U.S. patent application number 11/792170 was filed with the patent office on 2008-10-30 for microfluidic sieve valves.
This patent application is currently assigned to California Institute of Technology. Invention is credited to Carl L. Hansen, Joshua S. Marcus, Stephen R. Quake.
Application Number | 20080264863 11/792170 |
Document ID | / |
Family ID | 36565825 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080264863 |
Kind Code |
A1 |
Quake; Stephen R. ; et
al. |
October 30, 2008 |
Microfluidic Sieve Valves
Abstract
Sieve valves for use in micorfluidic device are provided. The
valves are useful for impeding the flow of particles, such as
chromatography beads or cells, in a microfluidic channel while
allowing liquid solution to pass through the valve. The valves find
particular use in making microfluidic chromatography modules.
Inventors: |
Quake; Stephen R.;
(Stanford, CA) ; Marcus; Joshua S.; (Altadena,
CA) ; Hansen; Carl L.; (Vancouver, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
California Institute of
Technology
Pasadena
CA
|
Family ID: |
36565825 |
Appl. No.: |
11/792170 |
Filed: |
December 5, 2005 |
PCT Filed: |
December 5, 2005 |
PCT NO: |
PCT/US2005/043833 |
371 Date: |
July 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60633121 |
Dec 3, 2004 |
|
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|
Current U.S.
Class: |
210/651 ;
210/198.2; 210/200; 210/321.65; 210/650 |
Current CPC
Class: |
B01J 2220/54 20130101;
G01N 30/603 20130101; F16K 99/0026 20130101; G01N 30/6095 20130101;
G01N 30/6004 20130101; F16K 2099/0074 20130101; B01J 20/286
20130101; F16K 2099/008 20130101; F16K 99/0034 20130101; F16K
99/0059 20130101; F16K 2099/0084 20130101; F16K 99/0001 20130101;
B01L 3/502738 20130101; B01L 3/502761 20130101 |
Class at
Publication: |
210/651 ;
210/321.65; 210/198.2; 210/200; 210/650 |
International
Class: |
B01D 15/08 20060101
B01D015/08; B01D 61/18 20060101 B01D061/18 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Work described herein has been supported, in part, by
National Institutes of Health (NIH) grant NIH IRO1 HG002644-01A1.
The United States government may have certain rights in the
invention.
Claims
1. A microfabricated sieve valve structure comprising an
elastomeric membrane that separates a first channel lumen and a
second channel lumen, wherein pressurizing the first channel over a
wide range of pressures causes the membrane to be deflected into
the second channel lumen and reduce the cross-sectional area of the
second channel lumen by not more than 90% and not less than 50% of
the cross-sectional area when the membrane is not deflected.
2. The structure of claim 1 wherein the cross-sectional profile of
the second channel is rectangular.
3. The structure of claim 1 wherein pressurizing the first channel
over a wide range of pressures causes the membrane to be deflected
into the second channel lumen and reduce the cross-sectional area
of the second channel lumen by not more than 90% and not less than
75% of the cross-sectional area when the membrane is not
deflected.
4. The structure of claim 1 wherein the range of pressures is a
range of at least 7 psi.
5. The structure of claim 3 wherein the range of pressures
encompasses a range of 18-30 psi.
6. The structure of claim 1 wherein the sieve valve has a retention
size of from 1 micron to 20 microns.
7. A microfluidic device comprising two or more sieve valves.
8. The device of claim 7 wherein a chromatographic separation
medium is disposed between two sieve valves thereby forming a
separation column.
9. The device of claim 7 that comprises more than 20 separation
columns.
10. A microfluidic device comprising a microfluidic chromatography
column, said column comprising a chromatographic separation medium
disposed behind a sieve valve, and optionally disposed between two
sieve valves.
11. The device of claim 10 wherein the chromatographic separation
medium comprises a polymeric bead coupled to a ligand.
12. The device of claim 11 wherein the beads have been derivatized
to bind a nucleic acid.
13. The device of claim 12 wherein the beads have been derivatized
with oligo(dT).
14. The device of claim 10 wherein the beads have been derivatized
with a protein, optionally an antibody.
15. The device of claim 7 that contains five or more sieve valves
paired with conventional valves.
16. A microfluidic device comprising two or more sieve valves
paired with conventional valves.
17. A method of making a microfluidic column in a microfluidic
device, wherein the device comprises a flow channel and a sieve
valve positioned to reduce the cross-sectional area of the lumen of
the flow channel when closed, the method comprising providing a
suspension of chromatography beads in the flow channel ante to the
sieve valve, wherein the valve is closed and the beads are of a
size that is retained by the closed sieve valve; flowing the
suspension through the flow channel, whereby the movement of the
beads is impeded by the closed sieve valve and the solution in
which the beads are suspended flows through the flow channel,
thereby producing a column of beads in the flow channel.
18. The method of claim 17 wherein the device comprises two or more
sieve valves each positioned to reduce the cross-sectional area of
the lumen of the flow channel when closed, said method comprising:
providing the suspension of chromatography beads ante to a second
sieve valve, wherein said second sieve valve is open and is ante to
the closed sieve valve, and wherein the beads are of a size that is
retained by the second sieve valve; flowing the suspension of
chromatographic beads through the flow channel through and past the
second sieve valve, wherein the flow of the beads is impeded by the
closed sieve valve and the solution in which the beads are
suspended flows through the flow channel, thereby producing a
column of beads in the flow channel; and, closing the second sieve
valve, thereby trapping the beads betwixt the sieve valves.
19. A method for trapping particles in a microfluidic flow channel
of a microfluidic device, the method comprising: providing a
suspension of the particles in a flow channel ante to a closed
sieve valve, wherein the particles are of a size that is retained
by the closed sieve valve; flowing the suspension through the flow
channel, whereby the movement of the particles is impeded by the
closed sieve valve and the solution in which the particles are
suspended flows through the flow channel, thereby trapping the
particles in the flow channel.
20. The method of claim 19 wherein the particles are living cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/633,121, filed Dec. 3, 2004, the entire contents
of which are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to microfabricated devices and
microchromatography.
BACKGROUND OF THE INVENTION
[0004] Microfluidic devices may be used in a variety of biomedical
and pharmaceutical applications, including analysis, preparation
and synthesis of chemical compounds and analysis and manipulation
of cells, proteins and nucleic acids. In many applications, the
ability to concentrate or chromatographically separate compounds in
a microfluidic environment enhances the utility of microfluidic
devices. Thus, a rapid, inexpensive and effective method for
performing chromatography and for microfluidic chromatography
columns would be of great benefit. The present invention meets
these and many other needs.
