U.S. patent application number 09/965448 was filed with the patent office on 2003-04-10 for sample evaporative control.
Invention is credited to Bjornson, Torleif Ove, Boone, Travis, Gibbons, Ian, Hooper, Herbert H., Singh, Sharat, Ullman, Edwin F., Xiao, Vivian.
Application Number | 20030068646 09/965448 |
Document ID | / |
Family ID | 27384430 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068646 |
Kind Code |
A1 |
Singh, Sharat ; et
al. |
April 10, 2003 |
Sample evaporative control
Abstract
Devices and methods are provided using microfluidic devices for
manipulating small volumes and determining a variety of chemical
and physical events. The devices rely upon an opening to the
atmosphere of a small volume in a zone, where a sample is placed in
the zone where evaporation can occur. The zone is maintained in
contact with a liquid medium that serves to replenish the liquid in
the zone and maintain the composition of the mixture in the zone
substantially constant. The diffusion of components in the zone is
restricted during the course of the determination by the liquid
flux into the zone.
Inventors: |
Singh, Sharat; (San Jose,
CA) ; Xiao, Vivian; (San Jose, CA) ; Gibbons,
Ian; (Portola Valley, CA) ; Boone, Travis;
(Oakland, CA) ; Bjornson, Torleif Ove; (Gilroy,
CA) ; Hooper, Herbert H.; (Belmont, CA) ;
Ullman, Edwin F.; (Atherton, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
27384430 |
Appl. No.: |
09/965448 |
Filed: |
September 27, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09965448 |
Sep 27, 2001 |
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09568786 |
May 10, 2000 |
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09568786 |
May 10, 2000 |
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09470677 |
Dec 23, 1999 |
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60133448 |
May 11, 1999 |
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60140180 |
Jun 18, 1999 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
B01L 2200/142 20130101;
B01L 2300/0861 20130101; B01L 3/5025 20130101; B01J 19/16 20130101;
B01L 3/5027 20130101; B01L 2400/0688 20130101; B01L 2200/0642
20130101; B01L 2400/0406 20130101; B01L 2400/0415 20130101; B01J
2219/00317 20130101; B01L 3/502723 20130101; C40B 60/14 20130101;
G01N 2035/00237 20130101; G01N 2035/00287 20130101; G01N 1/34
20130101; B01F 33/30 20220101 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 033/53 |
Claims
What is claimed is:
1. A method for performing operations in small volumes with a
volatile solvent, said method comprising: adding a component for
said operation to a liquid zone exposed to the atmosphere and
containing said volatile solvent subject to evaporation, wherein
said liquid zone is in contact with a replenishing medium in a
capillary channel; whereby during said operation said solvent
undergoes evaporation and is replenished by said replenishing
medium from said capillary channel.
2. A method according to claim 1, wherein said capillary is
connected to a reservoir of said replenishing medium.
3. A method according to claim 1, wherein said liquid zone is in a
well through a wall of said capillary channel, optionally at least
a portion of the wall of said well is non-wettable, and said
capillary channel is connected to two reservoirs.
4. A method according to claim 1, wherein said liquid zone is
expressed from the end of said capillary channel.
5. A method according to claim 1, wherein said capillary is at
least partially hydrophilic.
6. A method according to claim 1, wherein the total volume of
liquid in said liquid zone is not more than about 5 .mu.l.
7. A method according to claim 1, wherein after said operation, at
least one component in said liquid zone is transferred through a
capillary channel to an electrokinesis system.
8. A method according to claim 1, wherein said operation is an
enzyme assay.
9. A method according to claim 1, wherein said operation is a
ligand-receptor binding assay.
10. A method according to claim 1, wherein said operation is a
reporter gene assay.
11. A method for performing a determination in a small volume,
wherein said determination comprises the interaction between at
least two entities, said method comprising: adding at least a
portion of said at least two entities to a liquid zone comprising a
liquid exposed to the atmosphere and subject to evaporation, said
liquid zone in contact with a replenishing medium in a capillary
channel and having a substantially fixed meniscus position, wherein
said liquid zone comprises any remaining entities necessary for
said operations or such additional entities are added to said
liquid zone; and detecting the interaction of said at least two
entities in said liquid zone.
12. A method according to claim 11, wherein said at least two
entities comprise an enzyme, an enzyme substrate capable of
producing a detectable product, and a compound being tested for its
effect on the activity of said enzyme.
13. A method according to claim 11, wherein said at least two
entities comprise a ligand, a ligand receptor and a compound being
tested for its effect on the binding of said ligand to said ligand
receptor.
14. A method according to claim 11, wherein said liquid zone is at
least in part in a well through the wall of a capillary, said
capillary channel is horizontal and connects two reservoirs with
said well between said reservoirs.
15. A method according to claim 11, wherein said compound is added
to a reservoir in a non-aqueous solvent and is homogeneously
distributed between said reservoirs and said capillary channel.
16. A method according to claim 11, wherein said zone is at least
in part in a well having a diameter of less than about 2 mm and
said capillary has a cross-sectional area of less than about
one-half of said well
17. A method according to claim 11, wherein said zone is less than
about 500 nl and adding comprises additions of less than about 300
nl.
18. A method for performing a determination in a small volume,
wherein said determination comprises the interaction between at
least two entities, said method comprising: adding at least a
portion of said at least two entities and optionally additional
entities necessary for said determination to a liquid zone in a
well comprising a liquid exposed to the atmosphere and subject to
evaporation, said well being in liquid exchange relationship with a
replenishing medium in a capillary channel, and said liquid in said
well having a substantially fixed meniscus position during said
determination, wherein said liquid comprises any remaining entities
necessary for said determination; and detecting the interaction of
said at least two entities in said liquid zone.
19. A method for performing a determination in a small volume,
wherein said determination comprises the interaction between at
least two entities, said method comprising: adding at least a
portion of at least said two entities and any additional entities
necessary for said determination to a liquid zone exposed to the
atmosphere between the termini of two capillary channels forming an
unenclosed bridge, in contact with a replenishing liquid in said
capillary channels, wherein liquid in said zone is subject to
evaporation and said liquid zone comprises any remaining entities
necessary for said operations; incubating said volume for
sufficient time for said interaction to occur; and detecting the
interaction of said at least two entities in said liquid zone.
20. A microfluidic device comprising: a solid substrate comprising
a plurality of microstructures comprising reservoirs, capillary
channels and wells, each well connected to at least one reservoir
by a capillary channel, wherein said capillary channels and
reservoirs are at least partially wettable, wherein said well has a
cross-sectional area not greater than the cross-sectional area of
said reservoirs and greater than the cross-sectional area of said
capillary channel.
21. A device according to claim 20, wherein said substrate is
comprised of plastic.
22. A device according to claim 20, wherein said device comprises a
substrate having said channels and reservoirs and a cover enclosing
said channels and comprising said wells, said cover comprising a
wettable surface over said channels, said wettable microstructures
wetted by aqueous solutions.
23. A device according to claim 20, further comprising an
electrokinetic capillary channel in fluid connection with said
well.
24. A microfluidic device comprising: a solid substrate comprising
a plurality of microstructures comprising reservoirs, capillary
channels and wells, wherein at least a portion of said reservoirs
are connected to a common manifold, each of said wells is connected
to a capillary channel linking said well to at least one reservoir,
wherein said capillary channels and reservoirs are at least
partially wettable, wherein said wells have cross-sectional areas
not greater than the cross-sectional areas of said reservoirs and
not less than the cross-sectional area of said capillary
channel
25. A microfluidic device according to claim 24, wherein said
channel is connected to from 1 to 2 reservoirs of at least 1.2
times greater cross-section than said well.
26. A microfluidic device according to claim 24, further comprising
an electrophoretic system comprising said well as part of said
electrophoretic system, with a reservoir at a terminus of a channel
of said electrophoretic system for receiving an electrode.
27. A microfluidic device according to claim 24, wherein said
microfluidic device comprises a central reservoir connected to a
plurality of wells.
28. A microfluidic device comprising: two opposed capillary
channels with confronting orifices and an open space between said
orifices, each channel connected to a reservoir; and means for
moving liquid from the channels to the space between the
channels.
29. A microfluidic device according to claim 28, further comprising
a platform between said capillary channels and in fluid
communication with said capillary channels.
30. A microfluidic device according to claim 28 having at least one
row of a plurality of said two opposed channels, each channel
connected to a reservoir; and means for moving liquid from each of
said capillary channels to the space between said capillary
channels, wherein said capillary channels are at least partially
wettable, while said channels have cross-sectional areas not
greater than the cross-sectional areas of said reservoirs.
31. A method for confining a solute within a small region of a
liquid volume partially confined by a non-wettable border to form a
meniscus, with said liquid volume in contact with said small region
confined to a capillary channel, said method comprising: adding
said solute to said small region while liquid from said small
region evaporates and liquid from said capillary flows into said
small region to maintain said meniscus and said solute in said
small region.
32. A method according to claim 31, wherein said solute is added as
a solution, wherein said meniscus equilibrates in relation to said
non-wettable border after said adding.
33. A method for performing a determination where binding of a
first entity to a second entity results in a change in a detectable
signal, in a medium subject to evaporation under the conditions of
said determination, said method comprising: adding, in a volume of
not more than about 300 nl, a component for said determination to a
liquid in a zone to form a reaction mixture of not more than about
500 nl in liquid exchange with said liquid in a capillary channel,
wherein other components necessary for said determination are added
or contained in said liquid; wherein evaporation occurs during said
addition; incubating said reaction mixture for sufficient time for
binding to occur; and detecting said detectable signal in said
reaction mixture.
34. A method according to claim 33, wherein said first and second
entities are an enzyme and a candidate compound.
35. A method according to claim 33, wherein said first and second
entities are a protein and a candidate compound.
36. A microfluidic device comprising: a solid substrate comprising
a plurality of microstructures comprising reservoirs, capillary
channels and wells, each well connected to at least one reservoir
by a capillary channel, wherein said capillary channels and
reservoirs are at least partially wettable, wherein said well has a
cross-sectional area not greater than the cross-sectional area of
said reservoirs and not less than the cross-sectional of said
capillary interface with said well and in liquid exchange
relationship with said capillary, a side channel connecting said
well to a capillary electrokinetic system comprising an analytical
channel connected to said side channel and having reservoirs at its
termini.
37. A microfluidic device comprising: a solid substrate comprising
a channel connecting two reservoirs having volumes of less than
about 5.multidot.1 and a cover plate enclosing said channel and
having openings for said reservoirs and a well between said
reservoirs in liquid connection to said channel; said well having a
cross-sectional area not greater than said channel; and said cover
having a hydrophilic surface above said channel.
38. A microfluidic device according to claim 37, wherein said solid
substrate is hydrophobic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 09/470,677, filed Dec. 23, 1999, and claims priority to
provisional application No. 60/133,448, filed on May 11, 1999 and
No. 60/140,180, filed Jun. 18, 1999, which disclosures are
incorporated herein by reference.
INTRODUCTION
[0002] 1. Technical Field
[0003] The field of this invention is manipulation of small volumes
comprising a volatile liquid.
[0004] 2. Background
[0005] Microfluidic devices comprise small capillary channels in a
solid substrate, where the channels are usually present as a
network. Various orifices are provided for communicating with the
channels. Because of the small volumes of the networks and the
individual channels many benefits adhere. The small volumes require
less reagent and sample, frequently being limited by the level of
detection available. In addition, because of the small volumes,
reactions are very rapid. The networks allow for efficient movement
of the components from one site to the next and with little loss of
the components. Also, various components may be brought together,
separated by different operations and the individual fractions used
for various purposes.
[0006] The microfluidic devices lend themselves for various assays
involving candidate compounds, where binding events are measured,
enzyme activity measured, or metabolic processes measured. In this
way, the effect of the candidate compounds on the indicated events
may be determined. Where one is interested in comparing the effect
of different candidate compounds, it is necessary that the amount
of the candidate compound and other components, which affect the
measured outcome, be reasonably known. For the most part, solutions
that will be used are aqueous. Unless one uses relatively drastic
measures, the water will rapidly evaporate. Transfers of aqueous or
other solutions involving manipulative steps where the solution is
exposed to the atmosphere for any length of time will invariably
result in some evaporation, particularly where there are sequential
additions, and the solvent from the earlier additions is
evaporating while adding the next addition and during the interim
between additions. In addition, incubations can result in
evaporation, even where the container is covered. The problem is
exacerbated where one is interested in high throughput screening,
which may involve many very small aliquots of different solutions
to multiple sites on a microfluidic device. Using foreign
substances to diminish the evaporation can lead to contamination,
require repetitive cleaning and create other detrimental
issues.
[0007] Various methods have been tried, such as cooling the
liquids, so as to substantially reduce evaporation, adding a lower
volatility liquid over the surface of the sample, ambient humidity,
adding droplets of solvent to the sample after its deposition to
maintain the volume, and the like. All of these approaches are not
generally useful and have severe disadvantages for use with small
volumes, which must be transferred to a reaction vessel. There is a
need for improved methods for manipulating nanoliter volumes when
dealing with microfluidic devices, particularly associated with
high throughput screening of compounds, diagnostic assays or other
investigative procedures.
BRIEF DESCRIPTION OF THE PRIOR ART
[0008] U.S. Pat. Nos. 5,576,197 and 5,282,543 disclose the use of
wax and other flexible materials, respectively, to inhibit
evaporation. Microfluidic devices are described in U.S. Pat. Nos.
5,885,470; 5,858,195; 5,750,015; 5,599,432; and 5,126,022. Methods
of evaporative control are disclosed in WO98/33052 and
WO99/34920.
SUMMARY OF THE INVENTION
[0009] Methods and devices are provided for the manipulation of
small volumes in association with determinations employing
microfluidic devices, where a substantial portion of the liquid is
subject to evaporation during the operation. The microfluidic
devices comprise a partial enclosure for a zone for receiving a
small amount of a component of the operation, usually as a solution
comprising a component of a reaction. The zone is bounded by a
meniscus, whose position is affected by the nature of the zone,
which zone may have a non-wettable border, which may be made
wettable by addition of a detergent or may be wettable. During the
operation, the liquid in the zone is subject to evaporative loss of
liquid, and the zone is in fluid exchange relationship with a
channel housing a replenishing liquid. The channel liquid
replenishes the liquid in the zone and may serve as a source of a
second or more components of the operation. During the operation,
the position of the meniscus will be relatively fixed in a number
of embodiments, while in other embodiments be subject to the
movement of liquid into and out of a capillary channel. Either
substantially immediately upon entering the zone, the component is
in contact with the channel liquid, so that any solvent lost by
evaporation in the zone can be replenished, or the component is
placed at a site where evaporation of any liquid may occur and the
residue is dissolved in a liquid discharged from a capillary
channel, where contact is maintained with the solution which forms
the zone and the solution in the capillary channel. The reaction
volume is substantially maintained in the zone defined by a major
portion of the components of interest being present in the zone,
comprising the region between a meniscus and the region of liquid
exchange between the zone and the channel.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 is a fragmentary perspective view of a microfluidic
device according to this invention;
[0011] FIGS. 2A, 2B and 2C are diagrammatic cross-sectional views
of units of a subject microfluidic device, having two channels and
a central chamber, at various stages in the process of using the
device;
[0012] FIG. 3 is a diagrammatic plan view of a device with a
plurality of units with fluid supplied by a manifold;
[0013] FIG. 4 is a fragmentary perspective view of an alternative
embodiment of a microfluidic device with two channel blocks joined
by a platform;
[0014] FIGS. 5A, 5B and 5C are perspective diagrammatic views of a
device according to this invention employing two channels at
different stages in their use;
[0015] FIG. 6A is a plan diagrammatic view of a device according to
this invention, with
[0016] FIG. 6B a cross-sectional view along line B-B and
[0017] FIG. 6C a cross-sectional view along line C-C;
[0018] FIG. 7A is a diagrammatic plan view of a network according
to this invention.
[0019] FIG. 7B is a cross-sectional view of a device corresponding
to a portion of the network of FIG. 7A;
[0020] FIG. 8A is a diagrammatic plan view of a network according
to this invention.
[0021] FIG. 8B is a cross-sectional view of a device corresponding
to a portion of the network of FIG. 8A;
[0022] FIG. 9 is a diagrammatic plan view of an assembly of device
units according to this invention having common channels along a
row of device units;
[0023] FIG. 10 is a diagrammatic plan view of an assembly of device
units with a common assay well channel and shared reservoirs.
[0024] FIG. 11 is a diagrammatic plan view of an assembly of
devices with a plurality of units, each unit having a plurality of
assay wells sharing a common reservoir, with the assay wells on a
96-well microtiter plate footprint;
[0025] FIG. 12 is a diagrammatic plan view of individual units
comprising a combination of an assay system joined to an
electrokinesis system, with an exploded view of one of the
units;
[0026] FIG. 13 is a diagrammatic plan view of an alternative
embodiment of a combination of an assay system and an
electrokinesis system, with an exploded view of one of the
units;
[0027] FIG. 14 is a diagrammatic plan view of a card with three
different organizations of channels for the combination of an assay
system and an electrokinesis system;
[0028] FIG. 15 is a diagrammatic plan view of a single unit
indicating the sites of the electrodes and the detection site;
[0029] FIG. 16 is a calibration curve for fluorescein in a subject
device;
[0030] FIG. 17 is a series of electropherograms of an alkaline
phosphatase assay taken at different times;
[0031] FIG. 18 is a calibration curve of the effect of varying
alkaline phosphatase concentration;
[0032] FIG. 19 is a series of electropherograms of an alkaline
phosphatase assay using different concentrations of an
inhibitor;
[0033] FIG. 20 is a calibration curve of the alkaline phosphatase
assay using the data set forth in FIG. 19
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0034] Improvements are provided for performing reactions in
microfluidic devices, using methods and devices allowing for
efficient manipulation of small volumes of solutions comprising
evaporative solvents. The reaction components will normally be in
one or more additions to the zone and optionally a liquid in a
channel in liquid exchange relationship with the zone. The channel
liquid may have one or more components, or all of the components of
the reaction may be added to the zone. Microfluidic devices are
provided comprising at least one unit having a partial enclosure
defining at least a portion of the zone and connected to a
capillary channel, so that the zone is open to the atmosphere
during additions to the zone, which enclosure may be sealed after
each manipulation or after all manipulations are complete. The
devices have microstructures, which are for the most part channels,
reservoirs and wells, but may include other microstructures, such
as barriers, salt bridges, projections in the channels, etc. The
liquid containing capillary channel is in liquid transfer
relationship with the zone, replenishing liquid lost by evaporation
and creating a liquid flux in the channel toward the zone. The
opening permits convenient addition of solutes and solutions to the
zone, where evaporation of liquid into the atmosphere may occur
during the transfer of the solution into the zone and thereafter.
The conditions of the addition will usually be at below or at
ambient or elevated temperature and pressure, although higher
temperatures may be employed during the addition.
[0035] The zone has a border, a meniscus, as a result of a
wettable/non-wettable border on the surface of the enclosure, a
sharp change in direction of the wall of the enclosure, the
termination of the zone or the hydraulic head of the system. The
height of the meniscus will be controlled so that, after addition
of liquid to the zone, particularly the assay well, the position of
the meniscus will be restored to its equilibrium level, due to
evaporation and fluid movement into the capillary channel. Where
the zone is connected to a reservoir, and is parallel to the
reservoir, the hydrostatic head is selected to avoid pushing the
meniscus significantly past the border. For a non-wettable/wettable
boundary on the surface of the enclosure, which may be at either
end of the enclosure, the meniscus will normally form at the
boundary. The meniscus at the boundary will normally be convex. For
a wettable border, where the border is wettable due to the wall
being hydrophilic (for a polar medium) or the addition of a
detergent, where the wall is hydrophobic (for a polar medium), the
border will usually be at the termination of the zone. With a
wettable border, the meniscus will usually be concave. The method
permits the formation of the product of the reaction to be retained
within a small volume for ease of detection.
