slip to overlap the two areas again to
combine metered volumes of different solutions. The solutions can
also be incubated between fillings. The user can control the number
of solutions and the volume of each solution filled into a reactor
area. For example, an array of small areas with different volumes
can be used to meter exact volumes of different solutions into a
reactor area.
[0357] If the volume of a reactor area is larger than the volume of
the droplets that are metered into it, there is room for thermal
expansion. This is useful for applications where the temperature is
increased (as in thermocycling for PCR, for example), because the
solution will not spill out when thermal expansion occurs. If the
reactor area is full when a metering area is brought into contact
with it, the solution in the metering area will mix with the
solution in the reactor area. When the metering area is slipped
away, it will transport a metered volume of the mixed solution.
Mixing techniques can be integrated with the SlipChip to ensure
good mixing of the two solutions.
[0358] An embodiment of a SlipChip which uses small areas to meter
and transfer solutions to larger areas for mixing is described.
This device contains 10 rows, where each row contains 20 larger
areas, 20 smaller areas, and a duct, and each row can be filled
with a different solution. The larger areas (620 .mu.m.times.240
.mu.m size, 60 .mu.m deep, 6.8 nL volume) and duct (300 .mu.m
width, 60 .mu.m deep) are in the bottom plate, and the smaller
areas (620 .mu.m.times.120 .mu.m wide, 35 .mu.m deep, 2 nL volume)
are in the top plate. The SlipChip device was assembled under
fluorocarbon. The fluorocarbon oil filled the areas, ducts, and the
gap between the two plates. A red food dye solution was filled into
the fluidic path formed by the smaller areas and the duct. The
device was slipped to align the smaller areas with the larger
areas. Due to the surface tension of the aqueous solution, the
solution was transferred from the smaller areas into the larger
areas. The device was slipped back to its original position to form
a continuous fluidic path through the smaller areas and the duct,
and a blue food dye solution was filled into the smaller areas. The
device was again slipped to align the smaller areas with the larger
areas, and the red and blue food dye were combined in the larger
area and mixed. Between fillings the different food dye solutions,
the fluidic path was washed with water and FC40 to reduce
contamination.
[0359] In some embodiments the SlipChip can be used for metabolism
profiling. All people metabolize drugs differently. Determining
personal metabolism is possible with genetics but is expensive, and
faces challenges when trying to deal with combinatorial
interactions. Additional challenges are present because liver
enzymes can be induced and also inhibited. A functional test is
therefore useful. A SlipChip based device for metabolism profiling
can be used in the office or at home. There is also a need to
characterize nicotine metabolites to optimize smoking cessation.
The SlipChip is useful for this, since it can be used away from a
laboratory. It can for example, be used at drugstores with a
SlipChip equipped with thin-layer chromatography capabilities, to
aid in selecting among nicotine patches with different doses.
Shear-driven chromatography can be used to improve thin-layer
chromatography on a SlipChip. Detection and quantification can be
performed on such devices using, for example, a cell phone or
visual detection device.
[0360] A SlipChip for measuring substance metabolism is useful, for
example, in situations where a simple device is needed to determine
the concentration of the drug or the ratio between a drug and one
or more of its metabolites, or where dosing is important. Measuring
the concentration of a substance in saliva is preferred. Measuring
the concentration of a substance that partitions into saliva
regardless of the source of the saliva is preferred. Such a device
can also be useful in situations where monitoring, rather than
diagnosing, is important, for example over long term periods of
time where a patient is already taking one or more drugs and some
metabolic enzymes may be inhibited. In such a case, it is useful to
monitor the metabolism of the one or more drugs over time to avoid
overdosing. This kind of device is also useful during phase I, II
or III drug trials to minimize side effects and improve outcomes,
or for detecting pesticides on foods. Two-dimensional separations
by thin-layer chromatography or other techniques can be used to
improve concentration and separation (for example, one can
concentrate the sample in one dimension and then separate in the
other, using different solvent phases).
[0361] In some embodiments, a SlipChip for metabolism profiling can
include a discontinuous bridging duct to enable multi-step slipping
without cross-contamination, on-chip serial dilution, and
patterning of the device to create hydrophilic areas.
[0362] SlipChip is applicable to a multitude of approaches and
techniques to enable personalized medicine. The applications
include testing patient samples for diagnostics and drug
development and treatment monitoring.
[0363] SlipChip may be used to evaluate kidney function of
patients, including by analysis of blood, urine, saliva and other
samples. It includes analysis of creatinine and analysis of other
markers such as Neutrophil gelatinase-associated lipocalin (NGAL),
Cystatin C and other markers. Markers can be analyzed using
immunoassays, enzyme assays, and other assays as described
elsewhere in this application.
[0364] SlipChip can be used to evaluate liver function, including
enzymatic assays tests and immunoassay tests. Targets include
Alanine transaminase (ALT), Aspartate transaminase (AST), Alkaline
phosphatase (ALP), Gamma glutamyl transpeptidase (GGT),
Beta-Hexosaminidase (.beta.-HEX), Lactate dehydrogenase (LDH), 5'
Nucleotidase (5'NTD). Additional tests, such as coagulation tests
(e.g. INR), serum glucose, total and direct bilirubin (BIL), Serum
albumin can be performed on SlipChip using the methods described in
this application.
[0365] From the foregoing, it will be observed that numerous
variations and modifications may be effected without departing from
the spirit and scope of the invention. It is to be understood that
no limitation with respect to the specific embodiment illustrated
herein is intended or should be inferred. It is, of course,
intended to cover by the appended claims all such modifications as
fall within the scope of the claims.
[0366] In certain embodiments, the SlipChip can be used to generate
concentration profiles by serial dilution. Serial dilution is one
of the most common and fundamental laboratory techniques, with
applications including immunoassays, cell culture assays, and
determining the kinetics of enzymatic assays. Several microfluidic
methods exist to create dilutions, including simple diffusional
mixing of laminar flow, multi-step fluid-dividers that split and
recombine multiple streams, and mixing multiple streams with flow
rates proportional to the desired final concentration. However,
many microfluidic devices rely on continuous flow, which suffers
from large dead volume, adsorption, pressure drop limit, and other
limitations. In certain embodiments, the SlipChip is capable of
robustly handling multiplexed multi-step reactions in parallel
without using complex instruments. The inventors developed a simple
approach that uses the SlipChip to perform serial dilutions. The
inventors have designed a SlipChip to incorporate multi-step
slipping and multiple mixing ratios, controlled by adjusting area
sizes. This method can handle many samples in parallel, can
require, in certain embodiments, small volumes of sample
(nanoliters for each area), and is useful for quantitative
multiplexed assays. In one embodiment, a serial dilution SlipChip
is designed to perform eight serial dilution steps in parallel. It
contains two parts: a row of shallow areas that contains sample and
an array of deep areas that are filled with buffer solutions for
dilution. Using the SlipChip to perform serial dilutions involves,
in certain embodiments, three general steps: (a) loading buffers,
(b) loading samples, and (c) multi-step slipping to dilute. After
filling the SlipChip by, for example, pipetting, the two plates of
the chip are slipped to separate ducts from areas. As the ducts are
separated from the areas, they are also moved out of the slipping
path. The areas containing sample are brought into contact with the
areas containing buffer, and the sample is diluted. The mixing
ratio, or dilution factor, is determined by the ratio of area
volumes. Further steps of slipping operate by the same principle
and thus serial dilutions are performed. In one example, the serial
dilution SlipChip was composed of two layers of microfabricated
glass: The top layer contains all the inlets and outlets, ducts for
the sample, and areas for the buffer solution. All areas are 76
.mu.m deep and ducts are 30 .mu.m deep. The bottom layer contains
10 .mu.m deep areas for the sample and 30 .mu.m deep ducts for the
buffer solution. The surfaces of the device were silanized to be
hydrophobic while keeping the 10 .mu.m deep areas hydrophilic. The
inventors used relatively thin, 10 .mu.m deep areas to decrease
diffusion time in and out of the area. The inventors made the area
hydrophilic to control the shape (and the volume) of the water
droplet within the hydrophilic area, and also to prevent de-wetting
from the shallow area. The 10 .mu.m deep areas were temporarily
masked during silanization to maintain a hydrophilic surface.
Fluorescent dye was used to quantify the dilution using this
SlipChip. After 4 slipping steps, a .about.10.sup.4-fold dilution
was observed.
[0367] For fabrication of the SlipChip with hydrophilic areas the
inventors followed the glass etching fabrication of SlipChip
procedure described elsewhere in this application with the
following modifications. A blank glass plate (Soda-lime glass,
thickness: 0.7 mm; chromium coating: 1025 .ANG.; AZ 1500
photoresist: 1 .mu.m) was first cut to be 3 in.times.1 in.
[0368] After the photomask was removed from the glass plate, the
glass plate was developed by immersing it in 0.5% NaOH solution for
2 min. After the glass plate was taped and immersed in the etching
solution, a 25.degree. C. constant-temperature water bath shaker
was used to control the etching speed. By controlling the etching
time (.about.30 min), areas that were 46 .mu.m deep were etched
into the glass plate. The depth of the areas was verified using a
specially designed structure that indicates, without magnification,
that a certain etch depth has been passed. This structure consists
of an array of squares with a width equal to double the distance to
be etched. The squares are originally covered with chrome. After
the desired etch depth has been reached, the chrome is removed,
producing an obvious contrast difference that can be seen with the
naked eye. The plate was taken out and thoroughly rinsed with
Millipore water and dried with nitrogen gas. Using another
photomask containing the design for the ducts and an etching time
of .about.20 min, 30 .mu.m deep ducts were etched into the glass
plate. The plate was thoroughly rinsed with Millipore water and
dried with nitrogen gas. The inventors used the same protocol to
make 10 .mu.m deep areas and 30 .mu.m deep ducts in the bottom
plate.
[0369] After the glass plate was rinsed with ethanol to strip the
undeveloped photoresist, the glass plate was piranha cleaned (1
part 30% hydrogen peroxide, 3 parts sulfuric acid), washed twice
with Millipore water, and then dehydrated on a 220.degree. C. hot
plate for more than 2 hours. The plate was cooled down to room
temperature and spin-coated with OmniCoat (MicroChem, USA) and
baked at 200.degree. C. for 1 min. The plate was cooled down to
room temperature and spin-coated with a 20 .mu.m thick layer of SU8
2025. The plate was next covered with a photomask that protected
the areas on the plate that were to be hydrophobic. UV light was
shined from the back of the glass plate, to take advantage of the
preexisting chrome mask. In the area exposed by the photomask, UV
light only passed through the plate where the chromium coating was
removed, so only the SU8 in the areas remained after developing.
The SU8 in the areas protected the areas and prevented them from
being made hydrophobic. OmniCoat on the exposed surface was
developed by immersion in MF-319 for 30 sec and rinsed with
Millipore water for 2 minutes.
[0370] Finally, the glass plate was immersed in the chromium
etchant to remove the chromium coating. The glass was then rinsed
with ethanol and Millipore water and dried by baking in a
120.degree. C. oven overnight.
[0371] The glass plates were cleaned and subjected to an air plasma
treatment at 300 mTorr for 100 seconds, and then the surfaces were
rendered hydrophobic by silanization in a vacuum desiccator for 5
hours with tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane
as previously described. After silanization, the glass plates were
rinsed by (in this order) 3.times.20 ml anhydrous toluene,
3.times.30 ml anhydrous ethanol, 3.times.30 ml ethanol/H.sub.2O
(50%:50%, v:v), and 3.times.30 ml Millipore water. The plates were
baked in a 120.degree. C. oven for 15 minutes. Finally, the SU8 in
the areas was stripped by immersing the glass plates in Remover PG
at 80.degree. C. for 30 min. The plates were then rinsed with
chloroform, acetone, and then ethanol and blown dry with
nitrogen.
[0372] The SlipChip was assembled under FC-40. The bottom plate was
first immersed into FC-40 in a Petri dish, with the patterns facing
up. The top plate was then laid on top of the bottom plate, with
the patterns facing up. After .about.3 mins, the top plate was then
flipped carefully to prevent trapping of air bubbles when
assembling the SlipChip. If necessary, air bubbles can be removed
by quickly placing the chip in a vacuum desiccator. The two plates
were aligned by moving them relative to each other and were then
fixed by using four micro binder clips. The SlipChip was kept in
FC-40 during the process of loading.
[0373] All the fluorescent dye solutions were filtered with a 0.22
.mu.m PVDF syringe filter (Millipore) before use. Alexa Fluor 488
hydrazide (1.6 mM, Invitrogen) in PBS buffer (1.times., pH 7.4) was
loaded by pippetting into the sample duct. 1.times.PBS buffer
solution was loaded into the buffer duct. The SlipChip was slipped
under a Leica MZ 16 stereoscope to first form isolated droplets.
Then the sample areas were combined with the buffer areas
sequentially. After each slipping step, the inventors waited for 3
minutes to allow for the diffusion of the fluorescent dye. After 4
steps of slipping, the device was quickly transferred to a Leica
DMI6000 microscope (Leica Microsystems) with a 20.times.0.7 NA
Leica objective and a Hamamatsu ORCAER camera. A L5 filter with an
exposure time of 30 ms was used to collect Alexa Fluor 488
fluorescence. Images were acquired and analyzed by using Metamorph
imaging system version 6.3r1 (Universal Imaging). To calibrate the
microscope, the fluorescence intensity of a fluorescence reference
slide for the L5 filter was recorded and used for background
correction. 80 nM, 160 nM, 400 nM and 800 nM Alexa Fluor 488
hydrazide solutions in PBS buffer were used to obtain the
calibration curve to determine the concentration of fluorescent
dyes after four slipping steps. Area depth was measured with a
Veeco Dektak 150 profilometer and volume of the areas was
calculated with the assumption that etching is isotropic.
[0374] Certain embodiments of the SlipChip can also be used to
perform other multi-step reactions, including but not limited to
determining IC50, EC50 and other concentration curves (e.g. CP450,
etc). IC50 assays can be performed via serial dilutions of DMSO
compound libraries to achieve dilutions of 2.times.10.sup.7 in 100%
DMSO or DMSO/water mixtures and assays can be performed with each
dilution to ascertain the IC50 of the compound of interest. The
SlipChip is an ideal platform to make dilutions of libraries and
perform subsequent screening on the generated dilution library in a
multiplexed manner. Other multi-step reactions which can be
performed on the SlipChip include measuring enzymatic kinetics and
quantifying concentrations by PCR by serial dilution (either
combined with real time PCR or using end point PCR). For example,
the user can perform an HIV viral load test by PCR. The user can
serially dilute an unknown sample over a wide dynamic range and
extract the concentration from PCR results with the assumption that
the HIV virus follows a Poisson distribution. Other multi-step
reactions which can be performed on the SlipChip include
sensitivity testing, both drug and toxin, using serial dilutions of
the substance of interest which are then administered (on-chip or
off-chip) to a test organism or human subject, and isolation of
rare cells or molecules, especially from samples with unknown
initial concentration: in a dense cell population/high
concentration mixture, a rare cell or molecule will be difficult to
find. Serial dilution offers a convenient method to obtain the
concentrations necessary for stochastic confinement (or digital
PCR, etc.) when the initial concentration is unknown. Multi-step
reactions which can be performed on the SlipChip include bacterial
culture density (or the concentration of particles in solution)
which can be quickly estimated through serial dilution and
back-calculation. Determining antibody titer and serial dilution is
one possible method to either eliminate non-specific binding, or
identify it as a false-positive.
[0375] In certain embodiments of the present invention, high
throughput nanoliter digital PCR can be performed on the SlipChip.
The SlipChip has been shown to be free of cross contamination, and
it has been previously validated by performing protein
crystallization and immunoassays. The inventors have also
demonstrated the SlipChip can be applied to high throughput
multiplex PCR. The inventors have used the SlipChip platform for
performing digital PCR. In certain embodiments, over one thousand
nanoliter compartments can be formed simultaneously by one slipping
step after a sample has been introduced via pipetting. When a low
concentration of nucleic acid is loaded into the device, there can
be less than one copy of nucleic acid per compartment. In this
case, a "yes-or-no" digital readout of end-point fluorescence can
be used to detect the presence of nucleic acid in each compartment,
and the concentration of nucleic acid in the original sample can be
calculated. Such a digital PCR SlipChip" has been used to amplify
Staphylococcus aureus genomic DNA. It has also been used to amplify
RNA from HIV via RT-PCR. The digital PCR SlipChip offers a new
strategy for quantification of nucleic acids, study of cell
heterogeneity, diagnostics of prenatal disease, and improvement of
point-of-care devices. When combined with isothermal reactions and
visual readout, the PCR SlipChip platform is a powerful tool for
diagnostics in resource-limited settings.
[0376] Manipulating volumes of fluid is the basis of modern
laboratory practice. It is critical in research and development,
from new biomarkers and drugs to new materials and processes. It is
a critical part of analytical science in diagnostics, food and
water safety, and biodefense. In these areas, SlipChip technology
can be useful. SlipChips can be designed to encode a complex
program for parallel manipulation of many fluid volumes. The
SlipChip, in certain embodiments, comprises two plates that can
move relative to one another. The program is encoded into each
SlipChip as a pattern of areas imprinted into the plates. Each area
remains isolated until it overlaps with an area on the opposite
plate. The encoded program is executed by moving--or slipping--the
two plates relative to one another. As plates move, areas in the
two plates come in and out of contact in a precisely defined
sequence, creating and breaking up transient fluidic pathways, and
bringing reagents in and out of contact. One or multiple samples
can be introduced via such fluidic pathways. The program is
executed by bringing the samples into contact with reagents either
loaded by the user into a transient pathway, or pre-loaded onto the
SlipChip. Very complex programs can be executed on thousands of
areas very easily for the user. The smaller the volumes, the more
precious are the samples and reagents, the larger the number of
interacting samples and reagents, and the more complex the
manipulations, the more beneficial the SlipChip. The SlipChip
satisfied the seven unmet needs that are framing development of new
fluidic technologies: it enables miniaturization, smoothly and
precisely scalable from picoliter to nanoliter to microliter
volumes. The SlipChip enables experiments that simply cannot be
done on large scale, e.g. interrogating individual cells, both for
human cells and microbial cells, or counting molecules. The
SlipChip minimizes consumption of reagents and samples, reduces
waste, especially relevant for expensive reagents, rare samples
(biopsy samples, rare cells, banked samples), toxic, radioactive,
and biohazard waste. Certain embodiments of the SlipChip enable
multiplexed experimentation, with thousands of experiments easily
performed in parallel in miniaturized format. SlipChip enables "one
sample, one assay, many times", as required for detecting
variability in properties of individual cells or genotypes.
Examples include diagnosing cancer by analyzing biopsies and
circulating tumor cells, analyzing rare and drug resistant
genotypes in HIV, and emerging methods for diagnosing stroke.
Certain embodiments of the SlipChip enable massively simplified
"one assay, many samples" testing as is done by central labs such
as Quest Diagnostics. Certain embodiments of the SlipChip enable
"one sample, many assays", as in multiparameter diagnostics
necessary for diagnosis of complex conditions. Certain embodiments
of the SlipChip enable "many samples, many assays" experiments
required for biomarker discovery and validation. The SlipChip
satisfies demand for speed: cutting test-to-result time. By
performing analysis at a single-cell level and removing the
requirement for cell culture, certain embodiments of the SlipChip
accelerate microbiological testing critical for diagnosis of sepsis
and food, water, and environmental safety. By providing a
simplified platform, certain embodiments of the SlipChip enable
portable point-of-use devices critical for diagnosis of acute
conditions (such as stroke and heart attack). Certain embodiments
of the SlipChip satisfy demand for sensitivity down to
single-molecule level in "digital" formats. Detecting molecules one
at a time is attractive for sensitive detection and for
quantification, for nucleic acids and proteins. The SlipChip is an
ideal platform for such "digital" formats that require thousands of
experiments (to get an accurate count) in small volumes (to get
each molecule to a high enough concentration so the detection
chemistry works) and multi-step manipulations (e.g. for
heterogeneous immunoassays). This capability has wide-ranging
implications, from counting gene copy number in individual cancer
cells to diagnosing heart attacks and traumatic brain injury (TBI).
The SlipChip minimizes manual labor, reduces errors, increases
reproducibility, increases throughput, essential in well-equipped
laboratories, point-of-care, and resource-poor settings. It enables
complex pre-programmed multistep procedures on the microscale,
including sample preparation and processing, as required for
genetic testing. It supports reagents loaded by the user, or
preloaded at the factory and stored on board, minimizing handling.
A SlipChip with preloaded reagents functions as a "liquid-phase
microarray", with the potential of revolutionizing multiplexed
solution-phase assays just as gene-chips revolutionized DNA
hybridization assays. It enables these complex procedures to be
performed outside of the laboratory, as required for bench-top
discovery work, point of care and home testing, and resource-poor
environments. The SlipChip platform supports all common
experimental methods. For example, it supports PCR and other
nucleic-acid testing, immunoassays and handling of beads, enzyme
assays and cell-based assays, required for a broad range of
applications. It supports commonly used readout mechanisms
(optical, magnetic and electrical). It is uniquely suitable for
chemistry with, for example, simple Teflon and glass devices.
Certain embodiments of the SlipChip decrease costs. Certain
embodiments require no valves and are simple to manufacture by
standard plastics technologies. The platform can be simple to
operate with no or minimal equipment needed. This combination of
low cost and simplicity makes the SlipChip superior to other
microfluidic lab on a chip technologies, which can require complex
instrumentation to run the chips (robotics, pumps, actuators), and
can require complex chips with integrated valves. The SlipChip is
superior to the robotic workstations which are capital-intensive
and cannot match the performance of the SlipChip at the low-volume
range due to problems with evaporation and precision.
[0377] Certain embodiments of the SlipChip can be used on many
single cells/single particles/single samples/single molecules at a
time. They can also be used for 3D tissue models, with associated
fluidics for maintenance, perturbing, and analysis.
[0378] The SlipChip can be used in academic, pharmaceutical,
diagnostic segments, providing reagents and equipment. Certain
embodiments of the SlipChip allow the miniaturization and
simplification of standard laboratory protocols, measurement of
concentration of nucleic acids DNA/RNA ("digital PCR"), protein
crystallization, and unique capabilities in single-cell analysis.
The SlipChip can also bring value to companies selling libraries of
compounds by packaging them in SlipChips. Currently, these
libraries can be sold only to screening centers, and testing is
expensive. 10,000 times less, for example, of each compound can be
loaded on a SlipChip and tested. The SlipChip can reducing the
barrier for users testing reagents; preloaded SlipChips with a
panel of reagent formulations can be distributed to users who can
rapidly and efficiently test which of the formulations is optimal
for their application, and order that formulation in larger
quantity (perhaps also on SlipChips). Certain embodiments of the
SlipChip can also be used for genetic testing in forensics which
needs highly simplified approaches.
[0379] Other areas where the SlipChip may be useful include food,
water and environmental safety. Current methods of bacterial
detection require overnight culture before testing. Certain
embodiments of the SlipChip can provide answers in .about.1 hour
without culture, overcoming this costly time lag. Certain
embodiments of the SlipChip enable on-site testing, critical for
remote locations (for example, space missions, rural areas). It can
also be used for livestock diagnostics/agricultural testing.
Certain embodiments of the SlipChip allow miniaturizing and
accelerating existing diagnostic tests in home, point of care and
clinical testing. The simplicity of the technology makes it
attractive for point of care, home and military use; high
performance could make the same platform attractive for central lab
instruments, saving money in FDA costs needed to approve currently
disparate platforms used for clinical lab and point of care
technologies. Certain embodiments of the SlipChip can be used for
accelerated microbiological testing in sepsis, a cause of death for
over 100,000 people just in the US. Many of these deaths are
preventable by a more rapid diagnosis. Diagnostics include genetic
testing and screening for drug resistance, as in MRSA and
drug-resistant HIV genotypes, phenotypic testing for drug
resistance, testing for coagulopathies and associated testing and
monitoring of blood coagulation, cell-based
immunodiagnostics/allergy profiling, diagnostics for the developing
world (for example, HIV, malaria, TB, etc.), home and point of care
diagnostics for monitoring organ function and treatment, especially
with expensive biologics, general metabolic tests for monitoring
drug treatment and drug metabolism, to ensure safety and efficacy.
These are important both to consumers and, potentially, drug
developers to monitor clinical trials. Warfarin is the best known
example, but there are many more. The SlipChip can also be used for
new diagnostics approaches including but not limited to discovering
new biomarkers via single-cell analyses as in cancer, prenatal, and
stroke diagnostics; discovering new panels of biomarkers using
multiplexed analyses as in Alzheimer's disease and cancer (possibly
in conjunction with the chemistrode for pulse-chase diagnostics);
it can enable clinical studies and validation of these biomarkers,
enable the use of these biomarkers in diagnostics in point of care
and clinical formats. It can also be used for maintaining proper
mental status for patients with psychiatric disorders--including,
for example, for home testing, remote monitoring, patient networks,
and other non-traditional approaches. The SlipChip can also be used
for new therapeutic approaches including but not limited to
integration of biomarker discovery and validation with drug
discovery and diagnostics and complex tissue culture models with
integrated analysis.
[0380] The SlipChip can also be used as a test to modify behavior
or help people make choices, rather than offer a medical treatment.
For example, the nicotine patch and other smoking cessation
products are available without a prescription. It is well-known
that the metabolic rate of nicotine strongly affects the success
rate of smoking cessation and should guide the kind of patch the
person should be buying. Such a test can be implemented on a
SlipChip and sold to people who are quitting smoking, classifying
people into three classes of low-medium-high nicotine metabolizers
and suggesting appropriate smoking cessation products. A test,
taken daily, suggests the dosing of the patch to provide a smooth
cessation experience. A kit can include such tests with smoking
cessation products. SlipChip tests can also optimize performance:
levels of hydration and dehydration, diet, caffeine and other legal
substances, exercise levels can all be monitored and/or modified to
achieve top performance with the guidance of proper tests. Such
tests can by used by those whose performance at a given time
matters: competitive athletes, military, students, sports
enthusiasts and people whose time is too valuable to be wasting in
the afternoon lull. A "Stress chip" can be used for analyzing for
markers of acute and chronic stress in the general population.
Managing stress is perhaps one of the most important avenues of
improving life satisfaction. Such a test would provide more
immediate feedback to individuals on their life style to reduce the
possibility of stress-related health conditions. This is much less
expensive than waiting for development of chronic inflammation or
cardiovascular conditions, and then interfering. To employers,
military and law enforcement, and insurers it is valuable to
evaluate and manage human resources. An "addiction SlipChip" could
be used to perform panels, or combinations of panels, testing for
liver damage associated with alcohol consumption, nicotine and its
metabolite levels associated with smoking, levels of blood glucose
and glycosylated hemoglobins associated with metabolic and eating
disorders, levels of caffeine and its metabolites, and levels of
drugs of abuse. A "baby chip", "organic chip", and "chronic chip"
are additional applications to satisfy the need-to-know of
first-time parents, health fanatics, or people at high risk for
chronic conditions who are most likely to be proactive in wanting
information and monitoring. For a simple SlipChip with a cell-phone
readout, partnering with, for example, Google or Microsoft is
attractive, as a way for people to organize results of their tests
and optionally link them with blogs that reflect diet, exercise and
behavior. Healthy people can use their Google Health service to
mine for an incredible wealth of information, which can be used to
improve tests and to offer advertisement and new products. Certain
embodiments of the SlipChip can be used for maintaining health and
performance, in addition to treating disease. Analyzing testable
salivary markers linked to performance (both short term, and long
term as in health status) and individualized to each person via
simple testing platform, can improve quality of life and
productivity of the society.
[0381] In certain embodiments, the SlipChip can be used to
implement genetic algorithms (GA) to discover new homogeneous
catalysts using the oxidation of methane by molecular oxygen as a
model system. In one example demonstrated by the inventors, the
parameters of the GA were the catalyst, a cocatalyst capable of
using molecular oxygen as the terminal oxidant, and ligands that
could tune the catalytic system. The GA required running hundreds
of reactions to discover and optimize catalyst systems of high
fitness, and microfluidics enabled these numerous reactions to be
run in parallel. The small scale and volumes of microfluidics offer
significant safety benefits. The microfluidic system included
methods to form diverse arrays of plugs containing catalysts,
introduce gaseous reagents at high pressure, run reactions in
parallel, and detect catalyst activity using an in situ indicator
system. Platinum (II) was identified as an active catalyst and iron
(II) and the polyoxometalate H.sub.5PMo.sub.10V.sub.2O.sub.40
(POM-V2) were identified as active cocatalysts. The Pt/Fe system
was further optimized and characterized using NMR experiments.
After optimization, turnover numbers of approximately 50 were
achieved with approximately equal production of methanol and formic
acid. The Pt/Fe system demonstrated the compatibility of iron with
the entire catalytic cycle. This approach of GA-guided evolution
has the potential to significantly accelerate discovery in
catalysis and other areas where exploration of chemical space is
preferred. Kreutz, et al., J Am Chem Soc. 2010 Mar. 10;
132(9):3128-32 is incorporated in its entirety by reference.
[0382] In certain embodiments, the SlipChip platform can be used to
perform digital PCR with instrument free sample loading and small
sample volume. In one example, the PCR master mixture was
introduced into the SlipChip by pipetting. The fluidic path was
formed by overlapping elongated areas, and broken by simple sliding
to generate 1280 reaction compartments (2.6 nL each)
simultaneously. After thermal cycling, end-point fluorescence
intensity was used to detect the presence of nucleic acid. Digital
PCR on the SlipChip was validated using Staphylococcus aureus
genomic DNA. When the template in the PCR master mixture was
diluted, the fraction of positive areas decreased proportionally,
as expected by a statistical distribution. No cross contamination
was observed during the experiments. Digital reverse transcription
PCR (RT-PCR) was also demonstrated on a SlipChip by amplifying RNA
from HIV. The SlipChip provides an easily available strategy to
count nucleic acids by using PCR and RT-PCR, as well as to perform
single cell analysis, prenatal diagnostics, and point-of-care
diagnostics. With isothermal PCR and visual readout, the digital
PCR on the SlipChip can be designed to be instrument free, and can
be widely applied for research and diagnostics in resource limited
area.
[0383] The general idea behind digital PCR is to separate the
molecules of nucleic acid by placing one molecule or less into a
compartment. As the number of compartments is increased and the
size of the compartments is decreased, the probability of trapping
a single molecule in each compartment increases. At the single
molecule level, confining the molecule in a small volume also
increases the relative concentration, thus increasing the
sensitivity. The number of positive areas can be counted and the
total number of target molecules in the sample can be
calculated.
[0384] Digital PCR has been previously demonstrated on a multiwell
plate, and a number of groups have shown how to implement this
method in a microfluidic format. Valve-controlled microfluidic
chips have adapted digital PCR for various applications, for
example cell analysis and prenatal diagnosis; however, this method
still requires a complex multilayer soft lithography fabrication
process and a electrical pneumatic system to accurately control the
"open/close" state of the valves. Another system for digital PCR
uses picoliter droplets in a microfluidic device for single-copy
PCR and RT-PCR. Although a large number of picoliter droplets can
be generated by using a microfluidic T-junction, this method
requires high-precision pumps to accurately control the flow rate
in order to form droplets of uniform size. Emulsion PCR,
microdroplet, and engineered nanoliter droplets can be also
potentially be used for digital PCR, but these systems require
either mechanical agitation or pumps to generate the small volume
droplets. A microfluidic chamber for high throughput nanoliter
volume qPCR can also be adapted for digital PCR, but it still
requires mechanical loading and manual sealing operations. To date,
digital PCR is still restricted to high-end users. A simple,
inexpensive platform is still an unmet need to make digital PCR a
routine procedure in laboratory or resource limited settings. The
inventors have demonstrated such a system based on the SlipChip
platform. The SlipChip is an advantageous platform for digital PCR
due to its inherent simplicity. All samples can be loaded by simple
pippetting. The SlipChip can handle multistep processes on many
small volumes in the context of protein crystallization and
immunoassays. Multiplexed PCR was successfully demonstrated in
SlipChip: no cross-contamination was seen when different pre-loaded
primers were used to screen a sample to identify the presence of
pathogens, and the design of the SlipChip was modified to allow
room for thermal expansion of an aqueous PCR solution during
thermocycling. The SlipChip has also been shown to be capable of
performing digital PCR by dividing a sample into thousands of
nanoliter areas.