BRIEF SUMMARY
[0005] In one aspect the invention provides a microfabricated sieve
valve structure having an elastomeric membrane that separates a
first channel lumen and a second channel lumen, where pressurizing
the first channel over a wide range of pressures causes the
membrane to be deflected into the second channel lumen and reduce
the cross-sectional area of the second channel lumen by not more
than 90% and not less than 50% of the cross-sectional area when the
membrane is not deflected. In certain embodiments the
cross-sectional profile of the second channel is rectangular. In
certain embodiments pressurizing the first channel over a wide
range of pressures causes the membrane to be deflected into the
second channel lumen and reduce the cross-sectional area of the
second channel lumen by not more than 90% and not less than 75% of
the cross-sectional area when the membrane is not deflected. In
certain embodiments the range of pressures is a range of at least 7
psi, optionally at least 10 psi, optionally at least 15 psi or
encompasses a range of 22-26 psi, optionally 20-28 psi, and
optionally 18-30 psi. In certain embodiments the sieve valve has a
retention size of from 1 micron to 20 microns.
[0006] In a related aspect the invention provides a microfluidic
device having two or more sieve valves. In an embodiment, a
chromatographic separation medium is disposed between two sieve
valves thereby forming a separation column. In an embodiment the
device has more than 20 separation columns.
[0007] In a related aspect the invention provides a microfluidic
device comprising a microfluidic chromatography column, the column
comprising a chromatographic separation medium disposed behind a
sieve valve, and optionally disposed between two sieve valves. In
an embodiment the chromatographic separation medium comprises a
polymeric bead coupled to a ligand. For example, in certain
embodiments beads have been derivatized to bind a nucleic acid
(e.g., oligo(dT)) or a protein (e.g., an antibody).
[0008] In a related aspect the invention provides a microfluidic
device with two or more sieve valves paired with conventional
valves. In a related aspect the invention provides a microfluidic
device that contains five or more sieve valves paired with
conventional valves.
[0009] In a related aspect the invention provides a method of
making a microfluidic column in a microfluidic device, wherein the
device comprises a flow channel and a sieve valve positioned to
reduce the cross-sectional area of the lumen of the flow channel
when closed. The method involves providing a suspension of
chromatography beads in the flow channel ante to the sieve valve,
where the valve is closed and the beads are of a size that is
retained by the closed sieve valve; flowing the suspension through
the flow channel, whereby the movement of the beads is impeded by
the closed sieve valve and the solution in which the beads are
suspended flows through the flow channel, thereby producing a
column of beads in the flow channel.
[0010] In one embodiment, the device has two or more sieve valves
each positioned to reduce the cross-sectional area of the lumen of
the flow channel when closed, and the method of making a
microfluidic column in a microfluidic device includes providing the
suspension of chromatography beads ante to a second sieve valve,
wherein the second sieve valve is open and is ante to the closed
sieve valve, and wherein the beads are of a size that is retained
by the second sieve valve; flowing the suspension of
chromatographic beads through the flow channel through and past the
second sieve valve, wherein the flow of the beads is impeded by the
closed sieve valve and the solution in which the beads are
suspended flows through the flow channel, thereby producing a
column of beads in the flow channel; and, closing the second sieve
valve, thereby trapping the beads betwixt the sieve valves.
[0011] In a related aspect the invention provides a method for
trapping particles in a microfluidic flow channel of a microfluidic
device, by providing a suspension of the particles in a flow
channel ante to a closed sieve valve, wherein the particles are of
a size that is retained by the closed sieve valve; flowing the
suspension through the flow channel, whereby the movement of the
particles is impeded by the closed sieve valve and the solution in
which the particles are suspended flows through the flow channel,
thereby trapping the particles in the flow channel. In one
embodiment the particles are living cells.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIG. 1. Schematic representations illustrate the operation
mechanisms of (A) a regular valve having a round-profiled fluidic
channel and (B) a sieve valve having a rectangular-profiled fluidic
channel. When pressure is introduced into the control channels, the
elastic membranes expand into the fluidic channels. In a regular
valve, the fluidic channel is completely sealed because of the
perfect fit between the expended membranes and the round profile of
the fluidic channel. In a sieve valve, the square-profiled fluidic
channel is only partially closed, which allows fluid to flow
through the two edges. Sieve valves can be used to confine solid
objects within the fluidic channel, but allow liquid to flow
through it. (C) Schematic illustration of the loading of anion
exchange beads into a column module incorporating one fluidic
channel and five sieve and five regular valves. [.quadrature.],
open valve; [X], closed valve. A suspended solution of anion
exchange beads is introduced into the column modules where five
sieve valves and five regular valves operate cooperatively to trap
anion exchange beads inside the fluidic channel (total volume: 10
nL). A miniaturized anion exchange column for fluoride
concentration is achieved when the fluidic channel is fully loaded.
(D) A snapshot of the bead-loading process in action.
[0013] FIG. 2. A schematic representation corresponding to the
column module of FIG. 1.
[0014] FIG. 3. Microfluidic filter valve. a. Sampling of parameter
space sufficient to build functional bead columns. Triangles
represent the filter valve method (200.times.200 um valve, 13 um
tall) and circles (100.times.100 um valve, 13 um tall) represent
the previous approach using slightly opened valves (Hong et al.,
2004, Nat Biotechnol. 22:435-9). The flow channel pressure is the
pressure applied to the bead inlet and the column pressure is the
pressure applied to the column valve's inlet. Parameter space is
measured by if the beads escape to waste. b.-d. Optical micrographs
of the filter valve. Scale bars are 100 .mu.m. b. Top-down view of
an actuated filter valve. c. Cross section of the valve and open
channel above it. d. Cross section of the actuated filter valve and
pinched off channel.
[0015] FIG. 4. A microfluidic device with 20 columns (yellow) in
multiplex format. Flow channels are shown in white and control
channels in purple. Scale bars are 800 .mu.m.
DETAILED DESCRIPTION
[0016] In one aspect the present invention provides a microfluidic
device having at least one "sieve valve." In a related aspect the
invention provides a microfluidic device having at least one
chromatography module comprising a chromatographic separation
medium held in place in a microfluidic channel by one or more
"sieve valves," or a method for making such a module. In another
related aspect, the invention provides a device adapted for forming
a chromatography module as described. Other aspects of the
invention will be apparent upon review of the disclosure. The sieve
valves of the invention have an elastomeric component and, in a
preferred embodiment, the microfluidic channel is fabricated from
an elastomeric material.