[0036] Assays may be carried out for extended times with nanovolume
reaction mixtures comprising a volatile solvent, while the reaction
mixture is exposed to the atmosphere.
[0037] Reaction volumes of greater than 10 nl, usually in the range
of about 50 nl to 2.mu.l, more usually up to 500 nl, are employed,
where one or more components are added to the reaction zone
containing the reaction volume, where the components or their
products are substantially retained in the zone. The components are
added as solutions of from about 10 pl to 300 nl, more usually of
from about 10 to 200 nl, and preferably not more than about 100 nl.
The reaction mixture is bounded by a meniscus and the solution
directly under the meniscus. The additions are made directly onto
or through the meniscus, which may be surrounded by a wall forming
a well or passageway. Of particular interest are binding assays
involving proteins, where a candidate compound is tested and the
binding level of the candidate compound to the protein is
determined. The assay protocol involves a reaction mixture having a
meniscus exposed to the air, where the candidate compound may be in
the liquid of the reaction mixture with the meniscus border or
added to the reaction mixture. At least one other component of the
reaction is then added to the reaction mixture, in accordance with
the requirements of the determination, e.g. substrate for an
enzyme, competitive labeled compound for a binding protein, etc.
Depending on the nature of the label and the protocol, the label
may be detected in the reaction mixture.
[0038] The zone is defined functionally as comprising at least
about 50% of a component of interest, usually at least about 50% of
the components added to the zone, preferably at least 60%, more
preferably, at least about 80% and up to 95 or 100%. The zone will
always be a very small volume and where the operation of interest
provides a detectable signal, will usually be the region from which
the signal is detected. Desirably, the zone will be easily
addressable to maximize the signal for the determination, so that
the zone may approximate a cylinder. As will be described, the zone
need not be significantly enclosed and may be confined by solid and
liquid barriers, in addition to being open to the atmosphere, at
least initially during the operation.
[0039] The zone may have a portion of the zone at a
non-wettable/wettable interface or border, at a site of an abrupt
change of direction of the wall of the enclosure, which may include
the end of the enclosure or at the abrupt change, e.g. expansion
having a shelf, or extend to the end or beyond the end of the
enclsoure. (By wettable is intended that the surface will be coated
with the liquid and in a capillary the liquid will be drawn into
the capillary by surface tension. For a non-wettable border, in the
case of a polar solvent, particularly an aqueous solvent, the
surface will be hydrophilic, while the non-wettable surface will be
hydrophobic. Where the solvent is non-polar, e.g. hydrocarbon, the
reverse will be true for wettable and non-wettable.) This interface
may be at a region in an enclosure, at the edge of a capillary,
where the outer portion of the capillary is non-wettable, or other
structure where migration of the liquid in the zone is inhibited
from moving into another area as a result of the surface tension or
contact angle between the liquid and the non-wettable area.
[0040] In referring to microfluidic devices it is intended that the
devices comprise capillary channels having cross-sections of less
than about 5 mm.sup.2, usually less than about 1
mm.sup.2,frequently less than about 0.5 mm.sup.2, more frequently
less than about 0.1 mm.sup.2 and frequently as small as about 0.005
mm.sup.2 or less, generally being at least about 0.025 mm.sup.2,
more usually at least about 0.01 mm.sup.2. In addition, the devices
have a zone in which the reaction of interest occurs, which when
partially enclosed, so that a volume can be defined, the volume of
the zone that comprises the liquid of interest will be less than
about 5 .mu.l, usually less than about 1 .mu.l, and frequently less
than about 0.5 .mu.l, and may be as small as about 50 nl or less,
usually at least about 10 nl. At a non-wettable border, the
reaction volume will include the volume under the meniscus and
above the non-wettable border, where the meniscus may extend beyond
the non-wettable border. The reaction volume may also include a
volume in the capillary channel under the meniscus and extending a
short distance from the area under the meniscus. The partial
enclosure, when present, may have a substantially larger volume
than the volume of the zone, usually not more than about 10.times.
larger, more usually not more than about 5.times. larger, than the
volume of the zone. The zone, when partially enclosed, such as a
well, may have a cross-sectional area smaller than the channel
cross-sectional area, but will usually have a cross-sectional area
larger than the cross-sectional area of the channel, being at least
twice the area, conveniently at least about 5 times, and more
conveniently may exceed 20 times. Where the zone is not bordered by
a non-wettable boundary, a partially enclosed zone will usually be
the volume of the enclosure and may include a portion of the region
of the channel beneath the partial enclosure.
[0041] The capillary channel may be round, rectangular,
frusto-conical, truncated pyramid, normally inverted, or other
shape, preferably a regular shape. Of particular interest is when
the capillary channel is formed in a substrate, e.g. a plastic
card, and the channel enclosed with a film which is adhered to the
body of the substrate. In this case, the channel will not be
circular and will have a depth and width. In addition, the width
and/or depth may not be constant the length of the channel. In
referring to width and/or depth, it is intended the average width,
although differences from the average will usually not exceed more
than by 100%, usually by not more than about 50%.
[0042] For the non-circular channel, the depth of the capillary
channel will generally be in the range of about 10 .mu.m to 2 mm,
usually in the range of about 25 .mu.m to 1 mm, more usually in the
range of about 25 .mu.m to 500 .mu.m, preferably less than about
250 .mu.m, and at least about 10 .mu.m, usually at least about 20
.mu.m, particularly where the capillary channel serves as the floor
of the zone. For the circular capillary, the diameter will
generally be in the range of about 10 .mu.m to about 2 mm, more
usually at least about 20 .mu.m to 2 mm. The device may have one or
more capillary channels in liquid exchange relationship with the
zone, where the channels may be in the same or different planes, so
that there may be liquid contact at two or more different
interfaces. Conveniently, the signal may be determined without
having to view the signal through the material with which the
device is composed.
[0043] By having a network of channels, where some or all of the
channels may interconnect, substantial flexibility is achieved. It
is understood that for the purposes of this invention, channels and
capillaries may be used interchangeably, where capillary (includes
channel, unless it is clear from the context that channel intends a
cross-section greater than a capillary or is open along its length)
intends that there is liquid movement upon introduction of liquid
into one end of a capillary due to surface tension. The channels
may serve to deliver and remove agents from one or more zones,
simultaneously or successively, depending on the plumbing employed.
One may provide for miniaturized pumps, separation walls, gates,
etc., so as to be able to direct liquids to specific zones. One may
provide for successive replacement of liquids in the channels,
whereby different reagents may be directed to the zones, which
allows for modification of reactions, stepwise performance of
reactions, removal of agents from the zones, etc. By modulating the
temperature of the liquid in the channels one can modulate the
temperature of the liquid in the zones. Thus, one could provide for
heating and cooling of the mixture in the zone.
[0044] The zones provide opportunities for the introduction of one
or a few particles, such as beads, colloidal particles, cells,
organelles, microsomes, and the like. The small volumes allow for
enhanced signals from the particles, allowing for investigations or
determinations, where only a few particles need be present. For
cells, one may provide 1 cell or more, usually more than about 50
cells for statistically significant results, and generally fewer
than 1,000 cells, usually fewer than about 500 cells. Cells may be
dispersed in the zone, adhered to the surface of the zone, as a
wall of a well or channel, or the like. The small volume of the
wells allows for growing cells in the wells, where the reservoirs
may serve as a source of nutrients. Where one is interested in
unique events, such as mutagenesis of a genome, a single cell can
be maintained in a well and the occurrence of the unique event
assayed. For example, if one were interested in mutagenizing an
enzyme to be resistant to inhibition by a known inhibitor for the
wild type enzyme, each well containing a single cell could be
assayed with substrate and inhibitor and production of a product
would indicate that the enzyme had been successfully mutagenized.
Alternatively, cells may be genetically modified to have a reporter
gene, e.g. an enzyme that produces a detectable product from its
substrate, a fluorescent protein, etc., so that the operation
either turns the reporter gene on or off. This type of assay has
found extensive use in studying transcription factors, as well as
other cellular pathways.
[0045] In one embodiment, one has an orifice forming a well through
the wall of a capillary channel, where the partial enclosure is at
least the height of the thickness of the wall of the capillary. The
well may be at any angle in relation to a reference point to which
the position of the capillary may be related. For example, where
the capillary is in a solid substrate, particularly having a groove
or trench in a plate and a cover enclosing the plate, the orifice
may be in the cover or in the side of the plate or in the substrate
opposite from the plate, or any angle in between. However, for the
most part the orifice will be vertical and above the capillary
during operation. In this embodiment, where the wall is
non-circular, the well is normally in the cover enclosing the
channel in the substrate. The well can be varied in accordance with
the thickness of the cover, which up to a degree may be arbitrarily
chosen. Thus, covers may be from about 0.05 to 2 mm in thickness,
where the height of the well would be the same. Alternatively, one
may fuse or form a tube or collar to the substrate to obtain any
length for the partial enclosure. The partial enclosure serves as a
container, generally having a cross-sectional area at least about
one-half, frequently at least about equal and desirably greater
than about the cross-sectional dimension of the channel. The volume
of liquid in the zone, comprising at least a portion of the well
and optionally a portion of the channel under the well, will be
controlled in part by the nature of the wall of the partial
enclosure of the zone, where none or a portion of the wall will be
non-wettable by the liquid in the zone. (By "non-wettable" is
intended that the liquid in the zone will not migrate past the
region that is non-wettable when no force is applied to the liquid
to drive the liquid past such region. In effect, the contact angle
between the liquid and the wall is such as to inhibit the rise of
the liquid in the partial enclosure. Conversely, "wettable" intends
that the liquid will wet the surface and rise in a capillary in the
absence of a negative force.) Where the partial enclosure is
wettable, the zone may encompass the enclosure, depending on the
hydrostatic forces between the zone and the reservoir(s).
[0046] In this embodiment it appears that the evaporation from the
zones results in the movement of liquid from the channel into the
zone to retain the height of the meniscus. The liquid in the
channel is, of course, maintained by the reservoir(s), whose volume
will generally be large compared to the volume of the channel and
the liquid in the zone. Evaporation from the zone may be further
enhanced by having: a temperature differential between the liquid
in the zone and the liquid in the reservoir; a differential air
flow; a differential humidity; or the like, where the condition at
the zone is to enhance the evaporation at the zone, as compared to
the reservoir. The temperature during the time of addition may be
ambient, reduced or elevated, generally in the range of about
10.degree. C. to about 65.degree. C., more usually in the range of
about 20.degree. C. to 500.degree. C., so long as the rate of
evaporation is not unduly great to interfere with the
replenishment.
[0047] In other embodiments, one may have a discontinuity between
the liquid in the zone and the liquid in the channel, where liquid
from the channel may be brought into contact with liquid in the
zone. In this instance, the zone may be substantially open and only
have a floor or be substantially enclosed, where the channel could
be connected to the zone through an orifice at the bottom or at the
side of the zone. One has a channel in proximity to the zone, where
the liquid in the channel may be expressed into the zone and
optionally withdrawn to reduce, but not completely terminate,
evaporation during subsequent operation.
[0048] Depending upon the nature of the operation, different
protocols may be employed.
[0049] In one protocol, a liquid, normally a solution, is added to
the zone and upon introduction into the zone comes into
substantially immediate contact with liquid from a capillary
channel. The liquid may be added to the zone, where the channel
liquid may be the floor of the zone, a droplet between two channels
or may be in a side channel, where the channel may be vertical or
horizontal in relation to the zone. The solution may be retained in
the zone or withdrawn into the capillary channel during the course
of the reaction. After sufficient time for reaction to occur, the
resulting product may be processed in accordance with the
operation, and, as appropriate, a signal determined. As an
illustration, with a volume of the zone of about 200 nl, with a
capillary channel having a cross-sectional area of 450.times.100
.mu.m, the zone would be withdrawn into the capillary about 4-5 mm,
assuming all of the reaction mixture in the zone was withdrawn into
the channel.
[0050] In a second protocol, a solute or solution may be added to a
surface in the zone and any evaporation of the solvent ignored. (In
referring to a solution, it should be understood that any liquid
mixture of two components is intended, such as a mixture of liquids
or a solute and a solvent. In some instances, dispersions are also
included, such as colloidal dispersions, as may be understood from
the context.) Liquid for the reaction mixture is then discharged
from the channel to dissolve the residue, liquid or solid, to form
the reaction mixture. The reaction mixture solution is maintained
in contact with the liquid in the channel to replenish any solvent,
which evaporates, or the reaction solution is withdrawn into the
channel to substantially inhibit any evaporation. After sufficient
time for reaction to occur, the resulting product may be processed
in accordance with the operation, and, as appropriate, a signal
determined.
[0051] Evaporation helps keep the zone of the reaction mixture
defined. Despite the diffusion of small molecules, the liquid flux
into the zone during the operation inhibits the loss of the small
molecules into the channel away from the zone. Based on this
consideration, preferably the zone will be designed to have a
relatively short vertical path from the meniscus to the end of the
zone. Furthermore, depending on the height of the partial
enclosure, one can add various solutions, where the solutions will
mix in the partial enclosure and as the height of the meniscus is
restored through evaporation and the liquid moving into the
channel, the liquid at the bottom of the zone is moved back into
the channel.
[0052] In performing the reaction there will be at least one
component of the reaction added through the opening into the zone
and, as described, conveniently, at least one component of the
reaction in the solution in the channel. Frequently, components
added to the zone will be higher molecular weight components of the
reaction, generally exceeding 2 kD, frequently exceeding 5 kD, and
may exceed 10 kD. Where small organic molecules are being screened
for activity, they may conveniently be added to the zone and will
have a molecular weight in the range of about 150-2500 Dal or may
be added to the reservoir(s). One or more additions may be made
into the zone, adding one or more components to the zone. To
minimize the additions, mixtures of components may be added. By
virtue of the contact between the solution in the zone (zone
solution) and the solution in the channel (channel solution),
components in the channel solution will diffuse into the zone
solution to equilibrate the concentration of the component(s) in
the channel solution between the two solutions, while the small
cross-section of the channel, the capillary forces in the well
and/or evaporation keep the zone defined. Upon completion of the
addition(s), one can then determine whether the desired reaction
occurred.
[0053] A plurality of additions may be made concurrently or
consecutively, where the time between additions may be very short,
bordering on simultaneous addition, or require relatively long
intervals, e.g. 30 sec or more, where the intermediate reaction
mixtures may be incubated, processed, e.g. heated, or withdrawn
into the channel to inhibit evaporation. Generally, the volume of
the solution added to the zone will be less than 0.005 ml,
frequently less than about 1 .mu.l and more frequently less than
about 0.5 .mu.l usually being at least about 10 pl, more usually
being at least about 1 nl, frequently at least about 10 nl,
depending on the ability to accurately transfer liquids to the
zone.
[0054] Additions may be achieved using piezoelectric devices, e.g.
ink-jet devices, pins, slotted pins, pipettes, capillary
electrokinesis injection, etc. Preferably, the delivery devices
will not require contact with the solution in the microstructure or
the subject device. The particular manner of transfer will depend
on the volume to be transferred, the nature of the composition to
be transferred, the speed with which the composition can be
transferred, the accuracy required for dispensing the composition,
and the like.
[0055] Usually, the solution in the channel will be a buffered
solution, where the formality of the buffer, which may include
other ions, will be not more than about 200 mM, more usually not
more than about 100 mM, and frequently less than about 75 mM,
usually greater than about 5 mM, more usually being greater than
about 10 mM. Buffers which may find use include phosphate,
carbonate, borate, MOPS, HEPES, Tris, tricine, etc., the buffer
generally being selected in accordance with the nature of the
reaction. Where capillary electrokinesis is used, the buffer in the
channel may be selected to be suitable for the capillary
electrokinesis, may be modified after performing the operation or
may be transferred to the electrokinesis system and modified there.
The concentration of the components, which are added, may vary
widely depending on the volume of the solution. Concentrations may
vary from about 1 .mu.M to 0.1 M, usually being in the range of
about 1 pM to 0.01M, the concentration and volume depending on the
level of detection of the detectable signal and the manner in which
the signal is generated. Since the volumes added to the zone are
small compared to the volume of solution in the system comprising
the channel and reservoirs, the area of interface between the zone
and channel is small, and the evaporative flux inhibits diffusion
of components of the zone from leaving the zone, there will be
limited equilibration between the added solution and the liquid in
the channel.
[0056] Desirably, the buffer solution in the channel will be the
same as the buffer solution in the added solutions, where the
difference will then be as to the components and any non-aqueous
solvents. One can enhance fluid flow toward the zone by having the
added solution at a higher formality than the solution in the
channel, although an increased formality of the added solution will
occur as a result of evaporation, except for the compensation
provided by the solution in the channel. Where a component,
particularly the test compound, is added as a non-aqueous solution,
it may be desirable to include the test compound in the reservoir
and channel, rather than adding the solution to the opening in the
zone. This avoids problems of dissolving the test compound in the
buffer solution, where the test compound is only moderately soluble
in water. In this way, the non-aqueous solvent becomes equilibrated
in the reservoir(s) and the test compound is instantaneously
diluted into the buffer, preventing separation of the test
compound.
[0057] The subject device can allow for sample dilution, for
example, where the sample comprises a solvent that may interfere
with an intended operation. One can add the sample solution to a
reservoir prior or subsequent to introduction of the reservoir
solution into the reservoir. In the former case, one may have to
wait for equilibration of the test sample compound through the
unit. In the latter case, one can inhibit the movement of the
sample solution until diluted with the reservoir solution and then
distribute the sample containing solution throughout the unit.
Pneumatics, removable barriers, valves, etc may govern movement of
the sample and the sample solution. This operation may be achieved
by using a central dilution vessel into which the sample and
diluent are added. The dilution vessel may have an interface with
liquid in a channel for replenishment of liquid, which has
evaporated.
[0058] Capillary channels would lead from the dilution vessel to
one or more, usually a plurality of zones, where the diluted sample
would migrate by capillary action to the individual zones. As
appropriate, pneumatics, including a hydrostatic head, may be used
to direct the flow of the liquids. The liquid from the dilution
vessel would mix with other liquid(s) in the zone. In this way,
small volumes of a reagent or candidate compound would be
distributed among a number of zones for a subsequent operation,
without initially having to manipulate small volumes. The same
mechanism may be used to distribute an expensive reagent to a
plurality of zones. In this situation, it may not be necessary to
dilute the reagent, where the reagent may be directly added to the
central vessel. The reagent would then be distributed from the
vessel to the various zones. Desirably, the capillary channels will
be relatively short, usually less than 1 cm, more usually less than
about 0.5 cm and more than about 0.1 mm. The volume of the vessel
will usually be at least 100 nl, more usually at least about 300 nl
and less than about 1 ml, usually less than about 0.5 ml, depending
on the amount of the solution to be transferred to each of the
zones and the number of zones. By having a central vessel for
distribution to a plurality of zones, one can reduce errors in
transferring small volumes and provide for substantially equivalent
transfer to a plurality of zones, allowing for direct comparison of
results in each of the zones.
[0059] One may also have one or a multiplicity of vertical
capillary channels comprising a terminal region having a larger
cross-sectional area than the capillary channel which may comprise
a non-wettable region at or above the interface between the
terminal region and the channel. The capillary would be placed in a
reservoir to replenish liquid lost from the zone formed in the
terminal region. As one added new liquid to the terminal region,
initially the meniscus would be raised. Both evaporation and
movement of the meniscus downward would occur, so that displacement
of solution containing an active component would be minimized,
keeping the volume of the zone minimal. The terminal region could
be cylindrical, conical, or the like. Generally, the capillary
channel would be circular, so that the terminal region would have
at least about 1.2 times the capillary channel, frequently at least
about 1.5 times the diameter of the capillary channel and up to
about 20 times.