Example.
[0385] All solvents and salts purchased from commercial sources
were used as received unless otherwise stated. SsoFast EvaGreen
Supermix (2.times.) was purchased from Bio-Rad Laboratories
(Hercules, Calif.). Bovine serum albumin (BSA) was purchased from
Roche Diagnostics (Indianapolis, Ind.). All primers were ordered
from Integrated DNA Technologies (Coralville, Iowa). Mineral oil
(DNase, RNase, Protease free) and DEPC-treated nuclease-free water
were purchased from Fisher Scientific (Hanover Park, Ill.).
Dichlorodimethylsilane was purchased from Sigma-Aldrich (St. Louis,
Mo.). Staphylococcus aureus genomic DNA (ATCC number 6538D-5) was
purchased from American Type Culture Collection (Manassas, Va.).
Soda-lime glass plates coated with chromium and photoresist were
purchased from Telic Company (Valencia, Calif.). Spectrum food
colors (red food dye) were purchased from August Thomsen Corp (Glen
Cove, N.Y.). PCR tubes and barrier pipette tips were purchased from
Molecular BioProducts (San Diego, Calif.). Small binder clips (clip
size 3/4'') were purchased from Officemax (Itasca, Ill.).
Mastercycler and in situ adapter were purchased from Eppendorf
(Hamburg, Germany). Teflon tubing (O.D. 250 .mu.m, I.D. 200 .mu.m)
was purchased from Zeus (Orangeburg, S.C.). Teflon tubing (I.D. 370
.mu.m) was obtained from Weico Wire & Cable (Edgewood, N.Y.).
Photomasks were obtained from CAD/Art Services, Inc. (Bandon,
Oreg.). Amorphous diamond coated drill bits were obtained from
Harvey Tool (0.030 inch cutter diameter, Rowley, Mass.).
[0386] The procedure for fabricating the SlipChip was based on the
glass etching fabrication of SlipChip procedure described elsewhere
herein, with the following modifications. The soda-lime glass plate
coated with chromium and photoresist was aligned with a photomask
containing the design for the areas (both circular and elongated)
of the SlipChip, and exposed to UV light for 40 seconds. After
removing the exposed photoresist and chromium layers, the glass
plate was immersed in a glass etching solution for 35 min at
40.degree. C. to produce areas that were 50 .mu.m deep.
[0387] The glass plate with an etched pattern of areas was
thoroughly cleaned with Millipore water and ethanol and dried with
nitrogen gas. The glass plate was oxidized in a plasma cleaner for
100 seconds and immediately placed in a desiccator with 50 .mu.L of
dichlorodimethylsilane. A vacuum was then applied for one hour for
gas-phase silanization. The silanized glass plate was rinsed with
chloroform, acetone, and ethanol, and then dried with nitrogen gas.
In order to be reused, the glass plate could be cleaned with
piranha acid (3:1 sulfuric acid:hydrogen peroxide) and silanized
again as described above.
[0388] The mineral oil was filtered and degassed before using. The
SlipChip was assembled under mineral oil. The bottom plate was
first immersed into the oil in a Petri dish, with the patterned
side facing up. The top plate was then laid on top of the bottom
plate with the patterned side facing down. The two plates were
aligned and fixed using binder clips.
[0389] Two primer sequences for the nuc gene found in S. aureus
were selected from a previous publication:
5'-GCGATTGATGGTGATACGGTT-3' (primer 1) and
5'-AGCCAAGCCTTGACGAACTAAAGC-3' (primer 2). The reaction master
mixture consisted of 10 .mu.L of 2.times. SsoFast EvaGreen
Supermix, 0.5 .mu.L of primer 1 (10 .mu.mol/L), 0.5 .mu.L of primer
2 (10 .mu.mol/L), 2 .mu.L of 10 mg/mL BSA solution, 5 .mu.L of
RNase free water and 2 .mu.L of S. aureus gDNA solution. The S.
aureus gDNA solution was serially diluted using 1.times.BSA
solution (0.01 mg/mL) to give a range of final template
concentrations: 10 ng/.mu.L, 1 ng/.mu.L, 100 pg/.mu.L, 10 pg/.mu.L,
1 pg/.mu.L, and 100 fg/.mu.L. The amplification was performed using
a PCR thermocycling machine (Eppendorf). To amplify the DNA, an
initialization step of 2 min at 94.degree. C. was used to activate
the enzyme. Next, a total 35 cycles of amplification were performed
as follows: a DNA denaturation step of 1 min at 94.degree. C., a
primer annealing step of 30 sec at 55.degree. C., and a DNA
extension step of 30 sec at 72.degree. C. After the final cycle, a
final elongation step was performed for 5 min at 72.degree. C. Then
the PCR products were stored in the cycler at 4.degree. C. before
imaging.
[0390] There are 2.9 million total base pairs in S. aureus gDNA
(GenBank accession number NC_009632). The average molecular mass
per base pair was approximated to be 660 to simplify the
calculation. Therefore, 1 ng of S. aureus gDNA has
3.15.times.10.sup.5 copies. The volume of reaction solution in each
compartment was 2.6 nL, and the total number of areas in the device
was 1280. Thus, each area contained on average 944 copies when the
starting concentration of S. aureus gDNA was 1 ng/.mu.L.
[0391] Bright field images were acquired by Leica stereoscope. All
fluorescence images were acquired by using a digital camera (C4742,
Hamamatsu Photonics, Japan) mounted to a Leica DMI 6000 B
epi-fluorescence microscope with a 5.times.0.15 NA objective and L5
filter at room temperature. All fluorescence images were corrected
by a background image obtained with a standard fluorescent slide
and then stitched together using MetaMorph software (Molecular
Devices, Sunnyvale, Calif.). The intensity levels were adjusted to
the same values for all images.
[0392] The design of the device was symmetric to increase the
density of areas. Arrays of circular areas filled with oil were
designed in both the top and bottom plates, and overlapping
elongated areas in both top and bottom plates were used to
introduce the sample. Upon slipping, isolated compartments were
created, and an aqueous droplet of uniform size was generated in
each individual compartment. This SlipChip contained no ducts;
instead, each plate contained rows of elongated areas and circular
areas for a total of 1,280 reaction compartments. The elongated
areas were 400 .mu.m long, 200 .mu.m wide, and 50 .mu.m deep, and
the circular areas were 200 .mu.m in diameter and 50 .mu.m in
depth. In the initial configuration, the elongated areas in the top
and bottom plates overlapped to form a continuous fluidic path. By
using overlapping elongated areas instead of areas connected by
ducts, the pressure drop in the device was small, allowing many
areas to be filled by simple pipetting, and a 3.4 .mu.L sample
filled the entire device. By slipping the top plate relative to the
bottom plate a short distance, the elongated areas were separated
and each was centered on top of a circular area containing a layer
of lubricating fluid (mineral oil). For digital PCR, the primer was
added to the PCR mixture instead of being preloaded into the
circular areas. The elongated areas were designed so that the width
of the elongated areas was the same as the diameter of the circular
areas. Advantages of this design include: (1) The design enables
the droplets to be centered in the areas, allowing for better
imaging. (2) The design also produces droplets of consistent volume
(.about.2.6 nL), (3) The design creates an aqueous droplet
surrounded by oil within the area, as in the previously described
multiplexed PCR SlipChip, allowing room for thermal expansion
during thermal cycling.
[0393] During thermal cycling, the lubricating fluid and the
aqueous PCR mixture expand more than the glass material of the
SlipChip due to the different thermal expansion coefficients of the
three materials. When using certain embodiments of a SlipChip with
overlapping areas of the same size and geometry, the aqueous
solution will completely fill the areas. During the temperature
increase required for thermal cycling, the aqueous solution can
expand and leak out of the areas, resulting in a loss of material
and changes in the concentration of reagents. For the multiplexed
PCR SlipChip, this problem was solved by overlapping a square area
containing an aqueous PCR mixture with a circular area containing
oil, producing a droplet suspended in oil and centered in the
square area. For digital PCR, the inventors achieved the same
result by using elongated areas containing the aqueous PCR mixture
centered over circular areas filled with the lubricating fluid.
[0394] The inventors demonstrated digital PCR on the SlipChip with
10 fg/.mu.L of S aureus gDNA. At this concentration, there was less
than 1 copy of gDNA per 100 areas on average, and PCR amplification
of a single copy of gDNA was achieved. A linescan of the digital
PCR SlipChip before and after thermal cycling shows that the
fluorescence intensity for areas containing a single DNA template
increased significantly while the fluorescence intensity for areas
without a DNA template did not increase. This linescan also
verified that there was no cross-contamination in the SlipChip, as
the fluorescence intensity for empty areas adjacent to an area
containing DNA template did not change.
[0395] The inventors quantified the performance of this device
using five concentrations of genomic DNA from S. aureus. The
digital PCR SlipChip was able to detect template DNA at
concentrations as low as 1 fg/.mu.L. The inventors determined that
single copy target DNA amplification was achieved when less than
one-third of the total areas showed the signal of amplification.
The expected concentration of the DNA template was presented as
number of copies per area (cpw), and the concentration of the
original DNA stock solution was measured spectrophotometrically by
NanoDrop (Thermo Scientific). The detailed method for calculating
the cpw is presented elsewhere herein. As the DNA template in the
PCR master mixture was diluted, the fraction of positive areas
decreased proportionally. The inventors saw no evidence of
contamination, as a control sample containing no template DNA did
not give any positive results.
[0396] The inventors repeated the experiments for each
concentration (n.gtoreq.3), and generated a calibration curve to
relate the fraction of areas showing a positive PCR result and the
expected copy number of template per area. A Poisson distribution
was assumed to calculate the expected fraction of positive areas.
The fraction of positive areas was slightly lower than the expected
value from the Poisson distribution; this could be caused by
non-specific absorption during sample preparation. The inventors
performed reverse-transcription PCR (RT-PCR) using the digital PCR
SlipChip to quantify viral load with RNA purified from HIV. They
demonstrated that the SlipChip was capable of quantifying the
amount of nucleic acid present in a sample using standard thermal
cycling PCR techniques. The SlipChip contained 1,280 areas designed
to separate a 3.4 .mu.L sample into 1,280 droplets of .about.2.6 nL
each, and was capable of detecting the template DNA at single copy
level. The upper limit of concentration that could be detected
using this device can be increased by increasing the number of
areas on the SlipChip, and the sensitivity of the device can be
improved by decreasing the area volume. The inventors have
incorporated up to 16,384 areas of picolitre volume on a single
SlipChip with dimensions of 1.5 inch by 1 inch. Digital PCR
SlipChips can also be made to screen multiple samples on the same
chip as in SlipChips designed for protein crystallization
experiments and multiplex PCR. Multiplex digital PCR SlipChip can
be made to count multiple targets in one experiment without
interference by increasing the number of areas and using a
microarray technique to pre-load different dry primer sets in the
circular areas. Other improvements to the digital PCR SlipChip
design include incorporating non-thermal cycling methods such as
LAMP or NASBA, and increasing the dynamic range of the digital PCR
SlipChip by using a combination of large and small areas. For
example, in a device containing 2,000 areas, one would get a larger
dynamic range and higher confidence in the statistics if 1,000
areas contained 1 nL of solution and 1,000 areas contained 10 nL of
solution. The distribution of area sizes that gives the best
dynamic range and highest confidence interval can be predicted.
Additional improvements include incorporating real-time PCR and
multi-color probes, such as the Taqman system and molecular beacons
(using appropriate imaging devices known in the art). Multi-color
probes can be used to apply digital PCR for multigene detection
within a single cell to study heterogeneity and also to provide a
method to integrate internal positive controls. The SlipChip can be
made to perform nucleic acid (DNA/RNA) extraction and purification
on the same chip before digital PCR for "sample in, result out"
applications.
[0397] Another application of digital PCR on SlipChip is the
detection of rare cells in the presence of large amount of normal
cells, such as distinguishing between mutant and wild-type template
DNA. With traditional techniques, it is difficult to quantify the
faction of mutant due to the interference of a large population of
normal cells. Digital PCR on SlipChip is a robust and easy method
to increase the fraction of rare cells by confining them in areas
of small volume.
[0398] This platform makes digital PCR widely available, and
provides a very simple lab-based quantification of nucleic acids.
The SlipChip provides an easily available method to perform
prenatal diagnostics. The device can also be used for single cell
analysis such as detection of mutations, monitoring of gene
expression, and analysis of heterogeneity, as well as for
inexpensive diagnostics, especially in resource-limited settings.
Non-thermal cycling methods, nucleic acid purification methods, and
simple readouts can be incorporated into the digital PCR
SlipChip.
[0399] In certain embodiments, nanoliter multiplex PCR arrays can
be performed on the SlipChip. In one example, the SlipChip platform
was used to perform high throughput nanoliter multiplex PCR. The
advantages of using the SlipChip platform for multiplex PCR include
the ability to preload arrays of dry primers, instrument-free
sample loading, small sample volume, and high throughput capacity.
The SlipChip was designed to preload one primer pair per reaction
compartment, and to screen up to 384 different primer pairs with
less than 30 nanoliters of sample per reaction compartment. The
inventors used both a 40-area and 384-area design of the SlipChip
for multiplexed PCR. Both platforms were found to be free from
cross-contamination, and end point fluorescence detection was used
for readout. Multiple samples can also be screened on the same
SlipChip simultaneously. Multiplex PCR was performed on the
384-area SlipChip with 20 different primer sets to identify 16
bacteria and fungi species commonly presented in blood infections.
The SlipChip was able to correctly identify five different
bacterial or fungal species in separate experiments.
[0400] Since its introduction, multiplex PCR has been successfully
applied in many areas, including genetic analysis of cancer cells,
monitoring of genetic variability and clonal evolution, and
identification of infectious diseases caused by viruses, bacteria,
fungi, and parasites. The conventional method for performing
multiplex PCR is to load multiple primers to amplify multiple
target templates in one reaction compartment. The throughput of
this approach is generally limited to less than 10 targets per
compartment because of poor sensitivity or specificity and uneven
amplification rates of different targets, as well as interference
of different primers and the number of fluorescent probes required
for detection. Multiplex PCR can also be performed with PCR
microarrays, but this method usually requires a large amount of
reagent and samples. Another conventional strategy is to use many
miniaturized compartments each with primer set for different
target, but this approach is hindered by limitations in small
volume liquid handling and the cost of instrumentation.
[0401] Microfluidic technology has been demonstrated to have more
advantages over traditional PCR platforms, including, but not
limited to, small reaction volume, high-throughput capacity, and
portability. A number of groups have developed "Lab-on-a-Chip"
microfluidic platforms for PCR, and micro-droplet based PCR has
been demonstrated for single copy nucleic acid detection. However,
most microfluidic PCR systems still require complicated
fabrication, and rely on pumps or sophisticated valves to control
fluid flow. A microfluidic platform with pump-free easy loading,
small reaction volumes, and high-throughput capacity is still an
unmet need for multiplex PCR.
[0402] The SlipChip allows microliter solutions to be effectively
distributed to hundreds of nanoliter compartments with high
precision without requiring pumps or a loading machine. An
important feature of the SlipChip is that it allows preloading and
storage of multiple reagents without cross contamination. The
inventors made SlipChips to perform high-throughput, multiplex PCR.
An array of primer sets was directly deposited in the areas of the
SlipChip using manual deposition and allowed to dry at room
temperature. Methods for microarray fabrication, such as inkjet,
microjet deposition and spotting technologies, can also be applied
to fabricate of the array of primers on the SlipChip. Here, the
inventors describe a SlipChip that is capable of performing 384
simultaneous PCR reactions to identify up to 384 different
templates in a single 10 .mu.L sample with end-point fluorescence
detection. The SlipChip can be setup easily by users with simple
pipetting and PCR reactions are initiated by slipping without
relyin on pumps or other instruments.
Example.
[0403] All solvents and salts purchased from commercial sources
were used as received unless otherwise stated. All primers were
ordered from Integrated DNA Technologies (Coralville, Iowa). Primer
sequences are listed elsewhere herein. Bovine serum albumin (BSA)
was purchased from Roche Diagnostics (Indianapolis, Ind.). SsoFast
EvaGreen Supermix (2.times.) was purchased from Bio-Rad
Laboratories (Hercules, Calif.). Mineral oil (DNase, RNase,
Protease free), Agar, 100 by PCR DNA ladder, and DEPC-treated and
nuclease-free water were obtained from Fisher Scientific (Hanover
Park, Ill.). Dichlorodimethylsilane was purchased from
Sigma-Aldrich (St. Louis, Mo.). Staphylococcus aureus genomic DNA
(ATCC number 6538D-5), Candida albicans (ATCC 10231),
Staphylococcus aureus (ATCC 25923), methicillin resistant
Staphylococcus aureus (MRSA, ATCC 43300), Escherichia coli (ATCC
39391), and Pseudomonas aeruginosa (ATCC 27853) were purchased from
American Type Culture Collection (Manassas, Va.). YM Broth and LB
Broth were purchased from Becton, Dickinson and Company (Sparks,
Md.). Soda-lime glass plates coated with chromium and photoresist
were purchased from Telic Company (Valencia, Calif.). Spectrum food
colors (green, red, and blue food dye) were purchased from August
Thomsen Corp (Glen Cove, N.Y.). Barrier pipette tips and PCR tubes
were purchased from Molecular BioProducts (San Diego, Calif.).
Small binder clips (clip size 3/4'') were obtained from Officemax
(Itasca, Ill.). Mastercycler and in situ adapter were purchased
from Eppendorf (Hamburg, Germany). Teflon tubing (I.D. 370 .mu.m)
was obtained from Weico Wire & Cable (Edgewood, N.Y.), and
teflon tubing (O.D. 250 .mu.m, I.D. 200 .mu.m) was purchased from
Zeus (Orangeburg, S.C.). Photomasks were purchased from CAD/Art
Services, Inc. (Bandon, Oreg.). Red quantum dots (QDs), Qdot 655
ITK, and kit for pBad His B plasmid were purchased from Invitrogen
(Carlsbad, Calif.). Green QDs were obtained from Ocean Nanotech
(Springdale, Ark.). MinElute PCR Purification Kit was obtained from
Qiagen (Valencia, Calif.).
[0404] The procedure for fabrication of SlipChip from glass was
based on the glass etching fabrication of SlipChip procedure
described in detail elsewhere herein, with the following
modifications. The glass plate was aligned with a photomask
containing the design for the areas and the ducts, and exposed to
UV light for 40 seconds. The top slide for both the 40-area design
and the 384-area design contained the square areas that were etched
to be 70 .mu.m deep. The bottom slide for both the 40-area design
and the 384-area design contained the circular areas that were
etched to be 30 .mu.m deep. A through hole was drilled in the top
plate as an inlet for the solution. The final volume of a single
compartment (a pair of overlapping square and circular areas) for
the 40-area design was around 25.9 nL and for the 384-area design
was around 7.1 nL.
[0405] The glass slide with etched areas was thoroughly rinsed with
Millipore water and ethanol and then dried with nitrogen gas. The
glass slide was oxidized in a plasma cleaner for 100 seconds and
then immediately transferred into a desiccator. 50 .mu.L of
dichlorodimethylsilane was injected into the desiccator and a
vacuum was then applied to perform gas phase silanization for an
hour. The silanized glass slide was cleaned with chloroform,
acetone, and ethanol, and then dried with nitrogen gas. The
silanized glass slide was used for PCR experiments within one day.
The patterned glass slide could be re-used after it was cleaned
with piranha solution (3:1 sulfuric acid:hydrogen peroxide) and
silanized again as described above.
[0406] For the 40-area SlipChip design, the concentration of each
primer was 0.05 .mu.M. The solution of primer was flowed in Teflon
tubing (200 .mu.m ID) connected to a 50 .mu.L Hamilton glass
syringe. A volume of 0.1 .mu.L of primer solution, controlled by a
Harvard syringe pump, was deposited into the circular areas. The
solution was allowed to dry at room temperate, and the preloaded
SlipChip was used for experiments within one day.
[0407] For the 384-area SlipChip design, the concentration of each
primer was 0.1 .mu.M. All primer sequences are described in Table
1. A volume of 0.02 .mu.L of primer solution was deposited into the
circular areas on the bottom plate. The solution was allowed to dry
at room temperature, and the preloaded SlipChip was used within one
day.
TABLE-US-00001 TABLE 1 Name and sequence of deposited primer sets
in the 384-well SlipChip. Primer sets used in the 40-well SlipChip
are marked with asterisks. Name of primer sets Target DNA/pathogen
pBad GCGTCA CACTTT GCT ATG CC GCT TCT GCGTTC TGA TTT AAT CTG E coli
nlp ATA ATC CTC GTC ATT TGC AG {Palka-Santini, 2009 #20} GACTTC
GGGTGA TTG ATA AG S pyogene fah TTA AAT ACG CTA AAG CCC TCT
{Palka-Santini, 2009 #20} AGG GTG CTT AAT TTG ACA AG S pyogene OppA
CCC AGT TCA ATT AGA TTA CCC {Palka-Santini, 2009 #20} TTG ACT TAG
CCT TTG CTT TC S pneumoniae GGCTGT AGG AGA CAATGA AG cinASP
{Palka-Santini, 2009 #20} CTT TGT TGA CAG ACGTAG AGT G S pneumoniae
ATT TCG AGT GTT GCT TAT GG plySP {Palka-Santini, 2009 #20} GTA
AAGTGA GCC GTC AAATC E faecium bglB TCT TCA TTT GTT GAA TAT GCT G
{Palka-Santini, 2009 #20} TGG AAT CGA ACC TGT TTATC E faecalis ace
TAG TTG GAA TGA CCG AGA AC {Palka-Santini, 2009 #20} AGT GTA ACG
GAC GAT AAA GG P aerugino vic TTC CCT CGC AGA GAA AAC ATC {Qin,
2003 #17} CCT GGT TGA TCA GGT CGA TCT S agalactia CGA CGA TAA TTC
CTT AAT TGC cpsY {Palka-Santini, 2009 #20} TCA GGA CTG TTT ATT TTT
ATG ATT Pseu general GAC GGG TGA GTA ATG CCT A 16S {Qin, 2003 #17}
CAC TGG TGT TCC TTC CTA TA S aureus nuc ** GCGATTGATGGTGATACGGTT
{Brakstad, 1992 #18} AGCCAAGCCTTGACGAACTAAAGC S epid agrC GAT GAT
ATT AAT CTA TTT CCG TTT G {Palka-Santini, 2009 #20} TCA GGA CTG TTT
ATT TTT ATG ATT S mutans dltA AGATAT GAT TGC AAC AAT TGA A
{Palka-Santini, 2009 #20} CGC ATG ATT GAT TTG ATA AG P mirabil aad
CGCTAT TAA CCT TGC TGA AC {Palka-Santini, 2009 #20} CCT TTC TCA CTC
ACC ACATC MRSA mecA ** CAAGATATGAAGTGGTAAATGGT {Shrestha, 2002 #19}
TTTACGACTTGTTGCATACCATC C troplicalis CAA TCC TAC CGC CAG AGG TTA T
ctr {Luo, 2002 #16} TGG CCA CTA GCA AAA TAA GCG T C glabrata cgl
TTA TCA CAC GAC TCG ACA CT {Luo, 2002 #16} CCC ACA TAC TGA TAT GGC
CTA CAA C albicans TTT ATC AAC TTG TCA CAC CAG A calb {Luo, 2002
#16} ATC CCG CCT TAC CAC TAC CG K pneumonia AAT TTA ACC TGG TTT GAT
AAG AA cim {Palka-Santini, 2009 #20} CAA AAT ATG AAC TAT CAG AAA
GAT TG K pneumonia TAA CGG CAA AGA CGC TAA acoA {Palka-Santini,
2009 #20} TGA CCA GGG CTT CTA CTT C
[0408] Staphylococcus aureus, methicillin resistant Staphylococcus
aureus, Escherichia coli, and Pseudomonas aeruginosa were cultured
in LB broth for 6-8 hours to an exponential phase. Candida albicans
was cultured in YM broth for 8 hours. The cells were collected and
washed with 1.times.PBS buffer. The number of cells was counted
under a microscope and the concentration was normalized to be
approximately 1.times.10.sup.7 cfu/mL. The final concentration of
pathogens was 1.times.10.sup.6 cfu/mL after mixing with the PCR
master mixture.
[0409] The SlipChip was assembled under mineral oil, which was
filtered and degassed before experiments. The bottom plate was
first immersed into the oil in a Petri dish, with the patterned
side facing up. The top plate was then laid on top of the bottom
plate with the patterned side facing down. The two plates were
aligned and fixed using binder clips.
[0410] Thermal expansion was studied using a fluorescence
stereomicroscope, MZ FLIII (Leica, Germany), equipped with a GFP
filter set and 11.2 Color Mosaic camera (Diagnostic Instruments
Inc., MI). This stereomicroscope allowed simultaneous observation
of red and green quantum dots, both excited with a blue light. The
gap between the two plates of the SlipChip was filled with mineral
oil stained with green fluorescent quantum dots (QDs). To stain the
oil, the original 1% QDs solution in toluene was filtered through
0.22 micron microcentrifuge Amicon filters (Millipore, MA) and
sonicated in ultrasonic bath (Fisher Scientific, NJ) for 10 min. A
10% solution of QDs in mineral oil was thoroughly vortexed and kept
for at least 10 min under vacuum before filling the device.
[0411] Stained mineral oil was deposited between the slides of the
SlipChip; excess oil was removed by rinsing the assembled device
sequentially with chloroform, acetone, and ethanol. The SlipChip
areas were filled by injecting an aqueous solution of red QDs
through the fluidic path created by the areas and ducts. Red QDs
655 ITK were diluted 1:10 in 10 mM Tris-HCl buffer, pH 8.0,
containing 1 mM EDTA and 50 mM NaCl. The SlipChip was placed under
the stereoscope on the Mastercycler and multiple heating cycles
were performed to observe aqueous thermal expansion.
[0412] For reactions in the 40-area SlipChip, the reaction master
mixture consisted of 10 .mu.L of 2.times. SsoFast EvaGreen
SuperMix, 2 .mu.L of 10 mg/mL BSA solution, 6 .mu.L of RNase free
water, and 2 .mu.L of 1 ng/.mu.L S aureus gDNA (replaced with 2
.mu.L of RNase free water for control experiments). The final
concentration of gDNA template was 100 pg/.mu.L. For reactions in
the 384-area SlipChip, a 331-bp long piece of dsDNA amplified from
His B plasmid (pBad template) was used as a template for a PCR
control reaction (Primer 1: GCG TCA CAC TTT GCT ATG CC; Primer 2:
GCT TCT GCG TTC TGA TTT AAT CTG). The pBad template was purified
using a MinElute PCR Purification Kit (Qiagen). The reaction master
mixture for the 384-area SlipChip consisted of 10 .mu.L of 2.times.
SsoFast EvaGreen SuperMix, 2 .mu.L of 10 mg/mL BSA solution, 1
.mu.L of 100 pg/.mu.L pBad template, 2 .mu.L of cell suspension,
and 5 .mu.L of RNase free water. The PCR master mixture was
injected into the SlipChip by pipetting. The square areas on the
top plate were moved to overlay the circular areas on the bottom
plate. The SlipChip was then placed on an in situ adaptor in the
Mastercycler (Eppendorf) for thermal cycling. An initial step of 15
min at 94.degree. C. was used to lyse the cells and activate the
enzyme for reaction. Next, a total 35 cycles of amplification were
performed as follows: a DNA denaturation step of 1 min at
94.degree. C., a primer annealing step of 30 sec at 55.degree. C.,
and a DNA extension step of 30 sec at 72.degree. C. After the final
cycle, the DNA extension step was performed for 5 min at 72.degree.
C. Then the SlipChip was kept in the cycler at 4.degree. C. before
imaging.
[0413] Bright field images were acquired by using Leica
stereoscope. All fluorescence images were acquired using a Leica
DMI 6000 B epi-fluorescence microscope with a 5.times./0.15 NA
objective and L5 filter at room temperature. The intensity level of
fluorescence images was adjusted to be the same values for all
images. All fluorescence images were corrected by a background
image obtained with a standard fluorescent slide. Fluorescence
images were stitched together using MetaMorph software (Molecular
Devices, Sunnyvale, Calif.).
[0414] The inventors have performed PCR on the SlipChip with a
design containing forty areas and two inlets for two different
samples. This device can be used to simultaneously screen two
different samples with up to 20 different primer sets for each
sample. The top plate contained the fluid inlet, square areas (side
length of 640 .mu.m, depth of 70 .mu.m) and rectangular areas
(length of 570 .mu.m, width of 230 .mu.m, depth of 70 .mu.m). The
bottom plate contained circular areas (diameter of 560 .mu.m, depth
of 30 .mu.m) and the ducts for introduction of the sample (width of
150 .mu.m, depth of 30 .mu.m). Different primer sets were preloaded
into the bottom circular areas and allowed to dry under room
temperature. The top and bottom plates were then submerged under
mineral oil and assembled to form a continuous fluidic path. The
PCR master mixture, a solution containing SsoFast EvaGreen
Supermix, 1 mg/mL BSA, and template (or water for the control
experiments), was introduced into the SlipChip by pipetting. In
this geometry, the sample fluid spontaneously broke up into
discrete volumes even before sliding. This breakup of a continuous
stream into discrete volumes can be used for applications where
compartmentalization is required, such as stochastic confinement
and digital PCR. Immediately after injection of sample, the top
plate was slipped down to overlap the square areas with the
circular areas on the bottom plate, and the dry primers preloaded
in the circular areas dissolved in the sample introduced from
square areas. The rectangular areas on the top plate also aligned
with the middle of the duct on the bottom plate. The aqueous
solution formed a circular droplet in the areas due to surface
tension, and the volume of solution in each compartment was
estimated to be 25.9 nL by using AutoCAD software.
[0415] The inventors addressed the issue of thermal expansion
during thermal cycling by careful design of the SlipChip. The
material of the SlipChip (glass), the lubricating fluid (mineral
oil), and the sample (the aqueous PCR mixture) have different
thermal expansion coefficients. It is known that the mineral oil
and aqueous mixture should expand more than the glass when the
temperature of the SlipChip was increased from the annealing
temperature (55.degree. C.) to the dissociation temperature
(95.degree. C.). The unique design of this SlipChip held the
aqueous solution within the area by using area geometry.
Dichlorodimethylsilane was applied to render the surface of the
SlipChip hydrophobic. The inventors used an aqueous solution
containing red quantum dots and mineral oil containing green
quantum dots to study the fluid movement during thermal cycling.
When using the SlipChip with only square areas, the aqueous
solution filled the square area. After an increase in temperature,
the aqueous solution leaked out of the areas, resulting in a loss
of material and unpredictable changes in concentration. The
inventors found that when a smaller, circular area containing oil
in the bottom plate was brought into contact with a square area
containing aqueous solution in the top plate, the aqueous solution
would form a droplet surrounded by mineral oil within the
hydrophobic area due to the surface tension, providing room for
expansion during thermal cycling. When the temperature was
increased, the aqueous solution expanded to fill the reaction
compartment and the mineral oil expanded and moved through the gap
between the top and bottom plates in the SlipChip, serving as a
buffer material. Without this design, in certain embodiments
leakage has been observed during thermal cycling. The inventors
determined that the shape and size of the bottom area can be used
to form a single droplet of consistent size in the center of the
two areas. Consistent size of the droplets formed ensured that the
concentration of reagents within the droplets remained the same in
all droplets. The rectangular areas on the top plate overlapped
with the ducts on the bottom plate to address the issue of thermal
expansion of the solution remaining in the duct.