[0017] Microfluidic devices, both elastomeric and nonelastomeric,
are widely known. Thus, the ordinarily skilled artisan will be
familiar with such devices, their components and features, and
methods of fabrication. For purposes of the following discussion it
is assumed the reader is familiar with microfluidic devices
generally, and in particular is familiar with elastomeric devices
fabricated using multilayer soft lithography (MSL) methods and
comprising flow channels, control channels, valves, pumps and other
microfluidic and auxiliary components. There is ample additional
guidance in the scientific and patent literature (see Unger et al.,
2000, Science 288:113-116 and references below).
[0018] Fundamental components of the elastomeric devices of the
invention are flow channels, control channels and valves, each of
which will be described briefly to facilitate the discussion of
aspects of the invention.
[0019] The term "flow channel" refers to a microfluidic channel
through which a solution can flow. The dimensions of flow channels
can vary widely but typically include at least one cross-sectional
dimension (e.g., height, width, or diameter) less than 1 mm,
preferably less than 0.5 mm, and often less than 0.3 mm. Different
flow channels in a particular microfluidic device may have
different dimensions, depending on the function of the particular
channel. In some embodiments of the invention the flow channel has
a low aspect ratio (e.g., a height to width ratio of less than 1:5,
preferably less than 1:10; sometimes less than 1:15). For example,
in one embodiment, the height of the rectangular channel in which a
sieve valve is positioned is about 10 microns, with a width of 200
microns. In some embodiments of the invention, the height of the
(rectangular) channel in which a sieve valve is positioned is less
than about 30 microns, often less than about 20 microns, and very
often less than about 15 microns.
[0020] A "control channel" is a channel separated from a flow
channel by an elastomeric membrane that can be deflected into or
retracted from the flow channel in response to an actuation force.
The dimensions of control channels can vary widely but typically
include at least one cross-sectional dimension (e.g., height,
width, or diameter) less than 1 mm, preferably less than 0.5 mm,
and often less than 0.3 mm. For example, in one embodiment, a
control channel has dimensions of 250 micrometers wide by 250
micrometers high. In another embodiment, a control channel has
dimensions of 300 micrometers wide by 50 micrometers high.
[0021] Elastomeric valves (e.g., pressure-actuated elastomeric
valves) consist of a configuration in which two microchannels are
separated by an elastomeric segment that can be deflected into or
retracted from one of the channels (a flow channel) in response to
an actuation force applied to the other channel (a control
channel). In one embodiment the elastomeric segment has a
substantially constant thickness (i.e., the thickness does not vary
more than 25%, preferably not more than 5%). The elastomeric
segment is usually between 1 micron and 50 microns in thickness,
preferably between 5 microns and 20 microns in thickness. Examples
of elastomeric valves include, without limitation,
upwardly-deflecting valves (see, e.g., US 20050072946), downwardly
deflecting valves (see, e.g., U.S. Pat. No. 6,408,878), side
actuated valves (see, e.g., US 20020127736). The elastomeric
segment may be substantially In one embodiment the valve is a
push-down valve and the elastomeric segment has a convex shaped
membrane (thin in the center [e.g., 10 .mu.m] and thicker at the
edges [e.g., 46 .mu.m]. In one embodiment the valve is a push-up
valve and the elastomeric segment has a uniform thickness (e.g.,
5-15 microns). In conventional valves the flow channel has a
rounded surface opposite the elastomeric segment, so that the
deflected membrane can form a tight seal with the inner surface of
the channel. For example, in one embodiment, the flow channel
section is bounded by a circular arc 300 .mu.m in width and 50
.mu.m in height.
[0022] Valves can be actuated by injecting gases (e.g., air,
nitrogen, and argon), liquids (e.g., water, silicon oils and other
oils), solutions containing salts and/or polymers (including but
not limited to polyethylene glycol, glycerol and carbohydrates) and
the like into the control channel, a process referred to as
"pressurizing" the control channel. In addition to elastomeric
valves actuated by pressure-based actuation systems, monolithic
valves with an elastomeric component and electrostatic, magnetic,
electrolytic and electrokinetic actuation systems may be used. See,
e.g., US 20020109114; US 20020127736, and U.S. Pat. No.
6,767,706.
Sieve Valves
[0023] A sieve valve (also called a "filter valve") is a type of
elastomeric valve. Like a conventional valve, the sieve valve
consists of a configuration in which a control channel and a flow
channel are separated by an elastomeric segment that can be
deflected into the flow channel in response to an actuation force
applied to the control channel. However, in the sieve valve, small
gap(s) between the elastomeric segment and the flow channel walls
permit fluid to flow through the channel even with the elastomeric
segment is maximally deflected into the flow channel.
[0024] Sieve valves are represented schematically in FIG. 1. The
figure illustrates the operation mechanisms of (A) a regular valve
having a round-profiled fluidic channel and (B) a sieve valve
having a rectangular-profiled fluidic channel. When pressure is
introduced into the control channel to actuate the regular valve or
sieve valve, the elastic membranes deflect into the fluidic
channels. In general, when valves operate, the valve membranes
deflect in an elliptic shape. In the case of normal valve (FIG.
1A), the deflected membrane is fully compliant to the round-profile
fluidic channel lead to complete close of the valve. For a sieve
valve (FIG. 1B), a deflected membrane partially closes the valve,
for example generating two small gaps the two channel edges of a
rectangular-profile channel through which fluid can flow.
[0025] This property of sieve valves renders them useful in making
on-chip microchromatographic columns. Since the sieve valves can be
used to confine solid objects within the fluidic channel, but allow
liquid to flow through it, when a suspension of a particulate
chromatographic separation material ("beads" or "chromatography
beads") is introduced into the flow channel the beads are trapped
by the closed sieve valve and while the solution is allowed to pass
through. By using this design, a variety of miniaturized columns
filled with different type of beads (e.g., ion exchange resin,
affinity resin, size exclusion, etc.) can be produced for
applications such as ion extraction, filtration, purification,
concentration and separation, and chromatography. In some
embodiments the chromatography beads are roughly spherical and have
diameters of between about 1 micron and 15 microns, such as
approximately 1, approximately 2, approximately 3, approximately 4,
approximately 5, approximately 6, approximately 7, approximately 8,
approximately 9, approximately 10, approximately 11, approximately
12, approximately 13, or approximately 14 microns.
[0026] In a preferred embodiment, the elastomeric segment of the
sieve valve is upwardly deflecting, as shown in FIG. 1. However,
downward and sideways deflecting channels, for example, can also be
used.