[0060] In a first application, components are mixed and reduction
of the volume of the mixture due to evaporation substantially
precluded at the time of the addition by providing for contact with
a solution in a channel, where the interface between the solution
in the zone and the solution in the channel is relatively small,
usually having a cross-sectional area of less than about 5
mm.sup.2, usually less than about 2 mm.sup.2, while being at least
about 10 .mu.m.sup.2, more usually at least about 50
.mu.m.sup.2.
[0061] The solution added to the zone will normally involve a
volatile solvent and may also include a non-volatile solvent,
particularly where one or more of the components are not readily
redistributed into the volatile solvent, e.g. water. Various
non-volatile solvents include dimethyl sulfoxide, dimethyl
fomamide, hexamethylphosphoramide, liquid organic salts, such as
higher alkyl (>6) ammonium salts, polyethers, particularly
polyalkylene glycols (alkylene of from 2-3 carbon atoms), such as
dimethyl cellosolve, etc., where the volatility is in relation to
the vapor pressure of water, where the vapor pressure of the
non-aqueous solvent is generally less than half of that of water at
ambient conditions. The solution may be introduced into the zone as
described previously, where the method desirably assures a
consistent amount of the solution being transferred. Alternatively,
as described above, the solution may be distributed from a central
vessel through capillary channels to a plurality of zones.
[0062] Depending on the protocol, the zone, which defines the
reaction volume, may be contained in a region, e.g. space or gap,
between two capillaries, on a platform, in a cylinder, a portion of
a capillary channel, a vessel, such as a well, port, passageway or
chamber, etc. The zone may be contained in a vessel of sufficient
depth to serve as a receiving vessel and/or a portion of the
channel, underneath and/or adjacent to the vessel. The significance
of the zone is that it provides the area of liquid exchange between
the components of the added solution and the channel solution
during the reaction. The zone has an opening that allows for access
for addition of solutions, provides for liquid exchange between
liquid in the zone and liquid in the channel, and permits
evaporation. The channel will have a source of liquid for filling
the channel, usually a reservoir, and normally be filled with the
liquid prior to addition to the zone, which liquid will usually be
buffer, including electrokinesis buffer, containing a component of
interest, and/or reagent(s) or additive(s), or the like, necessary
for the reaction to occur. The liquid will usually be an aqueous
liquid, having at least 20 vol. % of water, usually at least 50
vol. % of water and may be solely water as the solvent. While one
could add all of the components to the zone, so that there need not
be components, e.g. reagents or compound of interest, present in
the liquid in the channel, it will usually be more efficient to
provide at least one component in the channel solution,
particularly where such component is relatively inexpensive, is
provided in a non-aqueous solvent or as a matter of
convenience.
[0063] In an embodiment where the channel serves as the floor of
the zone or there is a floor to the zone, where a capillary channel
outlet is in close proximity to the floor, a spatially restricted
region will frequently be present extending upwardly beyond the
periphery of the channel outlet. The region may have walls that
extend beyond the top of the wall of the capillary channel. The
zone may be all or partially contained in a receptacle that has a
lower surface, usually a floor, and an adjacent portion of the wall
that can be wetted, and desirably, but not necessarily, at least a
portion of the walls, mainly a portion distal to the channel
interface will be non-wettable, so that aqueous media will be
primarily restricted to the lower portion of the receptacle.
[0064] Depending on the nature of the walls of the receptacle or
partial enclosure, the walls may have to be modified to provide the
different properties. Non-wettable walls may be made wettable by
coating with an appropriate hydrophilic composition, e.g. polymers,
such as polyacrylates, having hydroxy- or aminoalkyl substituents,
hydrolysis of hydrophobic polymers having functionalities which can
be hydrolyzed to polar functionalities upon hydrolysis, proteins,
polysaccharides, polyalkyleneoxides, etc., oxidizing the surface
with ozone or other oxidizing agent, functionalizing the surface by
the introduction of hydroxyl, carboxyl or amino groups, etc. For
creating a non-wettable surface from a wettable surface, one may
coat with a higher hydrocarbon or hydrocarbon derivative, such as
grease, wax, lipid, oil, etc., a hydrophobic polymer, such as
polyethylene, polyamide, polyimide, polyester, etc.
[0065] In operation, a component of interest is provided in the
zone, usually being added as a solution, where during the
operation, none, all or part of the solvent may have evaporated.
Alternatively, one may add a powder, gel or other form of the
component of interest. The component may be obtained in a variety
of ways being accessed from a robotic source of a large number of
different components, a dispenser of a common component, or the
like. In some instances, two or more components may be combined and
incubated prior to addition of the mixture to the zone. In some
instances, solutions may be obtained from microtiter plate wells,
where the inlets and zones are positioned for receiving the
contents of the wells into the zones. Microtiter plate wells
usually have 96.times.n.sup.2 wells, where n=1-4. In this
situation, one may use pins, with surface contact transfer,
electrical fields, inertial forces, piezoelectric, electroosmotic
force or a pressure differential to transfer the liquid in the
wells to the subject zones. Generally, the volumes being
transferred from the microtiter wells will be very small, being in
the range described previously.
[0066] In view of the small volumes being transferred, evaporation
will frequently be rapid, and may leave a dry residue of the
components of the solution in the zone. The volume selected for
delivery may be small enough, and the zone size and zone bottom
large enough, that the solution will adhere to the bottom of zone
without significantly entering or even contacting the channel
inlet, where evaporation of the added solution is acceptable.
Preferably, the parameters will be selected so as to inhibit
evaporation to dryness.
[0067] In one embodiment, the microfluidic device will comprise a
layer or substrate of plastic, glass, silicon, or other convenient
materials, which may be hydrophilic, hydrophobic or combination
thereof. The device will usually have a network of various channels
and receptacles formed in the substrate and conveniently enclosed
with a cover of the same or different material. Orifices can be
provided in the cover or substrate, which orifices may serve as
receptacles. There are many different methods of fabrication of a
microfluidic network, which have been described in the literature.
One may have a common source of liquid, which includes a manifold
having a plurality of branches which provides liquid to a plurality
of common channels, much in the way risers are used in plumbing in
apartment buildings.
[0068] The channels may have a surface which is entirely
hydrophilic, entirely hydrophobic or portions may be one or the
other. For example, where there is a cover and a trench forming the
channel, the trench may be hydrophobic and the cover surface
enclosing the trench may be hydrophilic. It appears that having a
portion of the surface hydrophilic along the length of the channel
is sufficient to obtain capillary action and liquid replenishment
in the zone.
[0069] A zone which may be included in a partial enclosure and a
capillary channel, optionally in conjunction with other
microstructures may be considered a unit. Where the subject device
is to be used with microtiter well plates, each unit associated
with a microtiter well would have a zone comprising at least one
channel inlet, usually two opposed channel inlets. Depending on the
protocol and the means of transport of fluids, one may use
electroosmotic force, where there would be an independent pair of
electrodes for moving liquid, or have a common electrode associated
with a plurality of electrodes to provide the opposite polarity to
the common electrode, with the electrodes in contact with the
units. In an embodiment with individual pairs of electrodes at each
unit, the operations usually would be confined to individual units
having a single zone, rather than moving the composition to
different sites and carrying out additional operations, although
the individual pairs of electrodes could be used to provide a
moving wave electrical field as described in U.S. Pat. No.
5,750,015. Thus, the substrate would provide for electrokinetic
channels and the ability to receive electrodes or have the
electrodes painted, adhered or otherwise present on the
substrate.
[0070] However, one could provide for layered channels, where one
would have additional channels connected to the unit channels that
are normal to the plane of the unit channels. One would then have
an additional microfluidic network for addressing the units
individually and performing additional operations on the
compositions. When used with microtiter well plates, one can
provide for a microfluidic network having the zones positioned to
be in alignment with the wells of the microtiter well plates.
[0071] The component of interest may be all or partially dissolved
or dispersed and will reside in the zone. The liquid in the
capillary channel may be present in the zone or may be discharged
from the capillary to define the zone, where the liquid will retain
continuity between the liquid in the zone and the liquid in the
capillary channel. Various means can be employed for pumping the
liquid from the channel into the zone, including electrokinetic,
pneumatic, mechanical, sonic, capillary, thermal, or the like.
While the particular mode for moving the liquid into and out of the
capillary is not critical, many advantages accrue by using
electroosmotic or pneumatic pumping, where small volumes can be
moved in different directions by changes in direction of an
electrical field or by application of differential pressures. Where
electroosmotic pumping is used, one requires a channel with a
region where the walls are charged or the solution includes a
soluble charged polymer, such as an aminodextran, so that ions in
the liquid of opposite charge to the wall charge accumulate at the
wall. In the presence of an electrical field, the ions adjacent to
the wall will move toward the electrode of opposite charge and
carry liquid with them, providing a liquid pump. In this way, one
can push liquid with significant precision from the channel into
the area outside the capillary to define a zone and then withdraw
the liquid in the zone back into the channel. The pump can be used
to move liquid, which is not under the influence of an electrical
field, diminishing electrokinetic separation in the solution. By
this means, one may move liquid in defined volumes containing
components, which may be adversely affected, by an electrical
field. Alternatively, one may use pneumatic devices to move the
liquid.
[0072] In order to automatically determine when the desired liquid
volume has been introduced into the zone, rather than relying on
the parameters which were used to pump the liquid into the zone,
such as voltage, time, temperature, etc., one can provide for a
detection system. One system uses an ionic medium, conveniently
introduced into a channel connected to the zone, with a detection
electrode in the ionic medium connected to a voltage source or
ground. When electrokinetic pumping is employed, there will be an
electrical field in the fluid. When the fluid in the zone contacts
the ionic medium, a circuit will be formed with the detection
electrode, which can be detected and further pumping terminated or
the electrical field will be grounded and further pumping stopped.
One may simply have an electrode in the zone, which when contacted
with the liquid from the channel will act as described above.
Instead of an electrical detection system, one may use an optical
system, which detects the extent to which the liquid has penetrated
the zone. The particular mode of detection will depend to some
degree on the choice of the mode of transferring the fluid into and
out of the zone.
[0073] If desired, evaporation during the course of the reaction
may be impeded by closing the zone to the atmosphere, where
feasible, adding a solvated polymer to the solution, and the like.
A polymer may have the further advantage of reducing diffusion of
the components from the zone into the channel solution. Polymers,
which may be used, include polyethylene oxides, polypropylene
oxides, ethers and esters of such polymers, polyacrylamides,
dextran, modified dextrans, or other polymers which are water
soluble. Generally, such polymers would be present in less than
about 5 wt. % of the solution, preferably less than about 1 wt. %
of the solution.
[0074] In the situation where the solvent substantially evaporates
prior to dissolution in the channel liquid, the volume of liquid
discharged from the channel may serve to concentrate the components
from the well in the zone.
[0075] Where the zone is formed by expression of fluid from a
channel, the fluid in the zone, during the brief period after
introduction of the fluid from the channel into the zone, is
prevented from significant reduction in volume by the reservoir of
fluid in the channel. The fluid in the zone can be rapidly drawn
back into the enclosed channel with substantially the same volume
that was introduced from the channel into the zone and whatever
fluid was present from addition of fluid to the zone, which has not
previously evaporated. The zone solution may be withdrawn into the
channel as a defined volume. One now has a defined volume of fluid
as the zone in the channel, which will substantially retain its
composition, since diffusion can be relatively slow. Furthermore,
since some evaporation will occur at the channel outlet, the liquid
will flow in the channel toward the zone, reducing movement of
components away from the zone. In addition, by using microfluidics
and electrokinesis, the zone may be moved to any site in the
microfluidic network and be subject to various operations, such as
the addition of reagents, separation of components, heating,
cooling, etc., without significant change in its composition,
except for the added components.
[0076] In another mode, one may employ opposed capillary channels
to provide a continuous liquid fluid column as part of the
manipulations of the various components. In this embodiment, the
stream extends from one channel to the opposed channel through the
zone liquid during the operation of the unit. At one or more
different times, there may be a break in the column, particularly,
where the column may be interrupted in the zone area. One may
initially have liquid in one or both capillary channels and/or in
the zone area. There may be a plurality of zones, which are not
separated by walls from each other, being gaps between a plurality
of channel outlets. In this situation, the opposed capillary
channel outlets would be relatively close to each other, generally
spaced apart by not more than about 5 mm, usually not more than
about 2 mm, and preferably not more than about 1 mm. In this
manner, one may have a plurality of opposed capillary channels in a
block, which are separated by a gap, where liquid may be discharged
from one or both capillary channels to cross the gap and form a
continuous liquid column.
[0077] The openings of the channels at the gap are conveniently in
the range of about 10.sup.2 to 5.times.10.sup.5 .mu..sup.2. The
volume of liquid in the gap will usually be in the range of about 1
to about 10.sup.3 nl. The liquid droplet between the opposed
channels serves as the zone for addition of solutions. Various
methods may be used for addition to the liquid in the gap, as
described previously. Generally, each individual addition to the
gap liquid or zone will not exceed about 500 nl, more usually not
exceed about 250 nl. As appropriate, after each addition to the gap
liquid or zone, the solution in the gap may be withdrawn into a
channel and incubated and the signal then determined or discharged
from the channel and the signal determined without interference
from the device composition. The opposed channels may be provided
in blocks comprising a plurality of channels, where one could have
a planar array of opposed channels, as described in FIGS. 3 and 5,
where the chamber is substituted with a gap. Additions could then
be made at each gap from an array of devices for transferring
liquids in small volumes and the manifold could be as depicted, or
one could have different main channels providing different
solutions for the different rows of units. In this way, devices can
be provided which have 20 or more units, up to 2,000 or more
units.
[0078] The size of the zone will be affected by the sizes of the
ports, outlets and channels, volumes of the solutions added to the
zones, the amount of liquid in the channel into which the
components of the added solutions diffuse, by the nature (regions
of wettability and non-wettability) of the walls enclosing the
zone, the rate of evaporation, which may be related to the
humidity, depth of the zone and air flow above the zone, the time
of the reaction, the temperature, the composition of the solution
in the channel, particularly as to the solution viscosity, and the
like. Generally, these parameters will be selected to provide a
dilution in the zone of the sample component added to the zone in
the range of about 0.1 to 10:1, during the course of the reaction.
Incubations may involve from about 1 min. to 24 h, usually not
exceeding about 12 h. The reaction time will usually require at
least 1 min., usually at least about 5 mins, and not more than
about 6 h, usually not more than abut 2 h. Ambient conditions will
usually suffice, with temperatures below about 60.degree. C., more
usually not more than about 40.degree. C. In some situations where
thermal cycling is involved, temperatures may be as high as
95.degree. C., usually not exceeding about 85.degree. C., and
cycling between 45.degree. C. and 95.degree. C. Heating can be
achieved with lasers, light flashes, resistance heaters, infrared,
heat transfer, conduction, magnetic heaters, and the like.
[0079] Components of interest for use in many of the determinations
include small organic molecules about 100 Dal to 5 kDal in
molecular weight, more usually not more than about 2.5 kDal,
oligopeptides, oligonucleotides, and oligosaccharides, proteins,
sugars, nucleic acids, microsomes, membranes, cells, organelles,
tissue, etc., where the components may serve as ligands, receptors,
enzymes, substrates, cofactors, functional nucleic acid sequences,
e.g., promoters and enhancers, transcription factors, etc.
Reactions of interest will include binding reactions, which may
involve enzymes, receptors, transcription factors, nucleic acids,
lectins, and the like, where inhibition, activation, signal
transduction, antagonists, and chemical reactions may be involved.
Various protocols and different device structures may exemplify the
subject devices.
[0080] In one exemplification of the use of the subject devices
employing microtiter well plates, the microtiter well plate will
have solutions which are to be analyzed, but lack one or more
components necessary for the analysis. These solutions will usually
be constituted to determine a binding event, interactions between
two moieties, the presence of a particular moiety, and the like.
The solutions in the wells may involve a single compound to be
tested, a mixture of compounds including a test or control
compound, or the like. Normally, there will be different
compositions in different wells. The wells may involve
heterogeneous binding, where a component of the determination
method is bound to the surface of the wells and will be retained in
the well. For example, in a specific binding assay, one may have
receptors bound to the surface of the well and allow for a
competition between a test compound and a labeled analog for
binding to the receptor. After incubating the mixture in the well,
the mixture is transferred to the microfluidic device zone and the
label determined. Where the label is an enzyme, the liquid in the
zone could include substrate for the enzyme, where the product of
the substrate would provide a detectable signal. Alternatively, the
label could be a fluorescer, where one would read the fluorescence
in the zone. In both instances, the determination could be made in
the absence of bound label.
[0081] There is also the opportunity to perform a heterogeneous
assay in the zone. By having a non-diffusively bound entity, e.g.,
compound, cell, tissue, etc., for which the candidate and control
compounds compete, where the bound entity is in limited amount, one
can determine the activity of the candidate compound. By limited is
intended that it is insufficient to bind more than about 75%,
usually about 50%, of the total number of molecules of candidate
and control. In carrying out the determination, the candidate or
test compound and control are added to the zone. The bound compound
is in the zone, bound to any surface associated with the zone,
including walls, which includes the walls of the zone enclosure and
channel walls, particles and the like.
[0082] For example, one may coat the region surrounding the zone
with an entity, e.g., cell, compound, etc., where the entity
becomes bound in that region. The channel is then filled with a
solution and the candidate compound and control compound added into
the zone. The candidate and control compounds will compete for
available binding sites of the bound entity. After sufficient time
for reaction to occur, one may move the liquid in the zone. The
system allows for the addition of very small volumes to a reaction
mixture, where the dilution of the volume(s) may be controlled by
the size of the zone. During the competitive binding reaction, the
competitive compounds will be substantially retained in the region.
Removal of the control compound and washing of the region is
readily achieved by moving the liquid column in the channel, and
one can readily detect the signal in the channel.
[0083] By coupling of the assay system with an electrokinesis
system, where components can be separated, mixtures of candidates
may be put into a well to bind to a bound receptor in the presence
of a detectable binding compound. One could then transfer the
various candidate compounds and control to the electrokinesis
separation and determine whether any of the candidate compounds
displaced the control compound. If it appears that at least one
candidate compound has sufficient affinity for the receptor, the
candidate compounds may be separated into bands and the bands
analyzed, for example, by mass spectrometry. By knowing the
mobility of the individual compounds, one can time when the band
should be isolated and identified.
[0084] To enhance the surface area associated with the zone, one
may have a wettable porous membrane between the channel and zone
interface. The membrane may serve a number of functions, retaining
particles in the zone, providing surface for binding entities,
acting as a filter, and the like. Particles may be introduced into
the zone and held in position by a variety of ways, through
covalent or non-covalent bonding to the walls, barriers to
movements, such as protrusions, cross-bars, magnetic particles,
etc.
[0085] Instead of a heterogeneous system, namely a system requiring
binding to a surface and a separation, one may use homogeneous
assay protocols. Homogeneous assays may be exemplified by EMIT,
FRET, LOCI, SLFIA, channeling assays, fluorescence protection
assays, fluorescence polarization, reporter gene assays using whole
cells, particle labels, etc., where enzyme, particle, fluorescer
and chemiluminescer labels are employed. In these assays, one does
not require a separation, since the binding event changes the level
of observed signal. One would carry out the protocol in the same
manner, but for the binding of the bound compound and the
separation step, as the assay requiring the separation, where the
liquid in the channel could provide one or more reagents required
for the determination of the signal and/or provide a convenient
site for detection of a signal.