[0416] The inventors performed PCR in an embodiment of the SlipChip
by amplifying nuc gene in S. aureus genomic DNA. Primers for the S.
aureus nuc gene were preloaded into the circular areas of the
bottom plate of the SlipChip and allowed to dry under room
temperature. The PCR master mixture, containing EvaGreen supermix,
100 pg/.mu.L S. aureus genomic DNA (gDNA), and 1 mg/mL BSA, was
injected into the ducts to fill two rows of areas. Two other rows
of areas were filled with the same aqueous PCR mixture but replaced
gDNA template by RNase free water. The square areas in the top
plate and circular areas in the bottom plate were overlapped by
sliding the two plates of the SlipChip relative to one another. The
SlipChip was placed into the thermal cycler on a flat in situ
adaptor for PCR amplification. The inventors showed that no cross
contamination occurred between different rows in the SlipChip as
only areas containing template showed amplification. Fluorescence
intensity increased significantly after thermal cycling only in the
areas containing gDNA, and all 20 areas containing template showed
amplification, verifying the robustness of the PCR SlipChip. After
thermal cycling, the solution in the SlipChip was flowed out and
collected, and a gel electrophoresis experiment was performed. The
image of the gel showed successful on-chip amplification and the
correct size of the amplification product (.about.270 bp).
[0417] Next, the inventors tested the cross contamination among
adjacent areas by preloading the primer sets for the nuc gene in S.
aureus and mecA in Methicillin-resistant Staphylococcus aureus
(MRSA) on the chip alternatively in the same row, and injecting PCR
master mixture containing 100 pg/.mu.L of S. aureus genomic DNA
into the SlipChip (primer sets can be found in Table 1). Because
the nuc gene is commonly present in S. aureus but the mecA gene is
not, all ten areas preloaded with the primers for the nuc gene
showed a significant increase in fluorescence intensity after
thermal cycling, and none of the areas loaded with primers for the
mecA gene increased in fluorescence intensity. Combined with the
results above, the inventors demonstrated each area was an isolated
reaction condition, and there was no communication among areas.
[0418] Furthermore, the inventors demonstrated that the SlipChip
containing 384 areas, which can be preloaded with up to 384
different primer sets, can be applied for high-throughput multiplex
PCR. The inventors designed this platform for 16 different
pathogens that are commonly present in blood infections by using 20
different primer sets preloaded on the SlipChip. Primer sequences
were selected from previous publications, and the PCR master
mixture was combined with cells at a final concentration of
approximately 10.sup.6 cfu/mL. This guaranteed the presence of
targeted cells in each individual area. The inventors have
demonstrated that PCR on SlipChip can detect a single molecule. A
SlipChip was made with 28 independent regions, and a primer set for
each pathogen was preloaded as 4 by 4 matrices for the convenience
of imaging. Primers for pBad template were preloaded in the two
columns of areas at the edges of the SlipChip as a positive
internal control. A purified pBad 331 bp template (final
concentration 1 pg/uL) was added to the PCR master mixture before
loading. Two columns next to the areas containing primers for pBad
were left empty as a negative control for leakage.
[0419] The SlipChip was able to robustly identify cells, as only
the regions preloaded with the appropriate primers showed a
significant increase in fluorescent signal. The regions for
positive controls showed an increase in fluorescent signal and
regions for negative controls did not. The SlipChip was able to
correctly identify S. aureus, MRSA, Candida albicans, P.
aeruginosa, and E. coli. The inventors demonstrated high-throughput
multiplex PCR on the SlipChip. In certain embodiments, the SlipChip
can perform 384 nanoliter-scale reactions for multiplex PCR with a
prefabricated array of primer sets. The PCR SlipChip can be loaded
simply by pipetting, avoiding any requirements for complex
injection methods. The inventors have shown that an embodiment of a
PCR SlipChip can screen one sample for 16 different pathogens on
the same SlipChip, and that there was no detectable cross
contamination. The inventors have also demonstrated that two
different samples can be introduced and tested simultaneously on a
single preloaded SlipChip. The multiplexed PCR SlipChip can be
designed with a larger number of inlets for simultaneous screening
of multiple samples, for use with non-thermal cycling nucleic acid
amplification methods such as LAMP, RPA or NASBA, and/or with a
larger number of areas to allow for more conditions to be screened
in a single experiment. PCR SlipChips can be made to use the primer
sets established by current PCR microarray technology, but with a
much smaller size and reaction volume. One can also adapt the
current technologies of microarray printing to preload primers and
to fabricate SlipChips.
[0420] In addition to distinguishing a large number of different
species in one experiment, certain embodiments of the SlipChip are
capable of providing quantitative results, by, for example,
integrating real-time imaging techniques for multiplex real-time
PCR, or using a large number of areas for each primer set to enable
counting the number of all amplicons in one experiment to perform
multiplex digital PCR.
[0421] In addition to being used for multiplex PCR for screening of
specific genes, certain embodiments of the SlipChip can be used for
additional applications. Multiplexed PCR and other nucleic acid
amplification chemistries on SlipChip can be used for
high-throughput DNA amplification before sequencing, for example
enrichment methods for targeted sequencing, that can currently
performed in well plates and by droplet-based methods (as
described, for example, in Microdroplet-based PCR enrichment for
large-scale targeted sequencing Tewhey, R. et al., Nat. Biotechnol.
2009, 27, 1025-1031). PCR on a SlipChip can also be used for the
detection of genomic diseases, genetic mutations, and food or water
contaminants. The current platform can also be adapted to perform
reverse transcription PCR for RNA amplification for, for example,
RNA virus detection, study of gene expression, and investigation
cell heterogeneity.
[0422] The SlipChip can be fabricated from inexpensive materials
such as glass or plastic, and, in certain embodiments, requires no
complex equipment or specialized knowledge to operate. When dried
reagents are preloaded onto the SlipChip, it is also easy to
transport and store. It can be integrated with isothermal
amplification methods and simple readouts.
[0423] The SlipChip can also be used in other applications that
require prefabricated arrays of reagents with multiplex and high
throughput capacity, such as, for example, protein crystallization,
immunoassays, DNA hybridization, DNA-protein interaction, and
chromatin immuno-precipitation (ChIP).
[0424] In certain embodiments, combinatorial biocatalysis can be
performed on the SlipChip. Combinatorial biocatalysis is similar in
concept to combinatorial synthesis in organic chemistry.
Combinatorial biocatalysis can provide a diverse library of
derivatives from a single lead compound by sequentially combining
biocatalytic reactions via enzymes. Combinatorial biocatalysis
enables the generation of a huge number of enzymatic products in
parallel sequence of either different substrates or different
enzymes. The lead compound bearing multi-functional groups (e.g.,
carboxyl group, hydroxyl group, acyl group, amine group, etc.) is a
potential molecule to apply. Combinatorial biocatalysis requires
many steps of sequential mixing and reactions. If the amount of a
lead compound is very tiny and expensive, a 96, 384 or even 1,536
well plate may require too much volume for the thousands of
reactions that may be needed. In addition, there can be limitations
in testing synthesized derivatives because of the limited amount
available. Analysis of products on a standard multiwell plate can
be difficult in terms of both concentration and volume. Certain
embodiments of the Slipchip can provide appropriate confined
volumes and sufficient numbers of reaction centers without a
complicated apparatus. The Slipchip is an attractive solution for
high throughput drug discovery/drug screening. Possible
applications of combinatorial biocatalysis include biocatalysis
(enzyme screening, enzyme evolution, optimization of reaction
conditions), bioengineering (system development, robotics,
industrialization), bioprocess engineering (reaction system
optimization, downstream process, scale-up, commercialization),
medicinal chemistry (novel drug candidates, derivatization, ADME
toxicity tests), food chemistry and engineering (natural colorants,
antioxidants, food additives), agricultural chemistry (functional
dairy products, emulsifiers) and environmental chemistry (natural
pesticides).
[0425] In certain embodiments of the present invention, high
throughput enzyme screening can be performed in the SlipChip.
Screening enzymes is a huge research/industrial field in worldwide.
Researchers typically apply their enzyme candidate to certified
chemical libraries. Typically robotics and manual labor are used,
but the amount of enzyme samples is typically a limiting factor.
Similar problems occur as in combinatorial biocatalysis (see
above). A variety of chemical libraries can be provided in a
Slipchip with different substrates. Chemical libraries as the
target substrates preferably cover a large spectrum of functional
groups as well as having a target-specific focus on the particular
enzyme being tested. The Slipchip can contain different ranges of
chemical libraries with appropriate amounts of reactants. Users can
then flow a small amount of enzyme solution into the device and
analyze each area in a Slipchip. For example, if someone has a
putative lipase/esterase sample, a Slipchip containing various
chemical libraries testing for hydrolysis (e.g., one can contain C2
ester, C3 ester, C4 ester, . . . C14 ester, C16 ester . . . ,
etc.). Possible applications of high throughput enzyme screening
include, but are not limited to, determining stereo-specificity,
regio-specificity, hydrophobicity, hydrolysis and/or
reverse-hydrolysis reactivity, the pH range, the temperature range
for hyper-thermostable enzymes, the pressure range for
hyper-barostable enzymes, the ionic strength range, and tolerance
for high-salt conditions.
[0426] In certain embodiments, the SlipChip can be used for
enzymatic tests for the screening of novel enzymes. Once a
potential enzyme is isolated from a microorganism, it is typical to
run enzymatic reactions in a 96-well plate to evaluate the
substrate specificity, reactivity, selectivity, and stability. For
this analysis, typically one tests the enzyme against a chemical
library. A pre-loaded chip can be provided that contains multiple
substrates for use as a simple test screening kit for enzyme
samples.
[0427] In certain embodiments of the present invention, the
SlipChip can be used as a platform that reduces the complexity of
sample collection, concentration, and preparation (SCCP); processes
diagnostically relevant samples with viscosities ranging from urine
to sputum; and allows processing of large, milliliter-scale sample
volumes to capture low concentrations of analytes, and
concentrating them to small, nanoliter-scale volumes for easy
detection, all in a manner compatible with a wide range of
amplification, detection, and readout components.
[0428] The SlipChip platform overcomes several key challenges that
face healthcare and diagnostic technologies in resource-limited
settings. Diagnostic assays require a complex sequence of steps,
from sample preparation to amplification to detection and readout.
These steps are difficult to perform in resource-limited settings,
as they require either highly skilled technicians or complex
automation. The difficulty increases further for assays requiring
high sensitivity (little room for error and contamination),
quantification (complex protocols and equipment), and multiplexing
(the process must be repeated multiple times for multiple
analytes). The SlipChip platform can encode for all the steps
necessary for a complete diagnostic device, from sample collection,
concentration, and preparation, to amplification, detection, and
readout.
[0429] The SlipChip platform can ease sample preparation, and open
new assay techniques to point-of-care (POC) applications. It can,
for example: (i) accept small or large volumes (allowing for high
sensitivity) of diagnostically relevant samples such as blood,
sputum, urine or feces; (ii) manipulate them through many sample
preparation steps to isolate the molecules of interest; and (iii)
concentrate them into smaller volumes that can be used directly by
an amplification or detection component.
[0430] In certain embodiments, the SlipChip can be used for the
rapid, simple extraction of diagnostically relevant biomarkers from
raw sample inputs. Certain embodiments of the SlipChip can be used
to address many areas of current significant unmet need including
but not limited to the following: (1) sample preparation of whole
blood and plasma for isolation of viral RNA for quantification of
HIV viral load (for monitoring antiretroviral therapy and for
diagnosing infants, for example); (2) sample preparation of sputum
for isolation of both RNA and DNA nucleic acids from pathogens that
cause pneumonia (for determining when antibiotic treatment should
be administered, for example).
[0431] Quantitative monitoring of HIV viral load during treatment
in resource-limited settings to prevent widespread drug resistance
has been identified as a major barrier to HIV/AIDS care worldwide.
Diagnosis of HIV infections in infants over few weeks of age can be
performed by quantifying viral load, and is preferred in
resource-limited settings, as early diagnosis of HIV infection and
administration of HIV antiretroviral drug treatment drastically
reduces the rate of infant mortality.
[0432] At present, no HIV viral load quantification platform is
available that can be used in resource-limited settings;
centralized testing of viral load is not universally suitable.
Existing centralized viral load assays require significant
technical expertise and instrumentation. Installing complex
instruments in resource-limited settings has generally failed, and
transporting samples to centralized labs has also proven
problematic. Dried blood spots (DBS, spots of whole blood dried on
filter paper) are the only realistic option for transporting
samples in these settings. In addition to technical issues of
isolating viral RNA quantitatively from DBS, this approach is not
well-suited for traveling clinics, as results must be obtained
without delay so the result of the test can be actionable.
Moreover, the use of DBS still requires quantitative RNA testing
that assumes sophisticated equipment and technical expertise.
[0433] In certain embodiments, the SlipChip can be used for
quantitative and sensitive measurement of HIV viral load in
resource-limiting settings by performing multistep sample
processing in a self-contained format. Certain embodiments of the
SlipChip can accept, for example, 100-200 .mu.l of whole blood or
plasma, and produce purified viral RNA with >30% yield in 20-50
.mu.l with quality sufficient for subsequent isothermal
amplification performed on the Digital SlipChip or another
amplification component.
[0434] Accurate diagnosis of the cause of acute lower respiratory
infections (ALRIs) such as pneumonia could save hundreds of
thousands of lives every year and preferably involves concurrent
multiplexed detection of bacteria and viruses, and quantification
to distinguish lower levels (corresponding to bacterial
colonization) from higher levels (corresponding to bacterial
infections). In developing countries ALRIs, particularly pneumonia,
are the leading cause of death in children under 5 years of age
(>2 million/year), due to inadequate treatment caused by the
lack of accurate, low cost, readily available diagnostic tools.
Poor diagnostic capabilities also lead to overuse of antibiotics,
advancing the emergence of drug resistant strains. Bacterial
infections, particularly Streptococcus pneumoniae and Haemophilus
influenzae type b, which can be easily treated with antibiotics,
must be distinguished from viral or other causes. A major challenge
is differentiating bacterial infection from colonization of the
upper respiratory tract, and a simple qualitative yes/no test is
not effective. Diagnosis can be dramatically improved by
implementing a quantitative multiplexed test of sputum for, for
example, 16 common bacterial and viral pathogens. For example, a
medium level of S. pneumoniae bacterium in the absence of
significant levels of other pathogens is likely to indicate S.
pneumoniae infection, while a medium level of S. pneumoniae
bacterium in the presence of a very high level of respiratory
syncytial virus (RSV) would indicate RSV infection as a more likely
cause.
[0435] In certain embodiments, the SlipChip can be used to isolate
RNA and DNA of pathogens that cause pneumonia from sputum in
>30% yield with >5-10 fold increase in concentration for
downstream quantitative and sensitive detection on a Digital
SlipChip or another component.
[0436] Certain embodiments of the SlipChip can be programmed to
perform complex manipulations of volumes from mL to nL. They can be
used to easily process hundreds or thousands of nanoliter volumes
in parallel by simple slipping, for example, two plates. The
inventors have demonstrated that larger volumes can be incorporated
into this platform (e.g., 200 .mu.L of whole blood). This
multi-scale capability is useful. For example, to capture 50 HIV
viruses at 500/mL HIV viral load, one needs to handle at least 100
.mu.L of plasma, while concentrating samples into smaller volumes
reduces losses during processing and provides output more suitable
for amplification and quantification (e.g., using a digital PCR
SlipChip). Serial dilution by 105-fold and washing by dilution have
been demonstrated on the SlipChip. Quantitative handling of beads
has been demonstrated in nL-volume immunoassays in the pM range,
handling and detecting a few thousands of protein molecules. Local
heating and cooling can be programmed into the SlipChip via simple
chemistry, by programming heat- or cold-generating reagents to
combine at the required step. Temperature control can be also
achieved via external or internal on-chip means, including
electrical and thermoelectric heating and cooling, and a number of
approaches used to conduct PCR reactions. These features can be
used for reliable isolation of target nucleic acids with at least
30% yield, with 10-fold concentration, and can optimize the
trade-offs between yield and concentration.
[0437] In certain embodiments, the SlipChip can be encoded to
extract HIV RNA from whole blood or plasma for downstream HIV viral
load analysis. The SlipChip can use 100 to 200 .mu.L of plasma
(prepared on chip or off chip) with 500 to 10.sup.6/mL HIV viral
load and isolate viral RNA into 10-30 .mu.L of solution with
>30% yield. This output is sufficient for a digital SlipChip to
measure the viral load with a dynamic range from 500 to 106 copies
per mL, and less than 3 fold error with 95% confidence. Quality and
quantity of isolated HIV RNA can be quantified by real time
RT-PCR.
[0438] In certain embodiments, the SlipChip can be encoded to
extract RNA and DNA from sputum for identification and
quantification of pathogens that cause pneumonia. They can handle,
for example, 200-500 .mu.L of sputum for RNA and DNA isolation in
>30% yield, concentrating it in, for example, 20-50 .mu.L of
amplification-ready solution. The highly parallel processing on the
SlipChip enables optional enhancements that include: (i) parallel
purification of DNA and RNA simultaneously from the same sample,
and (ii) multiple sputum samples processed on the same device from
a single patient to ensure at least one high-quality sample, or
from multiple patients to increase throughput. These features,
combined with SlipChip components for amplification, readout, and
integration, provide solutions to urgent global diagnostics
problems including quantification of HIV viral load and multiplexed
quantitative analysis of pneumonia pathogens. The sputum sample
processing protocol can be easily adaptable to the isolation of DNA
from Mycobacterium tuberculosis for molecular diagnosis of TB and
identification of drug resistant strains, and is expandable to
isolation of nucleic acids from feces. HIV protocols are adaptable
to isolation of Plasmodium DNA from blood for diagnosis of
malaria.
[0439] In certain embodiments, the SlipChip can be used to provide
signal amplification and improve detection, in a manner compatible
with a wide range of existing and future amplification chemistry
components.
[0440] In certain embodiments, the SlipChip can be used to
dramatically enhance signal amplification and detection chemistries
by leveraging the advantages of the SlipChip platform to implement
the principle of "stochastic confinement". The SlipChip can be used
to, for example, (i) increase sensitivity of existing technologies
to the single-molecule or single-cell level; (ii) increase
specificity and reduced interference and background reactions;
(iii) robustly quantify over a large dynamic range; iv) perform
multiplexed experiments.
[0441] In certain embodiments, the SlipChip can be used as an open
platform that component builders can use to enable robustness,
quantification, sensitivity, and specificity of amplification
chemistry components for diagnostic applications in
resource-limited settings. The SlipChip can be used to address many
areas of current significant unmet need including but not limited
to the following: (1) quantification of HIV viral load (for
monitoring antiretroviral therapy and for diagnosing infants, for
example), and (2) multiplexed quantitative detection of bacterial
and viral pathogens that cause pneumonia (for determining when
antibiotic treatment should be administered, for example).
[0442] In certain embodiments, the SlipChip can be used for
quantification of HIV viral load for, for example, monitoring
antiretroviral therapy and for diagnosing infants. In certain
embodiments, the SlipChip can be used for highly quantitative and
sensitive measurement of HIV viral load by converting simple
qualitative amplification chemistries to a "digital" format with
end-point readout, this is sometimes referred to herein as a
"Digital SlipChip".
[0443] In certain embodiments, the SlipChip can be used for
multiplexed pathogen detection to diagnose the cause of pneumonia.
At the present, quantitative multiplexed diagnostics of pneumonia
pathogens is an unmet need under resource-limited settings.
Single-analyte tests can be done by isothermal techniques, but
their value is limited in the absence of quantification and
multiplexing. Multiplexed quantitative detection can be
accomplished by real time PCR, but this has not been useful in
point of care, resource limited settings. In certain embodiments,
the SlipChip can be used for quantitative and sensitive detection
of pneumonia pathogens by combining multiplexing and conversion of
amplification chemistries to a "digital" format with end-point
readout.
[0444] In certain embodiments, the SlipChip can be used for (i)
increased sensitivity of existing technologies to the
single-molecule or single-cell level; (ii) reduced interference and
background reactions; (iii) robust quantitation over a large
dynamic range; (iv) practically unlimited multiplexing
applications. Certain embodiments of the SlipChip can encode, as a
sequence of areas in the two plates, essentially any program to
manipulate fluid volumes.
[0445] In certain embodiments, the SlipChip can be used for
multivolume stochastic confinement. The SlipChip can split a sample
into, for example, hundreds or thousands of small volumes of
different sizes in a "digital" format (zero versus one or more
molecules of analyte per area), prior to amplification. Confinement
of molecules in small areas (i) increases concentration of
molecules, (ii) isolates these molecules from interfering
molecules, (iii) enables quantification from endpoint readout by
maximum likelihood estimation, with large dynamic range provided by
multiple volumes used simultaneously on the same chip.
[0446] The SlipChip is compatible with both digital PCR and digital
isothermal recombinase-polymerase amplification (RPA) amplification
technologies using commercially available stock reagents. Many
other isothermal techniques can be performed, including but not
limited to loop-mediated isothermal amplification (LAMP) and
nucleic acid sequence-based amplification (NASBA), for
quantification of analytes (even in the presence of
interference).
[0447] In certain embodiments, the SlipChip can have a dynamic
range of 500-10.sup.6/mL for analysis of HIV viral load using
multivolume stochastic confinement. In certain embodiments, the
SlipChip can be designed as a rotary multivolume Digital SlipChip.
This design can have hundreds of areas with volumes ranging from,
for example, 0.37 nL to 250 nL, and can quantify HIV viral RNA with
a dynamic range of 500-10.sup.6 copies/mL, in 3-fold changes with
95% confidence. The inventors have confirmed that HIV RNA can be
detected on the Digital SlipChip platform.
[0448] In certain embodiments, the SlipChip can be used for
detection and quantification in sputum samples of 16 pathogens
involved in pneumonia. In certain embodiments, the SlipChip can
contain preloaded reagents for isothermal amplification chemistry
for 16 pathogens (with an optional additional reverse transcription
step for detection of RNA viruses). The inventors have demonstrated
multiplexed detection of pathogens using preloaded reagents on a
384-plex uniform-area SlipChip platform. Different areas of the
proposed chip can be used to tune the dynamic range of the device
into the appropriate range, for example: outer areas with larger
areas for sensitive detection of CMV, HRV, and other pathogens in
the range of 10.sup.2-10.sup.5/mL, and inner areas with smaller
areas for detection and quantification in the 10.sup.2-10.sup.6/mL
range of colonizing pathogens such as S. pneumonia and H.
influenzae type b. The SlipChip's capabilities of sample
preparation, visual readout, and integration have the potential to
provide solutions to two areas of urgent global diagnostics
needs--quantification of HIV viral load and multiplexed
quantitative analysis of pneumonia pathogens. Diagnosis of
tuberculosis can be performed by stochastic confinement on the
SlipChip which would amplify physiological responses of
Mycobacterium tuberculosis and enable rapid detection and
phenotypic testing of drug resistance. Quantification of CD4 count
in AIDS patients can be performed efficiently using multivolume
stochastic confinement.
[0449] In certain embodiments, the SlipChip can be used for readout
and signal transduction. In certain embodiments, the SlipChip can
accept output from multiplexed amplification and detection
component technologies, e.g., 1000's of separate amplified nucleic
acid products produced during detection of pathogens, and convert
them to a readout for analysis and interpretation by eye or by
using a simple cell phone camera. The SlipChip can enhance signal
processing and readout by leveraging the advantages of the SlipChip
platform to implement multistep and multiplexed processing,
generating a visual readout for any diagnostic test. In certain
embodiments, the SlipChip can be used for (i) technically complex
processing without dependence on user expertise; (ii) access to
more diverse amplification, processing and detection chemistry than
currently available in POC, expanding the diagnostic tool box;
(iii) quantifiable visual readout without infrastructure.
[0450] In certain embodiments, the SlipChip can be used for rapid
visual analysis for diagnostics in two areas of current significant
unmet need in resource-limited settings: 1) quantification of HIV
viral load (for, for example, monitoring antiretroviral therapy and
for diagnosing infants), and 2) multiplexed quantitative detection
of bacterial and viral pathogens that cause pneumonia (for, for
example, determining when antibiotic treatment should be
administered).
[0451] In certain embodiments, the SlipChip can be used for
multiplexed visual readout for determination of HIV viral load in
regions with no infrastructure. In certain embodiments, the
SlipChip can be used for quantitative and sensitive measurement of
HIV viral load in regions without suitable infrastructure by
performing multistep processing in a self contained format and
generating an easy to interpret visual readout. It can accept
products of nucleic acid amplification technology (NAT) performed
on the SlipChip or another amplification component, and convert it
to a visual readout allowing for more immediate treatment plans to
take effect.
[0452] In certain embodiments, the SlipChip can be used for
multiplexed visual detection and analysis to determine the cause of
pneumonia.
[0453] In certain embodiments, the SlipChip can be used to (i)
Accept, for example, thousands of mixtures containing amplified
nucleic acids, e.g. from a Digital SlipChip or another component,
and (ii) Carry out multistep processing of these mixtures to
produce a quantitative visual readout from each. Other technologies
have not yet been able to meet these needs, which are preferred for
converting to visual readout the quantitative, highly multiplexed
"digital" tests, such as the HIV viral load test or the pneumonia
panel provided by the Digital SlipChip. Certain embodiments of the
SlipChip can encode, as a sequence of areas in the two plates,
essentially any program to manipulate fluid volumes. Certain
embodiments of the SlipChip can process multiple volumes, through
multiple steps of detection, amplification, and visual readout.
[0454] In certain embodiments, the SlipChip can accept, for
example, thousands of areas, for example, 0.3-300 nL in volume with
isothermally amplified nucleic acids in each area, and produce a
visual readout from each area by a multistep process. In certain
embodiments, an area larger than 250 .mu.m.times.250 .mu.m with
absorbance above 1 is easily visible on the SlipChip. For larger
areas with higher concentrations of nucleic acids, direct detection
by hybridization capture of gold or selenium particles can be used.
For smaller areas with lower concentrations, the user can transfer
samples into larger areas and perform additional amplification
chemistry. Amplification chemistry can be modified to provide
visual detection, and such modifications are already well
established for lateral flow readouts. Certain embodiments of the
SlipChip can support all of the preferably included steps (for
example, capture of molecules on beads and surfaces, magnetic
manipulation, optional washing by dilution) using preloaded
reagents by slipping, without requiring technical expertise of the
user. In certain embodiments, the SlipChip can be used for LAMP and
RPA and other isothermal amplification technologies.
Example.
[0455] For one RPA experiment, a TwistAmp Basic kit was purchased
from TwistDx. (Cambridge, United Kingdom) The RPA supermix was
prepared from a single tube containing RPA enzymes and reagents
(freeze-dried Basic reaction pellet), by addition of a mixture of
20 .mu.l rehydration buffer and 8 .mu.l control 1.times.
primer/probe mix. A positive control solution was prepared by
mixing 5 .mu.l positive control template (10 copies/.mu.l) with 14
.mu.l of RPA supermix, while 5 .mu.l water was added in 14 .mu.l of
RPA supermix as negative control solution.
[0456] A solution of 50 nl magnesium acetate (9.3 mM) was deposited
into each circular area on the bottom plate of a 40-area SlipChip
through the Teflon tubing (200 .mu.m ID) connected to a 50 .mu.L
Hamilton glass syringe controlled by a Harvard syringe pump. The
solution was let dry under room temperature for 10 minutes.
[0457] The SlipChip was assembled under degassed mineral oil. The
bottom plate was first immersed into the oil in a Petri dish, with
the patterned side facing up. The top plate was then laid on top of
the bottom plate with the patterned side facing down. The two
plates were aligned and fixed using binder clips.
[0458] Negative control solution (5 .mu.l) was injected into the
top two rows through one inlet by pipetting, while the positive
control solution (5 .mu.l) was loaded into the bottom two rows
through a separate inlet. The fluidic path was broken by slipping
and the top plate was moved to overlay with the circular areas,
which contain preloaded dry magnesium acetate, on the bottom plate.
The volume of the reaction mixture in each area was 27 nL with a
predicted Mg acetate concentration of 17 mM. The SlipChip was
immediately placed in a 39.degree. C. incubator, and the
fluorescence intensity was acquired using a Leica DMI 6000 B
epi-fluorescence microscope. The fluorescence images were acquired
using a 5.times./0.15 NA objective and L5 filter immediately after
slipping and 20 min after incubating at 39.degree. C.
[0459] Experiment one, with dry magnesium acetate, was performed as
described above. However, magnesium acetate solution can
alternatively be loaded into the SlipChip in then aqueous phase,
and then slid over to mix with RPA supermix to initiate the
reaction. In Experiment two, premixing magnesium acetate solution
with RPA supermix was done. A solution of 1 .mu.l of 280 mM
magnesium acetate was added to 19 .mu.l negative control solution,
and the solution was injected into the top two rows of a 40-area
SlipChip. Another solution of 1 .mu.l of 280 mM magnesium acetate
was added to 19 .mu.l positive control solution, and the solution
was loaded into the bottom two rows through a separate inlet. The
fluidic path was broken by slipping and the top plate was moved to
overlay with the circular areas containing mineral oil. The
SlipChip was immediately placed in a 39.degree. C. incubator, and
the fluorescence intensity was acquired by using a Leica DMI 6000 B
epi-fluorescence microscope. The fluorescence images were acquired
by using a 5.times./0.15 NA objective and L5 filter immediately
after slipping and 20 min after incubating at 39.degree. C. In
experiment one, out of 40 areas only two, corresponding to the
positive control solution lit up. In experiment two, out of 40
areas three, corresponding to the positive control solution, lit
up. In both experiments, the areas corresponding to the negative
control solution all remained dark.
[0460] The SlipChip is compatible with a wide range of visual
detection chemistries. Multistep processing on certain embodiments
of the SlipChip can utilize standard visualization chemistries
already established in the "dip stick" lateral flow devices (see
for example, U.S. patent application Ser. No. 12/425,121,
incorporated by reference herein in its entirety), or enable new
chemistries. The autocatalytic reduction of silver (I) ions,
initiated on the surface of gold nanopartices (AuNPs), provides a
very high degree of amplification, rapidly producing visually
observable silver deposition. In SlipChip experiments in a 55 nL
volume at 5 pM analyte (.about.165,000 molecules) this chemistry
produced a visible signal that was clearly distinguishable from
background. The signal was generated within 10 min. Additional
chemistries that can be performed on the SlipChip include but are
not limited to direct label capture, Alkaline Phosphatase (AP) to
generate a visual product from NBT and BCIP, and
polymerization-based amplifications.
[0461] In certain embodiments, the SlipChip can be used for visual
quantification of HIV viral load on the SlipChip with a dynamic
range of, for example, 500-106. The RPA products from HIV RNA from
the SlipChip or other amplification components can be processed
through additional steps on the SlipChip to perform, for example,
hybridization, purification and visual signal generation for direct
visual analysis of HIV viral load.
[0462] In certain embodiments, the SlipChip can be used for
visualizing detection and quantification of, for example, 16
pathogens involved in pneumonia. The additional areas for
hybridization and visual amplification can either be incorporated
within a two-layer device, or within a multilayer device to
increase density. The SlipChip can be used in other areas where
multiplexed, multistep readouts are needed. This includes, but is
not limited to, other diagnostic needs that rely on amplification
of nucleic acids (e.g., in identification and diagnosis of malaria
parasites or STDs), multiplexed immunoassays (e.g., in
identification of pathogens responsible for persistent diarrhea or
STDs), and in rapid visual detection and counting of Mycobacterium
tuberculosis bacteria.