[0027] In a sieve valve is comprised of an elastomeric segment that
can be deflected into a channel true "rectangular-profiled" fluidic
channel. In general, in a cross-sectional profile of the portion of
the channel opposite the elastomeric segment does not have the
shape of a section (arc) of a circle or ellipse or other conical
section (assuming an orientation in which the elastomeric segment
is located at the concave face of the circle or ellipse). In one
common embodiment, as illustrated in FIG. 1B, a sieve valve is
comprised of an elastomeric segment that can be deflected into a
true "rectangular-profiled" fluidic channel. "Rectangular-profiled"
means the cross-section has the profile of a rectangle, and
comprises first and second sides, which are opposite each other and
of approximately equal length, and a third side (floor) at right
angles to the first and second sides. It will be appreciated that
deflection of the elastomeric segment onto channels with somewhat
different profiles will also achieve the desired result of allowing
liquid to flow through will retaining particles. Thus, in
alternative embodiments the cross-sectional profile of the channel
is not truly rectangular but has a different shape that precludes
the elastomeric segment from entirely blocking the channel into
which it is deflected. For example, in one embodiment, the
cross-section has the profile of one-half of a rounded rectangle. A
rounded rectangle is the shape obtained by taking the convex hull
of four equal circles of radius (r) and placing their centers at
the four corners of a rectangle with side lengths a and b. The
rounded rectangle has area (A) and perimeter (p) as follows:
A=ab+2r(a+b)+.pi.r.sup.2 and p=2(a+b+.pi.r). In a preferred
embodiment the profile of the flow channel is rectangular. In one
embodiment, sieve valves are present on a portion of the flow
channel that is 200 microns wide, 13 microns high, and has a
rectangular profile.
[0028] As illustrated in FIG. 1A, when the sieve valve is closed,
the deflected elastomeric membrane contacts a wall of the flow
channel that lies opposite the membrane. This generates two small
gaps the two channel edges of a rectangular-profile channel,
through which fluid can flow ("flow gaps"). Both of the gaps are
considered in determining the cross-sectional area of the lumen
when the valve is closed. A particular advantage of the present
invention is the ability of sieve valves to function over a wide
range of actuation pressures. This represents a significant advance
over previous designs in which a "slightly opened" valve present on
a flow channel with a semicircular profile allowed fluid, but not
particles, to pass through (see Hong et al., 2004, Nat Biotechnol.
22:435-9; see US 2005/0053952). The flow gap generated by the
"slightly opened" valves varies continuously with changes in
actuation pressure (i.e., an "analog" filter valve that may be more
open or less open) while the sieve valve creates flow gaps of
more-or-less constant size over a broad range of actuation
pressures (i.e., an "digital valve" that is open or closed, with
gaps).
[0029] A sampling of parameter space sufficient to build functional
bead columns by the two methods is shown in FIG. 3. The experiment
either applied 1.5 psi pneumatic pressure to the bead inlet while
varying the valve's pressure, or kept the column valve's pressure
constant while varying the pressure applied to the flow inlet.
Using the sieve valves, flow pressure can be varied by an order of
magnitude more than the "slightly opened" valves, measured by
whether or not beads escape to waste (FIG. 3). Similarly, when
applying constant pressure to the bead inlet, the pressure applied
to the sieve valve used to stack the beads can be varied seven-fold
more than the "slightly opened" valves.
[0030] The proportion of the cross-sectional area of the channel
that remains open when the sieve valve is closed is another
characteristic feature of the valve, and can be adjusted by varying
the height and width of the flow chamber profile, the pressure
applied to the sieve valve, the length, width, and thickness of the
membrane, the flexibility of the membrane (Young's modulus), and
the applied actuation force. See US Pat. App. 2005/0053952 for a
discussion. When the membrane is fully deflected into the flow
channel lumen the cross-sectional area of the lumen is reduced but
is not fully blocked. Usually the cross-sectional area of the lumen
is reduced by at least 30%, more often at least 40% and preferably
by at least 50%. Preferably, when the membrane is fully deflected
into the flow channel lumen the cross-sectional area of the lumen
is reduced to from 5% to 50% (more often 10% to 50%, and very often
from 10% to 25%) of the cross-sectional area of the lumen when the
membrane is not deflected. That is, in some embodiments fully
actuating the sieve valve results in a reduction in the lumen size
by 50% to 90%, preferably from 75 to 90%. In the case in which two
small gaps are maintained at the two channel edges of a
rectangular-profile channel, both of the gaps are considered in
determining the cross-sectional area of the lumen when the valve is
closed.
[0031] As noted above, in general, a sieve valve of the present
invention will remain closed with gaps over a wide range of
actuation pressures and flow channel pressures. Thus, the present
invention provides a valve that remains deflected into the flow
channel lumen sufficient to reducing the cross-sectional area of
the lumen by from 50% to 90%, preferably by from 75 to 90% over a
wide range of flow pressures and/or actuation pressures. In this
context, "a wide range" means a range of at least 7 psi (e.g., from
16-23 psi, or 18 to 25 psi) and preferably a range of at least 10
psi (e.g., from 16-26 psi, or 20 to 30 psi), and most preferably a
range of at least 14 psi (e.g., from 16-30 psi, or 18 to 32 psi).
In other particular embodiments, the wide range is at least 7, at
least 8, at least 9, at least 11, least 12, at least 13, at least
15, or at least 16 psi. In particular embodiments, the range of
pressures encompasses a range of 22-26 psi, alternatively 20-28
psi, alternatively 18-30 psi, alternatively 16-20 psi. In certain
embodiments the sieve valve having these properties has a width of
from 50 to 300 microns, a length of from 50 to 300 microns, and is
deflected into a channel depth of 5 to 30 microns. Preferably the
sieve valve membrane has a width of from 100 to 300 microns, a
length of from 100 to 300 microns, and is deflected into a channel
with a depth of 10 to 20 microns. Preferably the sieve valve has a
width of from 100 to 200 microns, a length of from 100 to 200
microns, and is deflected into a channel with a of 10 to 20
microns. In one embodiment the sieve valve membrane is
approximately square and has width and length dimensions of 100 to
300 microns (e.g., 100.times.100, 150.times.150, 200.times.200, and
250.times.250 microns) and has a channel depth of 5 to 30 microns,
preferably 10 to 20 microns.
[0032] It will be apparent that in normal operation, the flow
channel lumen is not completely blocked by the membrane when the
sieve valve is fully actuated, e.g., when the control channel is
maximally pressurized. "Maximally pressurized" refers to the
maximum pressure applied to the control channel in the normal
functioning of the valve, or alternatively refers to the pressure
above which the device will fail (e.g., delaminate).
[0033] In particular embodiments valves (including valves with
dimensions as described above) do not completely block the flow
channel lumen with the membrane is fully actuated by a control
channel pressure of 30, 32, 34, 35, 38 or 40 psi.