[0086] In some instances one may wish to monitor the effect of a
test compound on enzyme activity. In this situation one may add the
test compound and enzyme to the zone comprising the channel
solution, which provides the substrate. After sufficient time for
reaction to occur, one may then determine the extent of the enzyme
activity in the presence of the test compound.
[0087] Other assays of interest involve the effect of a test
compound on the association of two other compounds, usually
proteins, as members of a complex. These associations include
transcription factors, cell surface receptors with other proteins,
e.g. G-proteins, proteins binding to nucleic acids, e.g., DNA,
lectins with sugars, subunit associations, etc. These assays may be
carried out in substantially the same way as the heterogeneous
assay, where one member of the complex is bound to the zone
surface. However, in this case, instead of using a labeled member
of the complex, the liquid in the channel could provide for an
assay of the complex member. First, one would combine the candidate
compound and the two members of the complex, either in a well or in
a zone. The amount of complex formation and, therefore, amount of
free uncomplexed members would be related to the effect of the
candidate compound on complex formation., Once there has been
sufficient time for complex formation, the determinations in each
zone could be performed. By performing assays where a common liquid
is used for all of the zones, one can perform a number of discrete
steps. For example, since the complex member to be measured would
be common to all of the assay determinations, one could provide for
capture of the complex member in the channel portion of the zone,
e.g. by having specific antibodies for the complex member. One
could then wash out all of the channels using buffer, and then add
a second solution comprising labeled specific antibody, which would
bind to any of the complex member captured in the channel. With a
fluorescent label, one could detect fluorescence. If one does not
wish to capture the complex member, one may use several of the
homogeneous assays and determine the level of the complex present
in the zone.
[0088] One may use cells or compounds that are bound to the surface
in the zone. These cells or compounds may serve a variety of
functions, such as local buffering, production of agents to
interact with agents in the zone, interacting with agents from the
zone, production of detectable signals, etc. For example, by using
polymers comprising buffering agents, the acidity or alkalinity of
the solution in the zone may be controlled. Where a product is
produced in the zone, which can bind to a surface membrane receptor
of the cell and transduce a signal resulting in expression of a
detectable product, the production of such product, may be
monitored by the signal produced by the cell. Various compounds are
known to bind to surface membrane receptors and transduce signals,
such as steroids, hormones, interleukins, growth factors, etc., and
biomimetric analogs thereof. By having a reaction in the zone that
results in an active ligand, diffusion of the ligand to the cell,
will result in the transduction of a signal. By having a regulatory
region, e.g. promoter and/or enhancer, responsive to the transduced
signal, where expression results in a detectable product, e.g.
green fluorescent protein, an enzyme that catalyzes a detectable
product, etc., one can monitor the rate at which the ligand is
produced. Where one is screening for compounds, which activate or
inhibit formation of the ligand, the production of the detectable
signal would indicate the activity of a candidate compound.
[0089] With appropriate controls, one may take aliquots from the
microtiter plate wells or other source of reaction components, so
that one may obtain a plurality of determinations from a single
mixture. In some situations, it may be feasible to control the
volume transferred to the zone by using the detection systems
described for determining the volume of liquid discharged from the
channel. Alternatively, one may have detection systems in the
zones. Other monitoring methods may also find use. One would then
carry out an individual operation with a first microfluidic device,
remove the device and replace it with a second fresh microfluidic
device, and so on. When dealing with rare agents, such as test
compounds, there would be minimal loss of the test compound during
the operations and one could obtain a plurality of determinations
concerning the test compound. One could directly move a test
compound in a microtiter plate well from the well through an
opening in the zone into the zone containing a reaction medium.
After sufficient time for reaction to occur, one may then read a
signal through the opening.
[0090] Of interest when measuring a signal is the presence of an
orifice above the liquid in the channel, which allows for
evaporation at the site of the determination, where the area in and
optionally below the orifice serves as the zone. This zone may
serve as an assay well, a reagent accepting well, a reaction
vessel, etc. The solution of interest in the zone is bordered by
liquid, so that the adjacent fluid acts as a reservoir for
replenishing the liquid, which is lost by evaporation. This results
in fluid flow toward the zone, which maintains the solutes in the
zone, so that there is less diffusion away from the zone of the
signal producing components during the time of measurement. By
having a region associated with the zone of diminished area at
which there is liquid exchange, diffusion is diminished, while
liquid replenishment occurs. For example, in the case of a
passageway through the wall of a capillary channel, which serves as
at least a portion of the zone, the cross-section of the capillary
channel is chosen to discourage significant diffusion from the
region underneath the passageway, namely be less than the
passageway cross-section. The reduction in the rate of diffusion of
components from the zone allows for accurate rate determinations,
since the change in signal will be substantially larger than the
reduction in signal resulting from diffusion away of the
signal-producing moiety.
[0091] Generally, one will have two entities interacting, where all
or a portion of the two entities may be added to the well and any
additional portion of the entities provided by the medium from the
capillary. By referring to portion is intended only one entity or a
portion of both entities, where the remaining amount of the two
entities comes from the capillary. Since one will usually not wish
to have any reaction between two entities involved in the operation
prior to initiation of the reaction in the zone, normally at least
one entity will be added to the zone immediately prior to
initiating the reaction. However, in some instances where the
operation cannot proceed except at an elevated temperature or in
the absence of light, then the entities may be combined prior to
addition or added at the same time.
[0092] The subject devices allow for a wide variety of
applications. In one application, where the zone is at the terminus
of the capillary channel, one may introduce a drop of a solution
containing one or more components or reagents from a channel into
the zone, prior, subsequent or concomitant with introducing a test
component into the zone, where one is interested in the binding of
the test component to a reagent in the liquid mixture. One would
then withdraw the liquid in the zone into the channel, diminishing
evaporation. The mixture could be incubated for a predetermined
period of time. By providing that binding of the test component to
the reagent results in a detectable signal, one can determine the
binding of the test component to its target. For example, a reagent
which is a complex of a protein target and a known ligand, where
the protein is conjugated with quencher and the ligand with a
fluorescer, release of the ligand will result in a fluorescent
signal. By measuring the increase in fluorescence as a result of
the test component binding to target protein and displacing the
fluorescent ligand conjugate, one can determine the binding
affinity of the test component to the target protein.
[0093] An alternative assay could use the opposed channels
separated by a gap having a floor. In the gap one would bind
different enzyme alleles at different spaces on the floor between
each of the pairs of opposed channels. A solution of a compound
would then be passed through the opening created by the gap and the
mixture allowed to incubate, while in contact with the liquid in
the channel. After sufficient time, a solution of the substrate
would then be directed from the other channel into the gap to join
with the liquid from the opposing channel. In this way substrate
would be continuously supplied from the other channel. The turnover
rate of the enzyme would be determined by detecting product in the
gap, where the turnover rate would be constant, or increase with
time. The rate would be related to the inhibitory effect of the
compound and its binding affinity. For different alleles, one could
have a single source or manifold of substrate solutions for
supplying the individual channels where electroosmotic force could
be used for pumping the substrate solution through the channels.
This device allows one to rapidly determine the effect of a
compound on different alleles. Rather than different alleles, one
could have different enzymes and have different substrates in the
different channels and any combination of related or unrelated
entities.
[0094] In another method, one would have a continuous liquid column
with opposed channels and gaps between the channels to define
zones. Mixtures of enzymes and candidate and control compounds
would be prepared and added to the zones, simultaneously or
consecutively. After sufficient time of incubation, the liquids in
the wells would be introduced to the zone. In the channels would be
an appropriate substrate buffer solution. The solutions would mix
with the buffer solution and evaporation would occur. The effect of
the evaporation is to maintain the product narrowly confined to the
zone as a result of liquid flow from the channels into the zone to
replace the liquid lost by evaporation. By providing for production
of a detectable product, one could determine the effect of the
compounds on the enzymes.
[0095] In a further method, one would transfer a solution into an
orifice, well or passageway in an otherwise enclosed channel into
the zone and allow the solvent to evaporate. The solution would
form a droplet on the surface of the channel and leave its
components on the surface as a small spot. The components could be
cells and a candidate compound for a cell surface receptor. The
cells would adhere to the surface. Liquid would then be expressed
from the channel into the zone, or a reservoir(s) filled to direct
liquid into the zone, where the channel liquid introduced into the
zone would have a ligand conjugate, for example, a fluorescent
conjugate. After allowing sufficient time for the fluorescent
conjugate to bind to any available receptor binding sites, the
liquid would be withdrawn into the channel away from the zone and
the fluorescence read. If liquid were necessary for the reading, a
different liquid could be introduced into the zone through the
orifice or from the reservoir. The binding of the candidate
compound would be determined by the reduction in fluorescence in
the zone. Where the well is an opening in a channel wall,
substantially the same process could be performed without
withdrawal of the liquid into the channel.
[0096] Obviously, there are too many operations which may be
carried out, employing different diagnostic assay reagents,
different targets and different protocols, to exemplify all of
them, so that only a few have been illustrated as exemplary of the
subject methodology.
[0097] The device may provide for heating and cooling of the zone.
By varying the temperature of the channel, a large heat sink or
source is provided for the zone. By having means for heating or
cooling the fluid in the channel, one can modify the temperature of
the zone, cycling the zone temperature in relation to the channel.
To provide for more rapid variation in temperature, one may provide
for heating and/or cooling solely in the zone, where once the
source of thermal variation in the zone is terminated, the zone
would rapidly equilibrate with the temperature of the channel. For
example, in thermal cycling, one could use microwave heating, RF
heating, laser heating, or the like, where the electromagnetic
heating source is focused on the zone, so as primarily to change
the temperature of the zone. In processes involving thermal
cycling, such as the polymerase chain reaction, one would rapidly
raise the temperature of the zone to 85-95.degree. C., while
maintaining the channel temperature at about 35-50.degree. C. Once
the DNA has been denatured, which would be a matter of not more
than about 2 or 3 minutes, usually less, by removing the source of
heat, the liquid in the zone would rapidly equilibrate with the
temperature of the liquid in the channel. By appropriate selection
of the temperature of the liquid in the channel, the temperature
profile during the cycling may be controlled to provide the desired
times for the different temperature stages of the cycle.
[0098] The amplification may occur in solution or on beads, as in
bridged amplification. See, for example, U.S. Pat. No. 5,641,658.
By having the source of the DNA in the channels, all of the zones
may include the same DNA or by providing different DNA in different
channels, different zones may have different DNA. Conveniently, the
channels may also provide the dNTPs and primers, or the dNTPs and
primers may be added to the zones, as well as other components,
e.g. ddNTPs. By adding the DNA polymerase to the zone through the
orifice to the zone, the reaction may be initiated and cycled to
amplify the DNA. After completion of the thermal cycling, the
amplified DNA may be used for sequence determination,
identification of particular sequences, using probes, snps may be
identified or other characteristic of the amplified DNA may be
identified. Various protocols exist for identification of complex
formation between a probe and target DNA, which may occur in the
zone or as a result of analysis outside of the zone.
[0099] The subject systems may be used with many other ancillary
systems to further enhance the flexibility and variety of
operations for the system. One combination is with electrokinesis,
where the zone would be part of a channel in which an electrical
field is employed. By having reservoirs at opposite ends of the
channel or using the zone as one reservoir, by applying an
electrical field across the zone, charged species could be moved
from the zone into the channel. Alternatively, one may use
electroosmotic pumping to move the liquid in the zone to another
site. By having crossed channels in the electrokinetic unit,
components of the zone may be moved to an intersection and a
defined volume injected into a second channel, where the defined
volume may be subjected to different operations. The defined volume
may be analyzed by electrophoretic separation, where the result of
the operation in the zone is to have two or more detectable species
having different mobilities in electrophoresis. One can provide for
a detector along the second channel to identify the detectable
species and quantitate the detectable species. Since one would be
able to quantitate the initial and final agents, one would have a
material balance.
[0100] In one embodiment, one has an assay system comprising the
hydrophobic zone or well connected to one or more hydrophilic
reservoirs through a hydrophilic channel, where the zone or
channel, usually the channel, is connected to a side capillary
channel for connection to an electrokinesis system, that is,
providing for electrophoresis and/or electroosmosis. The two
systems may be connected in the same substrate and be substantially
in the same plane of the substrate, where the size of the channels
may differ in relation to their function. Thus, the capillaries of
the electrokinesis system may be the same as or smaller than the
capillaries of the assay system, and the reservoirs of the
electrokinesis system may be the same, larger or smaller than the
reservoirs of the assay system. The components of interest of the
zone for analysis by the electrokinesis system will usually be
charged, so that they can be transported by an electrical field
from the assay zone to the electrokinesis system, where the
components may be further processed, e.g. separated into bands,
purified for further analysis, e.g. a mass spectrometer, etc.
Conveniently, the side channel may be connected to an analytical
channel, whose length will depend on the nature of the analysis and
may be as short as 1 mm and as long as 50 cm, usually being between
2 mm and 10 cm. The channels of the electrokinesis system will
terminate in reservoirs, usually serving as waste reservoirs or
buffer reservoirs. It should be understood that the electrokinesis
systems may take any configuration of any electrokinesis system as
may be required for the particular procedure. The components of the
zone may be moved to the intersection of the side channel with the
analytical channel, where a waste channel terminating in a waste
reservoir may be directly across from the side channel or offset
from the side channel to form a double-tee. In either event, the
components will be moved into and across the analytical channel by
means of electrodes providing an electrical field between the zone
and the waste reservoir. Once the desired composition of components
is in the analytical channel, which may be a constant composition
having the composition of the liquid in the zone, the electrical
field may be changed so as to have the strongest field along the
analytical channel, whereby the assay medium in the channel is
injected away from the intersection toward the analytical waste
reservoir. By providing for a medium in the analytical channel,
such as a sieving medium, the assay mixture may be separated into
components. Where the components provide a detectable signal, e.g.
fluorescence, electrochemical, etc., a detector may be provided at
an appropriate site along the analytical channel to detect the
components as they move past the detector.
[0101] In many situations one may wish to separation constituents
of an assay mixture. Where the substrate and product of an enzyme
assay or chemical assay both provide the same signal, e.g.
fluorescence, but have different mobilities, the substrate and
product may be readily determined by using electrophoresis. Where
multiplexed reactions are performed in the zone, one will have an
interest in detecting the plurality of events that may have
occurred. For example, one may have a plurality of reagents
carrying electrophoretic tags (labels which have different
mobilities in electrophoresis), where the result of the process in
the zone is to release an electrophoretic tag in the presence of a
target moiety. Where there may be a plurality of target moieties in
the sample, the ability to detect the presence of the target
moieties by the separation of released electrophoretic tags greatly
enhances the simplicity with which the process may be carried out.
Since the entire process may be automated, the addition of the
assay components, the processing of the assay, the movement of the
assay components into the electrokinesis system and the separation,
confusion between samples is substantially eliminated, direct
comparisons are achieved between samples and controls, component
handling is minimized and more accurate results can be
obtained.
[0102] The units may or may not have electrodes associated with
each unit. Electrodes may be provided by painting electrically
conductive wires on the surface of the card to be in contact with
the solutions in the reservoirs or a "bed of nails" may be used,
where a plurality of electrodes extend from the surface of a plate,
each electrode associated with a unit having individually
controlled voltage, and the electrodes may be introduced into the
reservoirs or zones simultaneously. The entire system may be
computer controlled, so that all or some of the steps may be
automated. These steps include rinsing the system, additions of
components, control of conditions, such as temperature, incubation
time, movement of assay components and electrokinetic analysis,
detection and analysis of results. The combination of systems finds
use with homogeneous and heterogeneous immunoassays, chemical
assays, high throughput screening of compounds, e.g. drugs,
pesticides, etc., nucleic acids analyses, e.g. identification of
sequences, sequencing, identification of snps, mutations, etc., and
the like.
[0103] The zone may be combined with other devices for separation,
analysis, etc. These devices may be HPLC columns, which may be
miniaturized, connectors to gas chromatographic devices, mass
spectrometric devices, spectrophotometers, fluorimeters, etc. By
providing for pneumatic movement of the liquid in the zone to a
channel, which directs the liquid to the other device, the liquid
in the channel may be moved from the zone to the site where it may
be analyzed. One can withdraw samples from individual zones, by
employing reduced pressure above the zone, which will withdraw
liquid from the zone into the device for analysis. One need only
have a small pressure differential between the channel and above
the liquid in the zone to have the liquid in the channel chase the
liquid in the zone to a different site.
[0104] For the devices, large networks of channels may be produced
in small integrated devices using a solid substrate, plate, block
or film, commonly referred to as a card or chip, having one
dimension ranging from about 5 mm to 10 cm and a second dimension
ranging from about 5 mm to 50 cm, usually not more than about 20
cm, and preferably not more than about 10 cm, where the thickness
may or may not be critical. In many cases, microstructures, such as
channels and reservoirs may be formed in one substrate and the
microstructures, enclosed as appropriate, with a cover or other
substrate. The thickness of the device will depend on a number of
factors, generally ranging from about 0.2 mm to about 5 mm, more
usually from about 0.5 mm to about 2 mm. The thickness of the
layers will determine, in part, the height of the ports and the
dimensions of the channels, particularly channel height. Depending
on the structures and protocols, there may be no orifice, the zone
open to its environment being present in a gap or being in a part,
channel or combination thereof. The part in the cover or base layer
may have a depth as small as 1 .mu.m and will usually be less than
about 3 mm, generally being in the range of about 100 .mu.m to 2.5
mm. Where there is a combination of a port or well and channel,
desirably the port or well will have a height of at least about 0.1
mm, and may be 2.5 mm or more, usually less than about 1 mm. One
may have as many individual units as space allows, desirably having
at least about 12, more usually at least about 36 and up to 2,000
or more.
[0105] When having ports in channels, where the port comprises at
least a portion of the zone, the chip will usually be comprised of
at least two layers, a base layer comprising depressions or
cavities, which may serve as channels, chambers, electrode contacts
or connectors, and optionally ports to the depressions and
cavities, and a cover layer, which encloses the depressions and
cavities and may alternatively provide ports to the depressions and
cavities. Additional layers may be present, laminated to the
substrate, such as heat transfer layers, supports, casings, where
films are used as the substrate and cover, and the like. The
substrates may be flexible or rigid, usually not elastomeric, and
may be composed of various materials, such as silicon, fused
silica, glass, plastics, e.g. acrylates, polybornenes,
polystyrenes, polydialkylsiloxanes, polycarbonates, polyesters,
etc.
[0106] In FIG. 1, a fragment of a device is shown in perspective.
The device 10 comprises a first layer substrate 12 of sufficient
thickness to accommodate the features for the operation of the
device 10. Sealed to the substrate 12 is base 14. Embodied in the
substrate are units 16. Each of the units comprises a reservoir 18
in which contact electrode 20 extends from surface wire 22. The
contact electrodes 20 and surface wires 22 may be wires,
electrically conducting paint, or other means of electrical
conduction. The surface wires 22 are connected to a controlled
voltage source for providing an electric potential in accordance
with a predetermined regimen. The reservoir 18 has port 24, for
allowing communication with the atmosphere, and may be employed for
introduction and removal of materials into and from the reservoir
18. Chamber 26 has port 28, where chamber 26 differs from reservoir
18 in its function, and will usually have different dimensions from
reservoir 18. For the most part, the cross-section of the chamber
26 will be smaller than the cross-section of the reservoir,
generally being smaller by at least about 10%, usually at least
about 25%, and not more than about 90%, and larger than the
cross-section of the capillary 36. Normally, there will not be an
electrical connection in chamber 26, although an electrode may be
employed for monitoring the presence and or amount of fluid in the
chamber. Adding an additional wire to the device can be readily
accomplished in the same manner as the electrical connections for
the reservoirs 18. Not shown is an optical detector, which could be
used for detection of the presence or amount of liquid in the
reservoir 18. Reservoir 30 is substantially the same as reservoir
18 in having contact electrode 32 in electrical connection with
surface wire 34. Reservoir 30 is optional, but may be present where
greater versatility is desired in the device, rather than only a
single chamber and a single reservoir per unit. Horizontal channel
36 provides fluid connection between the reservoirs 18 and 30 and
the chamber 26. Finally, electrode 38 extends through substrate 12
into horizontal channel 36 and is connected to surface wire 40 for
connection to a control device.