[0463] In certain embodiments, ELISA-based methods of determining
viral load, such as ExaVir Load (Cavidi AB), can be performed in
the SlipChip. ExaVir Load determines viral load based on
quantification of Reverse Transcriptase activity, it can measure
any HIV type or subtype, including O and N-group. Unfortunately
Exavir Load and similar assays are slow and not quantitative. Such
assays require a long incubation time for DNA synthesis to get
detectable amount of DNA at low viral loads. However, if the assay
is performed in certain embodiments of the SlipChip using
stochastic confinement and a digital readout, the high local
concentration allows shorter incubation times. For example, the
ExaVir Load measuring range is up to 600,000/mL. This is determined
by the synthesized DNA saturating all templates anchored on the
bottom of the well in the incubation time (typically, 1 day). In a
SlipChip area with, for example, 10 nL volume and a depth of 100
.mu.m, one viron in a well is 100,000/mL, but the area is only
about .about. 1/300 compared to a well in a 96-well plate. Assuming
the speed of consuming the templates depends on the concentration
of the viron, one would only need .about. 1/50 time, that is,
typically approximately one hour, to saturate the templates in
SlipChip.
[0464] Other methods can be performed in conjunction with ExaVir
Load such as radical initiation/polymerization amplification in
order to increase amplification. One can further enhance
amplification by adding a small amount of radical chain terminator
as an inhibitor to establish a threshold. This reduces the number
of washing steps required.
[0465] In certain embodiments, the SlipChip can be stackable. A
stackable SlipChip can be used in many areas of current significant
unmet need, including but not limited to the following: (1)
quantification of HIV viral load (for, for example, monitoring
antiretroviral therapy and for diagnosing infants), and (2)
multiplexed quantitative detection bacterial and viral pathogens
that cause, for example, pneumonia (for, for example, determining
when antibiotic treatment should be administered). A stackable
SlipChip can be used for a complete blood-to-answer diagnostic
solution for quantification of HIV viral load. In certain
embodiments, a stackable SlipChip can be used for quantitative and
sensitive measurement of HIV viral load by integrating different
SlipChip technologies, for example: (i) SlipChip for sample prep
and concentration to isolate HIV viral RNA from blood, (ii) Digital
SlipChip to quantify the viral load by isothermal amplification and
counting of RNA molecules, and (iii) a SlipChip to convert
amplified nucleic acids into a readout detectable by eye or, for
example, with a cell phone camera.
[0466] In certain embodiments, a stackable SlipChip can be used for
multiplexed pathogen detection for diagnosis of the cause of, for
example, pneumonia. In certain embodiments, a stackable Slipchip
can be used for quantitative and sensitive detection of pneumonia
pathogens at the point of care by integrating different SlipChip
technologies, including, for example: (i) a SlipChip for sample
prep and concentration to isolate from sputum RNA and DNA pathogens
responsible for pneumonia, (ii) a Digital SlipChip for multiplexed
identification and quantification of nucleic acids from a panel of,
for example, 16 pathogens responsible for pneumonia, (iii) the
SlipChip to convert amplified nucleic acids into visual readout
detectable by eye or, for example, with a cell phone camera.
[0467] In certain embodiments, the stackable SlipChip can be used
for integration of multiple SlipChip components among themselves or
with other chemistry and hardware components. Certain embodiments
of the SlipChip can encode, as a sequence of areas in the two
plates, essentially any program to manipulate fluid volumes. They
can be used for sample concentration and preparation, multiplexed
amplification for identification and quantification of nucleic
acids, and conversion of amplified nucleic acids to visual or, for
example, cell-phone readout. The stackable SlipChip can be used for
complete diagnostic tests by integrating these components with one
another or with other components developed by others. There are
many methods of integration for stackable SlipChips, including but
not limited to the following: stacking of pre-made component chips
to exchange a limited number of inputs/outputs, and fabricating
multiple stacked layers to create complete SlipChip components that
exchange hundreds or thousands of inputs/outputs. The stackable
SlipChip can control slipping, trapping of beads, and control fluid
movements through stacks, including capillary and pressure-driven
flow. Slipping the individual layers of a stackable SlipChip can
create and break up the fluidic paths through the stack, so even a
simple wick or pressure source can cause highly controlled
reconfigurable movement of fluids through the stack.
[0468] In certain embodiments, the stackable SlipChip can be used
for integrating pre-made component SlipChips with few
inputs-outputs. In certain embodiments, the stackable SlipChip can
be used for the integration of a concentrating SlipChip with a
Digital SlipChip. A single connection between the components of the
stack can be sufficient for HIV viral load measurements, and a few
connections (e.g., to separately handle solutions from RNA and DNA
preparation modules) can be sufficient to identify and quantify
nucleic acids from, for example, pneumonia pathogens. This approach
is attractive because as long as the input-output configuration
standards are established, the components can be individually
optimized and then easily integrated.
[0469] In certain embodiments, the stackable SlipChip can be used
for direct integration of SlipChip layers. This approach takes
layouts of component SlipChips, and integrates them in stacks that
allow direct sample transfer from areas in one layer into areas of
another layer. This approach is valuable for integration of
components that handle hundreds or thousands of sample volumes, for
example a Digital SlipChip to amplify nucleic acids and a readout
SlipChip to perform multistep processing of each volume to create a
visual readout. It also provides significant simplification of the
overall device, as a single Digital SlipChip layer can be
integrated with either one of several different types of readout
SlipChips, depending on the needs of the diagnostic device. In
certain embodiments, the stackable SlipChip allows a complete test
for measuring HIV viral load with dynamic range of 500-106/mL. In
certain embodiments, the stackable SlipChip can be used for
detection and quantification in sputum samples of, for example, 16
pneumonia pathogens.
[0470] In certain embodiments, the SlipChip can be used for the
amplification of cascades to count molecules of traumatic brain
injury (TBI) biomarkers. TBI is a major health issue in the
military. Mild TBI (mTBI) is of special significance as it
encompasses the majority of cases, is harder to diagnose, and can
result in long-term disability. Current diagnostic techniques,
magnetic resonance imaging and computer tomography, are impractical
in battlefield settings and are limited by cost and low
sensitivity. An unmet need is to diagnose and initiate appropriate
treatment for TBI at the point-of-care (POC).
[0471] Biomarkers can be used to diagnose TBI, but there is a need
for improved (1) detection and, especially, quantification of low
(pM) levels of panels of biomarkers in blood (Quantification is
critical because both absolute levels and time-dependent changes in
levels of biomarkers are needed for proper assessment of TBI), and
(2) function in a portable lightweight device without extra
equipment to perform and read the assay. Qualitative results can be
obtained from known dip-stick type devices, but quantification
requires a separate reader and can be unreliable. Quantification at
low concentrations enables detection of biomarkers in blood or
urine and potentially even saliva, which are simpler and safer to
collect in field settings than the current "gold standard"
cerebrospinal fluid.
[0472] Assays development has two opposing requirements:
amplification and quantification. Low starting concentrations and
the need for strong, easily detectable signal require a very high
degree of amplification. However, such amplification is generally
difficult to quantify and is too sensitive, with even small amounts
of spurious interference triggering the amplification cascade,
leading to errors and false positives. This challenge can be
addressed by single-molecule detection in thousands of areas of,
for example, nanoliter, picoliter, or femtoliter volumes, by
combining "stochastic confinement" and chemical amplification.
"Digital" approaches to count single molecules are routine for
nucleic acids by PCR (digital PCR) and have been demonstrated for
enzymes. These approaches use "stochastic confinement": the sample
is separated into, for example, hundreds or thousands of small
volumes, or areas, so statistically each area contains zero or one
molecule of the target analyte. Stochastic confinement has several
advantages, including: (1) Strong, qualitative yes/no amplification
chemistry leads to a quantitative result by counting positive
areas; (2) Artifacts, e.g. false initiation or inhibition of
amplification, are restricted to individual areas; (3) Sensitivity
and specificity of assays are increased because the effective
concentration of a single molecule is higher in a smaller volume
(increased signal) and interfering molecules are statistically
excluded (decreased noise). Amplification is initiated (e.g., by
thermal cycling in PCR), and the number of positive areas,
corrected by Poisson statistics, corresponds to the number of
molecules in the sample. One can use "stochastic confinement" and
chemical amplification for single molecule immunoassays for digital
biomarker detection.
[0473] Stochastic confinement and amplification can be used for
"barcoded" visual readout, to provide immediate measurement,
interpretation, and treatment suggestions. Visual readout is
important in, for example, far-forward military settings away from
the sophisticated imaging instruments of hospitals and
laboratories. Visual readout of multiplexed assay volumes can be
structured as a digital pattern of dots, like a barcode, so that
the pattern can be interpreted by eye or with a cell phone camera.
Capturing the image is useful for making time course measurements
and to automate or delegate decision-making. The pattern could be
analyzed and interpreted immediately via on-board software or a
central facility to instruct the best course of action. "Digital"
counting of TBI biomarkers, for example, enables diagnosis in
far-forward military settings. After a series of amplification
steps, a visual signal is generated that can be rapidly imaged and
analyzed using, for example, a cell phone camera to provide
immediate instruction.
[0474] Multistep processing of, for example, thousands of, for
example, nanoliter, picoliter or femtoliter volumes can be achieved
by using the SlipChip. Sophisticated fluid manipulation on a
SlipChip can be used to perform the multi-step heterogeneous
immunoassays and other chemistries that are preferred for detection
of TBI biomarkers at the single-molecule level. The SlipChip is a
microfluidic platform that can be used to encode a complex program
for parallel manipulation of thousands of small volumes.
Heterogeneous immunoassays can be performed quantitatively with
amplification on certain embodiments of the SlipChip. To detect low
levels of protein biomarkers in TBI, heterogeneous immunoassays are
useful because a large excess of capture antibody can be used to
drive binding. For these assays, multi-step processing is
preferred, including washing steps and addition of reagents for
signal amplification. The inventors have demonstrated a bead-based
immunoassay with pM-level sensitivity for the metabolic marker
insulin, using nanoliter volumes on SlipChip. Stochastic
confinement on the SlipChip can be used for the counting of single
DNA molecules after amplification by digital PCR. The inventors
demonstrated that single molecules of DNA can be detected and their
concentration quantified by counting the number of positive areas
out of 1,280 total areas, each 2.6 nL in volume.
[0475] In certain embodiments, the SlipChip can be used for: (1)
Quantitative detection of TBI biomarkers with high sensitivity
which can be accomplished by counting single molecules after a very
high degree of amplification. (2) A multistep amplification cascade
which can give the sensitivity needed to detect and count single
molecules of the TBI biomarkers of interest in visual readout. (3)
Stochastic confinement of molecules of TBI biomarkers in tiny (for
example, femtoliter to picoliter) volumes which can be used for
standard heterogeneous immunoassay chemistries for molecular
recognition.
[0476] Ubiquitin C-terminal Hydrolase-L1 (UCH-L1) is a marker for
neuronal cell body injury, and SBDP 150 is a product of
all-spectrin cleavage by calpain and a marker linked to axonal
injury. These are present at 4 to 130 pM and 7 to 70 pM,
respectively, in blood during TBI. There are commercial monoclonal
antibodies against them that can be used in these assays. Certain
embodiments of the SlipChip can be used to quantify a range of
.about.0.02-200 pM concentrations, or down to 0.001 pM with
coincidence detection.
[0477] Single-molecule microscopy can be used to verify the
presence of a single molecule of interest in areas used for
amplification and visual readout. Samples can be imaged on Alba
microscopy system (ISS, Champaign, Ill.). Target biomarkers,
antibodies, DNA probes and even gold nanoparticles (AuNPs) can be
labeled with quantum dots (QDs, enhanced by blinking). Lanthanide
dyes with long lifetimes can be used in a time-gated mode in
strongly fluorescent human samples of plasma.
[0478] In certain embodiments, the SlipChip can be used to
partition and manipulate samples in small volumes. The SlipChip
enables formation and manipulation of a wide range of volumes, for
example, from tens of nanoliters (area dimensions, for example,
500.times.500.times.50 .mu.m3) to tens of picoliters
(50.times.50.times.5 .mu.m.sup.3) to tens of femtoliters
(5.times.5.times.0.5 .mu.m.sup.3). Appropriate control reactions
on, for example, the microliter scale can be performed to confirm
that reagents and assays perform as expected. Stochastic
confinement can be used to isolate single molecules: for example,
at 9 pM concentration, areas 0.2 .mu.L in volume contain on average
a single molecule. The advantages and limitation for each size of
area, such as extremely rapid transport versus increased complexity
of fabrication for femtoliter areas can be determined for a given
application. If small volumes are less preferred for a given
application, short equilibration times can be used to capture
single biomarker molecules from larger volumes. Characterization by
single-molecule microscopy can be used to ensure improved
representation of the actual concentration.
[0479] To analyze single-molecule assays, Poisson statistics can be
used to calculate the initial concentration of analyte from the
number of positive and negative areas observed. To establish
accuracy and precision of the single-molecule immunoassay with
visual readout, the user can analyze biomarker concentrations in
buffer, artificial plasma, and archived normal human plasma
un-spiked and spiked with biomarker at 0.02 pM-200 pM in log-scale
concentration steps (n 5 samples per concentration). To perform the
assay in the context of TBI, archived human plasma from 12
individual TBI patients (Banyan) can be analyzed 3 times to assess
precision of the assay and measure levels of biomarkers in mTBI
patients.
[0480] In certain embodiments, the SlipChip can be used to achieve
multistep threshold amplification of single molecules with visual
readout in a multiplexing format. For simple visualization by eye
or a cell-phone camera, the optical properties (absorbance or
reflectance) of positive areas can be equivalent to an absorbance
of 0.5 to 1 in a 200 .mu.m.times.200 .mu.m area.
[0481] The autocatalytic reduction of silver (I) ions, initiated on
gold nanoparticle (AuNP) surfaces, provides a very high degree of
amplification producing visually observable silver deposition. For
use in immunoassays, an AuNP is conjugated to the detection
antibody. In the SlipChip this chemistry can produce a visible
signal that is clearly distinguishable from the background in a 55
nL volume with 5 pM AuNP (165,000 AuNP per area). Amplification
conditions can be modified to achieve lower detection limits and
the starting volume can be reduced to 200 fL so a 5 pM
concentration will produce an average of 0.6 molecules per area.
Robust amplification from single particles to generate a visual
signal from >95% of particles with <1% false positives, on a
device containing at least 200 areas can be achieved.
[0482] Dark field microscopy can be used to track anti-UCH-L1
antibody conjugated to 150 nm AuNPs at the single molecule level.
Alternatively, a fluorescence microscopy system (e.g., an Alba
system) can be used to track fluorescently labeled AuNPs.
[0483] For certain applications, two-stage amplification is
preferred to visualize the signal, as the signal from sub-picoliter
areas may not be intense enough to detect visually. The output from
the first stage of AuNP-catalyzed amplification can initiate
amplification in a second set of areas that are large enough to be
visually observed. As silver deposition is autocatalytic, potential
signal generation in the absence of AuNPs (background) is a
concern. During the first stage of amplification, true positives
generate a strong signal and background noise generates a weak
signal. A threshold can be introduced such that only an input
signal that is stronger than a critical level generates further
amplification. Passivating the gold surface by the addition of high
affinity thiols can suppress both background and sub-threshold
concentrations of AuNPs for over 1 hr. The use of a threshold can
ensure that visual signal appears after the second stage of
amplification only from areas that initially contained AuNP.
[0484] PCR can be used as an additional amplification step to
increase the signal-to-noise ratio from the amplification chemistry
and single-molecule amplification of nucleic acids by standard PCR
are well known. Single-molecule immuno-PCR combined with stochastic
confinement can be performed. The user can conjugate, for example,
anti-UCH-L1 detection antibody to a DNA sequence. The DNA serves as
a template for PCR amplification, producing many copies of the
sequence. Each copy of PCR product can be designed to hybridize to
two probes: one that immobilizes it on the surface of the area or a
bead, and one that links it to a signaling molecule. Alkaline
phosphatase (AP) with NBT/BCIP and AuNP-catalyzed silver deposition
have both been used to generate a visual signal from PCR
products.
[0485] In certain embodiments, the SlipChip can be used for
quantitative single-molecule PCR and isothermal DNA amplification.
Standard PCR can be performed on the SlipChip because it is robust
and well-characterized, but the required thermocycling is not
always preferred, for example, for field use. Isothermal techniques
are preferred for certain field applications. The inventors have
demonstrated single-molecule detection using isothermal recombinase
polymerase amplification (RPA) on the SlipChip platform.
[0486] Other chemistries for amplification combined with stochastic
confinement can be used on the SlipChip. Photoinitiated systems
have proven to be very sensitive and can function at the single
molecule level. Polymerizations can generate visual signals, and
attaching multiple radical photoiniators to an antibody has shown
promise for visual readout. Photoacid generators linked to
autocatalytic acid generation are used extensively in photoresists
that require extreme sensitivity.
[0487] A single sandwich-complex can provide a clearly observable
signal on the SlipChip, and the immunoassay for, for example, TBI
biomarkers (e.g., UCH-L1) can be used to detect down to the single
molecule level.
[0488] Stochastic confinement isolates single molecules of analyte
in SlipChip areas containing capture antibody: a device with, for
example, 1000 areas of, for example, 0.5 .mu.L volume can allow for
detection of single molecules in the range of 0.2-200 pM. Capture
efficiency can be assessed using, for example, target biomarker
labeled with fluorescent quantum dots (QD) by tracking
biomarker-containing areas before and after washing using, for
example, a fluorescence microscopy system, and comparing results to
calculated prediction. For example, a 1 .mu.L area containing one
biomarker molecule and 0.1 .mu.M capture antibody (K.sub.d=1 nM) is
predicted to capture the biomarker with 99% efficiency. The user
can label the detection antibody with a different QD, and directly
visualize binding. The user can measure colocalization of the two
labels to quantify formation of the immuno-sandwich complex and
quantify background off-target binding of detection antibodies.
This allows quantification of improvements from altering assay
conditions (concentrations, buffers, surface chemistries, etc) to
optimize single-molecule binding with low background.
[0489] Single molecule measurements often suffer from high
background and weak signal from single analyte molecules.
Stochastic confinement can increase the signal intensity in
positive areas, but this does not necessarily decrease the number
of false-positive areas due to non-specific binding of the
detection antibody (background binding), and may not be alleviated
using traditional methods described above. One solution to decrease
background binding is to attach the detection antibody to a
magnetic bead, so that unbound antibody can be more easily removed
from the areas and removal can be further enhanced using acoustic
techniques. For applications with a high background signal,
coincidence detection using colocalization of two detection
antibodies can be used to directly measure and correct for the
background signal.
[0490] After loading areas with single molecules of analyte and
obtaining predicted binding of antibodies, amplification chemistry
can be performed and then the user can evaluate the entire
immunoassay in buffer and in artificial plasma. The user can
stochastically confine samples to isolate single molecules, form
the immuno-sandwich complex, amplify using the chosen amplification
chemistry, and image to count the number of positive areas. The
user can evaluate sensitivity, specificity, linearity (or the
dose-response relationship in general), and deviation from expected
results over the range of concentrations.
[0491] The user can use a labeled biomarker to apply
pre-amplification visualization to confirm the presence and
location of single molecules. Tracking how many antigen-containing
areas result in visual signal and how many blank areas result in
visual signal can quantify the performance of the system.
[0492] The SlipChip single-molecule approach can be used for
coincidence detection to lower the signal due to background binding
and also directly distinguish background binding from on-target
binding, quantifying the background binding. The user can use two
detection antibodies, labeled with different fluorophores against
two different epitopes of the target biomarker (for example, three
antibodies are available for three distinct epitopes of UCH-L1
(Banyan Biomarkers)) and use two-color detection. The user can
detect coincidence initially directly with an appropriate
fluorescence microscope and then by amplification to give two-color
visual readout or conditional readout (e.g., requiring capture of
both horse radish peroxidase and glucose oxidase to generate color
after PCR). Background binding gives a signal with low coincidence,
predicted by Poisson distribution. On-target binding gives easily
distinguishable above-random coincidence (for example, >98%
confidence even for a 25-area chip). Without single-molecule
coincidence detection, these signals may appear indistinguishable,
with 5 units of binding for either detection antibody. If
necessary, the background signal can be lowered even further by
requiring that only tags in close proximity produce a signal. For
example, fluorescence resonance energy transfer or fluorescence
cross-correlation spectroscopy can be used for characterization
with paired detection aptamers initiating rolling circle
amplification (RCA) only when the two aptamers are in close
proximity, as when bound to analyte.
[0493] Human samples can have higher background signal from
nonspecific binding due to many other substances in plasma, and
varied background concentration in different human samples. The
user can conduct single-molecule immunoassays for biomarkers in
plasma samples, by first verifying that binding occurs specifically
using single-molecule fluorescence microscopy measurements and then
applying amplification chemistry to get visual readouts.
[0494] The user can detect biomarkers quantitatively and with
visual readout in human samples by counting single molecules after
multistep amplification in, for example, sub-nanoliter volumes. A
user can use aptamers instead of antibodies, isothermal
amplification instead of PCR, engineer devices to carry out the
assays under field conditions, and design software for
communication devices to interpret results of the assays and
suggest actions. The SlipChip can also accelerate drug discovery
for, for example, TBI, and discovery and testing of biomarkers. Its
high sensitivity can be used in the development of biomarkers in
more accessible fluids such saliva, urine or tear fluid. This can
also enable detection of non-protein biomarkers such as mRNA or
miRNA.
[0495] In certain embodiments of the SlipChip, stochastic
confinement can be combined with coincidence detection to allow
sample-specific background correction to quantify samples that have
a high level of nonspecific binding. To eliminate amplification of
noise, the user can introduce a threshold such that only input
signal that is stronger than a critical level generates further
amplification.
[0496] In certain embodiments, the SlipChip can be used for
combinatorial biocatalysis using many different organisms,
including but not limited to extremophiles. Many researchers use
plate assays for the screening of general thermophiles (preferring
about 45.degree. C. to about 80.degree. C.), whereas for the
hyperthermophile (preferring about 80 to about 122.degree. C.), it
is difficult to run standard plate experiments because of the
evaporation and melting of agar media. Most known hyperthermophiles
are anaerobic, sulfur requiring, and slow growing organisms. The
SlipChip can be used to culture many organisms including
thermophiles and hyperthermophiles. Many of the biocatalytic
reactions run by hyperthermophiles are biomass degradation (e.g.,
capable of cellulase production and reaction). The SlipChip can be
used for novel enzyme screening or culturing community-based
cultures.
[0497] The following patents and applications are herein
incorporated by reference in their entirety: U.S. Ser. No.
12/257,495 "Automated analyzer for clinical laboratory", U.S. Ser.
No. 12/411,020 "Integrated microfluidic assay devices and methods",
U.S. Pat. No. 3,996,345 "Fluorescence quenching with immunological
pairs in immunoassays", U.S. Pat. No. 5,686,315 "Assay device for
one step detection of analyte" and PCT/US2007/20810 "Integrated
microfluidic assay devices and methods".
[0498] In certain embodiments, the SlipChip may be used as
cell-cell communication devices, where the surface is wetted by
reaction fluid instead of lubricating fluid. Areas that connect by
very thin ducts, which may be nanopatterned, along the surface can
be used to monitor cell-cell interactions without contact, or to
filter solutions (if flow is induced from one area to another) for,
for example, sample preparation and bead-based chemistries. In some
instances, both surfaces will be hydrophilic, but in others, only
one surface can be hydrophilic. Hydrophilic nanopatterns can be
used.
[0499] Certain embodiments of the SlipChip can be used to analyze
plugs which can come from any plug making system or device,
including for example, the chemistrode. Reagents in certain
embodiments of the SlipChip can be bathed in a lubricant or carrier
fluid. Protein adsorption on certain embodiments of the SlipChip
can be controlled at interfaces by controlling surface chemistry
using, for example, flourous soluble surfactants. Certain
embodiments of the SlipChip can be used to store reagents without
risk of contamination.
[0500] In some embodiments, the SlipChip can be an opening and
closing device. This can be used for isolating and analyzing rare
cells, particles, and/or beads carrying cells or molecules of
interest, out of large volumes. This is relevant to a number of
different kinds of cells including but not limited to circulating
tumor cells, microbial cells in bodily fluids, purification of
other cells. This can be done using many different approaches
including but not limited to standard loading and capture and using
an open SlipChip that is used to capture, and then assembling
afterwards. One plate of the SlipChip can act as a filter or as a
capture surface, solving the problem of analyzing large volumes
with only a few cells of interest. Such devices can be used for,
for example, analysis of samples that may be difficult to load
otherwise, for example, aerosols of bacteria and viruses generated
during coughing, or tissue slides from which a user would like to
analyze the sample without losing track of spatial relationships
among cells, as is done for tumors and biopsies. In addition, a
user can open the chip for analysis by methods that benefit from
direct access (for example mass-spectrometry, including analysis of
areas of the SlipChip by DESI and MALDI techniques). When SlipChip
is constructed using materials that can be penetrated (including
PDMS, polyurethane, other elastomers, and sealing tape manufactured
by 3M), contents of areas can be accessed directly, for example by
puncturing the material with a needle.
[0501] In certain embodiments of the present invention, surface
tension can be used to prevent leakage ("surface tension seal").
Two halves of the device can be made of, for example, plastic which
are then made very hydrophobic using for example, plasma treatment.
A closed path around the chip can be made hydrophilic. The
hydrophobic areas are wetted with a hydrophobic liquid. To prevent
evaporation, there can be a liquid reservoir in contact with
appropriate areas. The two halves of the SlipChip can be clamped
together and the aqueous solutions added to the chip will not leak
between the plates because of capillary pressure. Similarly, the
hydrophobic solution is stopped by the hydrophilic layer. The
highest pressure the device can withstand is governed by the
capillary pressure.
[0502] When clamping the two sides of a SlipChip together, if the
layers are very thin, then it is preferred to apply pressure
uniformly over the surface. With a pre-strained holding device the
SlipChip can be made very thin, pre-clamped together at the
factory, and peeled apart. Alternatively, two rigid glass slides
can be used as holders and, if necessary, imaging can be performed
through them. The glass slides can be removed if x-ray diffraction
is to be performed. However, in certain embodiments, clamping is
not necessary. For example, two glass slides, if wet, stick
together very tightly; similar ideas can be used to keep the layers
of a SlipChip together. If the opposing surfaces are rigid and
flat, a very high capillary pressure is produced, and the rigidity
requires that when separating the slides the contact must be broken
over a large area simultaneously, requiring high force.
Applications include, but are not limited to protein
crystallization, for example for membrane protein
crystallization.
[0503] In certain SlipChip applications in which precise metering
of a sample is preferred, a well can be overfilled, and then excess
can be pushed away by the adjacent layer. Alternatively, the device
can have a set of redundant pathways, wherein each path for
purification and/or analysis takes, for example, 5 .mu.L, and as
the user loads the sample into the device, the first 5 .mu.L is
filled, then the second, etc. Such a device has a robust system
that can do quantitative analysis on, for example, 10 .mu.L and on
50 .mu.L of plasma.
[0504] In certain embodiments of the present invention, the
SlipChip may be in a centrifuge tube. This kind of device can be
used for reconcentration of cells/particles by sticking a SlipChip
at the bottom of a centrifuge tube.
[0505] The chemistrode, a microfluidic device that relies on
two-phase laminar flow, can acquire repeated samples and maintain
them for analysis. The chemistrode is a microprobe that performs
like an electrode (delivers and records signals) but uses chemical
rather than electrical signals. Chemistrodes for sampling
secretions from tissue in an isolated area, and needle-like
chemistrodes for sampling soil suspensions have been
demonstrated.
[0506] Chemistrodes are compatible with parallel chip-based
nanoliter assays down to single-molecule methods, ensuring that
many small volumes can be sampled and analyzed from a single
animal. Detection of single-molecules of the metabolic marker
insulin has been achieved using a competitive immunoassay and
fluorescence correlation spectroscopy (FCS). Droplets obtained by
the chemistrode also can be analyzed on a SlipChip. The
chemistrode, combined with FCS or SlipChip for analysis, can
continuously sample biofluids from live animals for quantitative
analysis.
[0507] Certain embodiments of the SlipChip can process many
nanoliter volumes from a chemistrode to perform, for example,
multi-step heterogeneous immunoassays at picomolar levels required
for detection of biomarkers (TBI biomarkers, for example).
[0508] Certain embodiments of the SlipChip can be used for
inexpensive and simple measurement of HIV viral load at the point
of care (POC). Such a test is urgently needed to provide proper
care to patients on antiretroviral therapies in resource-limited
settings, and to control the emergence and spread of drug-resistant
strains of HIV worldwide. While a number of qualitative yes/no
diagnostic tools have been developed, there is still an unmet need
for quantitative viral load measurement in resource-limited
settings. Although PCR-based assays with real-time readout are
quantitative, these assays require equipment and environments too
complex for POC in resource-limited settings. Also, isolation and
concentration of viral RNA from plasma is challenging for most POC
approaches. Certain embodiments of the SlipChip can encode a
complex program (algorithm) for manipulation of many fluid volumes
in parallel. Certain embodiments of the SlipChip consist of two
plates that move--or "slip"--relative to one another, lubricated by
inert fluid that is immiscible with the sample fluid and also
provides control of surface chemistry and prevents
cross-contamination. The program is encoded into the plates as a
pattern of areas containing reagents, and is executed by slipping.
Slipping brings areas (or wells) in the two plates in and out of
contact to execute a diagnostic assay. Manipulations on multiple
scales, e.g., from 100 pL to 100 .mu.L, can be performed on the
same chip. Such SlipChips facilitate integration of upstream sample
preparation to isolate and concentrate viral RNA and permit
quantification of viral particles via nucleic acid amplification
using "digital" (single molecule) detection with downstream signal
amplification to enable readout as simple as an image taken with,
for example, a cell phone camera.
[0509] One can target different HIV-1 subtypes, including the A, C,
and G subtypes predominantly found in India and Nigeria.
[0510] Currently available qualitative POC diagnostics tests are
not suitable for the quantitative monitoring needed. The HIV
antibody test has been incorporated into a dipstick format that can
be readily used in resource-limited settings. However, this test
does not reflect the effect of HIV antiretroviral therapy (ART) as
it only provides information on the patient's serostatus. The p24
antigen test has low sensitivity and works only at a very high
level of HIV viremia (>10.sup.5 particles/ml), and therefore
cannot be used to monitor ART. Methods for CD4 cell counts are
currently not widely available, and the counts can be low in a
number of illnesses and may not reflect HIV infection. In addition,
HIV viral dynamics and resistance to therapy can only be inferred,
since CD4 counts are slow to reflect changes in viral load that are
happening on a more rapid timescale. The ExaVirLoad from Cavidi AB
has potential for use in resource-limited settings, but testing
requires about 3 days, is expensive, and has an extra burden of
proof to connect it to the established clinical practice.
[0511] Quantitative measurement of HIV viral load by nucleic acid
testing is urgently needed for resource-limited settings. The main
goal of ART is formulated as to reduce the HIV RNA level in plasma
as much as possible for as long as possible. This requires
quantification, which is currently based on direct nucleic acid
testing (NAT) by real time reverse transcriptase-polymerase chain
reaction (RT-PCR), Nucleic Acid Sequence Based Amplification
(NASBA), and transcription-mediated amplification (TMA) on
automated machines in centralized laboratories. Quantification of
the HIV viral load is used to guide when to begin HIV
antiretroviral drug treatment, provide information on the degree of
initial antiretroviral effect achieved, assess the risk of disease
progression, and guide decision making on when to switch to a
different ART regimen.
[0512] At present, no HIV viral load quantification platform is
available that can be used in resource-limited settings, as
described elsewhere in this application. A preferred device has a
number of preferred characteristics: a wide dynamic range to
measure viral loads from, for example, 500 to 1,000,000
particles/mL in plasma; use, for example, 100-200 .mu.L of whole
blood or plasma; be quantitative enough to distinguish, for
example, 3-5 fold changes in viral loads with 90-95% probability;
be low in cost; be easy to use; provide results in under, for
example, 2-4 hours (within one visit); require only simple and
robust equipment; and have a simple readout.