[0034] "Retention size" is another characteristic feature of a
sieve valve. "Retention size" refers to the diameter of a spherical
particle, i.e., bead, that is retained by the sieve valve when
actuated. Accordingly, in preferred embodiments the retention size
of a sieve valve is about 1 micron, about 2 microns, about 3
microns, about 4 microns, about 5 microns, about 6 microns, about 7
microns, about 8 microns, about 9 microns, about 10 microns, about
11 microns, about 12 microns, about 13 microns, about 14 microns,
about 15 microns, or larger than about 15 microns. (It will be
apparent that a sieve valve with a retention size of 1 micron will
also trap larger beads). In general, the optimal diameters of beads
for use in chromatography are in the range of 2 .mu.m to as 50
.mu.m, depending on the specific geometry to the channels and
valves. Retention size can be calculated or measured. One way to
measure retention size is to use a roughly spherical polymeric bead
of known diameter (e.g., 3 microns) and determining whether or not
the bead is retained by the valve. Beads that may be used include
polystyrene beads. In one embodiment, the beads are monosized
polymer particles known as DYNABEADS (Invitrogen Corp. Carlsbad,
Calif.).
Microfluidic Column Module
[0035] A microfluidic column module of the invention has one or
more sieve valves, and a chromatographic separation medium disposed
adjacent to a sieve valve or between a pair of sieve valves. For
clarity, the terms "ante" or "ante-valve," "post" or "post-valve,"
and "betwixt" can be used to describe a position in a flow channel
relative to a sieve-valve and the direction of fluid flow in the
channel. "Ante" refers to a position upstream of valve. For
example, to produce a chromatography column, chromatographic
separation material is introduced ante (see FIG. 1C). "Post" refers
to a position downstream of valve. "Betwixt" refers to the region
of a microfluidic flow channel between two sieve valves (e.g., the
separation material-containing region of the flow channel in FIG.
1).
[0036] As described above, a microfluidic chromatography column can
be prepared by introducing a suspension (e.g., an aqueous
suspension) of a particulate chromatographic separation material
("beads") ante to a closed sieve valve, allowing the beads to be
trapped. In a preferred embodiment a second sieve valve ante to the
first is then closed to confine the separation material. Thus, the
chromatography beads lie between the two valves. The second sieve
valve is useful to contain the beads and permits fluid to be flowed
through the column in either direction. It will be immediately
recognized that the ability to flow solution in both directions
through a column has a number of applications, including
applications in chromatography.
[0037] A variety of different chromatography beads may used in the
column module. For example and without limitation, chromatographic
separation material can include a bead material (e.g., cross-lined
agarose or dextran beads, functionalized silica, polymer-coated
silica, or porous silica particles, resins such as copolymers of
styrene and divinylbenzen, and divinylbenzene and acrylic or
methacrylic acid, metal and other materials) which may be
derivatized, bound to or coated with a compound(s) that
specifically interacts with a compound in solution as it passes
through the column. For example and without limitation,
chromatographic separation material can be adapted for many types
of chromatography including gel filtration, anion exchange, cation
exchange, hydrophobic interaction, size exclusion, reverse phase,
metal ion affinity chromatography, IMAC, immunoaffinity
chromatography, and adsorption chromatography. For example and not
limitation chromatographic separation material that can be used in
the column module can be HEI X8 (BioRad Corp.).
[0038] In some embodiments, the region of the flow channel in which
the chromatographic material is disposed (betwixt two sieve valves)
is in fluidic communication with one, two, three or more than three
branch flow channels for which there are additional sieve valves
and/or conventional valves situated near the junction of the main
flow channel and branch flow channels, as shown in FIGS. 1 and 2.
This arrangement facilitates the use of the column for separations,
concentrations and the like.
[0039] In certain embodiments, a number of sieve valves are used.
As illustrated in the Figures, sieve valves and conventional valves
can work in concert in the construction and use of a microfluidic
column. FIG. 1C is a schematic illustration of the loading of anion
exchange beads into a column module incorporating one fluidic
channel and five sieve and five regular valves. [.quadrature.],
open valve; [X], closed valve. A suspended solution of anion
exchange beads is introduced into the column modules where five
sieve valves and five regular valves operate cooperatively to trap
anion exchange beads inside the fluidic channel (total volume: 10
nL). A miniaturized anion exchange column for fluoride
concentration is achieved when the fluidic channel is fully loaded
(See FIG. 1D).
[0040] With reference to FIG. 2, for example, to generate a
microfluidic column in flow channel 1, a suspension of
chromatographic beads introduced though flow channel 2 (column
inlet) through open conventional valve CV1 and open sieve valve
SV1. The suspension solution flows through closed sieve valve SV2
and open conventional valve CV2 to flow channel 3 (column outlet)
while the beads are retained by closed sieve valves SV2-5.
Conventional valves CV2, and CV4-6 are also closed. Flow channels
1-3 are segment of the same channel in this example. After loading
the beads, sieve channel SV1 can be closed to retain the beads. A
sample mixture may be flowed through the column, in either
direction, with all valves except CV1 and CV3 closed. Additional
reagents, eluants or the like may be introduced thorough flow
channel 4 through open valve CV2 and closed valve SV2 with valves
SV1-5, valves CV4 and CV5 closed and either or both of CV1 and CV3
open.
[0041] Sieve valves may be used in a column module to circulate a
solution thought the column (either to increase the efficiency of
loading of a sample or of elution into a small volume). With
reference to FIG. 2, by the action of peristaltic pump 10 a
solution can be circulated through closed path formed by flow
channels 1 and 5A-C when valves CV1-3, CV6 and SV1-5 closed, and
CV4 and 5 are open. The solution can then be removed through any of
flow channels 2-4 or 5D. Optionally the solution can be displaced
by introducing another solution through a different flow
channel.
[0042] As illustrated in the figures and discussion above, sieve
valves are often paired with a conventional valve to separately
control flow of particles and liquid. In one embodiment the
invention provides a device having at least one sieve valve paired
with a conventional valve. As used in this context, valves are
paired when they are proximal to each other. For example, in some
embodiments, no more than 200 microns (alternatively, not more than
150 microns, or 100 microns) separates the region of a flow channel
blocked by the conventional valve (when actuated) and the region
blocked by the sieve valve (when actuated), measured valve edge to
valve edge. In one embodiment many (i.e., at least 20%) or most
(i.e. at least 50%) functioning sieve valves in a device are paired
with a conventional valve.