[0107] Depending on the manner of the use of the device, the
surfaces of the various parts may vary, as to wettability and
charge. For example, the upper portion of the inner wall 42 of the
chamber 26 may be coated with a hydrophobic material to prevent
aqueous media from rising up the wall. The region 44 in the channel
36 under the chamber 26 will be desirably wettable, so that aqueous
solutions introduced into the chamber will wet the surface.
Depending on what form of electrokinesis is used, electrophoresis
or electroosmotic force (EOF), the surfaces of the channels will
differ. For electrophoresis, it is desirable that the surface be
neutral, while for EOF the surface should be charged, although by
using an electrically charged water soluble polymer in the aqueous
medium, where the charges are randomly distributed, neutral
surfaces can be used. Charged surfaces may be achieved by using
silicates, e.g. glass, charged coatings, covalently bonded or
adhering, to the surfaces, or modifying neutral surfaces chemically
to introduce charged species. Neutral species may be a variety of
polymers, both addition and condensation polymers, particularly
acrylates, although polystyrenes, polyolefins, etc. find use.
Different regions may have different charge and functional
characteristics. For example, a portion of a structural feature may
be charged to permit EOF and another portion be neutral, where the
charged portion is a conduit for movement of fluid under the urging
of the EOF flow. During operation, there will be a fluid in at
least one of reservoirs 18 and 30 and at least a portion of channel
36, and there may be fluid as well in chamber 26, where there would
be a continuous or discontinuous stream in the unit.
[0108] In FIGS. 2A, 2B and 2C, are depicted diagrammatic
cross-sectional views of a unit in a device. The unit device 200a
has substrate 202a, in which the various features of the unit
device are present, and cover 204a. The unit comprises a channel
206a, which may be connected to a common manifold for receiving a
medium common to all of the units. Each unit has two wells 208a and
210a, where either or both may serve as wells for introduction of
fluids. Situated in the channel 206a are two sets of electrodes,
212a and 214a, where the electrodes may be painted onto or over
204a and chamber 216a all communicate with channel 206a. The
surface 218a under chamber 216a, which is the surface of the cover
204a, is hydrophilic for acceptance of hydrophilic liquids. The
unit is shown prior to introduction of any liquid.
[0109] In FIG. 2B, liquid 220b is introduced into the wells 208b
and 210b. In the present configuration, the liquid is indicated as
being the same, but with different protocols the liquid could be
different. The liquid 220b from the wells 208b and 210b moves by
capillary action into channel 206b and halts at chamber 216b, due
to the absence of capillarity at the chamber 206b. A sample may
then be added to chamber 216b, which will wet the surface 218b.
Where the sample is small enough, it will not contact the inlet
ports 222b and 224b of channel 206b. Depending upon the nature of
the solvent added to the chamber 216b and the time interval in
which the solvent is allowed to stand, all or a portion of the
solvent may evaporate, so that upon total evaporation, only a
solvent free liquid or solid will be present.
[0110] In FIG. 2C, contact is made between the material in the
chamber 216c and the liquid 220c. Liquid 220c may be expressed into
chamber 216c using one or both pairs of electrodes 212c and 214c,
using EOF for moving the liquid 220c. As shown in FIG. 2C, the
channel 206c is filled with the liquid 220c, so as to form a
continuous stream of liquid. However, it is not necessary to have a
continuous stream, and if desired, the stream may be discontinuous,
where fluid is driven by only one set of electrodes and is stopped
before making contact with the fluid in the channel 206c on the
other side of the chamber 216c. In the latter situation, one may
wish to withdraw the liquid from the chamber into the enclosed
portion of channel 206c to inhibit evaporation of the solution.
[0111] In FIG. 3, a diagrammatic plan view of a device is shown
comprising a plurality of units and employing a common manifold for
delivering liquid to the wells. This device is distinguished from
the device depicted in FIG. 2 in having a common source of liquid,
rather than allowing for different liquids to be available for
different units. The device 300 comprises a substrate 302 and a
cover 304, on which the substrate 302 is supported. The device has
a common inlet port 306 and tributary channels 310. Each of the
tributary channels 310 is connected to a plurality of side channels
312, which serve to provide liquid to chambers 316. Each side
channel 312 is equipped with a pair of electrodes 314 for EOF
pumping of liquid into and out of chambers 316. Liquid introduced
into the inlet port 306 will move by capillary action through the
channels 308, 310 and 312 to fill the manifold, but not enter the
chambers 316. Different samples may be added by any convenient
means to each of the chambers 316 and the sample may be further
processed. Usually, with an aqueous sample there will be rapid
evaporation. By using the pairs of electrodes 314 associated with
one of the two side channels 312 associated with each of the
chambers 316, a small volume of the liquid in the manifolds may be
pumped into the chamber 316 to dilute the sample and then be
rapidly withdrawn back into the side channel as a defined volume to
allow for any incubation and inhibit further evaporation. The
presence of the fluid in the channel in contact with the defined
volume will replenish any of the solvent, which evaporates due to
the presence of the inlet from the channel 312 into the chamber
316. In this way the composition of the defined volume will remain
substantially constant in that the flow of solvent is into the
defined volume and diffusion away of the larger components from the
defined volume is discouraged. After sufficient time for any
reaction to occur between the sample components and the components
of the liquid, a reading may be taken of the defined volume in the
channel or the defined volume may be pumped into the chamber 316
for taking the reading, to avoid having to read through the cover
304 composition. If one wishes to make a plurality of readings in
the chamber 316, or even in the case where a single reading is
made, the defined volume may be introduced into the chamber 316 and
contact made with the liquid in the opposing side channel 312.
Contact may be made by pumping the liquid from the opposing channel
312 into the chamber 316 or by adding enough volume from the
channel containing the defined volume to bridge the floor of the
chamber and join the fluid in the opposing channel 312.
[0112] The presence of the sample in the chamber in contact with
the two side channels permits replenishment of liquid, which
evaporates from the solution in the chamber. Diffusion of the
components of interest is not significant, so that the loss of the
components of interest in the zone is minimal and the signal from
the solution in the chamber remains substantially constant over
extended periods of time, particularly within the time frame of the
usual measurements, generally under about 6 h, usually under 3 h.
Since one is dealing with very small volumes, generally less than
about 500 nl, substantial changes in composition could have an
effect on the observed signal. For example, where one is interested
in a binding affinity of a ligand to a receptor, a change in
concentration of the ligand and/or receptor would affect the
observed signal. Where one is interested in determining a rate, the
problem is exacerbated, if during the assay, the concentration of
all components of the solution are changing. Therefore, by
permitting evaporation to occur in a zone of an assay mixture,
while the zone is in contact with a solution which has
substantially the same composition, except for one or few, usually
not more than about 4, more usually not more than about 3,
components, generally being the components of interest, many
advantages ensue. Handling is easier, diffusion of the components
having concentration gradients between the assay mixture and the
liquid in the channel appears to be slower, and the solution can be
read without the interference of the composition of the device.
Generally, the liquid in the channel will be substantially the same
liquid of the defined volume, except for the differing components
of the sample introduced into the defined volume. Usually, the
dilution factor of the sample in the zone will be in the range of
about 0.1-10:1 during the course of the reaction.
[0113] In a further embodiment, as depicted in FIG. 4, instead of
having chambers isolated by walls, one has a platform between a
plurality of capillary channels, where desirably each area between
the channels on the platform is wettable and separated by a
non-wettable zone. The device 400 has a first channel containing
block 402, a platform 404, which may be open at its ends 406 and
optionally, a second channel containing block 408, where the first
and second channel blocks 402 and 408 are joined by the platform
404. The second channel-containing block is not necessary since all
of the operations may be performed with a single channel containing
block, although there are advantages in having a source of liquid
on both sides of a droplet on the platform. Each of the channel
containing blocks 402 and 408 have a plurality of channels 410 and
412, respectively. Each channel 410 and 412 terminates at a block
face 414 and 416, respectively, which is non-wettable, with outlets
418 and 420, respectively, allowing for liquid communication with
the platform. Each of the channels 410 and 412 has an orifice 422
and 424. Fitted near the respective orifices in the channels are
electrodes 426 and 428. Conveniently, the area 430 of the platform
between the channel outlets 418 and 420 is wettable, separated from
the next wettable zone by a non-wettable band 432. Into each
channel is extended a second electrode 434 and 436, which can be
used for controlling flow of liquid in the channels in conjunction
with electrodes 426 and 428, respectively.
[0114] The spacing between the blocks 402 and 408 will vary,
depending on the protocol, the size of the sample volume, the size
of the defined volume to be used for the reaction, the surface
tension of the liquid, the contact angle of the liquid, and the
like. The higher the surface tension, the smaller the gap. Usually,
the spacing will be at least about 0.05 mm and not more than about
2 mm, usually not more than about 1 mm. The spacing will affect the
volume of the reaction mixture and the volume of sample, which may
be set down without contacting the channel outlets. Generally,
volumes of sample will be not more than about 300 nl, usually not
more than about 100 nl, with the minimum amount being controlled by
the ability to transfer the volume. The spaces on the platforms may
be coordinated with a microtiter well plate, so that the sample may
be received from individual microtiter well plates at each
hydrophilic site. The sample may be pre-prepared, combining some,
but not all, of the reagents required for a determination. The
remaining reagents necessary for the determination would be
contained in the liquid in a channel or could be divided between
the two opposing channels.
[0115] In carrying out a determination, one exemplary protocol is
as follows: A sample is pre-prepared comprising a compound of
interest and some but not all of the reagents required for a
determination. While one could have all of the reagents necessary
for the determination in the sample mixture, using the subject
device solely for maintenance of a liquid medium, generally one
will prevent a premature reaction by withholding a necessary
reagent from the sample mixture, which is provided by the liquid in
one or both channels. The samples are placed on the wettable sites
430 and, as appropriate, evaporation occurs. The walls of the
capillaries 410 and 412 are appropriately charged or the medium
contains an appropriate additive to support EOF pumping. Liquid is
added to the capillary channels 410 through orifices 422 in
sufficient amount to allow pumping of the liquid to extend a
droplet from channel outlet 418 of sufficient volume to capture and
dissolve the sample mixture in the droplet to form a defined
volume. This is achieved by providing the appropriate polarity
between electrodes 426 and 434, depending on the charge of the wall
of the channel 410. While not necessary, it may be desirable to
withdraw the defined volume through outlet 418 into channel 410 to
substantially inhibit evaporation. As discussed previously, little,
if any, significant diffusion occurs, so that the defined volume
retains substantially the same composition. Withdrawal of the
defined volume into the channel 410 can be achieved by reversing
the polarity of the electrodes 426 and 434 that was employed when
expressing the droplet. The defined volume may be retained in the
channel for a sufficient time for a reaction to occur. Where the
reaction is completed in the channel, the defined volume may be
interrogated in accordance with the signal generated by the
reaction. Alternatively, to avoid interference from the block 402
composition, the defined volume may be expressed onto the surface
430 and interrogated directly. If desired, fluid may be introduced
into channels 412, in sufficient amount to extend to the outlet
420. The fluid in channel 412 may be expressed and withdrawn much
in the manner of the fluid in channel 410.
[0116] In some situations, one may wish to incubate the defined
volume in the channel 410 and then express the defined volume onto
the platform 404 at site 430. The defined volume may then be
separated from the liquid in channel 410 by mechanical action,
introduction of a physical barrier, or the like, and the solvent
allowed to evaporate. The liquid in channel 412 containing an
additional reagent necessary for the determination may then be
expressed and contacted with the assay mixture at site 430, the
assay mixture dissolved in the liquid to form a second defined
volume, which may then be read or withdrawn into channel 412 for
incubation. As described previously, the defined volume may be
interrogated in the channel 412 or expressed onto the site 430 and
interrogated at that site.
[0117] Quite clearly, depending upon the protocol, less or more
sophisticated devices may be used. By having two channel blocks,
which can be independently operated, highly complex and
sophisticated protocols may be performed.
[0118] In FIG. 5, a simple structure is depicted of how two
channels could be used in accordance with the subject invention.
While only two channels are shown, it is understood that the two
channels are only exemplary of a device having a plurality of
channels, where blocks or plates are provided in which the channels
are formed and main channels provided for carrying and removing
liquid from the channels. Each channel in one block has a
corresponding channel in the other block, which may be directly
opposite or offset. The distance between the centers of the channel
outlets will not exceed about 5 mm, where the distance between
related channels will always be shorter than the distance to any
other channel in the opposing block. As shown in FIG. 5A, a first
channel 510 is positioned opposite a second channel 512. Channels
510 and 512 have channel outlets 514 and 516, respectively. In
channel 510 is housed liquid 518. In FIG. 5B, a small droplet 520
of liquid 518 is discharged into the gap 522 between channel
outlets 514 and 516. Movement of the liquid can be achieved with
EOF, pneumatically or mechanical pumping. Micropipette 524 is used
to transfer a small volume of liquid to the droplet 520 to form a
reaction mixture. After the addition of the liquid to the droplet
520, the liquid 518 in channel 510 is pumped to cross the gap 522
and enter channel 512, where the droplet 520 comprising the
reaction mixture is contained within channel 512. If one wishes,
one could have prefilled channel 512, so that there would be a
continuous column of liquid extending through the channels and the
droplet 520 would be protected from any evaporation. As shown in
the figure, only a small amount of evaporation can occur, due to
the very limited interface between the liquid and the atmosphere in
the channel. After incubating the reaction mixture, the occurrence
of a reaction can be determined, where the reaction provides for a
detectable signal. The determination may be made while the reaction
mixture is in the channel, or the reaction mixture may be expressed
and the signal read without interference from the material forming
the channel. Alternatively, by moving the droplet 520 into the gap
522, all or a portion of the liquid in the gap 522 could be
isolated with the pipette 524 and the reaction mixture
analyzed.
[0119] In FIGS. 6A, 6B and 6C, a device 600 is depicted with three
reservoirs 602, 604 and 606, where reservoirs 602 and 604 are
connected through auxiliary channel 608 and through auxiliary
channel 608 to main channel 610. Reservoir 606 is at the terminus
of main channel 610 opposite to the terminus of main channel 610
joined to auxiliary channel 608. Above main channel 610 are a
plurality of ports 612 aligned and evenly spaced along the main
channel 610, extending through the upper layer 614. Channel 610 is
enclosed at its bottom by lower layer 616. While in the figure, the
channel 610 is shown as having a greater width than the diameter of
the port 612, this can be reversed, where the channel would have a
smaller dimension than the port, and the width of the channel would
control the size of the interface between the port and the channel.
The effect of having a smaller channel width than the width of the
port is to have a portion of the droplet in the port supported by
the lower layer and out of contact with the liquid in the channel.
Furthermore, smaller channels will enhance the linear velocity in
the liquid for comparable levels of evaporation in the port. In
using the device, an aqueous medium is introduced into the
reservoirs so as to fill the channels. By having the port walls
non-wettable, the aqueous medium does not rise up the walls, but
forms a small convex meniscus. Solutions may be added to each of
the ports and reactions performed at each port site. Preferably,
there would be only one port along a channel, where there could be
many main channels, each with a single port.
[0120] It should be understood that the level of the liquid in the
reservoir may be the same, higher or lower than the level of the
meniscus. While preferably the level will be higher, the salient
consideration is that the surface tension in the well is sufficient
to support the meniscus. Therefore, as long as the liquid in the
zone is maintained at a substantially fixed level during the
operation despite evaporation from the zone, the level of the
liquid in the reservoir is not critical.
[0121] In FIGS. 7A and 7B, diagrammatic plan and cross-sectional
views are depicted of a unit with electrokinesis capability for
analyzing the components in the zone, while having a central
distribution of reagent components from a reservoir to a plurality
of zones. The unit 700 comprises a central reservoir 702, which
serves to receive a solution of one or more reagents and act as a
distribution center for distributing the solution to a plurality of
zone enclosures 704 by means of channels 706. The solution in the
central reservoir 702 is conveniently maintained at a level above
the liquid level in the zone enclosure. In this situation a
solution of the reagent is added to a dry central reservoir under
conditions that retain the solution in the central reservoir. After
adding buffer or other diluent, the solution from the central
reservoir is released into the channels and to the zones. The
solution migrates from the reservoir 702 through the channels 706
and enters the zone enclosure 704. Where liquid is present in the
zone enclosure 704, the solution will mix with the liquid in the
zone enclosure 704 to provide a reaction mixture. The zone
enclosure 704 comprises an upper region 708 of the zone enclosure
704, into which the reaction mixture 710 extends, having meniscus
712, from which liquid evaporates. The zone enclosure 704 is
connected by channel 716 to a buffer reservoir 718 and by channel
720 to waste reservoir 722. Thus, buffer reservoir 718, channel
716, zone enclosure 704, channel 720 to waste reservoir 722 define
an electrokinetic channel, whereby charged components may be moved
by electrophoresis and both charged and uncharged components by
electroosmotic force. The channel 720 crosses channel 724, which
can serve as an analytical channel. For example, it may contain a
sieving polymer to separate components of different mobilities,
such as proteins and protein complexes, DNA of different lengths,
etc. The analytical channel 724 connects buffer reservoir 726 and
waste reservoir 728. Each of the reservoirs has electrodes, where
the buffer reservoir 718 has electrode 730, the complementary waste
reservoir 722, electrode 732, the buffer reservoir 726, electrode
736 and the complementary waste reservoir 728, electrode 738.
[0122] The device has an upper plate 740 and a lower plate 742. The
lower plate 742 has channels 716 and 720, which connect buffer
reservoir 718 and waste reservoir 722 with zone enclosure 704,
where the channel provides solution under the upper portion of the
zone enclosure 712 with liquid from the channels 7716 and 720.
While the diameters and the reservoirs are shown as approximately
equal in FIG. 7B, this is for illustration. In practice, the zone
enclosure diameter would normally not be greater, usually smaller
than the reservoir diameters. In this case, by having a
non-wettable wall 746 in the zone enclosure 708, a convex meniscus
712 is observed and the height to which the liquid in the zone can
rise is restricted.
[0123] While not necessary to fabricate the device of two plates,
the use of two plates will be of great convenience. The appropriate
channels may be formed in each of the plates, independently of the
other. The openings for the zones and reservoirs in the upper plate
740 may be formed to be in register with the corresponding portions
of the microstructures present in the lower plate 742, while the
channels in the upper plate 740 may be made independent of the
microstructures in the lower plate 742. In this way a network of
channels and reservoirs may be formed in the lower plate and access
to these channels and reservoirs provided in the upper plate.
[0124] In carrying out an operation, the channels in the lower
plate may be filled with buffer, where different buffers may be
present in different channels. The buffer may contain one or more
reagents and or the sample, depending upon the nature of the
operation. If one wished to carry out enzyme assays, where the
enzyme is an expensive reagent, one could have the enzyme provided
from the central reservoir 702. One could fill the channels with
buffer and enzyme substrate. The liquid from the channels will rise
into the zone enclosures 704 to form a meniscus 712 and define the
reaction mixture. If one is interested in the effect of a test
compound on the activity of the enzyme, one could add a different
test compound to each zone. One would then add the enzyme solution
to the central reservoir 702, whereby the enzyme solution would
move by capillary action through channels 706 to zone enclosures
704. Liquid moving from zone enclosures 704 into channels 706 may
be prevented in a variety of ways, including maintaining reservoir
702 sealed until the enzyme solution is added, providing a barrier
at the interface between channel 706 and central reservoir 702,
which is dissolved by the solution added to central reservoir 702,
and the like. Once the enzyme enters the zone enclosure 704 the
enzymatic reaction will occur and product will begin to be formed.