[0513] Digital direct nucleic acid testing (NAT) is a technological
advance that enables quantification of DNA or RNA levels with
higher sensitivity and does not require real-time readout. For
certain applications, real-time RT-PCR provides accurate viral
loads and can be used, but for others, it is too complex in regard
to required expertise and equipment. To obtain quantitative results
without the necessity for real-time measurements, single-molecule
detection has emerged as preferred. Digital NAT is based on the
concept of confining and visualizing single copies of nucleic acid
in a series of small volumes. The number of small volumes that
generate a nucleic acid product directly corresponds to the number
of molecules present in the original sample, making the results
highly quantitative. The detection sensitivity of samples with high
background is increased in digital platforms, since each molecule
being detected is partitioned into individual small volumes (or
stochastically confined), apart from inhibiting contaminants.
[0514] Certain embodiments of the SlipChip provide a simple way of
compartmentalizing a large number of small (for example, picoliter
to nanoliter) fluid volumes in parallel without external
instrumentation. The SlipChip can be used to perform digital NAT
for HIV treatment and diagnosis in resource-limited settings.
Certain embodiments of the SlipChip readily form thousands of
nanoliter reactor chambers while not requiring costly pump-based
filling systems--a series of connected wells can be simply filled
by a single pipeting step, and wells are subsequently separated
into individual nanoliter reactors by slipping one plate next to
the other. The SlipChip can be highly multiplexed but does not
require valves.
[0515] Certain embodiments of the SlipChip maintain
compartmentalization of all reactions even under stringent
conditions required during sensitive assays and thermal cycling. By
altering the geometry of the wells, an aqueous droplet can be
suspended in the well, surrounded by a lubricating fluid. In
certain embodiments, during temperature changes associated with
thermal cycling, the fluids expand but the aqueous droplet
containing the PCR reaction does not leak out of the wells.
[0516] Certain embodiments of the SlipChip facilitate the addition
of multiple reagents in separate steps to all compartmentalized
reaction volumes in parallel without external instrumentation or
cross-contamination between neighboring reaction volumes, as is
preferred for both digital isothermal NAT and subsequent
amplification of the NAT readout. Isothermal NAT is advantageous
for resource-limited settings because it does not require thermal
cycling, eliminating the need for a major piece of equipment.
However, it is not currently used commercially as POC because of
the technical difficulty of controlling the initiation of
amplification reactions, as the reaction is initiated immediatedly
upon mixing the PCR mixture with the template RNA. Because
amplification starts prior to loading the sample into the digital
platform, the digital readout is not necessarily an accurate
reflection of original target concentration of RNA. When template
nucleic acid is amplified prior to stochastic confinement, false
positives can occur. Certain embodiments of the SlipChip solve this
problem. First of all, reagents can be added instantaneously at any
user-specified start time after loading of the RNA template by
slipping the wells containing sample into contact with wells
containing the reagent. Secondly, digital PCR can utilize end-point
readout so reaction time is not critical. In addition, there is no
cross contamination between neighboring reaction volumes. The
SlipChip facilitates manipulation of varying reaction volumes, as
preferred for RNA isolation. The SlipChip can be fabricated in any
geometry with varying well diameters and varying depths, for
example depths ranging from several microns to millimeters. Each
reaction volume containing a single nucleic acid can truly be
digitally interpreted. As an alternative to using fluorescence
readouts, colorimetric enzymatic amplification reactions can be
used to detect NAT products. The SlipChip can also accomplish the
simultaneous addition of multiple reagents to all reaction
volumes.
[0517] In certain embodiments of the SlipChip, molecules or viral
particles can be captured by magnetic beads, and pulled from a
large volume into a small volume by use of magnets, enabling
on-chip concentration. Likewise, small volumes can be added to
large volumes, enabling on-chip dilution.
[0518] Interfacial chemistry in certain embodiments of the SlipChip
can be controlled at the interface of the lubricating fluid and the
reaction fluid, simplifying fabrication. The interfacial chemistry
between two immiscible liquids can be controlled by, for example,
adding surfactants. Because the lubricating fluids used in certain
SlipChips can be the same as the carrier fluids used in previous
droplet-based work (see for example, U.S. Pat. No. 7,129,091, and
PCT/US2009/046255, both incorporated in their entirety herein) the
interfacial chemistry can be controlled in an analogous manner. For
a non-fluorinated lubricating fluid (such as mineral oil), a
surfactant can be added to the aqueous reaction fluid; for a
fluorinated lubricating fluid, a fluorinated oil-soluble surfactant
can be added to the lubricating fluid. Examples of different
lubricating fluids that have been used in the SlipChip include
mineral oil for a single-molecule PCR SlipChip and fluorinated oils
for a SlipChip for immunoassays achieving pM detection limits.
[0519] Possible surface treatments for a SlipChip include, but are
not limited to, dichlorodimethylsilane (appropriate for glass
devices) and gas phase silanization.
[0520] A glass SlipChip with uniform well volumes can have, for
example, a dynamic range of detection of 5,000 to 100,000 HIV
particles/mL. An advantage of such a design is that every well is
an identical replicate if loaded with the same solution, since the
surface-to-volume ratio is kept constant. In one experiment, the
concentration of a dye loaded in a SlipChip with uniform well
volume had a coefficient of variation of 3.2%. A uniform well
device can have, for example, 1280 reaction volumes, with 640
elongated wells in the top piece and 640 elongated wells in the
bottom piece of the SlipChip to conserve space. The elongated wells
can initially overlap for filling. After filling, the elongated
wells can be slipped over circular wells containing, for example,
mineral oil. This design promotes the formation of an aqueous
droplet surrounded by a volume of mineral oil upon slipping. The
aqueous droplet can expand upon heating, displacing mineral oil
between the two plates of the SlipChip, and preventing the aqueous
phase from leaking out of the well and causing cross-contamination
due to thermal expansion. Each circular well can be, for example,
50 .mu.m in diameter and depth.
[0521] This device can be made from glass using standard
photolithographic and wet chemical etching techniques. Surface
chemistries can be controlled by rendering the surface of the
SlipChip hydrophobic by silanization with, for example,
dichlorodimethylsiloxane, which is amenable to PCR. Mineral oil can
be used as the lubricating fluid between the two plates of the
SlipChip and as the wetting layer surrounding the aqueous phase
containing the reaction mixture.
[0522] A commercially available Access RT-PCR kit from Promega,
with a known concentration of the commercially available HIV
standard (8E5 LAV deletion mutation strain of HIV-1) and EvaGreen
dye to detect product can be used. One can use primers for
amplification of the HIV-1 long-terminal repeat (LTR) region, which
contains sequences that are conserved between all HIV-1 subtypes in
M, N, and O groups. These primers are suitable for amplifying all
subtypes of HIV-1 found in India and Nigeria (A, C, and G) as well
as the subtype predominant in the US (B). The primers are: A1352
sense, position 607 in the published sequence alignment from the
Los Alamos HIV Sequence Database, GRAACCCACTGCTTAASSCTCAA; A1355
antisense, position 708, GAGGGATCTCTAGNYACCAGAGT.
[0523] In an experiment, reliable filling of 1280 wells was
achieved using 6.5 .mu.L of initial sample, and a reproducible
digital readout was attained for both PCR and RT-PCR. A 1280-well
SlipChip was characterized for digital PCR using Staphylococcus
aureus genomic DNA. Results were both reproducible and
quantitative. In addition, an experiment demonstrated that the
biochemistry of RT-PCR using the 8E5 LAV deletion mutation strain
of HIV-1 and the A1352 and A1355 sense and antisense primers is
compatible with digital SlipChip platforms.
[0524] Internal controls can be built into the SlipChip to validate
results that are obtained in the field. For example, 100 wells can
be preloaded with primers to detect control RNA that can be added
to the sample. The primers can be dispensed either manually or by
simple robotics prior to assembly of the two plates of the
SlipChip.
[0525] An embodiment of a circular SlipChip that generates multiple
reaction volumes on one chip has been demonstratedError! Reference
source not found. An advantage of this design is that its dynamic
range can cover a range of detection of, for example, 500 to
1,000,000 HIV particles/mL. In certain embodiments, the wells
initially overlap with ducts to enable filling and are then slipped
into discrete reaction volumes by rotating the device. Exemplary
dimensions for this SlipChip of varying well volumes are 128 wells
of 200 nL volume (39-1667 RNA molecules/mL), 128 wells of 20 nL
volume (391-16667 RNA molecules/mL), 256 wells of 2 nL volume
(1953-166,667 molecules/mL), and 512 wells of 0.5 nL volume
(7813-1,333,333 molecules/mL). These well sizes allow checking the
internal consistency of the SlipChip due to the overlap in dynamic
range of the larger and smaller volumes. The device can incorporate
internal controls.
[0526] Such a device can, alternatively, use surfactants in the
aqueous sample solution or use fluorinated oil instead of mineral
oil.
[0527] In certain embodiments, it can be preferable to achieve an
equivalently large dynamic range by on-chip serial dilution, which,
in certain embodiments, contains larger wells. Exemplary dimensions
for this design are five rows containing 100 wells in each row, and
a shallow well containing 20 nL of sample is slipped over a
preloaded well containing 180 nL dilution buffer, achieving a 10
fold dilution with each slip.
[0528] SlipChips can be made of many materials, including, for
example, glass, polycarbonate, polypropylene and other plastics.
Both polypropylene and polycarbonate are known to be compatible
with PCR. Plastic devices can be coated with different surface
coatings, surfactants and oils.
[0529] Certain embodiments of the SlipChip can be used to control
the initiation of HIV RNA transcription into cDNA by reverse
transcriptase (RT) and subsequent amplification reactions.
Initiation of cDNA synthesis and amplification is controlled by
slipping wells containing the reaction mixture and template RNA in
the upper piece of the SlipChip over preloaded dried primers in the
bottom piece of the SlipChip. The primers can, for example, be
loaded manually for initial testing using Teflon tubing with an
I.D. of, for example, 50 .mu.m, or using simple robotics.
[0530] A 384-well SlipChip preloaded with primers for the detection
of different bacterial species successfully distinguished
methicillin-resistant Staphylococcus aureus (MRSA) from
methicillin-sensitive S. aureus (MSSA). Two columns at either end
of the SlipChip were preloaded with pBad primer, and pBad template
DNA was loaded into all wells as a positive control.
[0531] Isothermal amplification technologies that can be used
including NASBA, and RT-RPA These amplification techniques can
operate at 40.degree. C. (a lower temperature preferred for certain
POC devices): NASBA (product: RNA), RT-RPA (product: DNA), RT-LAMP
using one of LAMP HIV-RNA 6-primer sets, transcription-mediated
amplification (TMA, 41.degree. C.), helicase-dependent
amplification (HAD, 65.degree. C.), and strand-displacement
amplification (SDA, 37.degree. C.),
[0532] Amplification methods preferable for POC are those that do
not require large temperature differences from ambient and can be
initiated in one mixing step, however, NASBA and RPA contain
heat-labile enzymes. Therefore, one can exclude the denaturation
step from standard protocols and adjust the primer annealing
temperature to 40.degree. C. If, for certain embodiments, annealing
at 40.degree. C. gives lower sensitivity, one can select a 100-120
nucleotide long amplification target in the genomic RNA
conservative in different HIV-1 subtypes that has weak secondary
structure which allows efficient primer annealing at 40.degree.
C.
[0533] In an experiment, a Mg.sup.2+ solution was preloaded into
all wells of a SlipChip, and all other reagents for RPA in solution
were used to fill remaining wells. The original concentration of
control template provided with the kit was 2 molecules/.mu.L.
Approximately 500 nL was analyzed.
[0534] Several diagnostic NAT tests incorporate an internal control
within the same tube or well as the RNA of interest, and quantify
the internal control by using a specific probe conjugated to a
different fluorophore than that of the probe recognizing the
amplified target RNA. One can incorporate an internal control using
control template RNA (e.g., 3,569 nt-long bacteriophage MS2 genomic
RNA) mixed with, for example, HIV RNA and all amplification
reagents into the SlipChip. One can independently and
simultaneously analyze HIV RNA and internal control template by
preloading three quarters of the wells on the chip with a dry
reaction mixture containing HIV primers, and the other quarter with
internal control, using, for example, SYBR Green detection to
quantify the load of HIV and the internal control.
[0535] Visual readouts are preferred for certain resource-limited
POC settings. One can modify the SlipChip to incorporate additional
steps and slips useful for a visual readout. Obtaining a visual
readout can comprise hybridization of a nucleic acid product to an
enzyme, washing to remove excess enzyme, addition of a substrate
that the enzyme will convert to a visual signal, and incubation to
amplify the visual signal. To make visualization easier, the well
size can be increased to allow more visual signal to be produced. A
cell phone camera, for example, can serve to record, analyze and
document the results.
[0536] Hybridization of single-stranded RNA (generated by NASBA)
can be achieved in one step using surface immobilization, magnetic
beads and shallow wells.
[0537] Alkaline Phosphatase can be used for enzyme-based detection.
Alkaline phosphatase has a well established BCIP/NBT
((5-bromo-4-chloro-3-indoyl phosphate, disodium salt)/(nitro blue
tetrazolium chloride)) substrate for visualization. This substrate
forms a very strong blue-colored precipitate at the site of
enzymatic activity. For certain applications, the expected 100 nM
of product R/DNA binds enough enzyme to easily and rapidly consume
the BCIP substrate to generate the approximately 1 mM of products
preferred for producing a dark, easily identifiable signal.
[0538] Gold nanoparticles or colored magnetic beads that can either
be concentrated from a larger well into a small spot or amplified
using silver amplification (for gold nanoparticles) can also be
used to generate a strong visual signal.
[0539] A cell phone camera can easily record data and provide rapid
analysis using simple software to count and calculate the desired
information. The camera preferably can resolve and identify spots.
By focusing, for example, a 1 megapixel camera on a 1280 well
layout, each well image contains approximately 80 pixels. Using a 2
megapixel camera, each well image contains approximately 200
pixels. This number of pixels is sufficient for reliable counting.
Both resolutions are common levels in many cameras, and are readily
available even in resource-limited settings. The samples can be
transferred to larger wells during visual signal development to
facilitate detection.
[0540] For certain applications, it is preferred to attain a final
concentration of purified RNA that corresponds to at least 40% of
the initial HIV viral load present in patient blood, or 200 to
400,000 molecules/mL isolated. Calculations based on Poisson
statistics indicate that 40% recovery is adequate to reliably
quantify initial viral loads from the patient at 500 to 1,000,000
molecules/mL.
Exemplary isolation protocols include:
[0541] Protocol 1: Modified Boom's isolating RNA from plasma via
lysis of viral particles with chaotropic salts followed by trapping
of RNA on silica magnetic beads (MagPrep.RTM. beads, Merck
KGaA).
[0542] Protocol 2: Modified Boom's protocol isolating RNA from
plasma via lysis of viral particles with chaotropic salts followed
by trapping of RNA on iron-oxide beads.
[0543] Protocol 3: Isolating RNA from whole blood via capture of
viral particles on antibody-coated magnetic beads (Viro-Adembeads,
Ademtech, France) followed by a soft lysis procedure (heating at
95.degree. C. or treating with a weak alkali).
[0544] For each protocol, on a SlipChip one can reduce the number
of washing steps to two.
Example:
[0545] To obtain the HIV RNA used in a 1280-well digital SlipChip,
HIV-1 was purified from Acrometrix OptiQual HIV-1 High Positive
Control (1.7.times.10.sup.6 mutant HIV-1 particles/mL; 18 pg HIV
RNA/mL, 1 OD260=37 .mu.g/mL) using a Qiagen QiaAmpViral
purification kit, which contains complete lysis, carrier RNA, and
silica minicolumns.
[0546] An embodiment of a circular SlipChip platform can
accommodate all steps of the RNA isolation process using magnetic
capture beads.
[0547] Certain embodiments of the SlipChip can sample whole blood
or plasma and yield a measurable readout indicating HIV viral
load.
[0548] Nanoliters of solution can be stored for greater than 6
months in fluorocarbon in a plastic SlipChip. One can store
SlipChips in blister packs. One can use, for example, Drierite-type
Cobalt-based solid dessicants to estimate water flux, and/or to
create a dry boundary between the regions of a SlipChip.
[0549] A sample can be pre-stored in a big well on certain
embodiments of a SlipChip. Because, in certain embodiments, the
sample is surrounded by the lubricating oil, such as fluorocarbon
(FC) or paraffin oil, evaporation is prevented. When pressure is
applied through an inlet, the sample flows into wells via the
fluidic path until it reaches a dead-end. Once the sample stops
automatically, the sample wells are slipped into reagent wells to
initiate reaction. When loading hundreds of wells with different
volumes, it is preferable to make sure all wells will be filled.
The dead-end filling facilitates doing so. All wells upstream of
the dead-end are filled completely and the user does not have to
determine when to stop since loading stops automatically when the
sample reaches the dead-end.
[0550] A stackable, rotary SlipChip embodiment offers additional
capability through modularity. Different reagent types, such as wet
and dry reagents, can be stored on different layers of the rotary
device. Further, if standard configurations are used, different
detection systems can be easily mixed and matched simply by
introducing a different rotary layer into the system. The RNA
purification cycle described below can also be used for other assay
types.
[0551] Filling methods include, but are not limited to, pressure
driven well filling, centrifugal force, and dead-end filling.
[0552] In certain embodiments, a sample, first collected in a
larger big well, is preferably transferred to a second step for
processing. Dead-end filling provides both a driving force and a
stopping mechanism to transfer the sample. That is, connecting such
a sample to a controlled pressure source drives it to desired
channels or wells from the first layer to the second layer and then
stopping automatically without leaking when it reaches the end. It
restarts when it is connected to an opening in a third layer. The
pressure source can be as simple as an air-loaded syringe. This
method is not limited to filling within one layer of a rotary
system. It can fill different layers through holes by controlling
the pressure.
[0553] Certain embodiments of the SlipChip provide a platform for
storing solutions and dry reagents for use for POC diagnostics.
[0554] Experiments show that nanoliters of solution can be stored
for greater than six months in fluorocarbon in a plastic
SlipChip.
[0555] Water adsorption can be reduced by adding an external drying
agent, or adding a desiccant trap in the chip between the wet areas
and the dry areas to minimize crosstalk through fluorocarbons.
Alternatively, one can modify the designs so the reagents are
loaded dry and the device is configured to allow later addition of
a solvent.
[0556] The reagents and enzymes used in amplification assays can be
freeze-dried, optionally in the presence of stabilizing agents
(e.g., at protein:trehalose:mannitol ratio as 1:20:100) using known
freeze-drying methods. Reagents can be stored, for example, dry,
under mineral or fluorocarbon oil, stored in air or sealed under
vacuum.
[0557] A possible design of a SlipChip: 4 stacked layers (numbered
1 to 4, starting on the top), layers 1 and 2 together form a
SlipChip, as do layers 3 and 4. Layer 2 has a hole in the bottom
for transferring samples into layer 3. One can use standard
positions of the through-hole inlets/outlets so any two SlipChips
can be integrated with one another.
[0558] In certain embodiments, a sample can be pre-stored in a big
well on-Chip. Surrounding the well with lubricating oil (such as FC
or paraffin oil) can prevent evaporation. When pressure is applied
through an inlet, a sample flows into wells via the fluidic path
until it reaches a dead-end. Once the sample stops automatically,
the sample wells can be slipped into reagent wells to initiate
reaction. When loading, for example, hundreds of wells with
different volumes, it is preferred to make sure all wells will be
filled. The dead-end filling design can be used to do so. In this
design, all wells are filled completely and a user does not have to
determine when to stop since loading stops automatically when the
sample reaches the dead end.
[0559] One can array the droplets into the wells in a SlipChip,
which may be done manually or robotically. One can use an
alternative self-arraying design where droplets from the
chemistrode, or from other sources of plugs (for example, those
formed using the techniques described in U.S. Pat. No. 7,129,091,
incorporated by reference herein in its entirety) are flowed into
the chip and trapped spontaneously by known droplet trapping
mechanisms. Wu, L.; Li, G. P.; Xu, W.; Bachman, M., Appl. Phys.
Lett. 2006, 89, Boukellal, H.; Selimovic, S.; Jia, Y. W.;
Cristobal, G.; Fraden, S., Simple, robust storage of drops and
fluids in a microfluidic device. Lab on a Chip, 2009. 9(2): p.
331-338 and Hong Shen, Qun Fang and Zhao-Lun Fang, A microfluidic
chip based sequential injection system with trapped droplet
liquid-liquid extraction and chemiluminescence detection, Lab Chip,
2006, 6, 1387-1389, describe methods for droplet-trapping, and all
are incorporated by reference herein in their entirety.
PCT/US2008/001544, published as WO2008097559A2, and U.S. Pat. No.
7,556,776 are also incorporated by reference herein in their
entirety Certain embodiments of the Slipchip can be used in
combination with these techniques, for example by creating discrete
volumes using these techniques and then slipping reagents on top of
them.
[0560] Possible applications of a SlipChip include, but are not
limited to: detecting viral pneumonias; using ELISA to detect
cardiac markers, including but not limited to GPBB, myoglobin,
CK-MB and Troponin T; testing food, including, for example, milk,
wine, baby formula, barley, beans, dried fruit, fruit juice,
grains, maize, milk, dairy food, nuts, rice, grain, wheat, beef,
meat, seafood, chicken, dog food; testing food for the presence of
antibiotics (for example, chloramphenicol), pesticides (including
for example organophosphate pesticides (assayed by cholinesterase
inhibition), endrin, perthane, carbaryl, tetradifon, diphenylamine,
aldrin, dieldrin, benzene hexachloride, chlordane, chlordecone,
DDT, DDE, TDE, dicofol, ethylene dibromide, heptachlor, lindane,
and/or mirex), natural toxins (including, for example, aflatoxin,
ochratoxin and/or mycotoxin), residues, and allergens (including,
for example, almond, egg, gliadin, hazelnut, milk, mustard,
seafood, peanut or soy residues); testing for sulfites in shrimp;
testing for salmonella, listeria, and/or E. coli; testing for
deoxynivalenol (DON), fumonisin, T-2/HT-2 toxins, zearalenone,
histamine, patulin; blood typing; using PCR for Influenza A
Subtyping (including H1N1) HAI Screens (including MRSA and/or VRE),
testing for cystic fibrosis, newborn screening, cancer prognosis,
gene expression clustering, ADME/Tox pharmaceutical R&D
screening, sepsis detection, HBV/HCV/HIV blood donor screening, HCV
quantitation, HIV subtyping, HIV quantitation, HIV drug resistance,
HPV subtyping, running the Ashkenazi panel, prenatal screening of
chromosomes, e.g., chromosomes 13, 18, 21, X and Y, avian flu
strain subtyping, cancer diagnosis, cancer recurrence detection,
organ transplantation typing, organ transplantation monitoring,
high-throughput screening; molecular testing of blood for
infectious diseases; genotype/viral load testing; quantitative
measurement of viral load in infected patients (HIV, HCV); testing
for sexually transmitted disease including chlamydia/gonorrhea/HPV
and drug resistance; prognostics (e.g., drug effectiveness);
pharmacogenomics and theranostics (pharmaceutical/diagnostic
pairings); using PCR to test for, for example, chlamydia and/or
gonorrhea, mycobacterium tuberculosis, HCV quantitation, HIV drug
resistance testing, HBV in blood donations, HCV/HIV in blood
donations, drug metabolizing enzymes, Factor II (prothrombin),
Factor V leiden, HPV genotyping, gardnerella, trichonomonas,
vaginalis and candida spp., legionella pneumophilia, MRSA,
Staphylococcus aureus, Group B Streptococci; using immunoassays to
test for Group A Streptococci, Group B Streptococci, West Nile
(WNV), Cytomegalovirus, Cystic Fibrosis Screening; B-Cell Chronic
Lymphocytic Leukemia Chromosomal 8 enumeration (CML, AML, MPD, MDS,
for example), HER-2 Status, initial diagnosis and recurrence
monitoring of bladder cancer, sex mismatched bone marrow transplant
testing, detecting mutations in HIV-1 virus associated with drug
resistance; real-time tests for infectious diseases and FISH tests
for certain types of cancer, including cervical, esophageal and
melanoma; active screening to identify patients colonized with
MRSA; genetic tests for hereditary diseases, including breast and
ovarian cancer, hereditary melanoma; testing for adenomatous
polyposis syndromes; testing for hereditary nonpolyposis colorectal
cancer (HNPCC); chemical Q&A testing, including testing active
ingredient presence and/or quantity and/or for contaminants;
testing pesticides; testing fertilizers; testing petroleum;
industrial fermentation process control; testing water, fruit,
vegetables, food, soap oils, milk, dairy foods, beverages, eggs;
screening for and/or analyzing irregular proteins or amino acids,
free fatty acids, lactic acid, peroxides, ammonia, chloride,
glucose, phenols, urea; test for campylobacter; analyzing marine
algae; testing in slaughter houses and farms; blood tests for
colorectal cancer monitoring; skin patch cocaine testing for
professional drivers; pneumonia panel testing (using, for example,
RT-PCR) for mycoplasma pneumonia, Chlamydia, pneumonia and
legionella pneumonia; screening newborns for, for example, common
phenylketonuria, sickle cell disease, and hypothyroidism; and
testing for BNP/Pro, hs-CRP, or homocysteine. In addition, a point
of care test for C-Reactive protein on SlipChip may be used for
monitoring pain during therapy and during clinical trials.
[0561] Organisms that can be detected in certain embodiments of the
SlipChip using, for example PCR and/or immunoassay methods known to
those skilled in the art include, but are not limited to:
Streptococcus pneumoniae, Haemophilus influenzae type b,
Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,
Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella
pneumophila, Streptococcus agalactiae, Mycobacterium tuberculosis,
Klebsiella pneumoniae, Moraxella catarrhalis, Chlamydophila
psittacci, Streptococcus viridans, Coxiella burnetii, Cryptococcus
neoformans, Enterobacter, respiratory syncytial viruses (RSV),
influenza viruses (A and B), Human parainfluenza viruses,
cytomegalovirus (CMV), Human rhinovirus (HRV), Coronavirus (e.g.,
SARS), adenovirus, metapneumovirus, Herpes simplex viruses, Human
bocavirus, Giardia lamblia, Cryptosporidium parvum,
enteroaggregative Escherichia coli (EAggEC), Vibrio cholerae,
Shigella dysenteriae type 1 (Sd1), enterotoxigenic E. coli (ETEC),
Entamoeba histolytica, Campylobacter, Salmonella, Clostridium
difficile, rotavirus, norovirus, adenovirus, and astroviruses.
[0562] Devices and methods that use certain embodiments of the
SlipChip to isolate or capture targets such as, for example, rare
cells or beads carrying cells of interest out of samples such as,
for example, bodily fluids are described. Such devices and methods
are preferred for the downstream analysis of captured targets and
the samples that carry them. For example, rare cells, particles
such as beads or aggregates, or molecules can be captured out of
bodily fluids including, for example, blood, saliva, breath vapor,
tears, CSF, or urine, or from other samples, including soil
suspensions, environmental water samples, tissue homogenates,
gasses, liquids, solids or gels. The approach is beneficial when
analyzing samples with low concentrations of analytes, for example
organisms, organelles, molecules, macromolecules, DNA, protein, and
carbohydrates, rare nucleic acids or proteins, markers and
biomarkers of genetic or infectious disease, environmental
pollutants, cells or vesicles, including host cells such as
epithelial cells, circulating tumor cells, cells of the immune
system, red blood cells, platelets, exosomes, microvesicles,
non-host cells, including fetal cells and sperm. (See e.g., U.S.
Ser. No. 10/823,503 incorporated herein by reference). Another
example includes the analysis of rare cells, such as circulating
cancer cells or fetal cells in maternal blood for prenatal
diagnostics. This approach may be beneficial for rapid early
diagnostics of infections by capturing and further analyzing
microbial cells in blood, sputum, bone marrow aspirates and other
bodily fluids such as urine and cerebral spinal fluid. Analysis of
both beads and cells may benefit from stochastic confinement (See
e.g., PCT/US08/71374 incorporated herein by reference).
[0563] Isolation or capture of targets is important for
characterization or analysis of a wide variety of systems. One
example is analyzing rare cells in bodily fluids--an example that
is important for applications including, but not limited to, cancer
(circulating tumor cells (CTCs), invasive tumor cells in draining
lymph nodes), immunity (CD4 counts, antigen-specific cells, etc),
infection (microbial cells), prenatal diagnostics (fetal cells or
nucleated red blood cells in maternal blood) and stroke
(transcriptionally-altered peripheral blood mononuclear cells
(PBMCs)). A "rare cell" can be either a cell of one type (e.g.,
CTCs) in a mixture with an abundance of cells of other types (e.g.,
stromal cells, lymphocytes), or can be a cell with an unusual
phenotype or genotype (e.g., upregulated transcription) in a
mixture of normal cells of the same type (e.g., PBMCs).
[0564] Isolation or capture of CTCs can be important for cancer
diagnostics and monitoring. Metastases are the major cause of death
from cancer because they are often resistant to conventional
therapies (there is much heterogeneity in cancer cells in
metastases). CTCs are cells that have detached from a main tumor
and circulate in the blood stream. When adhering to other tissues,
they can act as a seed for growth of additional tumors by creating
a microenvironment around themselves in the invaded tissue. CTCs
are observed at very low concentrations in the blood (between one
cell in 10.sup.6 to 10.sup.9). The amount of CTCs varies
considerably in different cancer types, with some cancers having no
CTCs in most cases (ovarian cancer) to others having CTCs in nearly
every case (breast cancer). Much recent research has focused on
methods for improving the detection of such cells, and much
progress has been made (see Current methods section below).
However, most of these methods provide only enumeration of CTCs,
while a few can provide analysis by PCR or staining. In the present
invention methods and devices that can be used to capture CTCs for
a wide variety of downstream analyses and manipulations are
described.
[0565] While CTCs can provide information from the blood stream,
analysis of solid tumor samples or lymph node biopsies can provide
information about the primary tumor. However, solid tumor biopsy
samples are often limited to those from a fine needle aspirate or
fine needle biopsy, due to the difficulty of accessing an
internally located tumor without risk or major inconvenience to the
patient. These samples may provide as few as 200 cells, including a
mixture of tumor cells and stromal or lymphoid (non-tumor) cells.
There is a need to capture or isolate the tumor cells from these
samples and provide multiplexed analysis, despite small numbers of
target cells. Similarly, there is a need for rapid capture and
analysis on an intraoperative timeframe (<40 min) to determine
whether samples such as sentinel lymph node biopsies are
metastatic, thus alleviating the need for a second surgery in
positive cases. The present invention provides methods and devices
to capture and isolate tumor cells from these samples despite the
presence of a large number of stromal or lymphoid cells, and
enables rapid analysis and manipulation such as PCR for mutations
such as in kRAS2 (common for solid tumors), or RT-PCR for specific
mRNAs including MUC1 for breast cancer.
[0566] Another important application of capture and isolation is
analysis of the immune system. The human bloodstream contains
several million cells per mL, including T- and B-lymphocytes,
monocytes, dendritic cells, neutrophils, and red blood cells, in
addition to >10.sup.8 platelets per mL. For conditions such as
cancer as well as autoimmunity, allergy, and infection, the
frequency of T-cells that are specific for a particular antigen
(tumor antigens, self-antigens, allergens, or antigens from
pathogens, respectively) is often predictive of disease
progression. However, these cells are quite rare, occurring at a
frequency of 0.002% to 0.2%, or 2 in 1,000 to 100,000 cells.
Current methods for analysis focus on enumeration (flow cytometry,
ELISPOT) and offer little further analysis. The present invention
provides devices and methods to capture and isolate such cells
(e.g., by affinity capture with MHC-antigen complexes, by screening
for antigen-stimulated cytokine secretion, etc.) and provides
downstream analysis including, but not limited to, PCR, further
stimulus-response assays, and culturing. Such methods can provide
insight into the molecular mechanisms of tumors, autoimmunity and
other conditions. For example, it can be used to determine whether
T-cells that are specific for self-antigens are also more sensitive
to stimulation by cytokines, thus aggravating autoimmune responses.
SlipChip may be used to perform assays for all of the applications
for which ELISPOT technique can be used. Stochastic confinement on
SlipChip would provide more rapid and sensitive assays.
[0567] Some current methods provide capture of targets but have
provided little or no downstream analysis or processing. Current
methods for capture include, filtration by size or morphology,
affinity capture, such as with antibody-coated magnetic beads or
rods (e.g., Cell Search, MagSweeper, or RoboSCell technologies),
microfluidic posts (e.g., CTC-chip or exosome capture), or
microfluidic channel walls, functional capture, by unique behaviors
including metastatic invasion of collagen adhesion matrices,
negative selection by removing all other targets, capture by
magnetic, optical, other properties (e.g., by dielectrophoretic
field-flow fractionation or photoacoustics), and screening all
targets visually and collecting those of interest, including by
flow cytometry, fiber optic arrays, or laser scanning
(Laser-Enabled Analysis and Processing, LEAP.TM., by
Cytellect).
[0568] In contrast to the above-listed methods, certain embodiments
of the SlipChip enable a multitude of upstream or downstream
applications, including combining upstream sample preparation with
capture and downstream multi- or single-cell analysis and
manipulation. Examples of the types of analysis that can be carried
out include, but are not limited to, PCR and other nucleic-acid
based tests, immunoassays, staining, including immunostaining,
histological staining, and mass-spectrometry. Procedures that can
be carried out after isolation include, but are not limited to,
cultivation, including cultivation of single cells, pure cultures
(one cell type), mixed co-cultures, or spatially-organized
co-cultures, stimulus-response assays, including but not limited to
antigen, pathogen, or cytokine challenges, receptor binding and
chemotaxis assays.
[0569] Targets can be selected by size or morphology, for example
by filtration. For example, samples can be passed through a
filtration device by a process such as aspiration or flow.
Filtration devices, such as sieves or porous membranes, retain
targets larger than the filtration in the capture area. They can be
used to isolate larger targets, or to remove material from smaller
targets of interest. Captured targets can then be slipped into an
analysis area for further manipulation. Reagents for detection of
targets of interest can be included at various stages, including
being mixed with the sample before filtration, or being preloaded
on the device, as shown below. A filter (for example one with
submicron sized pores) can be placed in a channel, in a channel,
the sample can be flowed through the filter, and then a wash,
preferably smaller in volume than the original sample, can be
flowed in the reverse direction to resuspend what the filter
collected.
[0570] Capture by hydrodynamics can be used, for example, for
samples containing targets with hydrodynamic properties distinct
from other constituents of the sample. For example, arrays of
nanopillars have been used to separate objects according to their
hydrodynamic and diffusion properties. Differences in hydrodynamics
of objects moving next to a boundary are also well established. A
skimming method in which small cells were able to go into narrow
side channels, and large cells were not (also useful for separating
plasma from cells) has been described. Encapuslation of cells into
droplets followed by sorting hydrodynamically to exclude empty
droplets and collect cells of desired size has been described.
Targets can be sorted by utilizing density changes. For example,
species could be encapsulated in droplets with a detection reagent,
such that targets in droplets produce a molecule that makes the
droplet less dense and cause the droplet to float to an upper
portion of the SlipChip. Any of these methods can be utilized on
SlipChip, and then the captured targets can be slipped to another
area for analysis and manipulation.
[0571] Capture by electrical, optical, magnetic and other
properties can be used, for example when targets themselves
inherently have distinct electrical, optical, magnetic properties,
or when those properties can be induced. For example, selective
binding of magnetic particles to microorganisms changes the
organisms' magnetic properties, and may be used to separate those
organisms from the rest of the sample using magnetic fields.
[0572] Targets of interest can be captured by their affinity for a
capture agent, which can be either specific or non-specific for the
target of interest. In certain embodiments, the bulk of the sample
is not captured by the device, while the desired targets, such
microorganisms, cells, or molecules can be preferentially bound and
enriched.
[0573] Capture agents (or capture elements) can include affinity
reagents, including antibodies, aptamers, non-specific capture
agents, including for example a hydrophilic patch to which a
droplet or cell can stick and others described herein. Several
capture elements can be patterned on the same plate. For example,
one row can be patterned with capture agents against bacteria,
another row with capture agents against fungi. After assembly of
the plates, detection reagents against bacteria and fungi can be
added to the corresponding areas, to detect bacteria and fungi from
the same sample.
[0574] Targets of interest can be captured by a unique behavior.
For example, cells can be loaded into SlipChip wells coated with a
substance such as a collagen adhesion matrix. Metastatic cells will
migrate into the gel, while other cells will not. Other cells can
be washed away and the gel dissolved, leaving metastatic cells
isolated in wells that can be slipped to another area for
analysis.
[0575] The SlipChip is also capable of arraying species (cells,
beads, etc) across the different areas of a chip, and then applying
detection agents to all of them in order to identify the location
of the desired targets. These can then be isolated by slipping to
another area for further analysis and manipulation. For example,
all cells in a sample can be loaded into wells, then identified
with a labeled affinity reagent (such as a fluorescently-labeled
antibody for markers of interest, such as CD4 or EpCAM. Those wells
containing labeled cells could then be slipped into an analysis
area for further analysis or manipulation, for example, by PCR,
cell culture and/or immunoassay.
[0576] One can carry out off-SlipChip binding to carriers with
subsequent capture of carriers on a SlipChip. Carriers can be, for
example, magnetic particles, particles coated with DNA, antibodies,
and/or other targeting molecules. In certain embodiments, the
target of interest binds to the carrier, and the carrier is
captured on a SlipChip by a capture area. Binding is accomplished
by methods including, but not limited to, those using affinity,
electrical, optical, magnetic, or other properties. Capture of the
carrier can be accomplished by methods such as those described
above for capture of targets. For example, rare cells in a sample
solution can bind to magnetic beads coated with antibodies, and the
magnetic beads are then captured in SlipChip wells adjacent to a
magnet.
[0577] Capture can be done in either a closed device (two or more
plates together) or an open device (one or multiple plates
separately exposed to sample). For a closed device, sample can be
loaded by several means, including, for example, though an open
hole, through induced flow or through aspiration into a channel.
For an open device, one plate of the SlipChip may act as a filter
or as a capture surface. An advantage of an open device is that
large volumes of sample can be rapidly processed and rare targets
quickly captured. This is useful for targets such as CTCs, which
may be present at rates as low as 0.5-50 cells per mL of blood. In
addition, open devices can be useful for analysis of samples that
may be difficult to load otherwise, for example, aerosols of
bacteria or viruses generated during coughing, or for analysis of
samples on tissue slides, for which keeping track of spatial
relationships among cells is preferred, as is done for, for
example, tumor biopsies. In addition, opening the chip for analysis
by methods that benefit from direct access (for example,
mass-spectrometry) is advantageous. An exemplary method of
collecting material on an open SlipChip comprises, exposing at
least one plate of a SlipChip to a sample, allowing at least one
target to transfer to the plate (for example, by affinity capture
or filtration capture), optionally removing the SlipChip from the
environment, bringing the second plate of the SlipChip into contact
with the first plate, and slipping the plates to bring at least one
area on each plate into contact with one another to induce a
reaction/interaction with the target, for analysis or
manipulation.
[0578] Capture methods can be combined with other techniques
including stochastic confinement, multistep amplification of
detection signals, and visual readouts. For example, targets such
as cells from the sample can be stochastically confined into
separate small volumes that accelerate detection and/or make it
more sensitive. An application includes the stochastic confinement
of immune cells from blood samples into, for example, nanoliter
volumes, followed by slipping the device to perform an immunoassay
for CD4 in order to identify CD4+ cells. This provides a CD4 count.
Identified CD4-positive cells can then be isolated and slipped to
another area for further analysis, such as PCR.
[0579] Capture methods can be combined with downstream analysis and
manipulation, including, for example, stimulus-response assays and
directed crawling assays. Stimulation-response assays are useful
for detection and characterization of cells whose phenotypes are
not apparent under resting conditions, for example for the
detection of liquid tumors. Captured cells can be stimulated, such
as with cytokines, and their response assayed by a set of parallel
analyses and manipulations including ELISA for secreted signals
including cytokines and proteases, staining for phosphorylation
status to determine signaling pathways, PCR, RT-PCR, and culturing.
Directional crawling assays may be used to distinguish cells with
varying phenotypes. For example, metastatic cells crawl rapidly and
directionally when mechanically confined; captured CTCs can be
slipped into channels such as long straight ducts in order to
assess this behavior.
[0580] Similarly, chemotactic gradients can be established, for
example by loading one well of certain embodiments of a SlipChip
with a chemotactic agent and slipping so that it is connected with
another well or a duct, establishing a gradient by diffusion (as in
FID devices and bridging devices). Flow can also be used to
establish gradients. These gradients can be used to analyze
chemotaxis of captured cells, which is relevant to inflammation,
tumor regression and metastasis, autoimmunity, and infections.
[0581] Captured targets that are isolated individually can be
monitored over time, with or without treatment or stimulation,
providing time-resolved single target information that cannot be
obtained in bulk cultures. For example, single cells can be
monitored for proliferation, expression of a reporter (monitored,
for example, by fluorescence) and/or secretion of a signal.
[0582] Wells containing captured cells can be manipulated to
analyze the behavior of the cells. For example, wells can be
analyzed for deposition of extracellular matrices. The surface of
the chip can be modified by micro- or nano-scale topologies, or
with modifications such as chemical surface treatments, to alter
the dynamics and products of extracellular matrix formation. In
another example, stimulants, including but not limited to chemical
or cellular stimuli, can be applied to induce behaviors such as
proliferation or differentiation; this is useful in the study of
many cell types including lymphocytes, monocytes, and stem cells.
Captured cells of different types can be brought together into a
co-culture, either mixed or spatially-defined, in order to analyze
cell-cell interactions. In one example, antigen-presenting cells
can be cultured with T-cells, in order to analyze the dynamics of
the T-cell response to antigen recognition. In another example,
antigen-activated memory T-cells can be cultured in a well that is
fluidically connected to another well via a duct too small for
cells to pass through. Other cells, for example, naive T-cells or
B-cells, or epithelial cells, can be cultured in the other well, in
order to analyze the effects of soluble signals such as
cytokines.
[0583] Hydrophilic bridges can be used in certain embodiments of a
SlipChip to allow for cell-cell interactions by connecting wells.
An experiment to screen antibiotic resistance is described. The
device and methods described here can be used, for example, for
screening antibiotic resistance, for studying cell-cell
communication without bringing cells into physical contact, for
building spatial confined microbial communities, for understanding
diversity and evolution of microecological systems, and for
extracting or separating viruses, bacteria, and/or cells based on
their size, motility and/or chemotaxis.
Experimental Section
Chemicals and Materials
[0584] All solvents and salts purchased from commercial sources
were used as received unless otherwise stated. FC-40 (a mixture of
perfluoro-tri-n-butylmethylamine and
perfluoro-di-n-butylmethylamine) was obtained from 3M (St. Paul,
Minn.). Food dyes were purchased from Ateco (Glen Cove, N.Y.).
Tridecafluoro-1, 1, 2, 2-tetrahydrooctyl-1-trichlorosilane was
purchased from United Chemical Technologies, Inc. (Bristol, Pa.).
Alexa Fluor.RTM. 488 dye (Alexa-488) was purchased from Invitrogen
(Eugene, Oreg.). Soda-.quadrature.lime glass plates with chromium
and photoresist coating were purchased from Telic Company
(Valencia, Calif.). Amorphous diamond coated drill bits were
obtained from Harvey Tool (0.035 inch cutter diameter, Rowley,
Mass.). Fluorescence reference slides were purchased from
Microscopy/Microscopy Education (McKinney, Tex.). Binderclips (
5/32' inch capacity, 1/2' inch size) were purchased from Officemax
(Itasca, Ill.). Pipettors were obtained from Eppendorf Inc.
(Westbury, N.Y.). Fisherbrand pipettor tips were from Fisher
Scientific (Hanover Park, Ill.).
[0585] Chip Design and Fabrication.
[0586] SlipChip was fabricated using glass etching fabrication of
SlipChip as described elsewhere in this application, with the
following modifications. About 25 minutes of etching yielded a
depth of about 30 .mu.m. After etching, the tape was removed from
the plates. The plates were then thoroughly rinsed with Millipore
water and dried with nitrogen gas. The hydrophilic bridge surface
was created by aligning a photomask containing the black patterns
of hydrophilic bridge parts only to the bottom plate, then
following the glass etching fabrication procedure described
elsewhere. Access holes were drilled with a diamond drill bit 0.035
inches in diameter. The surfaces of the etched glass plates were
cleaned with Millipore water, followed by ethanol and subjected to
an oxygen plasma treatment before silanization. As the glass
surface of the hydrophilic bridge pattern was not silanized, it
remained hydrophilic after removing the chromium layer on the
hydrophilic bridge pattern. The plate was then rinsed with
Millipore water and ethanol and dried with nitrogen gas
thoroughly.
[0587] Assembling the SlipChip.
[0588] The SlipChip was assembled under a mixture of FC-40 and 0.4
mg/ml RfOEG. A 50 .mu.l mixture of FC-40 and 0.4 mg/ml RfOEG was
spread onto the bottom plate in a Petri dish, with the patterns
facing up. The top plate was then laid on top of the bottom plate,
with the patterns facing down. The two plates were aligned into
position by moving them relative to each other and then fixed by
using two micro binder clips. The SlipChip was ready for use after
the extra FC-40 on the surface was removed.
[0589] Food Dye Experiments.
[0590] All the solutions used for food dye experiments were
filtered with a 0.45 .mu.m PVDF syringe filter before use. Two food
dyes (blue and yellow, Ateco, Glen Cove, N.Y.) were pipet-loaded
into 20 reagent channels. To load each channel, 10 .mu.L of dye was
pushed through the inlets using a pipette until the dye solution
emerged from the air supply channel. After loading reagents, the
Chip was slipped to align two reagent wells over the hydrophilic
part. The hydrophilic bridge was completely wetted by slightly
slipping the wells left and right. Then two wells were connected by
a wetting layer created by the reagents left on the hydrophilic
surface.
[0591] Diffusion Test Using Fluorescence Dyes.
[0592] The loading procedure was similar to that for the food dye
experiments. Alexa488 (44 .mu.M) and MPTS (400 .mu.M) were
dissolved in 10 mM TRIS buffer. The Alexa488 solution and MPTS
solution were loaded into the device. The 10 inlets in one half of
the device were loaded with Alexa488, each path containing 10
wells. 10 inlets on the other half of the device were loaded with
MPTS. After the wells with fluorescent dyes were connected with
hydrophilic bridges, the diffusion processes were imaged for 3 h in
the dark using a Leica DMI6000 fluorescent microscope with a
10.times.0.4 NA Leica objective and a Hamamatsu ORCAER camera. GFP
and DAPI filters were used to collect Alexa.quadrature.488 and MPTS
fluorescence. An exposure time of 30 ms for both Alexa488 and MPTS
was used.
[0593] Measuring Fluorescence.
[0594] Images were acquired and analyzed using Metamorph imaging
system version 6.3r1 (Universal Imaging). To extract the intensity
of the fluorescent signal, a region of 100 pixels by 100 pixels was
selected in the middle of every well of interest. To calibrate the
microscope, the fluorescent intensity of fluorescence reference
slides for GFP and DAPI were recorded and used for background
correction.
[0595] Data Analysis.
[0596] To calibrate the intensity measurements, the background
intensity was first subtracted from all the fluorescent images. The
intensity of each well was then extracted from the integrated
intensity of a 100 pixel by 100 pixel region located at the center
of each well.
[0597] Antibiotic Screening Experiments with Escherichia coli.
[0598] Escherichia coli with plasmid pDsRed was provided by
Professor Benjamin S. Glick (University of Chicago). Stocks of
cells were stored at -80.degree. C. Before each experiment, stocks
were streaked onto LB agar plates (Difco LB Broth, Miller,
containing 2% (wt/vol) Alfa Aesar agar powder) containing 100
.mu.g/ml ampicillin. Plates were incubated overnight at 30.degree.
C. Colonies were inoculated in culture tubes containing 3 mL of LB
with ampicilin (100 .mu.g/ml) and subcultured overnight at
30.degree. C., 160 rpm. The bacteria culture loaded into the device
was re-inoculated from the overnight culture and cultured to the
log phase. A bacteria cell density of 2.5.times.10.sup.7 cells/ml
was loaded via half of the inlets of the hydrophilic bridge device.
Different concentrations of Chloramphenicol and Kanamycin (0.01
.mu.g/ml, 0.1 .mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml and 100 .mu.g/ml
for each antibiotic) were loaded into the other half of the device.
Air supply channels were sucked dry to allow for air transport for
E. coli growth. After the wells with bacteria and antibiotics were
connected with hydrophilic bridges, the growth of E. coli was
imaged for 16 h in the dark using a Leica DMI6000 fluorescent
microscope with a 10.times.0.4 NA Leica objective and a Hamamatsu
ORCAER camera. A Texas red filter was used to collect DsRed
fluorescence. An exposure time of 40 ms was used. Images were
acquired and analyzed by using Metamorph imaging system version
6.3r1 (Universal Imaging). To compare and quantify the bacteria
growth, the threshold area percentage was measured for every pair
of wells. This was done by selecting the features in the image by
thresholding and measuring the `red` pixel numbers. The threshold
area percentage represents the percentage of red pixel number over
the whole pixel numbers in the measuring region. Here, the entire
measuring region for every image was the same.
Results
[0599] A SlipChip to perform 10 independent interaction experiments
at the same time was prepared. Each experiment contained 9
duplicate trials. In one trial, two wells (1.5 nL each) are
separated by a submicron-thick hydrophilic bridge which is 300
.mu.m.times.40 .mu.m in size. The top plate containing pairing
wells was aligned with bottom plate containing microchannels and
hydrophilic square patterns. Two rows with pairing wells were
separately loaded with blue solution containing cell A and yellow
solution containing cell B. After loading, the top plate wells are
slipped relative to the bottom plate to break the continuous stream
into compartments and generate pairing wells connected through
hydrophilic bridges to start diffusion. Small molecules diffuse
through the submicron thick hydrophilic bridges. At equilibrium,
both wells were green. Cells A and B do not cross the hydrophilic
bridge, but chemicals they secrete can be exchanged through the
hydrophilic bridge.
[0600] A hydrophilic bridge device was tested with food dye. Blue
and yellow dyes were loaded separately into 20 loading channels.
After slipping, two wells were connected by the hydrophilic bridge.
Bidirectional diffusion of two food dyes between two wells through
the communication hydrophilic bridge was evidenced by a uniform
green color in both columns of wells.
[0601] In another experiment, one set of wells was initially loaded
with MPTS and these were paired with wells filled with Alexa488
(Green). The two dyes diffused towards each other through
hydrophilic surface of connected bridge. Overlaid brightfield and
fluorescent images show diffusion of fluorescent dyes from one set
of wells to the other. Complete mixing was achieved after .about.55
minutes for Alexa488 and .about.45 minutes for MPTS.
[0602] Antibiotic screening was performed in a hydrophilic bridge
device Bright field and fluorescence images showed E. coli growing
in wells on one side of the hydrophilic bridge. Chloramphenicol
(CLR) and kanamycin (Kana) were loaded into the wells on the other
side. Concentrations for each antibiotic were 0.01 .mu.g/ml, 0.1
.mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml and 100 .mu.g/ml. E. coli cells
(density of 2.5.times.10.sup.7 cells/ml) were loaded into the first
set of wells in pairs of columns. Data were analyzed after 16 h
from when E. coli was first exposed to different concentration of
antibiotics. The threshold area for grown E. coli DsRed was
selected and the threshold area percentage was measured for each
pair of wells. The threshold area percentage indirectly represents
the growth difference under different antibiotics
concentration.
[0603] In certain embodiments, fabrication and operation of the
SlipChip does not require lubricating fluid. The SlipChip can be
operated without lubricating fluid dispensed between the plates.
For such "dry" operation, it is preferable that the reaction fluids
have a high contact angle (for example, an angle above 130 degrees)
on the surfaces of the device. This high contact angle can be
achieved via multiple approaches and their combinations, including
the use of nanoporous and microporous polymers, phase separation of
block copolymers, surface coatings, surface roughness and a number
of other approaches, for aqueous solutions these are known as
approaches for creating hydrophobic and superhydrophobic surfaces.
Porous polymers may be used to create superhydrophobic surfaces,
for example as described in Levkin P A, Svec F, Frechet J M J,
Advanced Functional Materials, 2009 19 (12):1993-1998. An example
of SlipChip operating without lubricating fluid is described.
[0604] SlipChips were made from plastics by hot embossing using
glass molds. Fabricating glass molds--A glass mold was prepared by
glass etching. The glass plate (3 mm thick) with chromium and
photoresist coatings (Telic Company, Valencia, Calif.) was covered
by a photomask containing the SlipChip design (patterns were shades
on clear background) and was exposed to UV light for 1 min.
Immediately after exposure, the glass plate was developed by
immersing it in 0.1 mol/L NaOH solution for 2 min. Only the areas
of the photoresist that were exposed to the UV light dissolved in
the solution. The exposed underlying chromium layer was removed
using a chromium etchant (a solution of 0.6:0.365 mol/L
HClO4/(NH.sub.4).sub.2Ce(NO.sub.3).sub.6). As a result, the
patterns in the design were still covered by chromium and
photoresist coatings. The plate was thoroughly rinsed with
Millipore water and dried with nitrogen gas, and the back of the
glass plate was taped with PVC sealing tape (McMaster-Carr) to
protect the back side of glass. The taped glass plate was then
carefully immersed in a plastic container with a glass etching
solution (1:0.5:0.75 mol/L HF/NH4F/HNO3) to etch the bare glass
surface of the plate (areas on the plate where both photoresist and
chromium coatings were removed). A 40.degree. C.
constant-temperature water bath shaker was used to control the
etching speed. By controlling the etching time (.about.55 min), the
etching depth was 60 .mu.m. The photoresist and chromium coatings
that covered the patterns were then sequentially removed by ethanol
and the chromium etchant. Consequently, the non-etched patterns
stood as 60 .mu.m-high pillars. The glass plate with positive
patterns was then coated with another chromium layer. An array of
holes (5 .mu.m by 5.quadrature..mu.m) was formed by ablating the
chromium layer using a Resonetics RapidX 250 excimer laser
operating at 193 nm. The fluence was adjusted to ablate a 150 nm
layer of Cr in a single pulse, without affecting the glass. The
glass was subsequently etched with HF using the Cr as an etch mask.
Resulating holes become posts in the hot embossed plastic piece,
which significantly increases the contact angle. Fabricating
plastic SlipChips--The glass mold was used to emboss the chip
pattern into 1/16'' fluorinated ethylene propylene (FEP,
McMaster-Carr). The chips were embossed at 260.degree. C., 400
lbs/in2 for 20 minutes in a Carver 3889 hot press. The chips were
rapidly cooled to room temperature before pressure was removed.
[0605] In certain embodiments, operation of plastic SlipChips can
be done without lubricating fluid. A dead-end filling method was
adopted to load a dry FEP SlipChip with aqueous solutions.
Following the assembly of the FEP SlipChip in the absence of any
lubricating fluid, the SlipChip was sandwiched between two glass
slides. The top glass slide had access holes aligned to the inlets
of the SlipChip. The "sandwich" was fixed with paper clips.
Solutions were all loaded by directly pipetting a 1 .mu.L volume
into the inlets. The pipette tips were pushed against the inlets
through the access holes in the top glass slide. The loading
process spontaneously stopped when the solution reached the
dead-end. 0.1 M Fe(NO3)3 was used as a reagent and 0.3 M KSCN was
used as a sample. After loading, the top plate of the SlipChip was
slipped relative to the bottom plate and solutions were combined
while the Chip remained sandwiched between the two glass plates
throughout the process. Reaction between Fe(NO3)3 solution and KSCN
solution produced red solution of various complexes including
Fe(SCN)3. No evidence was found for cross-contamination or liquid
residue left behind after slipping, and the red complex did not
form in the ducts.
[0606] In one example of a simple chemical reaction in a dry FEP
device, the two plates of the SlipChip were aligned in the absence
of lubricating fluid to form the fluidic paths for the reagent and
the sample. The reagent and sample solutions were loaded into the
SlipChip via pipetting. The SlipChip was slipped to combine the
reagents with the sample. The reaction progress was monitored by
observing the color change from clear to red.
[0607] In certain embodiments, multivolume stochastic confinement
can be performed on the SlipChip for digital detection by PCR and
other techniques. The inventors have developed a multivolume
stochastic confinement method on SlipChip for quantification of
target species or molecules over a large dynamic range using
digital detection. Detection can be achieved through various
methods, including PCR, cell culture, enzymatic and isothermal
amplification methods. The principal of stochastic confinement is
laid out in the patent application PCT/US/2008/071374, Stochastic
Confinement to Detect, Manipulate, and Utilize Molecules and
Organisms. Potential applications of multivolume stochastic
confinement include, but are not limited to, diagnosing, monitoring
or detecting disease biomarkers, testing environmental or food
samples, and isolating, characterizing, and analyzing cultures or
other biological samples.
[0608] Digital PCR commonly uses microwells or emulsions of the
same volume, so requires very high numbers of compartments (1000's
to millions) to achieve high precision and a large dynamic range.
SlipChip can be designed to perform digital measurements within
wells of multiple volumes. Some advantages of this method over
single volume methods include a large dynamic range with fewer
wells, and increased precision achieved by overlapping ranges for
the different sized wells. Arrays of wells with multiple reaction
volumes can be designed on a single chip to achieve the entire
desired range of detection. The approach is analogous to serial
dilution methods and the statistical analysis can be performed with
the same mathematical calculations. Instead of the multivolume
approach, SlipChip can be used to perform serial dilution followed
by analysis The multi-volume approach has been used in
microbiology, such as the IDEXX Quanti-Tray.RTM./2000, for
detection and enumeration of microbes. These and other applications
can also be implemented on SlipChip. The multi-volume approach can
be applied to digital PCR on SlipChip. Three possible modes of
operation include: (1) Injection of the sample premixed with PCR
reagents into the chip, then compartmentalization via slipping to
perform digital PCR. (2) Separately preloading or user-loading
reagents such as primers, optionally in a multiplexed format, and
then mixing with the sample via slipping to initiate the reaction.
(3) Combinations of the above.
[0609] In addition to standard PCR techniques, SlipChip is
compatible with isothermal amplification techniques such as
loop-mediated amplification (LAMP), recombinase polymerase
amplification (RPA), nucleic acid sequence based amplification
(NASBA), transcription-mediated amplification (TMA),
helicase-dependent amplification (HAD), rolling-circle
amplification (RCA), and strand-displacement amplification (SDA).
The multivolume SlipChip can be used to digitize such platforms.
The multivolume SlipChip could be applied to other systems that are
compatible with stochastic confinement (patent application
PCT/US/2008/071374, Stochastic Confinement to Detect, Manipulate,
and Utilize Molecules and Organisms), including analysis or
detection of cells.
[0610] One example of an application for the multivolume SlipChip
is the measurement of HIV viral load. For HIV viral load
measurements at the point of care, one desired goal is a dynamic
range of 500 to 1,000,000 HIV particles/mL of blood plasma with the
ability to distinguish concentration changes of at least 3 fold
over the entire range. An example of a system that satisfies this
was demonstrated by the inventors. This example is composed of 128
wells of 50 nL volume, 128 wells of 10 nL volume, 256 wells of 2 nL
volume and 512 wells of 0.4 nL volume. The larger number of smaller
volume wells can be used to increase resolution or alternatively
can be used with an internal standard to calibrate the system.
Accounting for two copies of RNA per HIV viral particle, this
design has a lower detection limit of 200 HIV particles/mL and a
dynamic range where 3 fold resolution can be achieved of
600-3,500,000 HIV particles/mL, and will greatly exceed that
resolution over much of the range. This calculation needs to be
adjusted for the effects of sample losses and concentration during
sample preparation, and this can be done for example using an
internal standard detected on the same device using a probe with a
different color, or using different primers preloaded into specific
wells.
[0611] This design could be applied to point of care testing.
Alternatively, measurements in the range of 40-10,000,000
particles/mL might be required. A device can be designed to achieve
this range. One example of such a device uses a total sample volume
of 75 .mu.L for the lower limit of detection and the smallest well
volumes to be on the order of 0.25 nL. Being able to preconcentrate
samples would allow for smaller volumes to be used.
[0612] The multivolume SlipChip method for digital measurements can
also be applied to other diseases where accurate information on
infection load is useful such as for hepatitis B viral load.
[0613] A similar layout to that described above can be used for
other applications such as diagnosing the cause of pneumonia.
Because pneumonia can be caused by many different species, accurate
diagnosis requires a highly multiplexed test to detect the majority
of potential pathogens. It also requires quantification to
differentiate lower levels (corresponding to normal bacterial
colonization of the upper respiratory tract) from higher levels
(corresponding to bacterial infection of the lower respiratory
tract). By splitting the design into 16 equal sections, 16
different species of bacteria and viruses can be detected over an
approximately 1000 fold concentration range. An alternative design
for pneumonia detection would allow for low detection limits for
potential viral species, and a sufficiently large dynamic range for
detection of potential bacterial causes and differentiation of
colonization vs. infection. The design would include eight sets of
12.times.200 nL wells and 12.times.50 nL wells for viral detection.
These sets would have a detection range of about 1000 particles/mL
to about 30,000 particles/mL. It would also include eight sets of
24.times.25 nL wells and 24.times.2.5 nL wells for bacterial
detection and more precise quantification. These sets have a
detection range of about 4000 bacteria/mL up to about 800,000
bacteria/mL, with 3 fold resolution over much of that range. The
detection ranges and designs can be adjusted as necessary to meet
the requirements of the test, including changing well size or
number or preconcentrating the samples being tested. As has been
demonstrated in existing digital PCR literature, this approach can
be used in any application where real-time PCR has been applied.
This approach can combine digital analysis with multiplexing on a
single device, for example, by adding multiple samples (such as
blood, urine, or sputum) or running multiple tests on the same
sample, or a combination.
[0614] To design the devices and analyze the results, several
methods or their combinations can be used. Device design is
dependent both on the desired detection range and the resolution
achievable over the range. One method uses statistical approaches
based on the poisson or binomial distributions, to calculate the
concentration in the form of a "Most Probable Number (MPN)", as
presented in the following equation:
s i v i = ( ( n i - s i ) * v i * ( - v i d ) ) 1 - ( - v i d )
##EQU00002##
Where n.sub.i is the total number of wells at the ith dilution/well
size, s.sub.i is the number of sterile/empty/unreactive wells at
that level, v.sub.i is the fraction of the original sample solution
contained at that level (so a 10 fold dilution or reduction in well
volume by a factor of 10 would give a value for v.sub.i of 0.1),
and d is the original concentration, so the equation needs to be
solved for d.
[0615] The lower limit of detection is dependent on the total
sample volume contained in all of the wells. The upper limit of
detection is set by the sample volume and number of wells at the
smallest volume. Several methods or their combination can be used
to establish the confidence intervals (CIs) for given results and
determine the resolution of the system. Equation-based
approximations are useful because CI values can be obtained
rapidly, but they are only average approximations so may not be
accurate for a given result. They are useful for directing
system/device design, to make sure that the desired performance is
reasonable to expect. Another set of methods that are commonly used
are known as "exact" methods, because they utilize the actual
probabilities for all potential results. These methods are
predominantly based on existing work applied to single
dilution/volume systems commonly referred to as the Clopper-Pearson
(CP) and Sterne methods named for their creators. The CIs can be
used to determine the resolution of a given system, and as this is
dependent on number of wells and the dilution factor, the desired
resolution will also govern well sizes and numbers. The following
inequality is used to determine the factor/fold of resolution:
d1+95% CI for d1.ltoreq.d2-95% CI for d2
When the two sides are equal then d1/d2=X, which is the factor/fold
of resolution, and is typically set to be at most 3 fold in the
examples described throughout.