[0043] In one aspect the invention provides a method of making a
microfluidic column in a microfluidic device by providing a
suspension of chromatography beads in the flow channel ante to a
closed sieve valve (where beads are of a size that is retained by
the closed sieve valve); flowing the suspension through the flow
channel so that the movement of the beads is impeded by the closed
sieve valve and the solution in which the beads are suspended flows
through the flow channel, thereby producing a column of beads in
the flow channel. In this context "providing a suspension" means
introducing the suspension ante to the sieve valve by flowing the
suspension in a flow channel in the microfluidic device. The
suspension may be introduced from an external reservoir, or from
another part of the device, for example. After the beads are
trapped, a second sieve valve can be closed to trap the beads (or
other particles) between two sieve valves.
Microfluidic Device
[0044] A device of the invention may have multiple chromatography
modules which may be function in the purification, concentration,
or separation of a variety of compounds including biomolecules
(e.g., nucleic acids, proteins, sugars), products and reactants of
chemical reactions, and the like.
[0045] Usually a device will have a combination of sieve valves and
conventional (fully closable) valves. In one embodiment the ratio
of conventional valves to sieve valves will be greater than or
equal to 2:1, 3:1, 4:1, 5:1, 6:1, 10:1 or higher. In one embodiment
the device has flow channels with a rectangular profile throughout
the length of the channel and also has non-rectangular flow
channels. In one embodiment the device has flow channels that have
a rectangular profile in certain regions of the channel and a
non-rectangular profile in other regions.
[0046] A device of the invention may have one column or more than
one column (e.g., 1-5 columns, 5-10 columns, 10 to 1000 columns or
more than 1000 columns). FIG. 4 shows schematic of a device with 20
columns arranged in parallel. The microfluidic device can be used
to conduct separations in a multiplexing format, thus allowing
multiple analyses to be conducted simultaneously. In the device
illustrated in FIG. 4, beads (e.g., paramagnetic beads derivatized
with oligo(dT).sub.25 sequence (Dynal Biotech) can be distributed
serially (1 to 4 at a time) into 20 columns (rectangular box) and
held in place with sieve valves. Reagent(s) can be directed over
each of the columns and a target molecule (e.g., RNA from an
individual cell) can be captured by the affinity beads. Waste
(loading solution, wash buffers, etc.) can flow through the column
to waste ports (small wagon wheels) for removal. Once the target
molecules have bound to beads in each column, the sieve valves can
be opened and the beads allowed to flow to ports (large wagon
wheels) for collection. Alternatively, the target molecules can be
eluted from the beads and collected. In still another approach, the
target molecules can be manipulated on column. For example, bound
RNA can be reverse transcribed on column by, for example, flushing
the columns with reverse transcriptase and dNTPs in a first strand
reaction buffer for 45 minutes, and bringing the chips to 40
degrees C. in a thermal microscope stage to activate the
polymerase. Oligo(dT) sequences on the beads act as primers. When
cDNA synthesis is complete, the bead:cDNA complexes are sent to the
output ports in PCR buffer and collected for analysis.
Uses of Sieve Valves
[0047] The sieve valves of the invention and microfluidic devices
containing have a wide variety of uses. In particular, the uses of
sieve valves are not limited to conventional chromatography
modules.
[0048] In one aspect, a sieve valve can be used to collect any sort
of particle and hold them in place. In one embodiment, the particle
is a chromatography separation medium such as, for example, a
polymeric bead coupled to a ligand. Such beads can be used to
capture the corresponding anti-ligand in a sample. In some
embodiments, the beads are derivatized to bind a nucleic acid
(e.g., coupled to a complementary RNA, DNA, PNA, or the like). In
some embodiments, the beads are coupled to an antibody, an antigen,
a protein, protein A, biotin, steptavidin, a receptor, a probe, or
any other molecule with an affinity for the desired target. Useful
polymeric beads are available from commercial suppliers. For
example, DYNABEADS (Invitrogen Corp., Carlsbad, Calif.) may be
used.
[0049] For example, beads coated with an anti-ligand (e.g.,
antibody) can be circulated through flow channels of a device and
captured in a sieve valve; a solution carrying the ligand can be
flowed through the captured beads and the ligand bound to the
surface via the anti-ligand. The trapped particles may be processed
in place without opening the valves and/or they may be released by
opening the sieve valve(s). For example, at a desired time the
sieve valve may be opened and the beads allowed to flow to other
locations on or off the chip, thereby delivering the ligand to the
new locations.
[0050] In another example, cells may be captured by a sieve valve.
In one example, a lysis solution flowed through the collected cells
and cell components (e.g., small soluble molecules) may then flow
through the sieve valve while unlysed cells or debris are retained.
In another example, cells may be captured by a sieve valve and then
a chemical or immunological stain is flowed through the collected
cells, staining all or some of the captured cells. The sieve valves
can then be released and the cells transported to other locations.
In another example, a solution containing cells or other particles
may be flowed through a sieve valve and the cells or particles
retained, thus concentrating the cells or particles. A more
concentrated solution of cells or particles may be captured by
opening the sieve valve or reversing the direction of flow (so that
solution flows through the sieve valve towards the cells or
particles. Numerous other applications will be apparent upon review
of the disclosure.
Elastomeric Fabrication
[0051] As noted above, microfluidic devices, both elastomeric and
nonelastomeric, are well known, and the ordinarily skilled artisan
will be familiar with such devices, their components and features,
and methods of fabrication. In preferred embodiments, the device is
fabricated using elastomeric materials. Methods of fabrication
using elastomeric materials, and devices made using such materials,
have been described in detail (see, e.g., Unger et al., 2000,
Science 288:113-116; US 2004/0115838; and PCT publications WO
01/01025; WO 2005/030822 and WO 2005/084191) and will only be
briefly described here.
[0052] Sieve valves can be constructed using standard optical
lithography processes followed by multilayer soft lithography (MSL)
methods (Unger et al., Science 2000, 288:113-16). For example, a
device with sieve valves, designed for the purpose of capturing
mRNA from single cells, has been constructed of three layers of the
silicone elastomer polydimethylsiloxane (PDMS) (General Electric)
bonded to a RCA cleaned #1.5 glass coverslip. The device was
fabricated as described in Fu et al., Nat Biotechnol 1999,
17:1109-11 with slight modifications (Studer et al., J. Appl. Phys.
2004, 95:393-98). Negative master molds were fabricated out of
photoresist by standard optical lithography and patterned with
20,000 dpi transparency masks (CAD/Art Services) drafted with
AutoCAD software (Autodesk). The flow layer masks (column portion
and channel portion) were sized to 101.5% of the control layer
masks to compensate for shrinking of features during the first
elastomer curing step. The flow master molds were fabricated out of
40 .mu.m AZ-100XT/13 .mu.m SU8-2015 photoresists
(Clariant/Microchem) and the control molds were cast from 24 .mu.m
SU8-2025 (Microchem).