After sufficient time for product to form, the electrokinetic
analysis may begin. The electrodes 730 in buffer reservoir 718 and
732 and in waste reservoir 722 are activated to begin the migration
of charged species from the liquid in the zone enclosure 704 toward
the waste reservoir 722. When the enzyme product reaches the
intersection 746 between channel 720 and channel 724, the defined
volume of product is injected into the analysis channel 724, by
using electrodes 736 and 738. The product may then be separated
from other components in the reaction mixture and read. Where the
product is fluorescent, the product may be read with a PMT or CCD
or other detection device.
[0125] In analogous manner, one could perform DNA sequencing, where
the DNA sample would be put in the central reservoir, dNTPs and
labeled ddNTPs in the buffer and different primers in the different
zones. One would then add the polymerase to the different zones and
initiate the extensions, with thermal cycling in the zones. Once
the sequencing was completed, the electrophoretic analysis could
begin, where the DNA fragments could be directed to the
intersection 746 and the channel 724 would contain sieving buffer,
to provide separation of the different length fragments.
[0126] In FIG. 8 a different arrangement is provided, where the
partially enclosed zone has only a single channel connection and a
central reservoir for replenishing the volatile liquid in a
plurality of zones. The plan view of the device 800 shows three
units 802, although there would normally be many more, where the
units would be distributed to provide for high density of the units
802. For clarity, each unit is shown to have only four vessels 804,
although in a commercial device there would be a much greater
number of vessels connected to each reservoir 806. The reservoir
806 is connected through channels 808 to the vessels 804. The
reservoir 806 would normally be filled with an appropriate liquid
810 to provide liquid for replenishment of liquid evaporating from
the liquid 805 in the vessels 804. The height 812 of the liquid in
the reservoir 810 would provide a hydrostatic head, which would be
insufficient to drive the meniscus 814 of the liquid 805 past the
non-wettable region 816 in the vessel 804. For example, if one were
dealing with an aqueous medium there would be a region 816 in the
vessel 804, which would be non-wettable. This would result in the
aqueous medium rising in the vessel 804 to the non-wettable region
816, where a convex meniscus 814 is formed. The surface tension of
the meniscus 814 prevents the liquid in the vessel 804 from rising
beyond the wettable portion of the wall of the vessel 804. The
result is that as the liquid 805 in the vessel 804 evaporates,
liquid from the reservoir 806 will replenish the liquid 805, so as
to substantially maintain the volume of the liquid in the vessel
804. Furthermore, the movement of the liquid in the channel 808 is
in the direction toward the vessel 804, so as to diminish diffusion
of solutes in the liquid 805 toward the channel 808.
[0127] In carrying out operations in the liquid 805, one can have
very small reaction volumes, which are maintained during the course
of the reaction, regardless of whether the vessel 804 is covered or
uncovered. Furthermore, during additions of solutes, where the
vessel is open to the atmosphere, the inevitable evaporation of a
volatile solvent is compensated by-liquid from the channel, so as
to maintain the volume of liquid 805 substantially constant.
[0128] In FIG. 9 is shown a diagrammatic array of a plurality of
units having common channels and reservoirs in a row. The device
900 is designed to have the same distribution of zones as for a
96-well microtiter plate. The plate 902 has reservoirs 904
positioned between units 906. Each unit 906 comprises zone chambers
908 and parallel distribution channels 910, which channels are fed
by reservoir connecting channels 912. Feeding channels 914 connect
the distribution channels to the zone chambers 908. One would carry
out determinations by filling all of the channels with the
appropriate liquid buffer, where meniscuses would form in the zone
chambers 908. One could fit the device to be under a microtiter
well plate, where the wells had fritted disk bottoms, so that the
wells are in register with the zone chambers 908. By pressurizing
the wells, liquid in the wells would be driven into the zone
chambers 908 and mix with the liquid in the meniscus in each of the
zone chambers 908. The reaction mixtures may then be incubated and
the results determined by interrogating each of the zone chambers
908.
[0129] In FIG. 10, a diagrammatic array of an alternative
embodiment of a plurality of units in a microfluidic device having
common channels and reservoirs is depicted. The device 100 is
designed to have the same distribution of zones a102 as for a 96
well plate. Internal reservoir units a104 are symmetrical about the
reservoir a106, which is connected by parallel channels a108 to
orthogonal channels a110. The zones a102, which are internal to the
device (not on the periphery or along the outer channels) are
organized so as to be equally spaced apart along the distribution
channels a112. The distribution channels a112 may be the same as or
smaller in cross-sectional area than the parallel channel a108
and/or the orthogonal channels a110. Each zone a102 is connected on
both sides of the zone a102 through a segment a114 to an orthogonal
channel a110. In this way, each of the zones is symmetrically
situated and is fed by two different reservoirs a106. The outer
zones a116 are positioned somewhat differently, since the terminal
reservoirs a118 connect two of the distribution channels a112,
except for the corner reservoirs a120, which are connected to only
one distribution channel a112. In addition, the top and bottom
reservoirs a122, instead of feeding two distribution channels a112,
feed into only one. The organization of the device a100 provides
many economies, while at the same time providing greater
flexibilities. By having each zone receiving fluid from two
different reservoirs and each reservoir feeding four different
zones, one can provide for different components in the reservoirs
between alternating distribution channels a112, so as to provide
greater diversity of reaction components. The organization further
provides for substantially equal movement of fluid to each of the
zones and allows for hydraulic equalization, so that all of the
reservoirs may be equilibrated to the same height before initiating
any reaction. The reservoirs and channels may be filled using
pressure or allowed to fill by capillary action. If different
components are to be introduced into reservoirs in different rows,
one could initially fill the device with a common buffer and then
add the different components to the different reservoirs, where
diffusion and liquid flow would carry the components to the
zones.
[0130] In FIG. 11 the diagrammatic array of a plurality of units
employs a different organization. In this array, device a150 has as
in previous organizations the footprint of a 96 microtiter well
plate. The device has 6 units a164. There are a few significant
differences from the other devices in that zones a152 do not have
two channels feeding the zone a152, but rather a single feeding
channel a154. A distribution channel a156 is connected to two
feeding channels a154, where each feeding channel a154 provides
liquid to two zones a152, so that a single distribution channel
a156 serves four zones a152. The distribution channels a156 are
symmetrically situated about reservoir a158, where 16 zones a152
are fed from the reservoir a158 through main conduits a160 and
cross conduits a162.
[0131] In each unit a164, the zones a152 are symmetrically
situated, so that the channel distance from the reservoir a158
through the main conduits a160, the cross conduits a162, the
distribution channels a156 and the feeding channels a154 are
substantially the same distance from the reservoir a158. The fluid
head in the reservoir a158 and the resistance to flow through the
flowpath of the liquid through the channels to the zones a152 will
be substantially the same for each zone a152. In this way, the only
difference between the state of the zones will be based on any
difference in components added to an individual zone. In addition,
one could use one zone as a control, so that for each unit a164,
the other zones would have substantially the same conditions as the
control, providing for a more accurate comparison of the results of
the controls and samples.
[0132] In FIG. 12, the diagrammatic plan view is of a device a200,
which combines the advantages of evaporative control with
electrokinesis. The units a202 have zones with the same footprint
as a 96 microtiter well plate. Each unit a202 has a zone a204,
which is connected by connecting channels a206 to reservoirs a208.
This portion of the unit a202 has substantially the same purpose
and manner of use as the other evaporative control units that have
been previously described. In this embodiment the connecting
channels a206, that connect under and to the zone a204 are
connected at a tee to a side channel a210. The side channel a210
serves to connect the zone a204 with an electrokinesis network. at
tee intersection a212 with analysis channel a214. While the
configuration shown is a double-tee configuration, where waste
channel a216 connects to analysis channel a214 at intersection
a128, one could have a cross-intersection, where the two channels
a210 and a216 meet at the same site of analysis channel a214. Waste
channel a216 terminates in waste reservoir a220. Analysis channel
a214 terminates at one end in buffer reservoir a222 and at the
other end in waste reservoir a224. In operation, there would be
electrodes in the two waste reservoirs a220 and a224, the buffer
reservoir a222 and in at least one of the zone a204 or the
reservoir a208.
[0133] In operation, one would first carry out a reaction in the
zone. All of the channels could be filled with the same buffer or
one could initially fill only the reservoirs a208 and channels
a206, blocking any significant liquid from entering analysis
channel a214. The entry of liquid could be prevented by first
filling analysis channel a214 and the waste reservoirs a220 and
a224 and the buffer reservoir a222 using an appropriate pressure
differential between the electrokinesis network and the reaction
zone system. Alternatively, one could use a vacuum in one of the
reservoirs a208 to pull liquid from the other reservoir a208
through the channels a206, while covering the reservoirs of the
electrokineisis network. The particular manner in which one
distinguishes the liquid in the reaction zone system and the
electrokineisis network is not critical and any convenient method
may be employed.
[0134] After appropriate addition of the reservoir liquid, where a
meniscus will be formed in the zone a204, one or more components
may be added to the zone to form a reaction. For example, one could
have a library of candidate substrates, where the zone a204
initially contains an enzyme. The candidate substrates would be
added to the zone and the reaction mixture incubated, where all or
some of the candidate substrates would react to form product.
Either or both the reactants and the products would have unique
mobilities, preferably both. After completion of the reaction,
electrodes could be added to the various reservoirs and the zone,
as appropriate. Initially, an electrode would be activated in the
reaction zone system, e.g. in the zone a204, and the waste
reservoir a220. The charged substrates and products would move from
the reaction zone a204 through side channel 210, through the
portion of the analysis channel 214 between intersections a212 and
a218 and into waste channel a216. The result is to form a slug of
material from the zone a204 in the region between the intersections
a212 and a218. When this region has a stable composition, the
electric field is changed by activating electrodes in the buffer
reservoir a222 and the waste reservoir a224. Depending on the
nature of the substrates and products one may provide for a sieving
medium in the analysis channel. The substrates and products will
then move down the analysis channel a214 toward the waste reservoir
a224 separating into bands in accordance with their respective
mobilities. A detector may be placed along the analysis channel
a214 for detecting the passage of the bands past the detector. By
providing for fluorescently labeled or electrochemical molecule
labeled substrate and/or product, one can readily detect a
reduction or increase in the amount of substrate or product,
respectively to determine the effect of a candidate compound on the
reaction, the activity of an enzyme, or the like.
[0135] FIG. 12 also exemplifies a combination of a reaction zone
system and an electrokinesis system in a 96 well format. The device
a300 has a plurality of units a302, with a reaction zone unit
comprising the reaction zone a304, a reservoir a306 and connecting
channels a308 connecting the reservoir a306 to the reaction zone
a304 on both sides of the reaction zone a304. In this embodiment,
there is a single reservoir a306 providing replenishment liquid to
the reaction zone a304 on both sides of the reaction zone a304.
Side channel a310 connects the reaction zone and, thus, the
reaction zone system to the electrokinesis system. The side channel
a310 is connected to the connecting channels a308 juncture at the
reaction zone a304. The side channel a310 connects to the analysis
channel a312 at the intersection a314 with the waste channel a316.
As distinct from the double-tee configuration, this configuration
has the side channel a310 directly across from the waste channel
a316, so as to connect the reaction zone a304 through the side
channel a310 and the intersection a314 and the waste channel a316
to the waste reservoir a318. By having electrodes in the reservoir
a306 and the waste reservoir a318, the components in the reaction
zone a304 will be directed through the flowpath, as described
above, to the waste reservoir a318. Once the composition from the
reaction zone a304 has become substantially constant, electrodes
placed in buffer reservoir a320 and analysis channel waste
reservoir a322 may be activated to direct the composition at the
intersection a314 into the analysis channel a312 for separation of
the components as described previously.
[0136] The combination of the reaction zone system and the
electrokinesis system is very powerful for performing a large
number of different operations.
[0137] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0138] The following experiments were performed using a device
substantially as depicted in FIG. 6. While the format of the device
was kept constant, in different experiments the dimensions of the
elements of the device were modified.
[0139] The device is comprised of a lower and upper plate. In the
upper plate is a main channel, which forms a T at one end with an
ancillary channel, which terminates in a reservoir at each end. The
other end of the main channel terminates in a reservoir. Along the
main channel are five evenly spaced ports formed in the upper
plate. The upper plate also has openings for each of the
reservoirs. The channels and reservoirs are enclosed by a base or
lower plate.
[0140] The upper plate is about 1 mm in height and the lower plate
is also about 1 mm in height. The port for introducing solutions is
1 mm in diameter and about 900 to 950 .mu.m in height, while the
channel substantially extends the remaining length of the upper
sheet. The channel was varied from about 0.2 mm to 3.0 mm in width,
where the interface between the port or well and the channel
varied, with either the port or the channel determining the area of
interface. The reservoirs have a diameter of about 2 mm. The
channels were treated with 2N sodium hydroxide for 5 mins. using a
vacuum pump to ensure that the basic solution extends through the
channels and reservoirs. The ports or wells appear to be unaffected
by this treatment, so that the channels and reservoirs have a
hydrophilic surface, while the ports have a hydrophobic surface.
One or more of the ports are used in each of the studies. Common to
each of the experiments is to fill the device with 1.mu.l of
25.mu.M fluorescein diphosphate in 50 mM Tris buffer (pH 10.0)
added to each of the inlet reservoirs, after prewetting the
device.
[0141] In the first study, the channel is 1-2 mm wide and 10-30 nl
of enzyme (alkaline phosphatase) is added to one of the ports and
the fluorescence in the port is monitored for 60 mins. using a CCD
camera. The fluorescence observed in the port increases with time,
with the fluorescence primarily confined to the port area; a round
fluorescent spot develops, which can be easily imaged with a CCD
camera.
[0142] In the next study, the width of the channel is about 300
.mu.m and 30 nl of 1 nM or 0.1 nM enzyme is added to a total of
four ports and the florescence monitored with a CCD camera for 30
mins. The fluorescence is primarily confined to the ports and round
fluorescent spots develop. The fluorescent signal can be easily
related to the concentration of the enzyme introduced into the
ports. Fluorescence is observed in the channel, which is
substantially dimmer than the spots.
[0143] In the next study, a 2 mm wide channel is employed and 30 nl
of 0. nM of enzyme was added to the ports and the increase in
fluorescence at 5 min. intervals was monitored. A progressive
increase in fluorescent signal is observed with the signal
substantially confined to the ports. The amount of fluorescence in
the channel is substantially less than in the previous experiment.
This study was repeated with enzyme being added to two ports with a
1 mm wide channel and again the signal is substantially confined to
the ports, with only dim fluorescence in the channel.
[0144] In the next study, the effect of enzyme inhibitor was
investigated. The channel was 1 mm in width. Approximately 30 nl of
pyridoxal phosphate (250.mu.M or 25.mu.M) is added to the ports
followed by the addition of 30 nl of 0.1 nM of enzyme and all of
the ports closed to diminish evaporation. The fluorescence
development is monitored with a CCD camera. Fluorescence is
substantially confined to the ports and the fluorescent signal is
related to the concentration of inhibitor introduced into the port.
The port in which 250.mu.M inhibitor was added is still very faint
at 30 mins., while the port with only 25.mu.M appears to be only
moderately inhibited.
[0145] In the next series of studies, a polyacrylic substrate was
fabricated with side reservoirs of 2 mm diameter and wettable, a
middle chamber of 1 mm diameter and non-wettable, with the
connecting channel 100.mu. deep and 300-500.mu. wide. The
hydrophilic surface treatment was performed as follows. The middle
chamber was sealed with Scotch.RTM. tape. The channel was filled
with 4N NaOH through either of the two reservoirs, and flushed
through the channel with vacuum aspiration. The treatment was
repeated a number of times, allowing the basic solution to stand in
the device for up to 0.5 h each time. The device was then rinsed
with deionized water several times. Upon adding buffer to the
reservoirs, the buffer would move through the channel by capillary
action. The capacity of the device was 10.mu.l.
[0146] In carrying out the determinations, one protocol was to seal
the middle chamber and fill the channel by adding buffer to one or
both of the reservoirs. The level of the reservoirs was then
allowed to equilibrate. The middle chamber was unsealed, while
holding the device steady. A Nanoplotter.RTM. (GeSim Corp.,
Germany) was used to dispense the reactants into the middle
chamber, dispensing from 40 to 100 nl in volume. Depending on the
nature and complexity of the dispensing, the time for dispensing
varied from under a minute to 10 mins.
[0147] The signal detection system employed an Argon ion laser
source, Nikon microscopic system with 4.times. objective, CCD
camera and image frame capture software Rainbow PVCR. Fluorescence
was excited at its optimal absorbent wavelength and its emission
was collected through the CCD camera and captured by software
Rainbow PVCR. The images were then analyzed using ImagePro Plus
software. The fluorescent intensity was then quantified.
[0148] The rate of diffusion from the middle chamber was studied as
follows. 100 nl of 50.mu.M of 5-carboxyfluorescein in 30% DMSO was
dispensed into the sample port (middle chamber). The reservoirs and
channel were filled with 10 .mu.l of 50 mM Tris buffer, pH 9.0.
Fluorescence was excited at 480.+-.nm and emission was at 530.+-.20
nm, using the signal detection system described above. The
fluorescent signals were recorded as a function of time. 80-90% of
the original fluorescence intensity was maintained in the sample
port region over 1 h. The fluorescent signal in the channel away
from the sample port was found to be close to background. The loss
of the fluorescein through the channel by diffusion is
insignificant, as demonstrated in the following table.
1 Time, Min Distance from port 0 5 15 30 60 A_340.mu.M 1 1.0399
1.03 1.04 0.86 B_450.mu.M 1 0.94 1.02 1.07 0.97 D_1600.mu.M 1 0.966
0.98 0.93 0.83
[0149] In the next study, enzyme kinetics were performed using
alkaline phosphatase and substrate providing a fluorescent product.
The channel was rinsed with AutoPhos buffer (JBL Scientific, Inc.,
San Luis Obispo, Calif.) and then filled with 10 .mu.l of 1 mM
AutoPhos substrate. 50 nl of alkaline phosphatase, at different
concentrations was then dispensed into the sample port. The
concentrations varied from 31.25 attomoles to 62.5 femtomoles with
2-fold dilutions. The fluorescent signals were recorded at
different time points as described above. The following table
indicates the results.
2 ENZYME KINETIC ASSAY RESULTS Conc., nM Time 1000 250 125 31.25 0
12 min. 13390.8 2913.84 1497.68 821.08 0 20 min. 20692.4 4698.56
2323.8 1055.88 0 30 min. 28981.6 7579 2892.68 1798.84 0
[0150] As evidenced by the above results, the rate of the reaction
is linear with the enzyme concentration in accordance with a 1 st
order reaction.
[0151] The next study evaluated the system using a competitive
inhibition assay, 4-Nitrophenyl phosphate (PNPP) (Sigma Chemical
Co., St. Louis, Mo.) was used as a non-fluorescent substrate for
alkaline phosphatase (20 femtomoles) competing with the AutoPhos
substrate. The channel was rinsed with AutoPhos buffer and filled
with 1 mM AutoPhos substrate. Into the sample port was introduced
100 nl of PNPP at concentrations varying from 0 to 10 mM and the
fluorescent signal was determined at different reaction time
points. The fluorescent signal was found to diminish with
increasing inhibitor concentration, the following table providing
the results.