[0616] Several SlipChip designs can be used to implement
multivolume stochastic confinement, including rotating SlipChip
devices, stacked multilayer SlipChip devices, and devices that
require sliding in one or two directions. Wells of different
volumes can be made in the same layer or by combining wells and
through holes in multiple layers. In addition, wells of different
volume can be made by creating wells of the same depth but
different lateral dimensions, or by varying the depth of the wells.
Keeping the volume constant but increasing the depth of wells
reduces their lateral dimensions and is useful for increasing the
density of wells. For applications that require thermal expansion,
devices can be optionally designed so wells are brought into
contact with reservoirs containing lubricating fluid or another
fluid, as described in recent papers.
[0617] In one example, the device includes 128 wells of 50 nL
volume, 128 wells of 10 nL volume, 256 wells of 2 nL volume and 512
wells of 0.4 nL volume. The larger number of smaller volume wells
can be used to increase resolution or alternatively can be used
with an internal standard to calibrate the system. When considering
a solution containing purified HIV RNA, use of a nucleic acid
amplification technique for detection, and accounting for two
copies of RNA per HIV viral particle, this design has a lower
detection limit of 200 HIV particles/mL and a dynamic range where 3
fold resolution can be achieved of 600-3,500,000 HIV particles/mL.
This design will greatly exceed that resolution over much of the
range. This calculation needs to be adjusted for the effects of
sample losses and concentration during sample preparation, and this
can be done for example using an internal standard detected on the
same device using a probe with a different color, or using
different primers preloaded into specific wells. For PCR
applications, this design optionally includes smaller wells
containing oil that are brought into contact with larger wells
containing aqueous solution. When the smaller wells are brought
into contact with the larger wells, the aqueous solution
spontaneously forms a droplet surrounded by oil in the compartment,
allowing for room for thermal expansion during thermal cycling. The
wells and ducts can be patterned separately on the top and bottom
plates. The wells can be fabricated by the techniques described
elsewhere in this application. In some designs, the wells initially
overlap with ducts to generate continuous fluidic path to enable
filling. Filling can be achieved by using pipetting or other
mechanical or chemical driven pressure. Dead-filling or through
holes as outlets can be used to evenly fill the entire chip. The
SlipChip can be slipped into discrete reaction volumes, for example
by rotational motion of the device, and compartments of different
volumes are generated simultaneously.
[0618] In one example of a multivolume device made in glass, the
device includes 15 wells of each volume, with 135 wells in total;
the volumes are: 0.25 nL, 0.72 nL, 1.95 nL, 5.24 nL, 14.1 nL, 38.1
nL, 103 nL, 278 nL, and 511 nL. This provides a detection limit of
about 200 particles/mL with a dynamic range of at least 3 fold
resolution from about 800-2,400,000 particles/mL. The procedure for
fabricating this SlipChip was based the procedure described in
previous work. In general, the structure was patterned by using
photolithography and then etched by using a glass etching solution
(1:0.5:0.75 mol/L HF/NH.sub.4F/HNO.sub.3). The device was silanized
by dichlorodimethylsilane to provide a hydrophobic surface. A
solution of orange food dye was injected into the device, and wells
of different volumes were generated after slipping.
[0619] In another design the device has 88 large wells, 272 medium
wells and 216 small wells. The design could be applied to
quantification of HIV viral load. Considering two copies of RNA per
HIV particle and well volumes of 50, 5 and 0.5 nL respectively this
design gives a detection limit of about 250 HIV particles/mL and a
dynamic range with at least 3 fold resolution from about
800-3,300,000 HIV particles/mL, with better resolution over much of
the range. The overall range for the individual well sizes has
higher precision due to overlap of detection ranges.
[0620] Two examples of circular SlipChips to perform digital
measurements are described. In the first example a SlipChip
designed to measure HIV viral load contains 88 wells of 50 nL each
(dynamic range 950-2.5.times.10.sup.4 particles/mL), 272 wells of 5
nL each (dynamic range 3.0.times.10.sup.3-3.5.times.10.sup.5/mL),
and 216 wells of 0.5 nL each (dynamic range
3.8.times.10.sup.4-3.3.times.10.sup.6/mL) to give a total dynamic
range (after 4 fold concentration) of 800-3,300,000 particles/mL
with at least 3 fold resolution. In the second example a SlipChip
designed to identify and quantify pneumonia pathogens and
distinguish between bacterial colonization and infection contains
16 regions: 8 regions containing 6.times.400 nL wells and
26.times.50 nL wells for a detection range of about
800-4.times.10.sup.5 particles/mL for detection of viral and
noncolonizing bacterial detection, and 8 regions containing
5.times.400 nL wells and 8.times.50 nL wells and 27.times.5 nL
wells with a detection range of about 10.sup.3-4*10.sup.6
particles/mL for bacterial detection. It can achieve 3 fold
resolution over at least the middle portion of the range to
distinguish infection from colonization.
[0621] For multiplexed detection, the device can be separated into
multiple regions. Different inlets for different samples can be
used to fill each region. In addition, different primers and
chemistries can be preloaded into different regions. The regions
may have the same sensitivity and dynamic range, or different
sensitivity and dynamic range. Different sensitivity is needed, for
example, for multiplexed detections of pathogens in pneumonia,
where a 800-10.sup.5/mL range is needed for low level detection and
moderate quantification, and detection in the range of
10.sup.2-10.sup.6/mL is needed for pathogens such as S. pneumonia
and H. influenzae type b, for improved quantification to
distinguish colonization from infection. For example, in one design
there are 16 regions: 8 regions containing 6.times.400 nL wells and
26.times.50 nL wells for a detection range of several hundred to
about 40,000 particles/mL to detect viruses, and 8 regions
containing 5.times.400 nL wells and 8.times.50 nL wells and
27.times.5 nL wells for a detection range of about 1000 to 400,000
particles/mL to detect bacteria and discriminate between
colonization and infection. The design can be applied to detection
and quantification of pneumonia-causing pathogens.
[0622] The SlipChip is compatible with various readout
technologies, including colorimetric or fluorescence readout. These
readout methods can be applied either in real time or at the end
point. In certain embodiments, the user can use the SlipChip
platform to enrich sample and perform sample preparation from
milliliter scale of sample for further analysis, such as PCR,
isothermal amplification and immunoassays. This method can be
applied together with other SlipChip application to provide means
for diagnostics, monitoring or detecting disease biomarkers, and
testing environmental or food samples. In certain embodiments, the
SlipChip can be used to synthesize composite particles in a
high-throughput or combinatorial manner. SlipChip may be used to
fabricate particles, including solid or hydrogel particles made
from different polymers and hydrogels with many applications,
including surface decoration and protection, food additives,
Sustained Release Capsules, chromatography, flow cytometry, drug
delivery and encapsulation of cells for implantations. Particle
with precise size, shape, and composition have found applications
in MEMS (micro electro mechanical system), photonics, diagnostics,
and tissue engineering. However, the synthesis of such particles
using existing techniques like seed polymerization is time
consuming and expensive. Microfluidics has proved to be a powerful
tool for making spherical particles or non spherical particles, or
even janus particles. However, it is difficult to form arbitrary
shapes or form composite particles with these methods. In general,
SlipChip can be used to make rather arbitrary particles, by using
SlipChip as a mold. Methods include using SlipChip to fill molds,
slip away ducts used for filling areas of the molds and forming
particles. Methods of inducing formation of particles may include
curing using thermal energy, optical, ultra-violet light, chemical
binding agents, and so on. Methods of forming particles, and
materials for fabricating or coating SlipChip molds, may be adapted
from those used by Liquidia Technologies. The use of lubricating
fluid, for example fluorinated lubricating fluid, in the SlipChip
during particle formation may substantially facilitate release of
particles after formation. Slipping of several areas of slipchip
filled with precursor of particle material in contact and then
inducing particle formation can be used to create composite
particles of complex shapes and compositions. Particles may be
released by slipping or by simply dis-assembling the SlipChip.
Particles with gradient properties may be created by bringing
together precursors with different properties.
[0623] In certain embodiments, a SlipChip platform, called the
matrix slipchip, can be used to perform n.times.m reactions with
n+m loading steps. SlipChip designs to mix two, three, and four
components are described. Two experiments with bacterial cells are
described: culturing bacterial cells on the matrix SlipChip and
screening bacteria-bacteria interactions on the matrix SlipChip.
Features to highlight include high throughput: 1024 parallel
experiment in <4 cm.times.4 cm space; save precious reagents and
samples; mixing multiple times with precise time and volume
control; the device is reusable and reconfigurable: after each use,
the device can be opened and washed for second use. An 8 inlet top
plate can be used with a different bottom plate containing a
different number of inlets, such as 8 inlets, 16 inlets and 32
inlets, based on need, since the central design is same; open the
device to extract the content of nanoliter droplets for scale up
culture, detection, etc. or using permeable layers, such as
tape-sealed layers, to access the results of the experiments;
nanoliter aerobic cell culture with sufficient air supply and
without evaporation. or anaerobic culture; air supply channel;
nanopost pattern for oxygen transport; easily generate duplication
for reproduce and improve data quality, a lot of duplication wells
make it possible to extract more products from the device for
further usage and analysis; transfer of beads, cells from wells on
one plate to wells on another plate by gravity or magnetic force;
the device and methods described here can be used for a number of
applications. In particular, the SlipChip could be used as a
platform for performing high throughput screening, especially of
protein crystallization, multiplex genome sequencing, cell-cell
interaction, protein-protein interaction, and drug screening,
etc.
[0624] Matrix SlipChip has a number of additional applications.
ThermoFluor Assays and other assays that reflect protein stability
(for example by monitoring fluorescence of hydrophobic dye akin to
1-anilinonaphthalene-8-sulfonic acid (ANS)) can be used to monitor
stability of protein molecules as a function of temperature or
changes in chemical conditions. These assays are useful to monitor
ligand binding in drug discovery, and optimization of ligand and
buffer conditions for crystallography. It will be obvious to those
skilled in the art that SlipChip and Matrix SlipChip will enable a
number of additional applications, including but not limited to
those marketed by Fluidigm, including measurements of Copy Number
Variation, Gene Expression, Protein Crystallization, Sample
Quantification for Next Gen Sequencing, Single Cell Gene
Expression, SNP Genotyping.
[0625] The Matrix SlipChip was composed of a top plate and a bottom
plate with complementary patterns. It was fabricated by using
soda-lime glass plates with chromium and photoresist coating (Telic
Company, Valencia, Calif.). Microchannels and wells on the glass
plates were made by using standard photolithographic and wet
chemical etching techniques. Briefly, the glass plate with
photoresist coating was aligned with a photomask containing the
design of the microchannels and wells and exposed to UV light for 1
min. The photomask was removed, and the glass plate was developed
by immersing it in 0.1 mol/L NaOH solution for 2 min. The exposed
underlying chromium layer was removed using a chromium etchant (a
solution of 0.6:0.365 M
HClO.sub.4/(NH.sub.4).sub.2Ce(NO.sub.3).sub.6). The plate was
rinsed with Millipore water and dried with nitrogen gas, and the
back of the glass plate was taped with PVC sealing tape
(McMaster-Carr) to protect the back side of glass. The taped glass
plate was then carefully immersed in a plastic container with a
glass etching solution (1:0.5:0.75 M HF/NH.sub.4F/HNO.sub.3) to
etch the glass surface that was exposed after the chromium coating
was removed. A 40.degree. C. constant-temperature water bath shaker
was used to control the etching speed. .about.25 minutes of etching
yielded a depth of .about.30 .mu.m. After etching, the tape was
removed from the plates. The plate was then thoroughly rinsed with
Millipore water and dried with nitrogen gas. Access holes were
drilled with a diamond drill bit 0.030 inches in diameter. The
surfaces of the etched glass plates were cleaned with Millipore
water, followed by ethanol and subjected to an oxygen plasma
treatment before silanization.
[0626] To culture aerobic cells in the SlipChip, a nanopost pattern
was fabricated on the top plate to improve the oxygen supply. To
make the nanopost pattern, after etching the 30 .mu.m patterns, the
top plate was cleaned with water and dried with nitrogen gas. We
utilized the original photoresist and chromium coating still cover
those areas that were not etched. The plate was aligned with a
nanopost photomask and the same procedure was followed as described
above, through the step that removed the exposed underlying
chromium. After removing the chromium coating, the top plate was
immersed in 1:0.5:0.75 M HF/NH4F/HNO3 mol/L HF/NH4F/HNO3 etching
solution, and etched for 30.about.90 s at room temperature
(.about.23.degree. C.) to produce the desired nanopost height over
the surface. Finally, the top plate and the bottom plate (which has
no nanoposts) were rinsed with ethanol to strip the undeveloped
photoresist, and immersed in the chromium etchant to strip off the
chromium coating. The glass was then rinsed with ethanol and
Millipore water and dried with nitrogen gas.
[0627] The glass plates were cleaned and subjected to an oxygen
plasma treatment, and then the surfaces were rendered hydrophobic
by silanization in a vacuum dessicator for 3 hours with
Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane as
previously described. After silanization, the glass plates were
baked in a 120.degree. C. oven for 30 min, rinsed by immersing in a
tank of FC-3283, and dried in a 60.degree. C. oven overnight.
[0628] Before use, the bottom plate and top plate of the matrix
SlipChip were cleaned with soap, Millipore water and 100% ethanol
sequentially, and dried with nitrogen, and placed in clean Petri
dish with the etched pattern facing up. 50 .mu.L FC-40 (3M)
fluorinated oil with 0.4 mg/mL RfOEG3 was spread onto the surface
of bottom plate, and then the top plate was placed (patterned side
down) onto the bottom plate. FC-40 totally wet the silanized
surface and spread between two plates. The two plates were aligned
by slipping them relative to each other and then fixed by using two
micro binder clips. The SlipChip was ready for use after the extra
FC-40 on the surface was removed. Both plates of the Matrix
SlipChip contained elliptical wells. The wells were 200 .mu.m wide
and 400 .mu.m long, etched to be 30 .mu.m deep, with volume of
approximately 2 nL. Connecting microchannels were 860 .mu.m long
and 80 .mu.m wide, with depth of 30 .mu.m. Before loading solution,
the oil in the channels and wells were sucked up by applying vacuum
at the inlet of the device. Four food dyes (red, orange, green, and
blue, Ateco, Glen Cove, N.Y.) were diluted .about.20 times from
their stock solutions and were filtered with a 0.45 .mu.m PVDF
syringe filter before use. Solutions were pipet-loaded into wells
in 32 columns from 8 inlets. To load each channel, 8 .mu.L of dye
was first pushed through the inlet using a pipette until the dye
solution emerged from the outlet. After loading reagents, the top
plate was first slipped down and then slipped left, to form
continuous fluidic paths in rows. The same four food dye solutions
were loaded through the 8 inlets from the left side to fill 32 rows
of wells. Using a pipette, 8 .mu.L of dye was loaded into the Chip
until all the channels in row were fully filled. Once the rows was
loaded, the top plate was slipped again to mix the 1024 wells in
columns on the top plate and 1024 wells in rows on the bottom
plate.
[0629] The inventors designed a following 3-component and
4-component matrix SlipChip to incorporate mixing of more than 2
components in one compartment. The food dye experiments were
performed with the similar procedure described for the 2-component
Matrix SlipChip, except an extra washing step was needed to load
two sets of adjacent wells using the same connection channels.
[0630] In the step by step operation of three components mixing
matrix SlipChip, a first set of wells in the bottom plate are
filled. Optionally, the chip is slipped and the same ducts are used
to fill the second set of wells in the bottom plate. SlipChip is
slipped (e.g. in X and Y directions) so that the horizontal rows
are aligned, and the wells in the top plate are filled and the
SlipChip is slipped so that the wells overlap, combining solutions
in the two adjacent bottom wells with the solution in the top
well.
[0631] In the step by step operation of four components mixing
matrix SlipChip, a first set of wells in the bottom plate are
filled. Optionally the chip is slipped and the second set of wells
is filled. SlipChip is slipped so that the horizontal rows are
aligned, and the first set of wells in the top plate is filled. The
SlipChip is slipped so that the second set of horizontal wells in
the top plate is filled. The connecting channels were first washed
with buffer. The SlipChip is slipped so that the wells overlap,
combining solutions in the two adjacent bottom wells with the
solutions in the two adjacent top wells.
[0632] In the step by step operation of four component matrix
SlipChip using food dyes the first step was loading the first set
of vertical wells. The second step was slipping to fill the second
set of vertical wells followed by slipping to align the horizontal
wells and ducts. Then the first set of horizontal wells were filled
followed by the slipping and filling of the second set of
horizontal wells and finally slipping to combine solutions in 4
wells.
[0633] The success of culturing different bacteria cells (including
aerobic or anaerobic strains) on SlipChip is fundamental for
further study of cell-to-drug screening, bacteria antibiotic
resistance, bacteria quorum sensing, and multi species community
interactions, etc. Compared with conventional methods, the matrix
SlipChip can use nanoliter volumes to observe single cell or small
group of cells, increase the throughput, and save time and
reagents.
[0634] To be able to culture and grow aerobic bacteria in nanoliter
wells in matrix SlipChip, the user needs to continuously supply
oxygen to these wells. This was achieved on the slipchip by the
following features: To culture cells in the isolated wells, the
inventors connected horizontal wells and channels and loaded the
resulting fluidic path with air to form a breathing channel. Each
isolated well could get its oxygen supply from 2 nearby breathing
channels. The distance between the well and breathing channel was
240 .mu.m. The matrix SlipChip used FC-40 as lubrication oil, which
has a very high solubility of oxygen and good oxygen permeability.
A nanometer to micrometer thick FC-40 film can support the
transportation of oxygen. Since oxygen supply efficiency of the
breathing channel is limited by the thickness of the FC-40 film
between two plates, the inventors fabricated a nanoposts pattern on
the top plate. This increased the thickness of the FC-40 film from
estimated 500 nm to 1.5 .mu.m. As shown in Error! Reference source
not found.D) to F), this increase efficiently increased the growth
of E coli DS red cell in SlipChip.
[0635] The homogeneity of culture in SlipChip was tested as
describe in the following:
[0636] Escherichia coli with plasmid pDsred was obtained. Stocks of
cells were stored at -80.degree. C. Before each experiment, stocks
were streaked onto LB agar plates (Difco LB Broth, Miller,
containing 2% (wt/vol) agar powder, Alfa Aesar) containing 100
.mu.g/ml Ampicillin. Plates were incubated overnight at 30.degree.
C. Colonies were inoculated in culture tubes containing 3 mL of LB
media with Ampicilin (100 .mu.g/ml) and subcultured overnight at
30.degree. C., 160 rpm. The bacteria culture that was loaded into
the device was re-inoculated from the overnight culture and
cultured to the log phase. When loading cells into the device, the
bacteria cell density was adjusted to 1.1.times.107 cells/mL to
obtain .about.22 cell per well.
[0637] The 32.times.32 matrix SlipChip was prepared as described
previously. The cell suspension was shaken before pipette loading
from 8 inlets in the top plate. 8 .mu.L of cell suspension was
loaded into each inlet. After loading, the device was slipped to
disconnect wells in column from channels and connect the channels
and wells in rows to serve as air supply channels. The oil in the
air supply channel was removed with a vacuum to allow for air
transport for E. coli growth.
[0638] The micro binder clips were removed, and the 32.times.32
matrix SlipChip was carefully placed into a Petri dish. 2 small
caps with 50 .mu.L FC-40 and one small cap with 100 .mu.L H2O was
kept in the Petri dish beside the SlipChip to supply moisture in
the dish. The Petri dish was wrapped with Parafilm to avoid escape
of moisture.
[0639] The growth of E. coli was imaged every 2 hours for 16 hours
in the dark using a Leica DMI6000 fluorescent microscope with a
10.times.0.4 NA Leica objective and Hamamatsu ORCAER camera. Texas
red filter was used to collect Dsred fluorescence. An exposure time
of 40 ms was used. To calibrate the microscope, the fluorescent
intensity of a fluorescence reference slide for the Texas red
filter was recorded and used for background correction. Images were
acquired and analyzed by using Metamorph imaging system version
6.3r1 (Universal Imaging) with multi-dimension acquisition
function. To compare and quantify the bacteria growth, a measure
circle was drawn to cover the well and the integrated fluorescent
intensity with background substrate was measured for every well.
The 32.times.32 matrix of wells were grouped as 16.times.16 units,
each with 2.times.2 wells, and the average intensity for each unit
was gathered for 3 different devices (no nanoposts, 426 nm
nanoposts, and 940 nm nanoposts, respectively). The results
qualified that the nanopost pattern can improve the growth of E.
coli on the matrix SlipChip.
[0640] For cell culture on a device with a breathing channel there
were vertical isolated wells loaded with bacteria culture and
horizontal wells and channels connected and loaded with air to
supply oxygen to the bacteria wells. The nanoposts on the top plate
accelerated oxygen exchange. The nanoposts are 20 .mu.m by 20 .mu.m
in size and 900 nm in height, the spacing between nanoposts are 80
.mu.m. The nanoposts will maintain a gap of greater than 1 .mu.m
that is filled with FC-40 oil within the device. This oil is air
permeable and accelerates the exchange of oxygen from breathing
channel and bacteria wells. Different nanopattern heights were used
to culture E. coli DS red: no nanoposts; 426 nm nanoposts; 940 nm
nanoposts. With increase of nanopost height, there is better and
more even growth in the device.
[0641] A 32.times.32 matrix SlipChip with 16 inlets (each inlet
distributed solution to 2 columns) was prepared as previously
described. The device was aligned so that the fluidic paths formed
in columns. Three antibiotics, Chloramphenicol, Kanamycin, and
Streptomycin, were dissolved in LB broth media with different
concentrations (0.01 .mu.g/mL, 0.1 .mu.g/mL, 1 .mu.g/mL, 10
.mu.g/mL and 100 .mu.g/mL for each antibiotic), and were loaded
into the wells in columns. The device was then slipped to connect
wells in rows to load Escherichia coli with plasmid pDsred. E. coli
was cultured as described in the previous part. The bacteria cell
density was counted and adjusted to .about.2.4.times.10.sup.7
cells/mL to obtain about 48 E. Coli cells in each well. Then the
device was slipped to bring wells on the bottom plate with E. coli
into contact with the wells on top plate with antibiotic solution.
The device was kept still for 30 min with top plate facing down, so
that majority of E. coli cells were settled down in the well in the
top plate by gravity. Then the device was slowly slipped so that
continuous fluidic paths were formed in rows again to serve as air
supply channels. The solutions in the air supply channels were
removed with vacuum to allow air transport for E. coli growth.
[0642] The matrix SlipChip was put into a Petri dish and growth of
E. coli was imaged for 16 hours as previously described. The same
data analysis was carried out for every well at time point of 16
hour and the intergrated fluorescent intensity from E. Coli cells
were plotted as a gray scale map. For each antibiotic concentration
and the control without antibiotics, there are 64 wells in 2
parallel columns. The average intensity of these 64 wells was
plotted.
[0643] A control experiment on 96-well plate was carried out for
the same cell sample and antibiotic concentrations. Basically, 100
.mu.L aliquot of cell suspension was added into the wells, then 100
.mu.L of antibiotics with different concentrations. The OD unit was
measured in a microplate reader at 0 h and after 16 hours. A E.
coli growth inhibition breakpoint similar to that obtained with the
matrix SlipChip was seen for all three antibiotics.
[0644] For the antibiotic screening in 32.times.32 matrix SlipChip,
after 16 hrs, integrated intensity indicate growth of E. coli on
32.times.32 SlipChip after 1:1 mixing with control (LB broth media)
and three antibiotics (Chloramphenicol, Kanamycin and Streptomycin)
with different concentrations. Concentrations for each antibiotic
were 0.01 .mu.g/ml, 0.1 .mu.g/ml, 1 .mu.g/ml, 10 .mu.g/ml, 100
.mu.g/ml. The initial E. coli cells density was 2.4.times.10.sup.7
cells/mL. Data were analyzed after 16 hours of incubation. The
average fluorescent intensity from E. coli after cultured 16 hour
with different antibiotics concentration. The breakpoint represents
the growth difference under different antibiotics
concentration.
[0645] In certain embodiments, analog-to-digital conversion of
concentration with visual or cell-phone readout can be performed on
the slipchip. Using chemistry with threshold can convert analog
readout to digital readout. A definition of a threshold can be
found in patent application Stochastic Confinement to Detect,
Manipulate, And Utilize Molecules and Organisms (Pub. No.
WO/2009/048673, International Application No. PCT/US2008/071374).
An analog readout, in the case of assays, is a signal that
corresponds to the amount of a certain substance, is expressed on a
continuous scale, and therefore requires equipment to read. A
digital readout is expressed as a digit, which, in this case, is a
yes/no value (yes being above the threshold value and no being
below the threshold value). Such analog-to-digital conversion, when
coupled with assays that give visual readout, can be performed in a
SlipChip without special equipment. The threshold-based
analog-to-digital conversion allows the result to be realized and
semi-quantified by the naked eye or to be captured by a simple
camera, such as a cell phone camera which can send the picture out
for further analysis or storage. This approach works with various
assays and various threshold chemistries. Particularly, the
inventors have demonstrated two types of threshold chemistries,
with enzymes and with gold nanoparticles (Au NPs). Enzyme: A
threshold exists when an inhibitor binds tightly to an enzyme and
inhibits the enzyme from performing the catalysis function. When
there is a small amount of enzyme, there will be enough inhibitor
to inhibit all the enzyme molecules from performing the catalytic
function. When there is a larger amount of enzyme, there will not
be enough inhibitor to suppress the enzymatic reaction. As a
result, for a certain amount of inhibitor, there will be a
threshold, meaning that only when the enzyme concentration exceeds
that threshold can the inventors observe a signal. Thus, the
threshold position depends on the amount of inhibitor. Here the
inventors used the inhibitor syn-(S)-TZ2PIQ-A5.sup.1 which binds
tightly to acetylcholinesterase (AChE) in a 1-to-1 ratio. The
threshold amount of AChE is set by the amount of inhibitor. AChE
hydrolyzes acetylthiocholine to give out thiocholine. Thiocholine
reacts with stach/I.sub.2 complex. The reaction causes a color
change from dark blue to clear. Gold nanoparticles (Au NP): Au NPs
can catalyze the reduction of silver (I) ion (in the presence of
hydroquinone), which is colorless, to silver (0) particles, which
are black precipitates. Via a tight Au--S bond, the thiol forms a
layer on the surface of Au NPs. The layer will block the
interaction between Au NPs surface and reactants in the solution.
When there is small amount of Au NPs, there will be enough thiol to
coat the surface of all the Au NPs, inhibiting the contact between
Au and silver and suppressing the silver enhancement reaction. When
there is larger amount of Au, there will not be enough thiol to
coat the entire surface of Au, and silver enhancement will take
place quickly. Only when Au NPs are in excess compared with the
amount of thiol would there be surface exposed to silver, thus the
threshold position depended on the amount of thiol.
[0646] The threshold chemistries can be coupled with assays. For
example, the threshold can be coupled to the reporter molecule of
an immunoassay. The inventors herein reported an experimental
result in which an immunoassay for cystatin C in the SlipChip gave
visual digital readout by utilizing the threshold of AChE, which is
the reporter molecule. The inventors also showed that the threshold
for Au NP worked in SlipChip to give visual digital readout, thus
demonstrating the potential of applying this threshold to assays
such as immunoassays.
[0647] A sandwich immunoassay for insulin has been successful
demonstrated in the SlipChip. However, the readout for the assay
still required a fluorescent microscope. Here the inventors
modified the assay for cystatin C, with AChE as the reporter
enzyme. The assay gave digital readout due to thresholds set by
different amounts of the inhibitor syn-(S)-TZ2PIQ-A5, and gave
visual readout by the color changing reaction of thiocholine (a
product of the enzymatic reaction) with the dark blue starch/I2
complex to make the mixture colorless. The amount of cystatin C
correlates linearly with the amount of AChE. By using different
amounts of inhibitor, the inventors can set different thresholds
for AChE. The concentration of AChE will make the reaction proceed
at certain threshold values and is inhibited at other threshold
values. Such result will indicate the range of concentration of
AChE, and thus, the range of concentration of cystatin C.
[0648] This SlipChip was similar to the one used to perform
bead-based immunoassays, with the modifications of larger
dimensions, a change in the number of wells in each row, an
additional row for reagents in the top plate, and varied depths in
that row to allow for multiple threshold concentrations to be
evaluated on a single SlipChip.
[0649] For the SlipChip for immunoassay with threshold, diamond
wells dimensions were 780 .mu.m.times.780 .mu.m. Ducts were 380
.mu.m wide and 90 .mu.m deep. The spacing between the rows and
columns were 2.5 and 1.5 mm, respectively. The bottom plate of the
SlipChip contained wells to hold sample and ducts to load the
reagents. In the top plate, the ducts were used to load the sample.
The wells in row 1 on the top plate were loaded with the capturing
mixture. Rows 2-5 were filled with buffers for washing, row 6 was
loaded with the inhibitor, and row 7 was loaded with the substrate.
The wells in row 6 were divided into 5 sets of [5, 6, 6, 6, 6]
wells with respective depths of [16, 21, 28, 51, 90] .mu.m. Other
wells on the top plate were 90 .mu.m deep. Wells on the bottom
plate were 7 .mu.m deep. For the immunoassay, the plates were
aligned to load the capturing mixture. The plates were slipped and
aligned many times to load reagents and then slipped and aligned to
load the analyte. The plates were slipped so that the row of wells
in the bottom plate came into contact with each row of wells in the
top plate sequentially, and then slipped to show the final
results.
[0650] Before performing the whole enzymatic immunoassay in
SlipChip, the inventors validated the use of the AChE threshold by
showing the simple threshold of just AChE and the inhibitor
syn-(S)-TZ2PIQ-A5 in a SlipChip. Indeed, at a final inhibitor
concentration of 5 nM, AChE showed a threshold at 5 nM (final
concentration), as expected. The reactions with concentrations of
AChE>5 nM gave almost clear solutions, while the reactions with
concentrations of AChE.ltoreq.5 nM remained dark blue.
[0651] For enzyme threshold chemistry in SlipChip, the top plate
had four rows of wells that were connected to the same inlet. The
bottom plate had four rows of wells with separate inlets and
outlets. The depth of the wells on the top plate was 80 .mu.m and
the depth of the wells on the bottom plate was 60 .mu.m. A solution
of inhibitor was loaded into the wells on the top plate. Four
different solutions of AChE with different concentrations were
loaded into the wells on the bottom plate. The bottom plate was
slipped relative to the top plate to allow the wells both plates to
overlap. After a 30-minute incubation, the two plates were slipped
back to the original position. The substrate mixture was loaded
into the wells on the top plate. The SlipChip was slipped again to
bring the wells of the top and bottom plate back into contact, and
the reaction was monitored with the stereoscope.
[0652] The inventors also obtained preliminary results for the
other type of threshold-generating reaction, silver reduction using
Au NPs. The inventors have shown the threshold in Au NPs on a well
plate. Here, the inventors demonstrated that this threshold can be
performed in a SlipChip. In this experiment the inventors used a
constant concentration of Au NPs while varying the amount of thiol
inhibitor. When the concentration of 2-mercaptoethanol was below
110 .mu.M, the thiol did not completely cover the surface of the Au
NPs, so the reduction of Ag (I) proceeded as indicated by the dark
color. But when concentration of 2-mercaptoethanol was above 330
.mu.M, the reaction was suppressed and no signal was observed. Au
NPs are commonly used tags in biological applications, enabling the
coupling of this method to a wide range of detection reaction.
[0653] For threshold of AChE and immunoassay in SlipChip
bioconjugation: bead-Ab: cystatin C antibody clone 24 (Genway,
cat#20-511-242278) was conjugated to tosylated paramagnetic beads
(Invitrogen, cat#65501) using the manufacturer's instruction.
Ab-biotin: cystatin C antibody clone 10 (Genway, cat#20-511-242277)
was conjugated to biotin using Lightning Link kit (Innova
Biosciences, cat#704-0010) using the manufacturer's
instruction.
[0654] The solutions were prepared as follows: Phosphate buffer:
sodium phosphate 0.1 M, pH 7 with pluronic F127 (BASF) 1 mg/mL.
BAB: pluronic F127 1 mg/mL in 1.times.DPBS (Gibco) pH 7. WB: BAB
with extra 0.2 M NaCl (0.337 mM NaCl total) Starch solution: A
suspension of cornmeal in phosphate buffer was boiled for 10
minutes and cooled down to room temperature. The supernatant was
then filtered through a syringe filter with a 5-.mu.m membrane to
give the starch solution. Substrate mixture 1: 45 .mu.L starch
solution, 5 .mu.L acetylthiocholine solution (0.4 M in phosphate
buffer), and 1 .mu.L of the 620 .mu.L solution of NaI (18.64 mg)
and I2 (1.55 mg) in water were mixed in a 600-4 microcentrifuge
tube by vortexing. Substrate mixture 2: 98 .mu.L starch solution, 1
.mu.L acetylthiocholine solution (0.4 M in phosphate buffer), and 1
.mu.L of the 4.016 mL solution of NaI (798.07 mg) and I2 (101.93 M)
in phosphate buffer were mixed in a 600-4 microcentrifuge tube by
vortexing. Capturing mixture: 2.5 mg/mL bead-Ab, 0.025 mg/mL
Ab-biotin and 25 mg/mL AChE-avidin (Cayman Chemicals, cat#400045)
in BAB.
[0655] The fabrication of features on the SlipChip was performed as
follows: The SlipChip for simple threshold was fabricated as
previously described. The dimensions of the wells were 1960
.mu.m.times.400 .mu.m.times.80 .mu.m on the top plate and 1920
.mu.m.times.360 .mu.m.times.60 .mu.m on the bottom plate. On the
SlipChip for immunoassay with threshold, all features except the
wells in row 6 of the top plate and the wells of the bottom plate
were fabricated as previously described. Wells in row 6 of the top
plate and wells in the bottom plate were formed using laser
drilling (Resonetics RapidX250 system, with demagnification of 7,
constant energy mode of 130 mJ, 75-mm lens, fluence of 2.5
J/cm2).
[0656] The coating of the SlipChip was performed as follows: The
surface treatment of the SlipChip for simple threshold was
performed as previously described. The SlipChip for immunoassay
with threshold was coated with FEP to have a robust coating to
prevent wetting of the areas not containing any features (wells or
ducts) by aqueous solution. The bare glass chips were cleaned in
H.sub.2SO.sub.4 98%: H.sub.2O.sub.2 30% (3:1 v/v) for 1 hour. They
were then dip-coated in FEP emulsion (Fuel Cell Earth LLC,
cat#TE9568-250) diluted 3 times with Millipore water with the
speeds of going in and out of the solution of 10.8 and 1.8 cm/min,
respectively. The coated chips were baked on a hot plate from room
temperature (21-23.degree. C.) to 250.degree. C., and at
250.degree. C. for 5 min, then cooling in air at room temperature.
The FEP layer in wells in row 6 of the top plate and in the bottom
plate were removed by layer drilling (70 mJ with 50% attenuator,
with other parameters the same as when drilling wells) and
subsequently, manual application of a needle (Beckton-Dickinson,
cat#305109) under a microscope.
[0657] The operation of the SlipChip was performed as follows: The
SlipChip was assembled by dropping 0.5 mL of FC-40 (3M) onto the
bottom plate, putting the top plate on top of the bottom plate, and
clamping the two plates with clothespins. Each row of the SlipChip
was loaded by sticking a 10-.mu.L pipet containing 10 .mu.L of the
solution in the inlet hole and pushing the solution out of the
pipet.
[0658] The loading of the reagents and sample into the SlipChip was
performed as follows: The SlipChip for simple threshold: First, the
inhibitor solution was loaded into 4 rows in parallel via an inlet
connected to 4 rows; AChE (SigmaAldrich, cat#C2888) solutions in
phosphate buffer were loaded into 4 rows one by one from 4 separate
inlets. The chip was slipped so that each row of the AChE solutions
overlapped with a row of the inhibitor solution. The chip was then
incubated for 30 minutes before being slipped back to the original
position. An excess amount (.about.100 .mu.L) of substrate mixture
1 was loaded into the wells in 4 parallel rows. The chip was then
slipped again so that the substrate mixture came into contact with
the mixture of AChE and inhibitor formed in the previous step. The
final concentrations of AChE were .about.4, 5, 6, and 7 nM, and the
final concentration of inhibitor was .about.5 nM. The reaction was
monitored a Leica MZ 16 stereoscope (Leica Microsystems) with a
Plan APO 0.63.times. objective. The SlipChip for immunoassay with
threshold: After the assembly of the two plates, they were aligned
so that wells in row 1 of the top plate were connected by the ducts
in the bottom plate. The capturing mixture was then loaded into the
first row of wells. The plates were then slipped relative to each
other so that the wells in the second row of the top plate were
connected by the ducts in the bottom plate, and WB was loaded into
the second row. Similarly, wells in rows 3 through 7 were loaded
with WB, phosphate buffer, phosphate buffer, inhibitor solution,
and substrate mixture 2, respectively. Then the plates were aligned
so that the wells in the bottom plate were connected by the ducts
in the top plate, and the cystatin C sample (in BAB) was loaded in
the wells of the bottom plate.
[0659] The SlipChip was slipped so that wells of the bottom plate
were overlapped with the first row of wells in the top plate. The
mixture of the sample and the capturing mixture were incubated for
30 minutes at room temperature (21-23.degree. C.). A magnet was
used to pull the beads to the bottom of the wells in the bottom
plate. The chip was slipped so that the wells in the bottom plate
overlapped with the second row of wells in the top plate, and was
incubated for 2 minutes. The wells in the bottom plate were
sequentially brought into contact with wells in rows 3 through 7 of
the top plate with incubation time of 2, 2, 2, 30, and 120 minutes.
Finally, the beads were pulled to the bottom of the wells in the
bottom plate and the wells were separated from the wells in row 7
of the top plate. The results were read in the wells in row 7 of
the top plate, and wells in the bottom plate which contained beads
were used as markers of positions of the wells in row 7 of the top
plate, in case the reaction proceeded in the wells. The picture of
the result was taken with an inexpensive cell-phone camera (Nokia
3555b).
[0660] The fabrication of SlipChip was performed as follows: The
inventors followed the fabrication procedure previously described
with the following modifications. The glass plate with photoresist
coating was aligned with a photomask and exposed to UV light for 1
min. The size of wells was 1920 .mu.m (length).times.360 .mu.m
(width) decided by the. The chip used for Au NPs threshold had 5
wells in each row and 20 wells in total. Immediately after
exposure, the photomask was removed from the glass plate and the
glass plate was developed in 0.1 mol/L NaOH solution and a chromium
etchant (a solution of 0.6:0.365 mol/L
HClO4/(NH.sub.4).sub.2Ce(NO.sub.3).sub.6) separately. The taped
glass plate was then carefully immersed in a plastic container with
a glass etching solution (1:0.5:0.75 mol/L HF/NH4F/HNO3) to etch
the glass surface that was exposed after the chromium coating was
removed. 80 .mu.m deep wells and ducts were etched into the glass
plate. Finally, the glass plate was rinsed with ethanol to strip
the undeveloped photoresist, and immersed in the chromium etchant
to remove the chromium coating. The etched patterns were verified
using a Veeco Dektak 150 profilometer. After subjected to an oxygen
plasma treatment, the surfaces were rendered hydrophobic by
silanization in a vacuum desiccator for 3 hours with
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane.
[0661] Preparation of silver enhancement solution before mixing on
SlipChip was performed as follows: Solution A: 3 .mu.L 200 mM
citrate buffer was mixed together with 15 .mu.L 100 mM AgNO3
solution and 82 .mu.L Millipore water. Solution B (B1-B4): 4 .mu.L
0.15 mM Au NPs was mixed with 30 .mu.L 100 mM hydroquinone solution
and different volume of 1 mM mercaptoethanol solution (0, 10, 30,
50 .mu.L), the total volume was fixed to 90 .mu.L by compensating
with Millipore water.
[0662] Experiment of Au NPs-based threshold on SlipChip was
performed as follows: The SlipChip was assembled, loaded and
slipped as described previously. First, solution A was pipetted
into 4 rows in parallel via an inlet connected to 4 rows; solution
B1 to B2 were pipetted into 4 rows one by one from 4 separate
inlets. Then one plate of glass was slipped relative to the other
for the wells in different plates to overlap with each other. The
whole chip was put into darkness after mixing and results were
examined every 5 minutes by taking microphotographs with a Leica MZ
16 Stereoscope (Leica Microsystems) with a Plan APO 0.63.times.
objective.
[0663] The idea of using threshold to get analog-to-digital
conversion of concentration can also be applied to other assays
(besides immunoassay as described herein) making it relevant to
many diagnostic needs. For example, a threshold in nucleic acid can
be set using set amounts of immobilized complementary fragment to
bind to the nucleic acid and physically removing the bound
molecules. Such threshold could be applied to give digital readout
in nucleic acid quantification relevant in HIV, HBV, HCV, and other
infections. The SlipChip, when combined with the analog-to-digital
conversion, could be commercialized and presents an attractive
platform for an equipment-free, point-of-care device that could be
widely utilized.
[0664] In certain embodiments, dead-end filling of SlipChip can be
performed including control of the surface chemistry and the gap
size between the plates for lubricated and dry SlipChips.
[0665] This describes some of the current work to load SlipChips
via dead-end filling. In the process which we call "dead-end
filling", the fluid that fills the SlipChip after assembly (either
lubricating fluid or air) is dissipated through the gap between the
two plates of the SlipChip. This SlipChip design has no outlets (in
the conventional sense) in the fluidic paths filled by dead end
filling.
[0666] This method can be used to make a slipchip with inlets
compatible with the standard SBS format, e.g. 96 or 384 or 1536
well plate; standard equipment can be used to dispense the
solutions into the plate and after pressurization desired volumes
would be formed inside the slipchip, slipping may be used to drive
the processes. The standard SBS plate, with appropriate openings,
can be used as one of the layers of the slipchip; may be designed
to inject solutions through one of the wells, and observe through
another well, etc.
[0667] Device fabrication was performed as follows: Soda-lime glass
plates with chromium and photoresist coating (Telic Company,
Valencia, Calif.) were used to fabricate devices. The standard
method to make glass SlipChip was used. Briefly, the
photoresist-coated glass plate was exposed to ultraviolet light
covered by a photomask with designs of the wells and ducts.
Following removal of the photoresist using 0.1 M NaOH solution, the
exposed chromium coating was removed by a chromium-etching
solution. The patterns were then etched in glass etching solution
in a 40.degree. C. shaker. After glass etching, the remaining
photoresist and chromium coatings were removed by ethanol and
chromium-etching solution, respectively. The surfaces of the etched
glass plates were cleaned and subjected to an oxygen plasma
treatment, and then the surfaces were rendered hydrophobic by
silanization in a vacuum desiccator as previously described. Inlet
holes were drilled with a diamond drill bit 0.035 inch in
diameter.
[0668] Surface tension was measured as follows: The surface tension
of aqueous solution in fluorocarbon was measured as previously
reported with some modifications. Briefly, droplets of an aqueous
solution of interest were formed at the end of a disposable droplet
extrusion tip. The tip was assembled by using quick-set epoxy to
glue polyimide-coated glass tubing to one 10 .mu.L disposable pipet
tip. The tip was then inversely inserted through an drilled hole of
a 1 mL polystyrene cuvette and fixed by using epoxy glue. The
polyimide tubing was connected to a 50 .mu.L Hamilton Gastight
syringe by using 30-gauge Teflon tubing. The syringe was then
filled with the aqueous solution and the 1 mL cuvette was filled
with fluorocarbon. The formed droplets were imaged using Model 250
Standard Digital Goniometer & DROPimage Advanced software
(Rame-Hart Instrument Co).
[0669] Viscosity was measured by using the Cannon-Fenske calibrated
viscometers manufactured by Cannon Instrument Company (State
College, Pa.). The instructions accompanying the product were
followed to take the measurements.
[0670] Contact angles were measured following the same protocol
reported previously..sup.3,4 Briefly, 4 .mu.L of a solution to be
measured was pipetted on the substrate of interest. The contact
angle of the droplet on the substrate was then measured by using an
optical contact angle meter (Rame-Hart Instrument Co., Model
500).
[0671] Measuring and controlling the gap between two plates of a
SlipChip was performed as follows: Gap measurements were done on
DMI6000 epi-fluorescence microscope manufactured by Leica (Germany)
equipped with Hamamatsu digital cooled CCD camera (Japan). This
cooled camera has linear response on light intensity, which allows
precise intensity measurements. Gap between the slides was measured
with using mineral oil (Fisher Scientific, NJ) stained with green
fluorescent quantum dots (QDs) (Ocean Nanotech, AR). Original 1%
QDs solution in toluene was filtered through 0.22 micron
microcentrifuge Amicon filters (Millipore, MA) and sonicated in an
ultrasonic bath (Fisher Scientific, NJ) for 10 min. 10% solution of
QDs in mineral oil was thoroughly vortexed and kept for at least 10
min under vacuum before filling the device.
[0672] Stained mineral oil was deposited between the two plates of
the SlipChip; excess oil was removed by rinsing the assembled
device sequentially with chloroform, acetone, and ethanol. The two
plates were clamped with 8 paper clips and kept for at least 1 hour
under pressure before the measurements. Image acquisition, image
processing, and measurements were done by using Metamorph software
(Universal Imaging Corporation). Images were acquired at reduced
field of illumination to avoid leaching of fluorescent light from
the much brighter features used as a reference to relatively dim
surrounding areas. Fluorescence images were treated according to a
standard procedure, which include subtraction of the background
camera noise and compensating for the uniformity of field of
illumination. SlipChip has features of known depths, allowing for
the estimation of the depths of unknown features, including the gap
between the slides, by simply comparing fluorescence intensities
from these features. To determine precise distance between the
slides we applied a self-recursive procedure according to the
formula:
d.sub.i+1=(w+d.sub.i).times.I.sub.s/I.sub.w
Here w is the depth of the known feature (well), d.sub.0=0;
d.sub.i--gap size, I.sub.s and I.sub.w are intensities acquired
from the surrounding surface and from the well. The inventors
usually conducted i=1-2 iterations to obtain reliable distance.
[0673] To validate this procedure and check for linearity we
performed fluorescence measurements from the series of wells of
known depths. These reference wells were made on a Laser Ablation
System (Resonetics, NH). Depths of all features were measured with
using a profilometer (Dektak 150, Veeco, Calif.). Fluorescence
intensities acquired from the wells were found to be linear with
the well depth. Difference in distances obtained with both
techniques was within .about.5%. Therefore, one can use
fluorescence intensities to measure gaps between the SlipChip
plates.
[0674] To control gaps between the slides the inventors use
fluorescent silica beads of two different sizes. In particular, the
inventors used beads with diameter of 1.5 .mu.m and 3.86 .mu.m
respectively, obtained from Corpuscular Inc., NY. These beads were
silanized before use to make them compatible with the hydrocarbon
oil. Silanization was performed as follows: beads were rinsed and
sonicated with acetone three times; 5% dichlorodimethylsilane was
added to beads in acetone and exposed for 30 min at room
temperature. Beads were rinsed once with acetone and twice with
chloroform. The appropriate amount of beads was added to
fluorescently stained hydrocarbon oil to obtain relatively uniform
bead distribution. The gap between the SlipChip plates was measured
as described above for each case.
[0675] Each device consists of two plates. Approximately 300 .mu.L
of the lubricant FC was pipetted on the bottom plate and the top
plate was slowly placed on top the bottom plate to avoid trapping
air bubbles in channels. The plates, in close contact, were then
aligned under microscope and fixed by paper clips.
[0676] Testing the physical model (change pressure (home source and
barometer) and observe leaking, solution) was performed as follows:
Pressure control. Pressure was provided by a adjustable N2 source.
The N2 source was bifurcated into two ends, one of which was
connected to a barometer indicating the output pressure in the
system and the other was connected to the SlipChip. Loading
solutions. 4 .mu.L of a green dye was pipetted on top of the inlets
of an assembled device. An O-ring, made from PDMS and .about.5 mm
in height, was then sandwiched between the assembled device and a
glass plate and fixed by paper clips. The glass plate bore a
nanoport assembly (Upchurch Scientific). The assembly was then
connected to the pressure source and solutions were loaded into the
channels in the SlipChip. Any solution leakage was observed in the
FC-receiving channel. Characterization of loading speed. The
channel part between two circles was used to characterize the
loading speed. The speed is the average volumetric flow rate,
defined as Qave=V/t. V (m.sup.3) is the volume of the channel part
to be filled with solution and t (s) is the time recorded to fill
the channel part.
[0677] 5 solutions were used to load the FC-lubricated device: a
green dye solution was used to load the fluidic path for sample;
red, blue, orange, and yellow dyes were used to load the 16 fluidic
paths for reagents. The surface of the plates was patterned with
wells (approximately 12 .mu.m long, 12 .mu.m wide and 2 .mu.m deep)
with .about.8 .mu.m spacing. Such wells facilitate dissipation of
lubricating FC. The same sample loading procedure that was used to
test the physical model was used to load the sample and multiple
reagent solutions simultaneously, except that all solutions were
first loaded into big reservoir wells ahead of the fluidic paths.
After loading, the top plate was slipped relative to the bottom one
to bring reagent wells in contact with sample wells and to mix the
solution inside.
[0678] In order to describe filling process in more details the
inventors use equations for the pressure balance. The pressure
applied at the inlet has to overcome the capillary pressure at the
interface between phase 1 and phase 2.
.DELTA.P.sub.flow=P.sub.0-.DELTA.P.sub.cap Equation 1:
[0679] .DELTA.Pflow is the pressure difference between the opposite
ends of the channel filled with the aqueous phase 1 generated by
the resistance of fluids flow, P0 is the pressure applied at the
inlet to drive phase 1 into the fluidic path; and .DELTA.Pcap is
the capillary pressure generated at the interface of phase 1 and
phase 2 inside the filling channel. Generally it is difficult to
determine precise shape of the interface even in rectangular
channels,5,6 especially if this interface is formed partially by
the solid surface, partially by the liquid interface, like in this
case. According to the Young-Laplace equation, the approximate
pressure difference at the interface between phase 1 and phase 2 in
rectangular channel will be
.DELTA. P cup = .sigma. ( 1 / R w + 1 / R h ) = 2 .sigma. ( 1 w + 1
h ) cos .theta. 7 . ##EQU00003##
Here .sigma. is the surface tension, R.sub.w (R.sub.w=w/2 cos
.theta.) and R.sub.h (R.sub.h=h/2 cos .theta.) are the interface
approximate curvatures in horizontal (width w) and vertical (height
h) directions; .theta. is a contact angle.
[0680] When P.sub.0 is larger than P.sub.cap, P.sub.flow is
positive and the channel is filled with phase 1. The larger the
difference between these pressures the faster filling. Viscous drag
forces will prevent the channel to fill out instantaneously. The
detailed analysis of the viscous drag during flow through the solid
rectangular channel has been discussed previously. The channel is
formed (at least partially) by the fluorocarbon oil surrounding
aqueous phase. The sealing pressure, P.sub.seaI (Pa) (Equation 2)
prevents phase 1 from leaking out of the channel.
P.sub.seal=-2.times..gamma..times.cos
.theta./d<2.times..gamma./d=P.sub.seal,max Equation 2:
Here: .gamma. (N/m) is the surface tension between the aqueous
solution (phase 1) and the FC (phase 2); .theta. is contact angle
between phase 1 and surface of the SlipChip in phase 2 and is
required to be larger than 90.degree. to prevent capillarity of
phase 1; d (m) is the gap distance between the two plates of the
SlipChip. The maximal pressure, P.sub.seal,max (Pa) exists assuming
.quadrature.=180.quadrature..quadrature. The inlet pressure must be
smaller than the sealing pressure (Equation 3) to avoid leakage
into the gap, if the pressure is higher, the aqueous solution will
flow between the plates, causing leaking.
P.sub.0<P.sub.seal,max Equation 3:
[0681] Dissipation of FC Limits the Filling Speed.
[0682] The inventors used equations to make the prediction, and
found that changing the related parameters affects loading speed
while changing the unrelated parameters does not. In the testing
SlipChip, .DELTA.P.sub.flow includes three terms (Equation 5):
.DELTA.P.sub.1, the pressure difference due to flow resistance of
the aqueous in the loading channel; .DELTA.P.sub.2, the pressure
difference due to flow resistance of phase 2 in the loading
channel; and .DELTA.P.sub.3, the pressure difference due to flow
resistance of FC between the two plates of a SlipChip. Equation 6,
obtained by combining equation 1 and equation 5, expresses the
pressure difference along the system. The pressure difference due
to flow resistance can be expressed in equation 7..sup.7 .mu..sub.i
is the viscosity of the corresponding fluid, so here .rho..sub.1
(PaS) is the viscosity of the aqueous phase, .mu..sub.2 and
.mu..sub.3 are the same, equal to the viscosity of the lubricating
phase; L.sub.i (m) is the average length of the fluid path. The
inventors assume L1 and L2 are the same, equal to half length of
the whole loading channel. L3 equals to the distance between the
loading channel and the large receiving channel; Q.sub.i
(m.sup.3/s) is the flow rate discharge. Due to mass conservation,
Q.sub.1, Q.sub.2 and Q.sub.3 are the same; h.sub.i is the height of
the fluid path, therefore, h1 and h2 are the same, equal to the
height of the channel. h3 equals to the gap of the SlipChip;
w.sub.i is the width of the fluid path. w.sub.1 and w.sub.2 are the
same, equal to the width of the loading channel. The inventors
assume w.sub.3 is half length of the loading channel because it is
difficult to determine the flow profile of the lubrication
fluorocarbon along the loading channel between two plates.
.DELTA. P flow = .DELTA. P 1 + .DELTA. P 2 + .DELTA. P 3 Equation 5
.DELTA. P inlet = .DELTA. P 1 + .DELTA. P 2 + .DELTA. P 3 + .DELTA.
P cup Equation 6 .DELTA. P i = .pi. 4 .mu. i L i Q i 8 h i 3 w i (
1 - 2 h i .pi. w i tanh ( .pi. w i 2 h i ) ) Equation 7
##EQU00004##
Hyperbolic tangent will asymptotically go to 1 when channel aspect
ratio will increase (height will decrease and/or width of the
channel will increase). At the same time pressure drop .DELTA.P in
the channel will change proportionally to 1/h.sup.3w.quadrature.
when aspect ratio w/h lasymptotically goes to .infin..
.DELTA.P.sub.3 is much larger than .DELTA.P.sub.1 or .DELTA.P.sub.2
because of h.sub.3<<h.sub.1and2<w.sub.i (equation 8). The
inventors designed the testing chip to make sure .DELTA.P.sub.inlet
was much larger than .DELTA.P.sub.cap. Therefore,
.DELTA.P.sub.inlet is approximately the same as .DELTA.P.sub.3. By
combining equation 7 and 10 with approximation at
h.sub.3<<w.sub.3, the inventors obtained equation 10, which
indicates that the loading rate of the aqueous solution, at a fixed
inlet pressure, was determined by the dissipation of the
lubricating fluorocarbon, including its viscosity, its dissipation
dimension.
.DELTA. P 3 .DELTA. P 1 .apprxeq. .DELTA. P 2 Equation 8 .DELTA. P
inlet .apprxeq. .DELTA. P 3 Equation 9 Q = Q 3 = 8 h 3 3 .times. w
3 .times. .DELTA. P inlet .pi. 4 .mu. 3 .times. L 3 Equation 10
##EQU00005##
The inventors experimentally tested the prediction by varying
h.sub.3 and .mu..sub.3 while keeping w.sub.3, L.sub.3 and
.DELTA.P.sub.inlet constant at 1.times.10.sup.4 .mu.m,
2.times.10.sup.3 .mu.m and 5.3.times.10.sup.3 Pa respectively.
Approximately, the loading rate increased with h.sub.3.sup.3 and
.mu..sub.3 independently. Furthermore, the inventors confirmed that
change of other parameters related to .DELTA.P.sub.1,
.DELTA.P.sub.2 and .DELTA.P.sub.cap did not have large effects on
the loading rate.
[0683] SlipChip can be loaded by dead-end filling. The inventors
used the physical model and designed a system to use dead-end
filling to load multiple solutions into SlipChips at the same time.
The inventors used a previously reported design, relevant to the
user-loaded SlipChip screening conditions for protein
crystallization with 16 different precipitants and 11 mixing ratios
for each precipitant. The inventors made the following
modifications to simplify the design: the ducts were made straight
without turns optimal for loading; no narrow channels were used to
balance the pressure. In addition, the inventors added an inlet
reservoir for each loading solution. It was designed not only for
buffering the flow as described in the testing SlipChip, but for
storage and preventing evaporation as well. The inventors also
designed smaller outlet reservoirs to prevent undesirable back
flow. To minimize the flow pressure generated by dissipation of
lubricating fluid between plates while maintaining the same sealing
pressure, receiving channels were designed near the fluidic path so
that the flowing distance of LF was be minimized. The inventors
made small patterns (.about.2 .mu.m in depth) on the contacting
surface of SlipChip to further lower the flow pressure between
plates.
[0684] Filling spontaneously ceased when the solution reached the
end of the fluidic path even though other solutions were still
being loaded. As a result, all the solutions can be loaded using a
single pressure source.
[0685] To simplify pipetting of solutions into the SlipChip and
allow for stable storage of solutions, the design can be modified
such that the reservoir next to the inlet has multiple access
holes. In this design, the lubricating fluid can exit the reservoir
through one of the access holes, decreasing the pressure resistance
to allow for easy loading. The solution remains surrounded by the
lubricating fluid, allowing for stable storage reducing
evaporation. To dispense the solution, pressure is applied at all
the access holes to push the solution into the channel to be
loaded. The shape of the reservoir can be designed such that the
aqueous droplet moves spontaneously away from the access holes and
into the proximity with the loading channel.
[0686] From the foregoing, it will be observed that numerous
variations and modifications may be effected without departing from
the spirit and scope of the invention. It is to be understood that
no limitation with respect to the specific embodiment illustrated
herein is intended or should be inferred. It is, of course,
intended to cover by the appended claims all such modifications as
fall within the scope of the claims.
Sequence CWU 1
1
48121DNAArtificial Sequencesynthetic primer 1gcgattgatg gtgatacggt
t 21224DNAArtificial Sequencesynthetic primer 2agccaagcct
tgacgaacta aagc 24320DNAArtificial Sequencesynthetic pBad primer
3gcgtcacact ttgctatgcc 20424DNAArtificial Sequencesynthetic pBad
primer 4gcttctgcgt tctgatttaa tctg 24520DNAArtificial
Sequencesynthetic E coli nlp primer 5ataatcctcg tcatttgcag
20620DNAArtificial Sequencesynthetic E coli nlp primer 6gacttcgggt
gattgataag 20721DNAArtificial Sequencesynthetic S pyogene fah
primer 7ttaaatacgc taaagccctc t 21820DNAArtificial
Sequencesynthetic S pyogene fah primer 8agggtgctta atttgacaag
20921DNAArtificial Sequencesynthetic S pyrogene OppA primer
9cccagttcaa ttagattacc c 211020DNAArtificial Sequencesynthetic S
pyrogene OppA primer 10ttgacttagc ctttgctttc 201120DNAArtificial
Sequencesynthetic S pneumoniae cinASP primer 11ggctgtagga
gacaatgaag 201222DNAArtificial Sequencesynthetic S pneumoniae
cinASP primer 12ctttgttgac agacgtagag tg 221320DNAArtificial
Sequencesynthetic S pneumoniae plySP primer 13atttcgagtg ttgcttatgg
201420DNAArtificial Sequencesynthetic S pneumoniae plySP primer
14gtaaagtgag ccgtcaaatc 201522DNAArtificial Sequencesynthetic E
faecium bglB primer 15tcttcatttg ttgaatatgc tg 221620DNAArtificial
Sequencesynthetic E faecium bglB primer 16tggaatcgaa cctgtttatc
201720DNAArtificial Sequencesynthetic E faecalis ace primer
17tagttggaat gaccgagaac 201820DNAArtificial Sequencesynthetic E
faecalis ace primer 18agtgtaacgg acgataaagg 201921DNAArtificial
Sequencesynthetic P aerugino vic primer 19ttccctcgca gagaaaacat c
212021DNAArtificial Sequencesynthetic P aerugino vic primer
20cctggttgat caggtcgatc t 212121DNAArtificial Sequencesynthetic S
agalactia cpsY primer 21cgacgataat tccttaattg c 212224DNAArtificial
Sequencesynthetic S agalactia cpsY primer 22tcaggactgt ttatttttat
gatt 242319DNAArtificial Sequencesynthetic Pseu general 16S primer
23gacgggtgag taatgccta 192420DNAArtificial Sequencesynthetic Pseu
general 16S primer 24cactggtgtt ccttcctata 202521DNAArtificial
Sequencesynthetic S aureous nuc primer 25gcgattgatg gtgatacggt t
212624DNAArtificial Sequencesynthetic S aureous nuc primer
26agccaagcct tgacgaacta aagc 242725DNAArtificial Sequencesynthetic
S epid agrC primer 27gatgatatta atctatttcc gtttg
252824DNAArtificial Sequencesynthetic S epid agrC primer
28tcaggactgt ttatttttat gatt 242922DNAArtificial Sequencesynthetic
S mutans dltA primer 29agatatgatt gcaacaattg aa 223020DNAArtificial
Sequencesynthetic S mutans dltA primer 30cgcatgattg atttgataag
203120DNAArtificial Sequencesynthetic P mirabil aad primer
31cgctattaac cttgctgaac 203220DNAArtificial Sequencesynthetic P
mirabil aad primer 32cctttctcac tcaccacatc 203323DNAArtificial
Sequencesynthetic MRSA mecA primer 33caagatatga agtggtaaat ggt
233423DNAArtificial Sequencesynthetic MRSA mecA primer 34tttacgactt
gttgcatacc atc 233522DNAArtificial Sequencesynthetic C troplicalis
ctr primer 35caatcctacc gccagaggtt at 223622DNAArtificial
Sequencesynthetic C troplicalis ctr primer 36tggccactag caaaataagc
gt 223720DNAArtificial Sequencesynthetic C glabrata cgl primer
37ttatcacacg actcgacact 203824DNAArtificial Sequencesynthetic C
glabrata cgl primer 38cccacatact gatatggcct acaa
243922DNAArtificial Sequencesynthetic C albicans calb primer
39tttatcaact tgtcacacca ga 224020DNAArtificial Sequencesynthetic C
albicans calb primer 40atcccgcctt accactaccg 204123DNAArtificial
Sequencesynthetic K pneumonia cim primer 41aatttaacct ggtttgataa
gaa 234226DNAArtificial Sequencesynthetic K pneumonia cim primer
42caaaatatga actatcagaa agattg 264318DNAArtificial
Sequencesynthetic K pneumonia acoA primer 43taacggcaaa gacgctaa
184419DNAArtificial Sequencesynthetic K pneumonia acoA primer
44tgaccagggc ttctacttc 194520DNAArtificial Sequencesynthetic primer
1 45gcgtcacact ttgctatgcc 204624DNAArtificial Sequencesynthetic
primer 2 46gcttctgcgt tctgatttaa tctg 244723DNAArtificial
Sequencesynthetic A1352 sense primer 47graacccact gcttaassct caa
234823DNAArtificial Sequencesynthetic A1355 antisense primer
48gagggatctc tagnyaccag agt 23
* * * * *