[0053] In order to implement sieve valves, the flow channel portion
where columns are to be constructed has a rectangular profile in
cross section. Therefore, in one embodiment, a multistep
lithography process is used for microfluidic devices composed of
both sieve valves and conventional valves (Unger et al., 2000,
Science 288:113-116). In one approach, for example, the column
resist is spun onto a silicon wafer and processed, followed by
processing the resist for the conventional fluid channels. The
fabrication of molds having a rounded flow structure is achieved by
thermal re-flow of the patterned photoresist. Negative
photo-resists such as SU8 rely on thermal polymerization of
UV-exposed regions, and therefore can not be reflowed. In order to
be compatible with membrane valves, flow channel sections are
defined using a positive photoresist such as AZ-50 (Clariant Corp.
Charlotte, N.C.).
[0054] Once the fluid channels are processed, the two layer mold is
heated (e.g., baked on a hot plate of 200 degrees C. for 2 hours)
so that the photoresist can reflow and form a rounded shape, which
is important for complete valve closure (see Unger, supra). A hard
bake step is also implemented between resist steps, in order to
make the column resist mechanically robust for downstream
processing. Most devices that have sieve valves also have
conventional valves, and have both rounded and non-rounded (e.g.,
rectangular) flow channels.
[0055] A large variety of elastomeric materials may be used in
fabrication of the devices of the invention. Elastomers in general
are polymers existing at a temperature between their glass
transition temperature and liquefaction temperature. See Allcock et
al., Contemporary Polymer Chemistry, 2nd Ed. For illustration, a
brief description of the most common classes of elastomers is
presented here:
[0056] Silicones: Silicone polymers have great structural variety,
and a large number of commercially available formulations. In an
exemplary aspect of the present invention, the present systems are
fabricated from an elastomeric polymer such as GE RTV 615
(formulation), a vinyl-silane crosslinked (type) silicone elastomer
(family). The vinyl-to-(Si--H) crosslinking of RTV 615 allows both
heterogeneous multilayer soft lithography and photoresist
encapsulation. However, this is only one of several crosslinking
methods used in silicone polymer chemistry and suitable for use in
the present invention. In one embodiment, the silicone polymer is
polydimethylsiloxane (PDMS).
[0057] Perfluoropolyethers: Functionalized photocurable
perfluoropolyether (PFPE) is particularly useful as a material for
fabricating solvent-resistant microfluidic devices for use with
certain organic solvents. These PFPEs have material properties and
fabrication capabilities similar to PDMS but with compatibility
with a broader range of solvents. See, e.g., PCT Patent
Publications WO 2005030822 and WO 2005084191 and Rolland et al.,
2004, "Solvent-resistant photocurable "liquid Teflon" for
microfluidic device fabrication" J. Amer. Chem. Soc.
126:2322-2323.
[0058] Other suitable materials include polyisoprenes,
polybutadienes, polychloroprenes, polyisobutylenes,
poly(styrene-butadiene-styrene)s, polyurethanes,
poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F),
poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene)
(nitrile rubber), poly(1-butene),
poly(chlorotrifluoroethylene-vinylidene fluoride) copolymers
(Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride),
poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton),
elastomeric compositions of polyvinylchloride (PVC), polysulfone,
polycarbonate, polymethylmethacrylate (PMMA), and
polytertrafluoroethylene (Teflon).
[0059] In an alternative embodiment, microfluidic devices can be
fabricated in a variety of nonelastomeric materials including
silicon, glass, metal, ceramic and nonelastomeric polymers in which
an elastomeric segment is deflected into a nonelastomeric channel.
Composite structures are described in, for example, US
20020127736.
[0060] Additional guidance can be found in the scientific and
patent literature including, but not limited to the following:
Unger et al., 2000, Science 288:113-116; Quake & Scherer, 2000,
"From micro to nanofabrication with soft materials" Science 290:
1536-40; Xia et al., 1998, "Soft lithography" Angewandte
Chemie-International Edition 37:551-575; Unger et al., 2000,
"Monolithic microfabricated valves and pumps by multilayer soft
lithography" Science 288:113-116; Thorsen et al., 2002,
"Microfluidic large-scale integration" Science 298:580-584; Chou et
al., 2000, "Microfabricated Rotary Pump" Biomedical Microdevices
3:323-330; Liu et al., 2003, "Solving the "world-to-chip" interface
problem with a microfluidic matrix "Analytical Chemistry 75,
4718-23," Hong et al, 2004, "A nanoliter-scale nucleic acid
processor with parallel architecture" Nature Biotechnology
22:435-39; Fiorini and Chiu, 2005, "Disposable microfluidic
devices: fabrication, function, and application" Biotechniques
38:429-46; Beebe et al., 2000, "Microfluidic tectonics: a
comprehensive construction platform for microfluidic systems."
Proc. Natl. Acad. Sci. USA 97:13488-13493; Rolland et al., 2004,
"Solvent-resistant photocurable "liquid Teflon" for microfluidic
device fabrication" J. Amer. Chem. Soc. 126:2322-2323; Rossier et
al., 2002, "Plasma etched polymer microelectrochemical systems" Lab
Chip 2:145-150; Becker et al., 2002, "Polymer microfluidic devices"
Talanta 56:267-287; Becker et al., 2000, "Polymer microfabrication
methods for microfluidic analytical applications" Electrophoresis
21:12-26; Terry et al., 1979, A Gas Chromatography Air Analyzer
Fabricated on a Silicon Wafer, IEEE Trans. on Electron Devices, v.
ED-26, pp. 1880-1886; Berg et al., 1994, Micro Total Analysis
Systems, New York, Kluwer; Webster et al., 1996, Monolithic
Capillary Gel Electrophoresis Stage with On-Chip Detector in
International Conference On Micro Electromechanical Systems, MEMS
96, pp. 491-496; and Mastrangelo et al., 1989, Vacuum-Sealed
Silicon Micromachined Incandescent Light Source, in Intl. Electron
Devices Meeting, IDEM 89, pp. 503-506; U.S. Pat. Nos. U.S. Pat. No.
6,960,437 (Nucleic acid amplification utilizing microfluidic
devices); U.S. Pat. No. 6,899,137 (Microfabricated elastomeric
valve and pump systems); U.S. Pat. No. 6,767,706 B2 (Integrated
active flux microfluidic devices and methods); U.S. Pat. No.
6,752,922 (Microfluidic chromatography); U.S. Pat. No. 6,408,878
(Microfabricated elastomeric valve and pump systems); U.S. Patent
Application publication Nos. 20050072946; 20050000900; 20020127736;
20020109114; 20040115838; 20030138829; 20020164816; 20020127736;
20040229349; 20040224380; 20040072278 and 20020109114; and PCT
patent publications WO 2005/084191; WO05030822A2; and WO
01/01025.