ENZYME INHIBITION ASSAY
[0152]
3 Inhibitor conc., mM Fluorescent 0.001 0.0025 0.005 0.01 0.02
0.3125 0.625 1.25 5 10 Signal .times. 103 4.5 4.0 4.0 3.5 2.6 2.0
1.6 1.6 1.6 1.5
[0153] In another series of studies binding assays were performed
using fluorescence resonance energy transfer. The procedure
employed is as follows. The channel was rinsed and filled with 25
.mu.l of rhodamine labeled streptavidin and 100 nl of fluorescein
labeled biotin dispensed in the sample port. The concentration of
the antigen varied from 0 to 100 .mu.M by 2-fold dilutions. The
signal detection system was as described, except that emission was
detected at 600.+-.20 nm. The energy transfer increased
corresponding to the increase in antigen. The study was repeated
varying the amount of labeled streptavidin while keeping the
biotin-fluorescein at 25 .mu.M. The background FRET signal
contributed by rhodamine-streptavidin alone was substantially
negligible, when the concentration of rhodamine-streptavidin was
greater than about 2.mu.M. The following tables provide the results
for the two studies.
BINDING ANTIGEN-RECEPTOR ASSAYS
[0154]
4 Conc. Of Fluorescein Labeled antigen, mM 100 50 25 10 5 0 FRET
Signal 2956 2327 1639 869 370 0
[0155] In the next study the channel was rinsed and filled with 25
mM fluorescein-labeled antigen, 100 nl of rhodamine-labeled
receptor dispensed into the sample port. Various concentrations of
the rhodamine-labeled receptor were employed, with excitation and
emission as described above. The following table indicates the
change in FRET signal with concentration of the rhodamine-labeled
receptor. The background signal contributed by rhodamine-receptor
alone is also indicated.
5 Conc. Of Rhodamine Labeled receptor, mM 0 0.25 0.5 1.5 3.5 5 6 8
12 FRET Signal 2192 3663 2264 3254 7619 10604 10882 11952 11552
Background Signal 1923 1430 2336 1312 556 211 516 759 1005
[0156] In the next study, the effect of inhibitor on the observed
signal was investigated. Fluorescein-biotin was maintained at
50.mu.M and rhodamine-streptavidin at 25.mu.M. the signal was read
at 600.+-.20 nm at varying concentrations of biotin as a binding
inhibitor, with 100 nl of the binding inhibitor being added to the
sample port. The energy transfer decreased with increase of binding
inhibitor.
[0157] In the next study, the channel was filled with varying
concentrations of biotin in the range of 0 to 5.mu.M and 100 nl of
rhodamine-labeled streptavidin (625 nM) followed by 100 nl of 1.0
.mu.M fluorescein-biotin added to the sample port. After incubating
for 60 min., the signal was detected at 520.+-.20 nm. The results
are reported as fraction inhibition. The following tables provide
the results.
INHIBITION OF BINDING OF ANTIGEN-RECEPTOR ASSAYS
[0158]
6 Conc. Of Inhibitor, nM 0 0 30 60 180 240 500 600 1000 5000
Fraction of 0 0.0177 0.0385 0.0310 0.050 0.0514 0.224 0.3664 0.950
1.0 Inhibition
[0159] In the next series of studies, a number of different assays
were performed in the subject devices, including a protease assay,
alkaline phosphotase assay, ligand-receptor binding assay,
homogenous time resolved fluorescence assay and fluorescence
polarization assay. Initially, the device was evaluated as to the
stability of a fluorescence signal over time, in the presence and
absence of a loose cover. The device employed has substantially the
same parameters as previously described. The reagents and protocol
are as follows:
[0160] Reagents:
[0161] 5'-carboxyl-fluorescein (Molecular Probe, Eugene, Oreg.)
[0162] 50 mM Tris buffer (pH=9.0)
[0163] Protocol:
[0164] 700 nl buffer is dispensed into assay well followed by
dispensing 3.2 .mu.l buffer into each side well and 100 nl
50.multidot.M fluorescein into the assay well by Nanoplotter (GeSim
Corp., Germany).
[0165] Fluorescein readings were taken at 0, 30 min and 60 min
using Fmax.RTM. microplate reader (Molecular Device). The same
experiment was repeated except for putting a loose lid on the
device.
[0166] The results are set forth in the following table.
[0167] Table: Fluorescence Signal as a Function of Time
7 RFU 0 min 30 min 60 min 60 min with Lid Mean 65.14 60.82 54.57
51.096 C.V. 6.89% 8.79% 10.72% 10.42% Number of Wells 27 27 27
27
[0168] In the next study a series of different enzyme assays were
performed. The first assay was a protease assay using Cathepsin L
protease as an exemplary protease and was chosen to demonstrate the
correlation between a conventional 100 .mu.l reaction in 96 well
microtiter plates and a 200 nl reaction in a 33-hole subject
device. This protease assay is a FRET based assay. The assay uses
an internal quenched fluorogenic oligopeptide substrate, which
incorporates the cleavage site for Cathepsin L protease. Incubation
of human liver Cathepsin L with the fluorogenic substrate resulted
in specific cleavage at the Arg-Val bond and a time-dependent
increase in fluorescence intensity. The increase in fluorescence
intensity is linearly related to the extent of substrate
hydrolysis. FRET based protease assay facilitates the
identification of novel inhibitors of various proteases such as HIV
protease or renin protease, etc.
[0169] Reagents:
[0170] Human liver Cathepsin L (Cat #219402, Calbiochem-Novabiochem
Corp., La Jolla, Calif. 92039).
[0171] Enzyme buffer: 100 mM NaOAc,1.5 mM DTT (pH 5.5).
[0172] Cathepsin L substrate:
FITC-LC-Glu-Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala--
Ala-Lys(.epsilon.-DABCYL)-OH (Cat # ABSS-2, AnaSpec Inc., San Jose,
Calif. 95131). The substrate was dissolved in anhydrous DMSO at
concentration of 800 .mu.M and further diluted in the same buffer
mentioned above. Seven different Cathepsin L inhibitors (Calbiochem
corp.) were dissolved in anhydrous DMSO at a concentration of 1 mM
and further diluted in the buffer solution mentioned above.
[0173] The Cathepsin L protease assays used 33-zone cards. These
cards have 3 rows of 11 wells on each. The diameter of the sample
well is 1 mm and 1.5 mm for the reservoirs. The channel connecting
the sample well and reservoirs is 450.mu. in width, 100 .mu.in
depth and 3.5 mm in length (total 7 mm in length). The depth of the
evaporation control well is 1 mm. The device was laminated with
Rohm film, which was plasma treated. The plastic for the substrate
is V825. All the protease assays were conducted on plasma treated
film laminated cards unless specified otherwise. These cards were
placed in cardholders. The design of the holder was customized so
as to accommodate the optimized optical reading for a 96 well
microtiter format under a fluorescence plate reader (Fmax,
Molecular Devices).
[0174] The protocol is as follows:
[0175] After placing the card in its holder, 700 nl of Cathepsin L
substrate is added to the sample well by contacting the bottom of
the sample well with the pipette tip, with flow of the liquid
toward the reservoirs, avoiding the formation of bubbles. 3.2 .mu.l
of the substrate is then added to the reservoirs. The fluorescence
intensities are recorded using an Fmax plate reader at 485 nm
excitation/535 nm emission to determine the assay background
fluorescent signals. The gain of the signal collection was set to
2.65, the integration time for each sample well was 20 msec and the
plate scanning speed was set at the highest mode which was 10 in
the scale of 1 to 10. The reactants were dispensed using a
Nanoplotter (GeSim Corp., Germany) through the sample port at 50 or
100 nl in volume.
[0176] The coefficient of variation was determined with two of the
cards, using the above protocol, except that the Cathepsin L
substrate was 40.mu.M and 50 nl of 46.8 mg/ml Cathepsin L was
dispensed into each sample well and the mixture incubated at room
temperature for 1 h.
[0177] The signal for card 1 and card 2 were 24.5.+-.2.3 (n=31) and
26.4.+-.4.2 (n=31), respectively. Therefore, the c.v. for card 1
and card 2 were 9.2% and 15.9%, respectively. One-way analysis of
variance was performed and it was found that there was no
significant difference (p=0.038, .alpha.=0.05) between assay
signals obtained from cards 1 and 2. The overall assay signals for
both LabCards were 25.5.+-.3.5 (n=62) with C.V. of 13.7%.
[0178] In the next study, a comparison was made of the results for
the same assay between the subject card and a 96 well microtiter
plate. The channel was filled with 40 .mu.M substrate by adding 700
nl into the sample well and 3.2 .mu.l into both reservoirs. The
assay background signals were measured. Then, 50 nl of Cathepsin L
at 4 different concentrations were dispensed into different sample
wells using a Nanoplotter. There were six replicates for each of
the four different concentrations and one negative control where no
protease was added. The following table shows the mean and standard
deviation of fluorescent signals corresponding to five different
amounts of protease. The relationship of the fluorescent signal
with the increasing protease concentration in the reaction was
RFU=4.522.times.[protease]+1.4 with R equal to 0.99.
8TABLE Fluorescent Signals at Different Amounts of Cathepsin L in
Cards Cathepsin L, ng Mean of RFU (n = 6) S.D. of RFU 0 2.246611
0.533557 0.47 3.477907 0.746098 1.17 6.03478 0.882803 4.68 27.39389
1.707562 11.70 52.56761 6.542091 Card Background 0.760415
0.442258
[0179] The protocol for the microtiter well plate comparison was as
follows. A black polystyrene U-bottom 96-well microtiter plate
(Dynex) was used. 78 .mu.l of Cathepsin L buffer was added into the
wells followed by 10 .mu.l of Cathepsin L at different
concentrations, and finally 200 .mu.M of substrate. Three
replicates were performed for each protease concentration including
the negative control. The reactions were incubated for 1 h before
measuring the fluorescent signals. The following table shows the
mean and standard deviation of the fluorescence signals at
different protease concentrations.
9TABLE Fluorescent Signals at Different Amount of Cathepsin L in
96-Well Plates Cathepsin L, ng Mean of RFU (n = 3) S.D. of RFU 0
1.6234 0.1047 40 2.6897 0.1563 342 38.8344 2.1132 585 54.7233
3.8047
[0180] The relationship of fluorescent signal with increasing
protease concentration in the reaction was
RFU=0.0951.times.[protease]+1.6 with R.sup.2 equal to 0.98. The
results from the card and 96-well plate were comparable.
[0181] To estimate the reduction in reagents, the required quantity
of the reagent for each assay can be derived from the above signal
as a function of enzyme concentration plot. To set the ratio of
assay signal to assay background the same for both 96-well plate
and card, the ratio of the required enzyme for the 96-well plate
and the card is as following: 1 M 96 well M OASIS = Slope OASIS
Slope 96 well * Int 96 well Int OASIS = 106
[0182] (OASIS intends the device according to this invention.) In
other words, when the assay reaction volume reduces to 250 nl in
cards from 100 .mu.l in a 96-well plate, the key reagent protease
is used in 106 times less amount.
[0183] The next study was a determination of the effect of
inhibitors on the protease assay. For each inhibitor, five
different concentrations of inhibitor were used (0.1 .mu.l-1000
.mu.l with one log increment), there were six replicates for each
concentration of inhibitor and three replicates for one negative
control, where no inhibitor was added. One card was required to run
one set of experiments for each inhibitor assay. For each
experiment, the card was placed in the cardholder and the channel
filled with 700 nl of 20 .mu.M substrate through the sample well
followed by 3.2 .mu.l of substrate at each reservoir. The assay
background signals were measured. 50 nl of inhibitor was dispensed
into the sample well followed by dispensing 50 nl of 23.4 ng of
Cathepsin L. The card was incubated for half an hour at room
temperature covered by a dark loose lid to avoid direct light. The
fluorescent signals were measured. In the data analysis, the assay
background signals were subtracted from the reaction signal at each
different concentration of the inhibitor. The fraction of the
control signal is the ratio of reaction signal over control signal.
The decreasing of the signal, or the smaller the fraction of the
control signal, indicated the inhibition of the Cathelpsin L
protease. The following table indicates the results.
10TABLE Fraction of the Control Signal vs. Inhibition Concentration
in Card Fraction of Original Intensity [Inhibitor], .cndot. M Inh_1
Inh_2 Inh_3 Inh_4 Inh_5 Inh_6 Inh_7 0.001 1.0 1.0 1.0 1.0 1.0 1.0
1.0 0.01 0.92 0.98 0.89 0.89 0.57 0.64 0.56 0.1 0.77 0.79 0.47 0.46
0.55 0.32 0.35 1 0.56 0.33 0.13 0.056 0.30 -0.002 0.097 10 0 0.14 0
0 0 0 0
[0184] For comparison, inhibition assays were carried out under
comparable conditions in a 96-well microtiter plate. For each
inhibitor, five different concentrations of inhibitor were used
(0.1.mu.M-1000 .mu.M with one log increment), there were three
replicates for each concentration of inhibitor and one negative
control where no inhibitor was added. In each well, 75 .mu.l of
Cathepsin L buffer was added followed by 10 .mu.l of protease (40
ng) and 5 .mu.l of inhibitor. 10 .mu.l of 200 .mu.M substrate was
added last. The reaction was also incubated for half an hour. The
data analysis was the same as above. The following table indicates
the results.
11TABLE Fraction of the Original Reaction Signal vs. Inhibition
Concentration in 96-well Microtiter Plate Reaction Fraction of
Original Intensity [Inhibitor], .cndot. M Inh_1 Inh_2 Inh_3 Inh_4
Inh_5 Inh_6 Inh_7 0.0005 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.005 0.96
0.90 1.01 0.94 0.58 0.021 0.49 0.05 0.92 0.76 0.92 0.88 0.13 0.036
0.32 0.5 0.17 0.29 0.093 0.10 0.049 0.0076 0.038 5 0.033 0.32 0.052
0.062 0.031 0.0030 0.036
[0185] The results showed the reaction performance between the
96-well plate and card were comparable, despite the large disparity
in the amount of the protease used in the card assay.
[0186] The correlation of the performance between cards and 96-well
plates is shown in the above plot. Inhibition of the cleavage of
the substrate by the protease was reflected by the decrease in the
fluorescent signal. The correlation between 96-well plate and card
systems was satisfactory with an r value of 0.96. From this
preliminary result, taking results from the 96-well plate as the
reference, if the cut off value for the first phase screening was
80% of the control signal, there would be 3 false negatives and
fewer than 10 false positives.
[0187] The next assay was another hydrolase assay, using alkaline
phosphatase as the enzyme. The reagents and protocol are as
follows.
[0188] Reagents:
[0189] Alkaline phosphatase (Sigma, St. Louis, Mich.)
[0190] AutoPhos buffer (JBL Scientific, Inc., San Louis Obispo,
Calif.)
[0191] 1 mM MgCl2
[0192] 4-Nitrophenyl Phosphate (PNPP) (Sigma Chemical, St. Louis,
Mich.)
[0193] Protocols:
[0194] The channel was rinsed with AutoPhos buffer and then filled
with about 10 .mu.l of 1 mM AutoPhos substrate. 50 nl of alkaline
phosphatase was then dispensed into the sample port. The amount of
enzyme dispensed into the sample port increased from 31.25 attomole
to 62.5 femtomoles by a factor of 2. Enzyme solutions of different
concentrations were prepared in individual wells of a 96-well
microtitre plate. Fluorescence was excited at 480 nm.+-.20 nm and
emission collected at 520.+-.20 nm. The signals were recorded at
different time points, 0, 5, 10, 15, up to 35 minutes.
[0195] The results are shown in the following table. The
fluorescent signal as a function of enzyme concentration at
reaction times of 12, 20, and 30 minutes respectively was shown to
be linear with the enzyme concentration in accordance with the
1.sup.st order reaction.
12TABLE Fluorescence Signal as a Function of Enzyme Concentration
and Reaction Time [Enzyme], nM Time, min 1000 250 125 31.25 12
13390.8 2913.8 1497.7 821.1 20 20692.4 4698.6 2323.8 0 30 28981.6
7579.0 2892.7 1798.8
[0196] In addition, as to each enzyme concentration, in the
presence of sufficient enzyme substrate, the rate is linear with
time.
[0197] Time Course of the Alkaline Phosphatase
Reaction--Determination of the Diffusion during Incubation of Large
Molecules such as Enzymes
[0198] Procedure:
[0199] After taking the image of the empty card with the lamp on,
5.multidot.1 of 1 mM AutoPhos was added to each reservoir followed
by adding 400 nl of 1 mM AutoPhos to the assay well. A card image
was taken with the lamp off followed by taking an image with the
lamp on. 200 nl of 2 .mu./ml of enzyme was added to the assay well
and images taken every minute with the lamp on.
[0200] The above images show the fluorescence from an alkaline
phosphatase reaction in a 1 mm assay well. The fluorescent signal
increased as the reaction proceeded. In addition, as seen, most of
the fluorescence is concentrated in the assay well, without
significant diffusion of the fluoroscer.
[0201] The next assay was a competitive inhibition assay using the
following protocol: 4-Nitrophenyl phosphate (PNPP) was used as a
non-fluorescent substrate for competing for alkaline phosphatase
with AutoPhos substrate. After rinsing the channels with AutoPhos
buffer, the channel was filled with 1 mM AutoPhos substrate. 100 nl
of PNPP was dispensed at different concentrations ranging form 0 to
60 mM. The fluorescent signal was measured at different reaction
time points. The fluorescent signal as a function of different
inhibitor concentrations is tabulated as following.
13TABLE Fluorescence Signal as a Function of Inhibitor
Concentration [Inhibitor], mM 0.001 0.0025 0.005 0.01 0.02 0.3125
0.625 1.25 5 10 60 RFU 4527 4000 4000 3500 2600 2000 1600 1600 1600
1500 750
[0202] The following study used the Receptor-Ligand Binding Assay
via Fluorescent Resonance Energy Transfer (FRET). The reagents and
protocol are as follows.
[0203] Reagents:
[0204] Fluorescein labeled biotin (Molecular Probe, Eugene,
Oreg.)
[0205] Rhodamine labeled strepavdin (Molecular Probe, Eugene,
Oreg.)
[0206] D(+)-Biotin (Molecular Probe, Eugene, Oreg.)
[0207] 50 mM Tris buffer (pH=9.0)
[0208] Binding Isotherm
[0209] Protocol:
[0210] The channel is rinsed and filled with 25 .mu.M of rhodamine
labeled receptor, and 100 nl of fluorescein labeled antigen is
dispensed into the assay well. The concentrations of fluorescein
labeled antigen were 0, 5, 10, 25, 50 to 100 .mu.M, respectively.
The fluorescence was excited at 480.+-.20 nm and the emission was
collected at 600 nm.+-.20 nm. Shown in the following table is the
fluorescence resonance energy transfer (FRET) signal vs.
concentration of fluorescein labeled antigen. The energy transfer
increased in relation to the increasing antigen-receptor
binding.
14TABLE FRET Signal as a Function of Fluorescein Labeled Antigen
[Fl-Antigen], .cndot. M 0 5 10 25 50 100 FRET Signal 0 370 869 1639
2327 2956
[0211] In the next study the channel was rinsed and filled with 25
.mu.M of fluorescein labeled antigen, followed by dispensing 100 nl
of rhodamine labeled receptor into the sample port. The
concentration of rhodamine labeled receptor was 0, 0.25, 0.5, 1.0,
1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, and 12 .mu.M respectively. The
fluorescence was excited at 480.+-.20 nm and the emission was
collected at 600 nm.+-.20 nm. Shown in the following table is
fluorescence resonance energy transfer (FRET) signal vs.
concentration of rhodamine labeled receptor. The energy transfer
increased corresponding to the increasing in antigen-receptor
binding. The background FRET signal contributed by
rhodamine-receptor alone was negligible.