EXAMPLE
Protocols for Making Device With Microfluidic Sieve Valve
[0061] Making Su8-2010 10 .mu.m/Spr220-7 15 .mu.m/AZ-50 40 .mu.m
flow molds &*& [0062] 1. Spin Su8-2025 at 3000 rpm for 45
s. with an acceleration of 10. [0063] 2. Soft bake mold for 1
min./3 min. at 65.degree. C./95.degree. C. [0064] 3. Expose mold 50
s. real time on MJB mask aligner (7 mW/cm2). [0065] 4. Bake mold
post-exposure for 1 min./3 min. at 65.degree. C./95.degree. C.
[0066] 5. Develop in Su8 nano developer. Rinse in fresh Su8 nano
developer and determine if developed by looking at mold under
microscope. [0067] 6. Once developed, hard bake mold at 150.degree.
C. for 2 hr. [0068] 7. Expose mold to HMDS vapor for 90 s. [0069]
8. Spin Spr220-7 (cold, straight from refrigerator) at 1500 rpm for
1 min. with an acceleration of 15. [0070] 9. Soft bake mold for 90
s. at 105.degree. C. [0071] 10. Expose mold under a 20,000 dpi
positive transparency mask (CAD/Art Services) for 3.2 min. real
time on MJB mask aligner (POWER). [0072] 11. Develop mold in MF-319
developer and rinse under a stream of H.sub.20. Determine if
developed by looking at mold under microscope. Spr develops rather
quickly, except for areas around the Su8 layer. Therefore, some
areas may get overdeveloped when trying to remove residual resist
around Su8 portions. [0073] 12. Hard bake 2 hr. at 200.degree. C.
[0074] 13. Expose mold to HMDS vapor for 90 s. [0075] 14. Spin
AZ-50 (cold, straight from refrigerator) at 1600 rpm for 1 min.
with an acceleration of 15. [0076] 15. Soft bake mold for 1 min./5
min./1 min. at 65.degree. C./115.degree. C./65.degree. C. [0077]
16. Expose mold under a 20,000 dpi positive transparency mask
(CAD/Art Services) for 4 min. real time on MJB mask aligner (7
mW/cm2). [0078] 17. Develop mold in 3:1 H.sub.20:2401 developer.
Rinse mold under a stream of H.sub.20. [0079] 18. Once developed
(determine by visualization under microscope), reflow/hard bake 3
hr. at 200.degree. C. Note: Temperatures for hard bakes are ramped
up and down from room temperature by either turning on or off the
hot plate. This will prevent resist cracking.
[0080] Making Su8-2025 23 .mu.m control molds [0081] 1. Spin
Su8-2025 @ 3000 rpm for 45 s. with an acceleration of 10. [0082] 2.
Soft bake mold for 2 min./5 min. at 65.degree. C./95.degree. C.
[0083] 3. Expose mold under a 20,000 dpi negative transparency mask
(CAD/Art Services) 1.2 min. real time on MJB mask aligner (7
mW/cm2). [0084] 4. Bake mold post-exposure for 2 min./5 min. at
65.degree. C./95.degree. C. [0085] 5. Develop in Su8 nano
developer. Rinse in fresh Su8 nano developer and determine if
developed by looking at mold under microscope. [0086] 6. Once
developed, bake mold at 95.degree. C. for 45 s to evaporate excess
solvent.
[0087] Fabrication of 3-layer RTV device with push-up valves [0088]
1. Prepare 5:1 GE RTV A:RTV B (mix 1 min., de-foam 5 min.). [0089]
2. Expose flow mold to TMCS vapor for 2 min. [0090] 3. Pour 30 g
5:1 GE RTV A:RTV B on respective flow mold. [0091] 4. De-gas flow
mold under vacuum. [0092] 5. Bake flow mold 45 min. at 80.degree.
C. [0093] 6. While flow mold is de-gassing, prepare 20:1 GE RTV
A:RTV B (mix 1 min., de-foam 5 min.). [0094] 7. Expose control mold
to TMCS vapor for 2 min. [0095] 8. Spin 20:1 RTV mix at 2000 rpm
for 60 s. with a 15 s. ramp. [0096] 9. Let RTV settle on control
mold for 30 min. before baking 30 min. at 80 [0097] 10. Bake
control mold 30 min. at 80.degree. C. [0098] 11. Cut devices out of
flow mold and punch holes with 650 .mu.m diameter punch tool
(Technical Innovations #CR0350255N20R4). [0099] 12. Clean flow
device with transparent tape and align to control mold. [0100] 13.
Bake 2-layer device for 45 min. at 80.degree. C. [0101] 14. While
2-layer device is baking, prepare 20:1 GE RTV A:RTV B (mix 1 min.,
de-foam 5 min.) to spin on blank silicon wafer. [0102] 15. Expose
blank(s) to TMCS vapor for 2 min. [0103] 16. Spin 20:1 RTV mix on
blank wafer(s) at 1600 rpm for 60 s. with a 15 s. ramp. [0104] 17.
Bake blank wafer for 30 min. at 80.degree. C. [0105] 18. Cut out
2-layer device(s) from control mold(s), clean with tape and mount
on blank wafer(s). Check for debris and collapsed valves. Collapsed
valves can be fixed by applying pneumatic pressure with a syringe
to the respective control channel(s). This should overcome valves
sticking to channels. Once pressure is applied and released, peel
device back from blank wafer and re-mount. [0106] 19. Bake 3-layer
RTV device(s) for 6-18 hr. Less is best (devices can still handle
30 psi without delaminating). [0107] 20. If output holes need to be
punched, do so with technical innovation titanium nitride coated
punch (#CR0830655N14R4). [0108] 21. Cut 3-layer device(s) out,
clean with tape and mount on RCA cleaned glass slide(s). Check for
collapse as in (18). [0109] 22. Bake finished devices overnight at
80.degree. C.
[0110] Although the present invention has been described in detail
with reference to specific embodiments, those of skill in the art
will recognize that modifications and improvements are within the
scope and spirit of the invention, as set forth in the claims which
follow. All publications and patent documents (patents, published
patent applications, and unpublished patent applications) cited
herein are incorporated herein by reference as if each such
publication or document was specifically and individually indicated
to be incorporated herein by reference. Citation of publications
and patent documents is not intended as an admission that any such
document is pertinent prior art, nor does it constitute any
admission as to the contents or date of publication of the same.
The invention having now been described by way of written
description and example, those of skill in the art will recognize
that the invention can be practiced in a variety of embodiments and
that the foregoing description and examples are for purposes of
illustration and not limitation of the following claims.
* * * * *