15TABLE FRET Signal vs. Rhodamine Labeled Receptor [Rh-Receptor],
.mu.M 0 0.25 0.5 1.5 3.5 5 6 8 12 FRET Signal 2192 3663 2264 3254
7619 10604 10882 11952 11552 Bkgd Signal 1923 1430 2336 1312 556
211 516 759 1005
[0212] Using the above reagents and protocol, an inhibition assay
was performed. The protocol was to fill the channel with 0, 30, 60,
180, 240, 500, 600, 1000, 5000 .mu.M of biotin, respectively,
followed by dispensing 100 nl of rhodamine labeled receptor into
the sample port. After dispensing 100 nl of 1.0 .mu.M fluorescein
labeled antigen into the sample port, the reaction mixture was
incubated for 60 minutes. The fluorescence was recorded by exciting
at 480.+-.20 nm and reading the emission at 520 nm.+-.20 nm As the
inhibitor concentration increased, the fluorescence intensity
increased, indicating an increased inhibition. The increase in the
fluorescent signal as a function of inhibitor concentration was
converted to the percentage of inhibition. The results are
displayed in the following table.
16TABLE Inhibition vs. Inhibitor Concentration [Inhib- itor], nM 0
30 60 180 240 500 600 1000 5000 % of 0 3.85 3.10 5.00 5.14 22.4
36.64 95.0 100.0 Inhibition
[0213] The following assay is a HTRF-FRET assay. In TRF, the
species are excited through a pulse of laser light, and the
emission is then collected in a delayed time protocol (typically 50
.mu.s). Therefore, the initial burst of the fluorescence mostly
from background (lifetime of the order of 10 ns) is eliminated. The
homogenous assay of TRF was based on fluorescence resonance energy
transfer (FRET). The donor fluorophore is europium cryptate
(europium ion caged in a tris-bipyridine struture) with a
long-lived emission (.about.milliseconds) at 620 nm upon excitation
at 380 nm. The acceptor fluorophore is a stabilized allophycocyanin
XL665 When XL665 is in proximity to europium cryptate as a result
of a biomolecular interaction, the energy is transferred to the
XL665 and is emitted as a long-lived 665 nm signal. The emission of
free acceptor XL665 is short-lived. This FRET pair has a high yield
energy transfer of 50% at 9.5 nm, and is the longest energy
transfer distance reported for a FRET pair.
[0214] The card employed was a white acrylic card laminated with
plasma treated Rohm film. The following are the reagents and
protocol.
[0215] Reagents:
[0216] Biotin-K, biotin labeled with europium cryptate ("Biot-K",
CIS bio international)
[0217] Conditioning buffer: phosphate 0.1 M, pH 7.
[0218] SA-XL, streptavidin labeled with XL665 (Allophycocyanin, CIS
bio international)
[0219] Conditioning buffer: phosphate 0.1M, pH 7
[0220] TR-FRET buffer: 50 mM TRIS, 100 nM KF, 0.1% BSA, pH 8.
[0221] A europium cryptate concentration standard curve was
prepared. Biotin labeled europium cryptate (Biotin-K) was diluted
in various concentrations shown in the table below. 500 nl of
different concentrations of Biotin-K was then added to the assay
well. There were three replicates for each concentration. The
instrumental setting was the same as the one for the previous-FRET
assay. The range of europium cryptate concentration was tested to
determine the desirable Biotin-K concentration for the FRET assay.
The average and standard deviation of the donor signals are shown
in the table. The acceptor signals were negligible compared to the
background. The donor signals are linear corresponding to the
europium cryptate concentration. Biotin-K concentration of 400
pg/well was selected for the further TR-FRET assay.
17TABLE Biotin-K Concentration vs. Donor Emission Signal BiotinK,
pg Mean STD 0 70584.5 20952.3 25 84582.67 26065.5 50 72946.33
2105.7 100 125456 13859.1 150 252819.3 18653.7 200 243819.3 17990.6
300 614849.3 102782.6 400 662512.7 160733.5 500 889204.5 308942.7
1000 1666774 33679.5 2000 2716030 354542.6
[0222] TR-FRET signals:
[0223] In the next assay, the channel was filled with 5 .mu.l of
TR-FRET buffer Then 500 nl of Biotin-K was added to the assay well
followed by a 500 nl addition of different concentrations of SA-XL
to the assay well. Six replicates for each concentration point were
performed The signals were detected using an HTS Analyst
manufactured by LJL BioSystems. It was observed that as As SA-XL665
concentration increased, more binding of biotin-K occurred,
resulting in increased energy transfer. Therefore, the donor
emission decreased with the increasing acceptor concentration
indicating energy transfer was occurring, while the acceptor
emission increased as energy was retained. As limited by the
available biotin-K, the energy transfer leveled off at higher
concentrations.
18TABLE Acceptor Concentration vs. FRET Signal SA-XL665, ng 0 0.1 1
5 10 50 Donor Mean 243819.3 186521 156683.8 132737.6 131324.8
122187.7 SD 17990.59 55422.59 61107.69 45203.64 43235.92 26462.03
Acceptor Mean 21721.89 34277 74506.4 99773 91039.4 84773 SD
3916.839 7590.534 28612.11 46441.94 18667.63 23051.49
[0224] The next assay was a fluorescence polarization assay.
[0225] Fluorescence polarization (FP) is a technique that is used
to monitor molecular interactions in a homogenous environment at
equilibrium. FP is based upon the theory that when a molecule is
excited with plane-polarized light of the correct wavelength, it
will fluoresce in the same plane after its characteristic emission
lifetime, which is typically a few nanoseconds. During this time,
the molecule will have tumbled randomly with respect to the
original plane of excitation. If the molecule tumbles quickly with
respect to the fluorescence lifetime, the fluorescence will be
depolarized. However, if the molecule tumbles slowly with respect
to the fluorescence lifetime, the observed fluorescence will remain
significantly polarized. In general, a molecule's rate of tumbling
is directly proportional to its molecular volume at constant
temperature and viscosity. Small molecules tumble rapidly while
large molecules tumble slowly. When a small fluorescent molecule is
bound to a large molecule, it will tumble slowly. Therefore, by
measuring the extent of fluorescence polarization, the binding
equilibrium and the competition for binding at a site can be
determined. The following are the reagents and protocols
employed.
[0226] Reagents:
[0227] PTK detection mix (anti-phosphotyrosine antibody,
fluorescent phosphopeptide tracer, NP40, sodium azide, in phosphate
buffer saline, pH 7.4)
[0228] PTK competitor (100 .mu.M phosphopeptide in DEPC-treated
water)
[0229] PTK standard curve dilution buffer (phosphate buffer saline
pH 7.4)
[0230] Protocols:
[0231] The competitor was diluted to the following concentrations
in the same buffer: 100 .mu.M, 10 .mu.M, 1 .mu.M, 0.1 .mu.M, and
0.05 .mu.M 1 .mu.l of detection mix was added to the assay wells,
followed by the addition of 3.2.mu.l of detection mix to the
reservoirs. 500 nl of competitor solution was added to the assay
wells. Six replications for each concentration point were perfomed.
The assay mixtures were incubated at room temp. for 5 min. and the
polarization measured using an LJL BioSystems' HTS Analyst
microplate reader. The results are as follows. The extent of
fluorescence polarization can be indicated as: 2 mP = s - p s + p *
1000 ,
[0232] where s is the signal from the same plane of the excitation,
while p is the signal from the perpendicular plane to the plane of
excitation. The extent of the fluorescence polarization will vary
in the range of 0 to 1000 with a higher value indicating a higher
degree of polarization. Shown in the table, when a small
phosphopeptide labeled with a fluorescence tracer
(F1-phosphopeptide tracer) was bound to the bigger phosphotyrosine
antibody, the polarization signal was high. As concentrations of
unlabeled phosphopeptides increased competing for the same binding
sites of the phophostyrosine antibody, more and more
F1-phosphopeptide tracers remained unbound and free in solution and
the signals were depolarized. The IC.sub.50 for the competition was
determined as .about.0.5.multidot.M in accordance with the
0.4-0.6.multidot.M value reported in the literature.
19TABLE Competitor Concentration vs. Polarization Signal
Competitor, 100 10 1 0.1 0.05 .mu.m mP 112.1649 136.3539 243.4099
276.376 333.9214
[0233] In the next study assays were performed in assay wells,
where the solution in the assay well could be transferred to a
capillary electrokinesis system for further processing. FIG. 14
shows the layout of the capillary electrophoresis card, the CE
card. As can be seen in this Figure, the CE 2 card has three
different patterns. Each pattern consists of two parts; evaporation
control assay system and injection/separation capillary
electrokinesis system.
[0234] The devices are shown as stick diagrams, where the
reservoirs at the ends of the lines, which depict the channel
pattern, are not shown. See, for example, FIG. 7A for an indication
of the channels and reservoirs. Device a400 has capillary channel
a402, with reservoirs at its termini, a502 and a504 as depicted in
FIG. 15, with an assay well at the intersection a404, as shown in
FIG. 15 at a506. The side channel connects capillary a402 with the
capillary electrokinesis system comprising analytical channel a408
and waste channel a410. The device a412 differs from the device
a400 in having the side channel a406 offset from the waste channel,
so that there is a region between the side channel a406 and the
waste channel a410 along the analytical channel a408, which serves
to define the size of the slug of the assay composition that will
be detected in the analytical channel a408. Device a420 differs
from the device a400 in having hydrostatic head control channels
a422 and a424 along side channel a406, to provide better control of
the hydrostatic head during long incubations in the assay system.
In FIG. 15, device a500, is analogous to device a400 with assay
system capillary channel a508 being connected to side channel a406.
The intersection a512 serves as the injector or injection site for
injection of the assay composition into the analytical channel.
HV.sub.1-4 intends the voltages of the electrodes during the
transfer of the composition from the assay well a506 into the
capillary electrokinesis system for transport to the intersection
1a512 and injection into the analytical channel a514.
[0235] The assay well system incorporates a wide channel (450 .mu.m
wide and 50 .mu.m deep) with two buffer reservoirs (2 mm in
diameter) and the evaporation control well (1 mm diameter) in the
middle of the channel. The second part of the CE 2 device which is
the injection/separation part consists of injection and separation
channels with dimensions of 120 .mu.m wide and 50 .mu.m deep. The
injection channel is connected directly to the evaporation control
well. As shown in the FIG. 15, some of the patterns have no offset
(simple cross) and the others have a 250 .mu.m offset (double-T
injector). The third pattern has two more side channels for the
purpose of controlling the hydrostatic flow within the channel
manifold if a long incubation time is needed. The channels are
closed by laminating a film (plasma treated Rohm or MT40) on the
card.
[0236] The experimental procedure was as follows: the assay well is
covered by tape. 5 .mu.l of buffer was added to the reservoirs. 500
nl of the fluorescein or assay mixture was pipetted into the assay
well. For the alkaline phosphatase assay, enzyme and substrate with
or without inhibitor was mixed in a tube and then 500 nl of the
assay mixture was pipetted into the assay well. The detection was
performed at 7 mm distance from the injector. The particular
conditions for each determination are set forth with the
figure.
[0237] The following table shows the voltage configuration for
these assays
20 Electrode 1 Electrode 2 Electrode 3 Electrode 4 Injection 220
500 155 0 Separation 0 280 1000 280
[0238] To perform the analysis of the maintenance of signal in the
assay well, 500 nl of fluorescein was added to the assay well and
the whole card covered by a 96 well plate for 75 min. Then the
fluorescein was moved to the intersection, consecutively injected
and separated for another 15 min. A CV of 7-13% was achieved for
these repetitive injections. FIG. 16 shows the calibration curve
for fluorescein using the card. As can be seen a linear calibration
curve was achieved in the concentration range of 250-100 nM.
[0239] FIG. 17 illustrates the alkaline phosphatase activity for
the different incubation times. As shown in the electropherograms,
two product peaks (the first peak is fluorescein mono phosphate and
the 2nd peak is fluorescein) are well separated from each other.
Additionally, the use of longer incubation time results in more
conversion of FDP (fluorescein di-phosphate as a substrate) to the
FMP (fluorescein mono-phosphate) and finally to fluorescein. FIG.
18 depicts a linear calibration curve for the alkaline phosphatase
using the card. For the inhibition study, PNPP which is a
non-fluorescent substrate for the alkaline phosphatase and competes
with FDP which is a fluorescent substrate for the enzyme, is added
to the assay mixture at a number of different concentrations. FIG.
19 shows different electropherograms from different assay mixtures
containing 1.3 mU/ml alkaline phosphatase, 3.33 .mu.M of FDP, and
different concentrations of PNPP as depicted in the figure. As can
be seen, an increase of the concentration of PNPP results in a
reduction of FDP alkaline phosphates activity. FIG. 20 shows a
linear calibration curve for PNPP concentration.
[0240] The following example illustrates the subject device and
method for a cytochrome P450 enzyme Reaction:
[0241] Reagents:
[0242] RECO System CYP3A4 Purified, Recombinant Human (Panvera Cat
No. P2305).
[0243] RECO System CYP1A2 Purified, Recombinant Human (Panvera Cat
No. P2304).
[0244] RECO System CYP2C9 Purified, Recombinant Human (Panvera Cat
No. P2362).
[0245] 7-Benzyloxyquinoline (BQ) (Gentest Cat No. B720).
[0246] 3-Cyano-7ethoxycoumarin (CEC) (Gentest Cat No. UC-455).
Substrate for 1A2.
[0247] 7-Methoxy-4-(trifluoromethyl)-coumarin (MFC) (Gentest Cat
No. B740).
[0248] Acetonitrile.
[0249] B-Nicotinamide Adenine Dinucleotide Phosphate, Reduced Form
(NADPH) (Sigma Cat No. 201-3).
[0250] Pluronic F68 (Sigma Cat No. P1300)
[0251] Cards:
[0252] Cards (Each unit comprised two reservoirs, a central well
and a channel connecting the reservoirs and well. See FIG. 1 as to
the configuration of the microstructures.) molded of black
polystyrene and ultra sonically welded with plasma-treated LCF 3001
film were employed. A single pattern which has two evaporation
control wells on a common channel, with an assay well centered on
the channel between the evaporation control wells was used. This
pattern has a 1 mm diameter assay well, tapering to 0.9 mm at the
bottom The reservoirs have a 2 mm diameter, tapering to 1.9 mm.
[0253] Protocols:
[0254] The reagent solutions were prepared as follows.
[0255] Dissolve 7-Ethoxy-3-cyanocoumarin (CEC) 20 mM
[0256] Add 8.61 mg 7-ethoxy-3-cyanocoumarin to 2.0 mL acetonitrile.
Invert to dissolve. Store at -20.degree. C.
[0257] Dissolve 7-Methoxy-4-trifluoromethylcoumarin (MFC) 25 mM
[0258] Add 12.21 mg 7-methoxy-4-trifluoromethylcoumarin to 2.0 mL
acetonitrile. Invert to dissolve. Store at -20.degree. C.
[0259] Dissolve Benzyloxyquinoline (BQ) 20 mM
[0260] Add 4.706 mg benzyloxyquinoline to 1.0 mL acetonitrile.
Invert to mix. Store at -20.degree. C. Dissolve NADPH 10 mM
[0261] B-Nicotinamide Adenine Dinucleotide Phosphate. Add 2.87 mg
NADPH to 344 ul of deionized water. Invert to dissolve. Store at
-20.degree. C.
[0262] Furafylline 2.5 mM
[0263] Add 1.3 mg furafylline to 2.0 mL acetonitrile. Invert to
dissolve.Note: Solution may precipitate upon storage at -20.degree.
C. but will redissolve when sonicated in warm water 5% Pluronic
F68
[0264] Add 5.0 gm Pluronic F68 and bring to 100 mL with deionized
water. Stir to dissolve.
[0265] Example: Cytochrome P450 1A2 Enzymatic Assay
[0266] A. Cyp450 1A2 enzymatic activity:
[0267] Procedures:
[0268] 1. Make 20 mM CEC substrate for Cyp450 1A2 enzyme.
[0269] 2. Make fresh 10 mM NADPH solution with water.
[0270] 3. Make Buffer Mix to be used to fill channels:
[0271] 20 .mu.l Water
[0272] 20 .mu.l 5% Pluronic F68
[0273] 20 .mu.l 5X CYP3A4 buffer
[0274] 20 .mu.l 20 mM CEC
[0275] 20 .mu.l 10 mM NADPH
[0276] 100 .mu.l Total vol. (enough for 10 reactions)
[0277] 4. Place card in holders.
[0278] 5. Add 5 .mu.l of the buffer mix to both of the side wells
of the channels. Because the solution contains Pluronic F68, the
middle assay mixture rises to the top of the well.
[0279] 6. Add 300 nl of various concentrations of CYP450 1A2 enzyme
to assay (middle) well.
[0280] 7. Cover with 96 well plate cover. Incubate at 37.degree. C.
for 35 minutes.
[0281] 8. Take RFU readings using Molecular Devices Fmax plated
reader. f-max settings: Filter pair 390/460; Int.20 ms;
speed10.
[0282] Results:
21TABLE CYP450 1A2 Enzyme Concentration vs. Reaction Signal [1A2],
nM 0 8 16 32 64 100 133 RFU(mean) 7.0 10.7 13.2 13.9 16.5 21.7 24.5
RFU(std) 1.5 1.8 1.3 1.0 0.6 3.1 4.2
[0283] The fluorescence signal increased linearly with the increase
of the CYP450 1A2 enzyme concentration.
[0284] B: Inhibition in CYP450 1A2 Assay:
[0285] Protocols:
[0286] 1. Make 500.multidot.M CEC substrate for 1A2.
[0287] 2. Make fresh 10 mM NADPH solution with water.
[0288] 3. Make serial dilutions of furafylline at 2500, 1250, 250,
125, 25, 12.5, 2.5, 0.multidot.M concentrations
[0289] 4. For each of the dilutions of furafylline make Buffer
Mix:
22 Water 14.85 .mu.l 5% Pluronic F68 9 .mu.l 500 uM CEC 0.9 .mu.l
CYP1A2 buffer (5X) 9 .mu.l 10 mM NADPH 11.25 .mu.l Furafylline
(from 0 to 2.5 mM) 1.8 .mu.l Total Volume 45 .mu.l (enough for 4
reactions)
[0290] 5. Place card in holders.
[0291] 6. Add 5 .mu.l of the buffer mix to both of the side wells
of the channels. Because the solution contains Pluronic F68, the
solution in the middle assay well rises to the top. Dilute CYP1A2
enzyme 2:1 with water.
[0292] 7. Add 300 nl of diluted enzyme to assay (middle) well.
[0293] 8. Cover with 96 well plate cover.
[0294] 9. Incubate at 37.degree. C. for 35 minutes.
[0295] 10. Take RFU readings using Molecular Devices F-max plate
reader. F-max settings: Filter pair 390/460; Int.20 ms;
speed10.
[0296] Results:
23TABLE Percentage of Inhibition vs. Inhibitor Concentration
[Inhibitor], .mu.M 133 66.5 13.3 6.65 1.33 0.665 0.133 % of
Inhibition 78.0 77.0 75.0 72.0 64.0 31.0 2.0
[0297] It is evident from the above results that the subject
devices and methods provide for efficient manipulations of small
volumes and determinations of a wide variety of events, such as
chemical reactions, binding events, enzyme reactions, and the like.
The subject invention has great flexibility in the variety of
protocols, which may be employed, with a single device allowing for
different protocols. In addition, the subject devices may be
combined with other devices, such as microtiter well plates, where
the subject device may be in registry with the wells, so that
samples may be readily followed and results recorded with
confidence as to the compound involved.
[0298] Each document, reference or patent application, cited herein
is incorporated by reference as if the reference was set forth
verbatim in the text of this specification.
[0299] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *