U.S. patent application number 11/983405 was filed with the patent office on 2008-05-29 for microfluidic systems including three-dimensionally arrayed channel networks.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Janelle R. Anderson, Oksana Cherniavskaya, Daniel T. Chiu, Rebecca J. Jackman, J. Cooper McDonald, George M. Whitesides.
Application Number | 20080122140 11/983405 |
Document ID | / |
Family ID | 39462850 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080122140 |
Kind Code |
A1 |
Anderson; Janelle R. ; et
al. |
May 29, 2008 |
Microfluidic systems including three-dimensionally arrayed channel
networks
Abstract
The present invention provides, in certain embodiments, improved
microfluidic systems and methods for fabricating improved
microfluidic systems, which contain one or more levels of
microfluidic channels. The inventive methods can provide a
convenient route to topologically complex and improved microfluidic
systems. The microfluidic systems provided according to the
invention can include three-dimensionally arrayed networks of fluid
flow paths therein including channels that cross over or under
other channels of the network without physical intersection at the
points of cross over. The microfluidic networks of the invention
can be fabricated via replica molding processes, also provided by
the invention, utilizing mold masters including surfaces having
topological features formed by photolithography. The microfluidic
networks of the invention are, in some cases, comprised of a single
replica molded layer, and, in other cases, are comprised of two,
three, or more replica molded layers that have been assembled to
form the overall microfluidic network structure. The present
invention also describes various novel applications for using the
microfluidic network structures provided by the invention.
Inventors: |
Anderson; Janelle R.; (New
York, NY) ; Chiu; Daniel T.; (Seattle, WA) ;
Jackman; Rebecca J.; (Boston, MA) ; Cherniavskaya;
Oksana; (New York, NY) ; McDonald; J. Cooper;
(Somerville, MA) ; Whitesides; George M.; (Newton,
MA) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
39462850 |
Appl. No.: |
11/983405 |
Filed: |
November 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10303642 |
Nov 25, 2002 |
7323143 |
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11983405 |
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PCT/US01/16973 |
May 25, 2001 |
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10303642 |
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09578589 |
May 25, 2000 |
6645432 |
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PCT/US01/16973 |
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Current U.S.
Class: |
264/401 ;
264/225; 264/405 |
Current CPC
Class: |
B81B 2201/058 20130101;
B01L 2300/0874 20130101; B29C 35/0888 20130101; B01D 61/18
20130101; B29C 33/52 20130101; B29C 33/3892 20130101; B29C 39/34
20130101; B01D 71/70 20130101; B29C 33/3857 20130101; B01D 2323/24
20130101; B29C 39/021 20130101; Y10T 436/11 20150115; B81C 2201/019
20130101; B01D 67/003 20130101; B29C 2035/0827 20130101; B01D
67/0034 20130101; B01L 2300/0887 20130101; B01D 69/02 20130101;
Y10T 436/2575 20150115; B81C 1/00119 20130101; B01L 3/502707
20130101; B01D 2325/08 20130101; B01D 2323/34 20130101; B33Y 80/00
20141201; B01D 2325/028 20130101 |
Class at
Publication: |
264/401 ;
264/225; 264/405 |
International
Class: |
B29C 35/08 20060101
B29C035/08 |
Claims
1. A method for forming a microfluidic network structure
comprising: providing at least one mold substrate; forming at least
one topological feature on a surface of the mold substrate to form
a first mold master, where at least one of said at least one
topological feature is a two-level topological feature
characterized by a first portion having a first depth or height
with respect to a region of the surface adjacent to the feature and
a second portion, integrally connected to the first portion, having
a second depth or height with respect to the region of the surface
adjacent to the feature, which is greater than the first depth or
height; contacting the surface with a first hardenable liquid;
hardening the liquid thereby creating a first molded replica of the
surface; removing the first molded replica from the first mold
master; and assembling the first molded replica into a structure
comprising a microfluidic network having at least a one fluid flow
path comprising a series of interconnected channels within the
structure, the series of interconnected channels including at least
one first channel disposed within a first level of the structure,
at least one second channel disposed within a second level of the
structure, and at least one connecting channel fluidically
interconnecting the first channel and the second channel, where at
least one of which channels has a cross-sectional dimension not
exceeding about 500 .mu.m and where the structure includes at least
one channel disposed within the first level of the structure that
is non-parallel to at least one channel disposed within the second
level of the structure.
2. The method for forming a microfluidic network structure as in
claim 1, wherein the topological features comprise protrusions from
the surface of the first mold master, and wherein the first portion
of the at least one two-level topological feature has a first
height with respect to the region of the surface adjacent to the
two-level topological feature and the second portion has a second
height with respect to the region of the surface adjacent to the
feature, which is greater than the first height.
3. The method for forming a microfluidic network structure as in
claim 2, wherein the first molded replica formed by the hardening
step and removing step includes a first surface forming the first
level of the microfluidic network formed in the assembling step,
and wherein the at least one first channel disposed in the first
level is molded by the first portion of the at least one two-level
topological feature.
4. The method for forming a microfluidic network structure as in
claim 3, wherein the first molded replica formed by the hardening
step and removing step further includes the at least one connecting
channel of the microfluidic network formed in the assembling step,
and wherein the connecting channel is molded, at least in part, by
the second portion of the at least one two-level topological
feature.
5. The method for forming a microfluidic network structure as in
claim 2, wherein the contacting step comprises: creating a layer of
the first hardenable liquid on the surface of the first mold
master, the layer having a depth exceeding the first height but not
exceeding the second height.
6. The method for forming a microfluidic network structure as in
claim 5, wherein the creating step further comprises the step of:
bringing a surface of a second mold substrate into contact with a
surface of the second portion of the at least one two-level
topological feature in the surface of the first mold master.
7. The method for forming a microfluidic network structure as in
claim 6, wherein the surface of the second mold substrate is an
essentially planar, featureless surface.
8. The method for forming a microfluidic network structure as in
claim 6, wherein the second mold substrate comprises a second mold
master and wherein the surface of the second mold master includes
at least one topological feature formed thereon.
9. The method for forming a microfluidic network structure as in
claim 8, wherein at least one topological feature in the surface of
the second mold master is formed by a photolithography process.
10. The method for forming a microfluidic network structure as in
claim 8, wherein the surface of the second mold master comprises a
molded replica of another surface including at least one
topological feature thereon.
11. The method for forming a microfluidic network structure as in
claim 10, wherein the second mold master is formed from an
elastomeric material.
12. The method for forming a microfluidic network structure as in
claim 8, wherein the at least one topological feature in the
surface of the second mold master comprises a protrusion from the
surface.
13. The method for forming a microfluidic network structure as in
claim 12, wherein the first molded replica formed by the hardening
step and removing step includes a second surface forming the second
level of the microfluidic network formed in the assembling step,
and wherein the at least one second channel disposed in the second
level is molded by the at least one topological feature in the
surface of the second mold master.
14. The method for forming a microfluidic network structure as in
claim 12, wherein the surface of the second mold master includes at
least one two-level topological feature thereon, which two-level
topological feature is characterized by a first portion having a
first height with respect to a region of the surface adjacent to
the feature and a second portion, integrally connected to the first
portion, having a second height with respect to the region of the
surface adjacent to the feature, which is greater than the first
height.
15. The method for forming a microfluidic network structure as in
claim 14, wherein at least a portion of the second portion of the
two-level topological feature of the second mold master is shaped
and positioned to mate with at least a portion of the second
portion of a two-level topological feature of the first mold master
when the mold masters are brought together in the bringing
step.
16. The method for forming a microfluidic network structure as in
claim 15, wherein at least a portion of the second portion of the
two-level topological feature of the second mold master is shaped
and positioned to interdigitate with at least a portion of the
second portion of a two-level topological feature of the first mold
master when the mold masters are brought together in the bringing
step.
17. The method for forming a microfluidic network structure as in
claim 15, wherein the first molded replica formed during the
hardening step and removing step further includes the at least one
connecting channel of the microfluidic network formed in the
assembling step, and wherein the connecting channel is molded, at
least in part, by the second portion of the at least one two-level
topological feature of the second mold master.
18. The method for forming a microfluidic network structure as in
claim 8, wherein at least one topological feature on the surface of
the first mold master comprises a first alignment element and
wherein at least one topological feature on the second mold master
comprises a second alignment element, the second alignment element
shaped to be matable with the first alignment element.
19. The method for forming a microfluidic network structure as in
claim 18, wherein both of the first and second alignment elements
comprise topological features that do not mold, during the
contacting and hardening steps, channels in fluid communication
with the at least one fluid flow path in the microfluidic network
structure.
20. The method for forming a microfluidic network structure as in
claim 18, wherein the first and second alignment elements comprise
topological features that mate together during the bringing step,
and wherein at least a portion of at least one connecting channel
of the microfluidic network structure is molded, at least in part,
from at least a portion of the mated topological features.
21. The method for forming a microfluidic network structure as in
claim 8, wherein the contacting step comprises: bringing the
surface of the first mold master into at least partial contact with
the surface of the second mold master; aligning the at least one
topological feature of the first mold master and the at least one
topological feature of the second mold master with respect to each
other to yield a desired alignment of features; applying the first
hardenable liquid in contact with a periphery of the interface
between the first and second mold masters; and allowing the first
hardenable liquid to flow into interstices between the first and
the second mold masters by capillary action.
22. The method for forming a microfluidic network structure as in
claim 8, wherein the contacting step comprises: forming a layer of
the first hardenable liquid on the surface of the first mold
master; bringing the surface of the second mold master into at
least partial contact with the surface of the first mold master;
and aligning the at least one topological feature of the first mold
master and the at least one topological feature of the second mold
master with respect to each other to yield a desired alignment of
features.
23. The method for forming a microfluidic network structure as in
claim 21 or 22, further comprising: interdigitating at least a
portion of the at least one topological feature of the first mold
master and at least a portion of the at least one topological
feature of the second mold master.
24. The method for forming a microfluidic network structure as in
claim 8, wherein the removing step comprises: applying a force to
at least one of the first and the second mold masters tending to
separate the masters from each other; removing the first molded
replica from the surface of the first mold master while leaving the
first molded replica in contact with and supported by the surface
of the second mold master; and removing the second mold master from
the first molded replica.
25. The method for forming a microfluidic network structure as in
claim 24, further comprising after the step of removing the first
molded replica from the surface of the first mold master while
leaving the first molded replica in contact with and supported by
the surface of the second mold master, and before the step of
removing the second mold master from the first molded replica, the
step of: contacting the first molded replica with a support
surface.
26. The method for forming a microfluidic network structure as in
claim 1, wherein the first hardenable liquid comprises a liquid
able to solidify to form a solid polymeric material.
27. The method for forming a microfluidic network structure as in
claim 26, wherein the first hardenable liquid comprises a curable
prepolymer of an elastomeric polymer.
28. The method for forming a microfluidic network structure as in
claim 27, wherein the first hardenable liquid comprises a curable
prepolymer of poly(dimethylsiloxane).
29. The method for forming a microfluidic network structure as in
claim 26, wherein the hardening step comprises applying heat to the
first hardenable liquid.
30. The method for forming a microfluidic network structure as in
claim 26, wherein the hardening step comprises applying ultraviolet
radiation to the first hardenable liquid.
31. The method for forming a microfluidic network structure as in
claim 1, wherein the assembling step comprises: providing a first
support substrate having at least one oxidizable surface; oxidizing
the oxidizable surface of the first support substrate and the first
molded replica; bringing the surface of the first support substrate
into conformal contact with at least a portion of a first surface
of the first molded replica; and sealing the first molded replica
to the first support substrate via chemical reaction between the
surfaces.
32. The method for forming a microfluidic network structure as in
claim 31, wherein the oxidizable surface of the first support
substrate is essentially planar having essentially no features
disposed thereon.
33. The method for forming a microfluidic network structure as in
claim 32, wherein the first support substrate is formed of a
different material than the material forming the first molded
replica.
34. The method for forming a microfluidic network structure as in
claim 32, wherein the first support substrate is formed of a
material that is the same as that forming the first molded
replica.
35. The method for forming a microfluidic network structure as in
claim 31, wherein the first support substrate comprises a second
molded replica.
36. The method for forming a microfluidic network structure as in
claim 35, further comprising before the oxidizing step the steps
of: bringing at least a portion of the first surface of the first
molded replica into contact with at least a portion of a surface of
the second molded replica; aligning molded features of the first
molded replica with molded features of the second molded replica to
yield a desired alignment of features; and separating the surfaces
of the first molded replica and the second molded replica from each
other without disrupting the desired alignment of features.
37. The method for forming a microfluidic network structure as in
claim 35, further comprising after the oxidizing step and before
the bringing step the steps of: placing a liquid that is
essentially non-reactive with the surfaces oxidized in the
oxidizing step in contact with at least one of the surfaces
oxidized in the oxidizing step; disposing the first surface of the
first molded replica and a surface of the second molded replica
adjacent to each other such that they are separated from each other
by a continuous layer of the liquid that is essentially
non-reactive with the surfaces oxidized in the oxidizing step;
aligning molded features of the first molded replica with molded
features of the second molded replica to yield a desired alignment
of features; and removing the liquid that is essentially
non-reactive with the surfaces oxidized in the oxidizing step from
between the surfaces.
38. The method for forming a microfluidic network structure as in
claim 37, wherein the step comprising removing the liquid that is
essentially non-reactive with the surfaces oxidized in the
oxidizing step from between the surfaces and the bringing step
comprise a single step.
39. The method for forming a microfluidic network structure as in
claim 37, wherein the liquid that is essentially non-reactive with
the surfaces oxidized in the oxidizing step is removed from between
the surfaces by evaporation.
40. The method for forming a microfluidic network structure as in
claim 31, wherein the assembling step further comprises: providing
a second support substrate having at least one oxidizable surface;
oxidizing the oxidizable surface of the second support substrate
and the first molded replica; bringing the surface of the second
support substrate into conformal contact with at least a portion of
a second surface of the first molded replica; and sealing the first
molded replica to the second support substrate via chemical
reaction between the surfaces.
41. The method for forming a microfluidic network structure as in
claim 40, wherein the oxidizable surface of the second support
substrate is essentially planar having essentially no features
disposed thereon.
42. The method for forming a microfluidic network structure as in
claim 41, wherein the second support substrate is formed of a
different material than the material forming the first molded
replica.
43. The method for forming a microfluidic network structure as in
claim 41, wherein the second support substrate is formed of a
material that is the same as that forming the first molded
replica.
44. The method for forming a microfluidic network structure as in
claim 40, wherein the second support substrate comprises a second
molded replica.
45. The method for forming a microfluidic network structure as in
claim 1, further comprising after the assembling step the steps of:
at least partially filling the at least one fluid flow path of the
microfluidic network with a second hardenable liquid; solidifying
the second hardenable liquid into a molded article having a
structure conforming to the flow path of the microfluidic network;
and removing the microfluidic network structure surrounding the
molded article.
46. A method for forming a molded structure comprising: providing
at least one mold substrate; forming at least one two-level
topological feature having at least one cross-sectional dimension
not exceeding about 500 .mu.m on a surface of the substrate to form
a mold master, which two-level topological feature is characterized
by a first portion having a first depth or height with respect to a
region of the surface adjacent to the feature and a second portion,
integrally connected to the first portion, having a second depth or
height with respect to the region of the surface adjacent to the
feature, which is greater than the first depth or height;
contacting the surface with a hardenable liquid; hardening the
liquid thereby creating a molded replica of the surface; and
removing the molded replica from the mold master.
47. The method for forming a molded structure as in claim 46,
wherein the molded replica formed by the hardening step and
removing step includes a first surface with at least one channel
disposed therein that is molded by the first portion of the at
least one two-level topological feature and further includes at
least one connecting channel fluidically interconnected to and
oriented essentially perpendicularly to the channel disposed in the
first surface of the molded replica, which connecting channel is
molded by the second portion of the two-level topological
feature.
48. The method for forming a molded structure as in claim 46,
wherein the mold substrate comprises a silicon wafer.
49. The method for forming a molded structure as in claim 48,
wherein at least one surface of the silicon wafer is coated with at
least a first layer of photoresist having a surface forming a
surface of the substrate on which the at least one topological
feature is formed in the forming step.
50. The method for forming a molded structure as in claim 49,
wherein the photoresist comprises a positive photoresist.
51. The method for forming a molded structure as in claim 49,
wherein the photoresist comprises a negative photoresist.
52. The method for forming a molded structure as in claim 49,
wherein the forming step comprises: providing a first photo mask
defining a first pattern; exposing the surface of the first layer
of photoresist to radiation through the first photo mask; coating
the surface of the first layer of photoresist with a second layer
of photoresist; providing a second photo mask defining a second
pattern; and exposing a surface of the second layer of photoresist
to radiation through the second photo mask.
53. The method for forming a molded structure as in claim 52,
wherein the first and second photomasks comprise printed
transparencies.
54. The method for forming a molded structure as in claim 53,
wherein the first and second patterns are designed by a computer
assisted design program and are printed onto the transparencies
with a high resolution printer.
55. The method for forming a molded structure as in claim 52,
further comprising after each of the exposing steps, the step of:
developing the photoresist layer with a developing agent that
selectively removes photoresist material based on whether the
photoresist material has been exposed to radiation through the
photomask to yield a positive relief pattern in photoresist with
topological features corresponding to the pattern of the photo
mask.
56. The method for forming a molded structure as in claim 52,
further comprising after the second exposing step, the step of:
developing the first and second photoresist layers with a
developing agent that selectively removes photoresist material
based on whether the photoresist material has been exposed to
radiation through either of the first or second photomasks to yield
a positive relief pattern in photoresist with topological features
corresponding to the first and second patterns of the first and
second photo masks.
57. The method for forming a molded structure as in claim 52,
further comprising after the step for providing the second photo
mask and before the second exposing step, the step of: aligning the
second photo mask so that the second pattern has a desired
orientation and position with respect to a prior orientation and
position of the first pattern of the first photo mask.
58. The method for forming a molded structure as in claim 57,
wherein features of the first pattern of the first photo mask
correspond to first portions of the at least one two-level
topological feature and wherein features of the second pattern of
the second photo mask correspond to second portions of the at least
one two-level topological feature.
59. A method for forming a molded structure comprising: providing a
first mold master having a surface formed of an elastomeric
material and including at least one topological feature with at
least one cross-sectional dimension not exceeding about 500 .mu.m
thereon; providing a second mold master having a surface including
at least one topological feature with at least one cross-sectional
dimension not exceeding about 500 .mu.m thereon; placing a
hardenable liquid in contact with the surface of at least one of
the first and second mold master; bringing the surface of the first
mold master into at least partial contact with the surface of the
second mold master; hardening the liquid thereby creating a molded
replica of the surface of the first mold master and the surface of
the second mold master; and removing the molded replica from at
least one of the mold masters.
60. A method for forming a molded structure comprising: providing a
first mold master having a surface including at least a first
topological feature with at least one cross-sectional dimension not
exceeding about 500 .mu.m thereon and at least a second topological
feature comprising a first alignment element; providing a second
mold master having a surface including at least a first topological
feature with at least one cross-sectional dimension not exceeding
about 500 .mu.m thereon and at least a second topological feature
comprising a second alignment element having a shape that is
matable to the shape of the first alignment element; placing a
hardenable liquid in contact with the surface of at least one of
the first and second mold master; bringing the surface of the first
mold master into at least partial contact with the surface of the
second mold master; aligning the first topological features of the
first and second mold masters with respect to each other by
adjusting a position of the first mold master with respect to a
position of the second mold master until the first alignment
element matingly engages the second alignment element; hardening
the liquid thereby creating a molded replica of the surface of the
first mold master and the surface of the second mold master; and
removing the molded replica from at least one of the mold
masters.
61. The method for forming a microfluidic network structure as in
claim 60, wherein both of the first and second alignment elements
comprise topological features that do not mold, during the
hardening step, any features of the final molded structure.
62. The method for forming a microfluidic network structure as in
claim 60, wherein the first and second alignment elements comprise
topological features that together mold, during the hardening step,
at least a portion of at least one feature of the final molded
structure.
63. The method for forming a molded structure as in claim 60,
wherein at least one of the first mold master and the second mold
master is formed of an elastomeric material.
64. A method for molding an article comprising: providing a first
mold master having a surface with a first set of surface
properties; providing a second mold master having a surface with a
second set of surface properties, wherein the surface of at least
the first mold master is formed of an elastomeric material, and
wherein at least one of the first and second mold master has a
surface including at least one topological feature with at least
one cross-sectional dimension not exceeding about 500 .mu.m
thereon; placing a hardenable liquid in contact with the surface of
at least one of the first and second mold master; bringing the
surface of the first mold master into at least partial contact with
the surface of the second mold master; hardening the liquid thereby
creating a molded replica of the surface of the first mold master
and the surface of the second mold master; separating the masters
from each other; and removing the molded replica from the surface
of the first mold master while leaving the molded replica in
contact with and supported by the surface of the second mold
master.
65. The method for molding an article as in claim 64, wherein the
separating step comprises applying a peeling force to at least one
of the first and second mold masters.
66. The method for molding an article as in claim 64, wherein the
surface of at least one of the first and second mold masters has
been silanized.
67. The method for molding an article as in claim 64, wherein the
elastomeric material comprises a silicone polymer.
68. The method for molding an article as in claim 67, wherein the
silicone polymer comprises poly(dimethylsiloxane).
69. The method for molding an article as in claim 68, wherein the
molded replica is formed of poly(dimethylsiloxane).
70. The method for molding an article as in claim 71, wherein the
surface of the second mold master is formed of a material other
than poly(dimethylsiloxane).
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/303,642, filed Nov. 25, 2002 which is a continuation of PCT
International Application No. PCT/US01/16973 filed May 25, 2001,
which is a continuation-in-part of U.S. application Ser. No.
09/578,589, filed May 25, 2000. Each application is hereby
incorporated by reference in its entirety.
FIELD OF INVENTION
[0002] The present invention involves microfluidic network
structures, methods for fabricating microfluidic network
structures, and methods for using such structures.
BACKGROUND OF THE INVENTION
[0003] The need for complexity in microfluidic systems is
increasing rapidly as sophisticated functions--chemical reactions
and analyses, bioassays, high-throughput screens, and sensors--are
being integrated into single microfluidic devices. Complex systems
of channels require more complex connectivity than can be generated
in conventional two-dimensional microfluidic systems having a
single level of channels, since such typical single-level designs
do not allow two channels to cross without fluidically connecting.
Most methods for fabricating microfluidic channels are based on
photolithographic procedures, and yield such two-dimensional
systems. There are a number of more specialized procedures, such as
stereolithography (see for example, K. Ikuta, K. Hirowatari, T.
Ogata, Proc. IEEE MEMS '94, Oiso, Japan, Jan. 25-28, 1994, pp.
1-6), laser-chemical three-dimensional writing (see for example, T.
M. Bloomstein, D. J. Ehrlich, J. Vac. Sci. Technol. B, Vol. 10, pp.
2671-2674, 1992), and modular assembly (see for example, C.
Gonzalez, R. L. Smith, D. G. Howitt, S. D. Collins, Sens. Actuators
A, Vol. 66, pp. 315-332, 1998), that yield three-dimensional
structures, but these methods are typically time consuming,
difficult to perform, and expensive, and are thus not well suited
for either prototyping or manufacturing, and are also not capable
of making certain types of structures. Better methods for
generating complex three-dimensional microfluidic systems are
needed to accelerate the development of microfluidic technology.
The present invention, in some embodiments, provides such improved
methods for generating complex three-dimensional microfluidic
systems.
[0004] It is known to use a stamp or mold to transfer patterns to a
surface of a substrate (see for example, R. S. Kane, S. Takayama,
E. Ostuni, D. E. Ingber, G. M. Whitesides, Biomaterials, Vol. 20,
pp. 2363-2376, 1999; and Y. Xia, G. M. Whitesides, Angew. Chem.
Int. Ed. Engl., Vol. 37, pp. 551-575, 1998; U.S. Pat. No.
5,512,131; International Pat. Publication No. WO 97/33737,
published Sep. 18, 1997). Most conventional soft lithographic
techniques, for example, microcontact printing (.mu.CP) (see for
example, C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, D. E.
Ingber, Science, Vol. 276, pp. 1425-1428, 1997; A. Bernard, E.
Delamarche, H. Schmid, B. Michel, H. R. Bosshard, H. Biebuyck,
Langmuir, Vol. 14, pp. 2225-2229, 1998) and micromolding in
capillaries (MIMIC) (see for example, N. L. Jeon, I. S. Choi, B.
Xu, G. M. Whitesides, Adv. Mat., Vol. 11, pp. 946-949, 1999; E.
Delamarche, A. Bernard, H. Schmid, B Michel, H. Biebuyck, Science,
Vol. 276, pp. 779-781, 1997; E. Delamarche, A. Bernard, H. Schmid,
A. Bietsch, B. Michel, h. Biebuyck, J. Am. Chem. Soc., Vol. 120,
pp. 500-508, 1998; A. Folch, A. Ayon, O. Hurtado, M. A. Schmidt, M.
Toner, J. Biomech. Eng., Vol. 121, pp. 28-34, 1999; A. Folch, M.
Toner, Biotech. Prog., Vol. 14, pp. 388-392, 1998), have been
limited to procedures that pattern one substance at a time, or to
relatively simple, continuous patterns. These constraints are both
topological and practical. The surface of a stamp in .mu.CP, or of
a channel system in MIMIC, is effectively a two-dimensional
structure. In .mu.CP, this two-dimensionality of the stamp limits
the types of patterns that can be transferred to those comprising a
single "color" of ink in the absence of a way of selectively
"inking" different regions of the stamp with different materials.
Patterning of multiple "inks" using conventional methods requires
multiple steps of registration and stamping. In MIMIC, the
two-dimensional channel system limits patterning to relatively
simple, continuous structures or requires multiple patterning
steps.
[0005] There remains a general need in the art for improved methods
for forming patterns on surfaces with soft lithographic techniques,
and for providing techniques able to pattern onto a surface
arbitrary two-dimensional patterns and able to form complex
patterns comprised of multiple regions, where different regions of
the pattern can comprise different materials, on a surface without
the need for multiple steps of registration or stamping and without
the need to selectively "ink" different regions of the stamp with
different materials. The present invention, in some embodiments,
provides such improved methods for forming patterns on surfaces
with soft lithographic techniques.
SUMMARY OF THE INVENTION
[0006] The present invention involves, in certain embodiments,
improved microfluidic systems and procedures for fabricating
improved microfluidic systems, which contain one or more levels of
microfluidic channels. The inventive methods can provide a
convenient route to topologically complex and improved microfluidic
systems. The present invention also, in some embodiments, involves
microfluidic systems and methods for fabricating complex patterns
of materials, such as biological materials and cells, on surfaces.
In such embodiments, the invention involves microfluidic surface
patterning systems and methods for fabricating complex,
discontinuous patterns on surfaces that can incorporate or deposit
multiple materials onto a surface. The present invention, in some
embodiments, can provide improved stamps for microcontact surface
patterning able to pattern onto a surface arbitrary two-dimensional
patterns and able to pattern multiple substances onto a surface
without the need for multiple steps of registration or stamping
during patterning and without the need to selectively "ink"
different regions of the stamp with different materials.
[0007] According to one embodiment of the invention, a microfluidic
network is disclosed. The microfluidic network comprises a
polymeric structure including therein at least a first and a second
non-fluidically interconnected fluid flow paths. At least the first
flow path comprises a series of interconnected channels within the
polymeric structure. The series of interconnected channels includes
at least one first channel disposed within a first level of the
structure, at least one second channel disposed within a second
level of the structure, and at least one connecting channel
fluidically interconnecting the first channel and the second
channel. At least one channel within the structure has a
cross-sectional dimension not exceeding about 500 .mu.m. The
structure includes at least one channel disposed within the first
level of the structure that is non-parallel to at least one channel
disposed within the second level of the structure.
[0008] In another embodiment of the invention, a microfluidic
network is disclosed. The microfluidic network comprises an
elastomeric structure including therein at least one fluid flow
path. The flow path comprises a series of interconnected channels
within the structure. The series of interconnected channels
includes at least one first channel disposed within a first level
of the structure, at least one second channel disposed within a
second level of the structure, and at least one connecting channel
fluidically interconnecting the first channel and the second
channel. At least one channel within the structure has a
cross-sectional dimension not exceeding about 500 .mu.m, and the
structure includes at least one channel disposed within the first
level of the structure that is non-parallel to at least one channel
disposed within the second level of the structure.
[0009] In yet another embodiment, a polymeric membrane is
disclosed. The polymeric membrane comprises a first surface
including at least one channel disposed therein, a second surface
including at least one channel disposed therein, and a polymeric
region intermediate the first surface and the second surface. The
intermediate region includes at least one connecting channel
therethrough fluidically interconnecting the channel disposed in
the first surface and the channel disposed in the second surface of
the membrane. At least one channel has a cross-sectional dimension
not exceeding about 500 .mu.m.
[0010] In another embodiment of the invention, a method for forming
a microfluidic network structure is disclosed. The method comprises
providing at least one mold substrate, forming at least one
topological feature on a surface of the mold substrate to form a
first mold master, contacting the surface with a first hardenable
liquid, hardening the liquid thereby creating a first molded
replica of the surface, removing the first molded replica from the
first mold master, and assembling the first molded replica into a
structure comprising a microfluidic network. The assembled
microfluidic network structure has at least one fluid flow path
comprising a series of interconnected channels within the
structure. The series of interconnected channels includes at least
one first channel disposed within a first level of the structure,
at least one second channel disposed within a second level of the
structure, and at least one connecting channel fluidically
interconnecting the first channel and the second channel. At least
one of the channels within the structure has a cross-sectional
dimension not exceeding about 500 .mu.m. The structure includes at
least one channel disposed within the first level of the structure
that is non-parallel to at least one channel disposed within the
second level of the structure.
[0011] In yet another embodiment, a method for forming a molded
structure is disclosed. The method comprises providing at least one
mold substrate and forming at least one two-level topological
feature having at least one lateral dimension not exceeding 500
.mu.m on a surface of the substrate to form a mold master. The
two-level topological feature is characterized by a first portion
having a first depth or height with respect to a region of the
surface adjacent to the feature, and a second portion integrally
connected with the first portion having a second depth or height
with respect to the region of the surface adjacent to the feature
that is greater than the first depth or height. The method further
comprises contacting the surface with a hardenable liquid,
hardening the liquid thereby creating a molded replica of the
surface, and removing the molded replica from the mold master.
[0012] In another embodiment of the invention, a method for forming
topological features on a surface of a material is disclosed. The
method comprises exposing portions of a surface of a first layer of
photoresist to radiation in a first pattern, coating the surface of
the first layer of photoresist with a second layer of photoresist,
exposing portions of a surface of the second layer of photoresist
to radiation in a second pattern different from the first pattern,
and developing the first and second photoresist layers with a
developing agent. The developing step yields a positive relief
pattern in photoresist that includes at least one two-level
topological feature having at least one cross-sectional dimension
not exceeding 500 .mu.m. The two-level topological feature is
characterized by a first portion having a first height with respect
to the surface of the material and a second portion, integrally
connected to the first portion, having a second height with respect
to the surface of the material.
[0013] In yet another embodiment, a method for forming a molded
structure is disclosed. The method involves providing a first mold
master having a surface formed of an elastomeric material and
including at least one topological feature with at least one
cross-sectional dimension not exceeding about 500 .mu.m thereon.
The method further comprises providing a second mold master having
a surface including at least one topological feature with at least
one cross-sectional dimension not exceeding about 500 .mu.m
thereon. The method further comprises placing a hardenable liquid
in contact with the surface of at least one of the first and second
mold master, bringing the surface of the first mold master into at
least partial contact with the surface of the second mold master,
hardening the liquid thereby creating a molded replica of the
surface of the first mold master and the surface of the second mold
master, and removing the molded replica from at least one of the
mold masters.
[0014] In another embodiment of the invention, a method for forming
a molded structure is disclosed. The method involves providing a
first mold master having a surface including at least a first
topological feature with at least one cross-sectional dimension not
exceeding about 500 .mu.m thereon and at least a second topological
feature comprising a first alignment element. The method further
comprises providing a second mold master having a surface including
at least a first topological feature with at least one
cross-sectional dimension not exceeding about 500 .mu.m thereon and
at least a second topological feature comprising a second alignment
element having a shape that is mateable to the shape of the first
alignment element. The method further comprises placing a
hardenable liquid in contact with the surface of at least one of
the first and second mold master, bringing the surface of the first
mold master into at least partial contact with the surface of the
second mold master, aligning the first topological features of the
first and second mold masters with respect to each other by
adjusting a position of the first mold master with respect to a
position of the second mold master until the first alignment
element matingly engages and interdigitates with the second
alignment element, hardening the liquid thereby creating a molded
replica of the surface of the first mold master and the surface of
the second mold master, and removing the molded replica from at
least one of the mold masters.
[0015] In yet another embodiment of the invention, a method for
aligning and sealing together surfaces is disclosed. The method
comprises disposing two surfaces, at least one of which is
oxidized, adjacent to each other such that they are separated from
each other by a continuous layer of a liquid that is essentially
non-reactive with the surfaces, aligning the surfaces with respect
to each other, and removing the liquid from between the surfaces,
thereby sealing the surfaces together via a chemical reaction
between the surfaces.
[0016] In another embodiment of the invention, a method for molding
an article is disclosed. The method comprises providing a first
mold master having a surface with a first set of surface properties
and providing a second mold master having a surface with a second
set of surface properties. At least one of the first and second
mold masters has a surface including at least one topological
feature with at least one cross-sectional dimension not exceeding
about 500 .mu.m thereon. The method further comprises placing a
hardenable liquid in contact with the surface of at least one of
the first and second mold masters, bringing the surface of the
first mold master into at least partial contact with the surface of
the second mold master, hardening the liquid thereby creating a
molded replica of the surface of the first mold master and the
surface of the second mold master, separating the mold masters from
each other, and removing the molded replica from the surface of the
first mold master while leaving the molded replica in contact with
and supported by the surface of the second mold master.
[0017] In yet another embodiment, a microfluidic network is
disclosed. The microfluidic network comprises a polymeric structure
including therein at least a first and a second non-fluidically
interconnected fluid flow paths. The first flow path comprises at
least two non-colinear interconnected channels disposed within a
first plane, and the second flow path comprises at least one
channel disposed within a second plane that is non-parallel with
the first plane. At least one channel within the structure has a
cross-sectional dimension not exceeding about 500 .mu.m.
[0018] In another embodiment of the invention, a microfluidic
network is disclosed. The microfluidic network comprises a
polymeric structure including therein at least one fluid flow path.
The fluid flow path is formed of at least one channel and has a
longitudinal axis defined by the direction of bulk fluid flow
within the flow path. The longitudinal axis of the flow path is not
disposed within any single plane.
[0019] In another embodiment of the invention, a method of
patterning a material surface is disclosed. The method comprises
providing a stamp having a structure including at least one flow
path comprising a series of interconnected channels within the
structure. The series of interconnected channels includes at least
one first channel disposed within an interior region of the
structure, at least one second channel disposed within a stamping
surface of the structure defining a first pattern therein, and at
least one connecting channel fluidically interconnecting the first
channel and the second channel. The method further comprises
contacting the stamping surface with a portion of the material
surface, and, while maintaining the stamping surface in contact
with the portion of the material surface, at least partially
filling the flow path with a fluid so that at least a portion of
the fluid contacts the material surface.
[0020] In yet another embodiment, a method of patterning a material
surface is disclosed. The method comprises providing a stamp having
a structure including at least two non-fluidically interconnected
flow paths therein including a first fluid flow path defining a
first pattern of channels disposed within a stamping surface of the
structure and a second fluid flow path defining a second pattern of
channels disposed within the stamping surface of the structure.
Each of the first and second patterns of channels is
non-continuous, and the channels defining the first pattern are
non-intersecting with the channels defining the second pattern. The
method further comprises contacting the stamping surface with a
portion of the material surface, while maintaining the stamping
surface in contact with the portion of the material surface, at
least partially filling the first flow path with a first fluid so
that at least a portion of the first fluid contacts the material
surface and at least partially filling the second flow path with a
second fluid so that at least a portion of the second fluid
contacts the material surface, and removing the stamping surface to
provide a pattern on the material surface according to the first
pattern, which is formed by contact of the material surface with
the first fluid, and according to the second pattern, which is
formed by contact of the material surface with the second
fluid.
[0021] In another embodiment, a method of patterning a material
surface is disclosed. the method involves providing a stamp having
a structure including at least one non-linear fluid flow path
therein in fluid communication with a stamping surface of the
structure. The method further involves contacting the stamping
surface with a portion of the material surface and, while
maintaining the stamping surface in contact with the portion of the
material surface, at least partially filling the flow path with a
fluid so that at least a portion of the fluid contacts the material
surface.
[0022] Other advantages, novel features, and objects of the
invention will become apparent from the following detailed
description of the invention when considered in conjunction with
the accompanying drawings, which are schematic and which are not
intended to be drawn to scale. In the figures, each identical or
nearly identical component that is illustrated in various figures
is represented by a single numeral. For purposes of clarity, not
every component is labeled in every figure, nor is every component
of each embodiment of the invention shown where illustration is not
necessary to allow those of ordinary skill in the art to understand
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1a is a perspective view of a schematic illustration of
a microfluidic network structure having a series of interconnected
channels arranged in a "basketweave" configuration;
[0024] FIG. 1b is a two-dimensional projection of the microfluidic
network structure of FIG. 1a;
[0025] FIG. 2a is a perspective view of a schematic illustration of
a second embodiment of a microfluidic network structure;
[0026] FIG. 2b is a two-dimensional projection of the microfluidic
network structure of FIG. 2a;
[0027] FIG. 3a is a perspective view of a schematic illustration of
a third embodiment of a microfluidic network structure;
[0028] FIG. 3b is a two-dimensional projection of the microfluidic
network structure of FIG. 3a;
[0029] FIG. 4a is a perspective view of a schematic illustration of
a five-level microfluidic network comprising a centrally disposed
straight channel surrounded by a coiled fluid flow path;
[0030] FIG. 4b is a two-dimensional projection of the microfluidic
network structure of FIG. 4a;
[0031] FIGS. 5a-5c are schematic illustrations of one embodiment of
the fabrication method for forming a microfluidic network structure
according to one embodiment of the invention;
[0032] FIGS. 6a-6c are schematic illustrations of one embodiment of
a self-aligning method provided by the invention;
[0033] FIG. 6d is a schematic illustration of a replica molded
layer of a microfluidic network having a perimetric shape for use
in one embodiment of a self-aligning method according to the
invention;
[0034] FIG. 7 is a schematic illustration of a second embodiment of
a microfluidic network fabrication method according to the
invention;
[0035] FIG. 8 is a schematic illustration of a method for forming a
two-level topological feature on a surface of the substrate by
photolithography provided according to the invention;
[0036] FIGS. 9a-9b are schematic illustrations of a third
embodiment for forming a microfluidic network structure according
to the invention;
[0037] FIG. 9c is a series of schematic, cross-sectional
illustrations of a modification of the third embodiment for forming
the microfluidic network structure of FIGS. 9a-9b.
[0038] FIG. 10 is a schematic illustration of a method for forming
a five-level microfluidic network structure including a straight
channel surrounded by a coiled series of interconnected
channels;
[0039] FIG. 11 is a schematic illustration of a pattern on a
material surface formed with a microfluidic stamp provided
according to the invention;
[0040] FIG. 12a is a perspective view of a schematic illustration
of a lower and an upper mold master for forming a basketweave
microfluidic network structure provided by the invention;
[0041] FIGS. 12b-12c provide photocopies of photomicrographs of a
microfluidic network characterized by a network of channels
arranged in a basketweave configuration in accordance with one
embodiment of the present invention;
[0042] FIG. 12d is a photocopy of an SEM image of a micromolded
structure produced according to one embodiment of the
invention;
[0043] FIG. 13 is a photocopy of a photomicrograph of a
microfluidic network comprising a straight channel surrounded by a
coiled fluid flow path comprising a series of interconnected
channels, according to one embodiment of the invention;
[0044] FIG. 14a is a schematic illustration of a microfluidic
stamping process according to one embodiment of the invention;
[0045] FIG. 14b is a schematic illustration of the fluid flow path
layout of the microfluidic stamp illustrated in FIG. 14a;
[0046] FIG. 14c is a photocopy of a photomicrograph of a patterned
surface produced using the microfluidic stamp illustrated in FIG.
14a;
[0047] FIG. 15a is a schematic illustration of the layout of fluid
flow paths in one embodiment of a microfluidic stamp provided
according to the invention;
[0048] FIG. 15b is a photocopy of photomicrograph of a stamped
pattern on a material surface produced using a microfluidic stamp
having the microfluidic network structure illustrated in FIG.
15a;
[0049] FIG. 16a is a schematic illustration of the layout of fluid
flow paths in one embodiment of a microfluidic stamp provided
according to the invention;
[0050] FIGS. 16b-16d are photocopies of photomicrographs of
patterned cells on a material surface deposited using a
microfluidic stamp having the microfluidic network configuration
illustrated in FIG. 16a;
[0051] FIG. 17a is a schematic illustration of the layout of fluid
flow paths in one embodiment of a microfluidic stamp provided
according to the invention; and
[0052] FIGS. 17b-17e are photocopies of photomicrographs of
patterned cells on a material surface deposited using a
microfluidic stamp having the microfluidic network configuration
illustrated in FIG. 17a.
DETAILED DESCRIPTION
[0053] The present invention is directed to fabrication methods for
producing three-dimensional microfluidic network structures,
polymeric microfluidic network structures having a
three-dimensional array of channels included therein, and various
uses for the microfluidic networks, for example as a template for
forming and depositing complex patterns on substrates. A
"three-dimensional microfluidic network," "three-dimensional
microfluidic network structure," or "three-dimensional microfluidic
stamp" as used herein refers to a structure capable of containing a
fluid and/or providing fluid flow therethrough, which includes at
least three channels therein, and may contain many more;
furthermore, the structure includes at least three channels that
are arranged with respect to each other such that there exists no
plane, or curved planar surface, which contains disposed therein
the longitudinal axes of the three channels. The microfluidic
networks provided according to the invention, because of their
three-dimensionality of structure, are able, for example, to
provide channels within the structure having longitudinal axes
(defined as the axial centerline of the channel aligned parallel to
the direction of bulk fluid flow within the channel) aligned along
each of the x, y, and z directional components of space. The
ability to produce microfluidic structures having channels arranged
in a three-dimensional network enables the systems provided
according to the invention to include therein a plurality of
channels providing one or more independent fluid flow paths, where
the channels and flow paths can be arrayed in arbitrarily complex
geometric networks since the channels of the structures have the
capability of crossing over and/or under each other within the
structure.
[0054] One way to analogize the capabilities of the microfluidic
networks, and methods for producing the microfluidic networks,
according to the invention, is to compare the channel systems of
the microfluidic networks to a knot in three-dimensional space. The
microfluidic networks provided according to the invention have the
ability to fabricate the physical realization of knots, and thus
can include channel systems of arbitrary topological complexity. In
mathematical terms, a knot is a closed, non-intersecting, curved
line in three dimensions. Knots are typically described in
mathematics in terms by their projections onto a plane. For non
trivial knots, these projections contain "double points", which are
points where the projected curve crosses itself. A knot can always
be slightly perturbed in three dimensions so that, in projection,
it has no triple or higher order points: that is, points where the
projected curve crosses itself three or more times. Hence, knots
can be described completely by giving such a two-dimensional
projection, together with information about which piece of the
curve crosses over or under the another piece at each double
point.
[0055] The microfluidic networks provided according to the
invention, because of their three-dimensional channel network
structure, are able to provide a physical realization of the
above-mentioned double point. In other words, the structures enable
one channel, comprising a flow path or a segment of a flow path, to
cross over or under another channel providing another flow path, a
segment of another flow path, or providing another segment of the
same flow path. Thus, the inventive microfluidic networks can
provide a physical realization of essentially any topological knot
system. Likewise, the inventive networks can provide a physical
realization of essentially any arrangement of interlinked knots and
of arbitrarily complex three-dimensional networks of interconnected
channels whose projections onto a plane or surface, as explained in
more detail below, can contain any arbitrary number of crossings.
As shown and explained in more detail below, in order for the
inventive microfluidic networks to avoid intersection of channels
at their points of crossing in the planar projection, there
typically are provided at least three identifiable "levels" within
the structure: a "lower" level that contains a channel disposed
therein that crosses "under" an "upper" level that contains
disposed therein a channel that crosses "over" the channel
contained in the bottom level, and an intermediate level that
isolates the channels of the lower and upper levels and contains
connecting channels penetrating therethrough that fluidically
connect the channels in the lower level and the channels on the
upper level in order to form a fluid flow path comprised of a
series of interconnected channels. It should be understood that the
terms "lower" and "upper" in the present context are intended to
suggest only the relative positions of the various levels of the
structure and are not meant to imply any particular orientation of
the structure in space. For example the structure can be flipped,
rotated in space, etc. so that the "lower" level is positioned
above the "upper" level or the levels can be positioned side by
side, etc. In yet other embodiments involving flexible structures,
the structure can be twisted or bent thereby deforming planar
levels into curved surfaces in space such that the "upper" and
"lower" levels of the structure may be positioned differently with
respect to each other at different locations in the overall
structure. In order to produce microfluidic networks with
arbitrarily complex channel networks, no additional levels are
typically needed because triple, or higher order points in the
projection are not necessary to allow the channels within the
structure to cross over or under each other and thus cross each
other in space without physical intersection of the "crossing"
channels within the structure.
[0056] FIG. 1a illustrates one exemplary embodiment of an
essentially infinite number of microfluidic network structures that
can be produced according to the invention. Microfluidic network
structure 100 includes a series of interconnected channels
providing seven non-fluidically interconnected fluid flow paths.
The channels are arranged in a "basket weave" arrangement. Channel
system 100, as illustrated, includes three non-fluidically
interconnected fluid flow paths, 102, 104, and 106 arrayed within
planes parallel to the y-z coordinate plane, and four
non-fluidically interconnected flow paths 108, 110, 112, and 114
arrayed within planes parallel to the x-z coordinate plane. Each
fluid flow path of the structure comprises a series of
interconnected channels (e.g. fluid flow path 102 comprises
interconnected channels 113, 124, 126, 116, 118, 120, 128, 122 and
123 within structure 100).
[0057] Flow path 102, for example, includes two channels 116 and
122 disposed within the first, lower level of structure 100 and two
channels 120 and 124 disposed within the second, upper level of the
structure. Flow path 102 also includes a number of connecting
channels, e.g. 118, 126, and 128 traversing a third, intermediate
level of the structure and interconnecting channels contained in
the first, lower level and second, upper level of the structure.
The microfluidic network provided by structure 100 is truly
three-dimensional because it cannot be produced by a
two-dimensional structure comprising a series of interconnected
channels disposed within a single plane or any stack or array of
such structures. In other words, network 100 includes channels
disposed within the first, lower level of the structure that are
non-parallel to channels disposed within the second, upper level of
the structure (e.g. channel 116 of fluid flow path 102 and channel
130 of fluid flow path 110). Another way to describe the
three-dimensionality of network 100, and distinguish the network
from those realizable in two-dimensional system, is to point out,
that, for example, flow path 102 comprises a series of non-colinear
interconnected channels disposed within a first plane of the
structure, which is parallel to the y-z coordinate plane, and a
second fluid flow path, for example, fluid flow path 108, is
disposed within a second plane (parallel to the x-z coordinate
plane as shown) that is not parallel with the first plane. Yet
another way in which the microfluidic networks provided according
to the invention differ from those realizable with two-dimensional
systems is that the inventive microfluidic systems can include a
fluid flow path therein having a longitudinal axis, defining a
direction of bulk fluid flow within the flow path, that is not
disposed within any single plane in space, nor is disposed within
any a surface that is parallel to any surface (such as surface 132
or 134) of the microfluidic structure.
[0058] A "level" of a structure, as used herein, refers to a plane
or curved surface within the structure, typically parallel to a top
surface and a bottom surface of the structure, which can have a
channel or series of channels disposed therein and/or penetrating
therethrough. It should be understood that in the discussion and
figures illustrated below, the microfluidic network structures are
generally shown as having planar surfaces (e.g. surfaces 132 and
134), such that the levels within the structure are planar;
however, many of the structures, as described in more detail below,
are fabricated from flexible and/or elastomeric materials that are
capable of being bent, twisted, or distorted from the illustrated
planar configurations. For such embodiments, the "levels" within
the structure will comprise curved surfaces that are parallel to
the distorted planar surfaces of the structure, and any discussion
herein with regard to "levels" of the structures should be
understood to encompass such curved surfaces as well as the planar
surfaces illustrated. "Parallel," when used in the context of
comparing the topology of two surfaces in space, has its common
mathematical meaning referring to the two surfaces being everywhere
spaced apart from each other equidistantly.
[0059] "Non-fluidically interconnected" fluid flow paths, as used
herein, refers to fluid flow paths each comprising one channel or
multiple, fluidically interconnected channels, where the channels
of different flow paths do not intersect and are physically
isolated from each other within the structure so that they can not
communicate fluid between each other through bulk mixing of fluid
streams. A "fluid flow path" as used herein refers to one channel
or a series of two or more interconnected channels providing a
space within the microfluidic structure able to contain fluid or
through which fluid can continuously flow. Each fluid flow path of
the structure includes at least one opening thereto able to be
placed in fluid communication with the environment external to the
microfluidic structure and some preferred embodiments of fluid flow
paths include at least two openings able to be placed in fluid
communication with the environment external to the microfluidic
structure, thus providing an inlet and an outlet. A "channel" as
used herein refers to a flow path or continuous segment of a flow
path, which is disposed within one or more levels of the
microfluidic network structure and/or penetrates through one or
more levels of the microfluidic network structure. "Interconnected
channels," as used herein, refers to two or more channels within
the structure that are able to communicate fluid between and
through each other. A "non-linear" flow path and/or channel, as
used herein, refers to such flow path or channel having a
longitudinal axis that deviates from a straight line along its
length by more than an amount equal to the minimum cross-sectional
dimension of the channel or flow path. A "longitudinal axis" of a
channel or flow path as used herein refers to an axis disposed
along the entire length of such channel or flow path, which is
coextensive with and defined by the geometric centerline of the
direction of any bulk fluid which would flow through the channel or
flow path should such channel or flow path be configured for fluid
flow therethrough. For example, a linear or "straight" channel
would tend to have a longitudinal axis that is essentially linear,
while a fluid flow path comprising a series of such straight
channels that are fluidically interconnected can have a
longitudinal axis, comprising the interconnected longitudinal axes
of the individual interconnected channels forming the fluid flow
path, which is "non-linear." A channel which is "disposed within,"
"disposed in," "contained within," or "contained in" a level or
multiple levels of the structure refers herein to such channel
having a longitudinal axis that is coplanar with or, in the case of
a level defined by a curved surface, is lying along a contour of
the surface, of the level(s) in which it is disposed or contained.
A channel that "penetrates," "penetrates through," or "traverses" a
level or multiple levels of the structure refers herein to such
channel having a longitudinal axis that is non-coplanar with or, in
the case of a level defined by a curved surface, is not lying along
a contour of the surface of the level(s) such that the longitudinal
axis of such channel is non-parallel with any line that can be
disposed within the level.
[0060] Fluid flow path 102 of microfluidic network 100 communicates
with the external environment through an inlet opening 136 in fluid
communication with bottom surface 134 and an outlet opening 138 in
fluid communication with upper surface 132. The other fluid flow
paths of the network have similar inlet and outlet openings, as
illustrated.
[0061] The channels of the microfluidic networks provided according
to the invention have at least one cross-sectional dimension that
does not exceed about 500 .mu.m, in other embodiments does not
exceed about 250 .mu.m, in yet other embodiments does not exceed
about 100 .mu.m, in other embodiments does not exceed about 50
.mu.m, and in yet other embodiments does not exceed about 20 .mu.m.
A "cross-sectional dimension," when used in the above context,
refers to the smallest cross-sectional dimension for a
cross-section of a channel taken perpendicular to the longitudinal
axis of the channel. While the channels of network 100 have
cross-sectional dimensions that are essentially equal to each
other, in other embodiments, the channels can have unequal
cross-sectional dimensions, and some channels can have depths
within the structure sufficiently great so that they are disposed
in two or all three levels of the structure, instead of being
disposed in only a single level, as illustrated. In addition, while
in network 100 the channels are straight and linear, in other
embodiments the channels can be curved within the level(s) in which
they are disposed.
[0062] The double points formed where the channels of the fluid
flow paths of network 100 cross over each other are more clearly
seen in the two-dimensional perpendicular projection shown in FIG.
1b. FIG. 1b shows microfluidic network 100 as projected onto the
y-x plane as viewed in the negative z-axis direction. Crossover
double point 140, for example, represents the double point defining
the cross over of channel 130 of fluid flow path 110 and channel
116 of fluid flow path 102. In general, microfluidic networks
provided according to the invention having fluid flow paths
including channels that "cross over" each other refers to
structures including channel networks wherein a perpendicular
projection of the channels onto a surface defining a level of the
structure, in which either of the channels are disposed, at least
partially overlap each other. A "perpendicular projection" refers
to a projection in a direction that is perpendicular or normal to
the surface being projected upon. "At least partially overlap" or
"at least partially overlapping," as used herein when referring to
projections of channels which cross over each other, refers to the
two-dimensional projection of the channels intersecting each other,
as shown by point 140 in FIG. 1b, or, if, for example, the channels
are arranged in a parallel direction with respect to each other
within the network structure, to their being at least partially
superimposed upon each other in the two-dimensional projection.
[0063] While the three-dimensional microfluidic network structures
described herein could potentially be fabricated via conventional
photolithography, microassembly, or micromachining methods, for
example, stereolithography methods, laser chemical
three-dimensional writing methods, or modular assembly methods, as
described in more detail below, the invention also provides
improved fabrication methods for producing the inventive structures
involving replica molding techniques for producing individual
layers which comprise one or more of the levels of the structures,
as discussed above. As described in more detail below, such layers
are preferably molded utilizing mold masters having various
features on their surface(s) for producing channels of the
structure. In some preferred embodiments, the features are formed
via a photolithography method, or can themselves comprise a molded
replica of such a surface.
[0064] The microfluidic network structures produced by the
inventive methods described herein can potentially be formed from
any material comprising a solid material that comprises a
solidified form of a hardenable liquid, and, in some embodiments,
the structures can be injection molded or cast molded. As will be
described in more detail below, preferred hardenable liquids
comprise polymers or precursors of polymers, which harden upon, or
can be induced to harden during, molding to produce polymeric
structures. For reasons described in more detail below,
particularly preferred polymeric materials for forming the
microfluidic networks according to the invention comprise
elastomeric materials.
[0065] For structures produced according to the preferred methods
described herein, the microfluidic networks provided according to
the invention will typically be comprised of at least one discrete
layer of polymeric material, and other embodiments will be
comprised of at least two discrete layers of polymeric material,
and in yet other embodiments will be comprised of three or more
discrete layers of polymeric material. A "discrete layer" of
material as used herein refers to a separately formed subcomponent
structure of the overall microfluidic structure, which layer can
comprise and/or contain one, two, or three, or more levels of the
overall channel network of the microfluidic structure. As described
and illustrated in more detail below, the discrete layers of the
structure can be stacked together to form a three-dimensional
network, or multiple three-dimensional networks, if desired, and
can also be, in some embodiments, placed between one or more
support layers or substrate layers in order to enclose and
fluidically seal channels of the lower and upper levels of the
microfluidic structure.
[0066] As described in more detail below, the methods for producing
microfluidic network structures provided by the invention can, in
some embodiments, produce discrete layers comprising a single level
of the overall structure, wherein the three-dimensional network
structure is formed by forming a first layer including a series of
channels disposed therein, forming a second layer including a
second series of channels disposed therein, and forming a third
layer having connecting channels traversing the layer, and
subsequently stacking the third layer between above-mentioned first
and second layers and aligning the layers with respect to each
other to achieve the overall desired three-dimensional network
structure. In another embodiment, the microfluidic network
structure includes two channel-containing layers: a first discrete
layer containing both a first level, including a series of channels
disposed therein, and a third, intermediate level of the structure
including the connecting channels traversing the level; and a
second discrete layer including the second level of the structure,
having a second series of channels disposed therein. In such a
method the first discrete layer and the second discrete layer are
stacked and aligned with respect to each other to produce the
overall desired three-dimensional microfluidic network structure.
And in yet a third embodiment, all three levels of the microfluidic
network structure can be produced in a single discrete layer, the
layer comprising a three-level microfluidic membrane structure.
[0067] FIGS. 2a and 2b illustrate a microfluidic structure 150
having an alternative three-dimensional arrangement of channels
therein. Microfluidic network 150 includes two non-fluidically
interconnected flow paths 152 and 154. Fluid flow path 152
comprises a series of interconnected channels 156, 158, 160, 162
and 164, which are non-linear and which define a plane parallel to
the y-z coordinate plane. Channels 156 and 164 are disposed within
a first, lower level of the structure, and channel 160 is disposed
within a second, upper level of the structure. Connecting channel
158 traverses a third, intermediate level of the structure from the
first, lower level to the second, upper level and fluidically
interconnects channel 156 to channel 160. Similarly, connecting
channel 162 traverses the third, intermediate level of the
structure connecting channel 164 and channel 160. Flow path 152 is
connected in fluid communication with the external environment via
inlet opening 168 in side wall 170 an outlet opening 172 in side
wall 174. Fluid flow path 154 comprises a single channel 176
disposed within the first, lower level of the structure, and is
interconnected to the environment via inlet opening 178 in side
wall 180 an outlet opening 182 in side wall 190. The perpendicular
projection of the microfluidic channel network, onto the first,
lower level of the structure is illustrated in FIG. 2b. FIG. 2b
shows double point 192 where channel 160 of fluid flow path 152
crosses over channel 176 of fluid flow path 154.
[0068] FIGS. 3a and 3b illustrate yet another simple microfluidic
network provided according to the invention but not achievable with
a conventional two-dimensional microfluidic network structure.
Microfluidic network 200 includes a single fluid flow path 202.
Fluid flow path 202 is comprised of a first channel 204 disposed
within a first, lower level of the structure; a second channel 206
disposed within a second, upper level of the structure; and a
connecting channel 208 traversing a third, intermediate level of
the structure and fluidically interconnecting channels 204 and 206.
Channel 204 disposed within the first level of the structure and
channel 206 disposed within the second level of the structure are
non parallel to each other and, in the illustrated embodiment,
happen to be perpendicular to each other. FIG. 3b illustrates the
perpendicular projection of microfluidic network 200 onto the
first, lower level structure along the negative z-axis direction.
As illustrated, microfluidic network 200 does not include any
crossover points in the projection.
[0069] As previously discussed, a microfluidic network need only
include three levels therein (a first and a second level including
channels disposed therein such that their longitudinal axes are
coplanar with a surface defining the level and a third intermediate
level having one or more connecting channels passing therethrough
fluidically connecting the channels of the first level and the
second level) in order to provide any arbitrarily complex network
of channels that pass over and under one another. However, certain
potentially desirable geometric configurations of channels may
require more than the three levels contained within the structures
discussed and illustrated above. For example, if it is desired to
produce a microfluidic network having channels disposed within
three or more non-coplanar levels of the structure, additional
levels are needed. In general, the number of levels required for
microfluidic structures produced according to the invention
required to produce n levels, each level having channels disposed
therein such that their longitudinal axis are coplanar with the
level, requires a total of 2n-1 total levels in the structure.
Thus, for the previously illustrated embodiments having two levels
therein in which channels are disposed, each structure requires a
total of three levels to form the overall network structure (an
upper and lower level in which the channels are disposed and an
intermediate level through which the connecting channels pass).
[0070] FIGS. 4a and 4b illustrate one embodiment of a microfluidic
structure, producible according to the methods of the invention
described below, including therein three levels having channels
disposed therein such that their longitudinal axes are coplanar
with each of the levels, and a total of five levels overall.
Structure 220 includes a microfluidic network comprising a fluid
flow path 222 arranged as a coil surrounding a second fluid flow
path 224. Such an arrangement may be especially useful for
particular microfluidic applications involving, for example, heat
transfer or mass transfer between components contained within fluid
flow paths 222 and 224, or for embodiments where electrical,
magnetic, optical or other environmental interaction between
materials in the respective flow paths is desired.
[0071] The first, lower level of structure 220 includes disposed
therein channels 226, 228, 230, and 232 of coil flow path 222. The
second level from the bottom of structure 220 includes disposed
therethrough the lowermost region 234 of connecting channels 236,
238, 240, 242, 244, 246, and 248 of fluid flow path 222. The third
level from the bottom of structure 220 includes channel 250 of
fluid flow path 224 disposed therein and also includes intermediate
region 251 of the connecting channels. The fourth level from the
bottom of structure 220 includes, traversing therethrough, upper
regions 252 of the connecting channels, and the uppermost level of
structure 220 includes disposed therein channels 254, 256, 258 and
260 of flow path 222.
[0072] FIG. 4b illustrates the perpendicular projection of
microfluidic network 220 onto a surface coplanar with the first,
lowermost level of the structure that is parallel to the y-x
coordinate plane, as viewed in the negative z direction. As
illustrated, structure 220 includes 8 double point crossovers 264,
266, 267, 268, 269, 270, 272, and 274 where either flow path 224
crosses over a channel of flow path 222 (e.g. crossover points 264,
267, 269, and 272), or where channel 250 of flow path 224 crosses
under a channel of fluid flow path 222, (for example, crossover
point 266, 268, 270, and 274.) It should be evident that the five
level structure illustrated by structure 220, in alternative
embodiments, can have flow paths therein comprising a series of
interconnected channels arranged so as to yield higher order
crossover points than the double points illustrated. For example,
in other embodiments, a five level structure can have channels
disposed therein including triple point crossovers wherein a
perpendicular projection onto a surface coplanar with a level of
the structure includes points where three levels of channels
intersect (i.e., where a channel disposed in the lowermost level, a
channel disposed in the third, intermediate level, and a channel
disposed in the uppermost level overlap and/or intersect each other
in the two-dimensional projection).
[0073] As discussed above, the present invention also provides a
variety of methods providing relatively simple and low cost
fabrication techniques for producing the inventive microfluidic
structures described herein. The preferred methods provided
according to the invention and described below are based upon
utilizing a hardenable liquid to create replica molded structures
that comprise, or are assembled with other replica molded
structures to form, the three-dimensional microfluidic network
structures provided by the invention.
[0074] FIGS. 5a-5c illustrate a first embodiment of a method for
forming the inventive microfluidic structures by utilizing a
replica molding process provided by the invention. The method
illustrated by FIGS. 5a-5c involves forming a number of replica
molded layers from a hardenable liquid, each of which structures
comprises a single level of the overall microfluidic network.
Following the fabrication of each of the replica molded structures
comprising layers of the overall microfluidic network structure,
the layers are stacked upon each other, aligned with respect to
each other so that the respective molded features in the layers
create the desired and predetermined microfluidic network pattern,
and, optionally, the layers can be permanently sealed to each other
and/or to one or more substrate layers, which substrate layers do
not comprise a level of the overall microfluidic structure, in
order to yield a finished microfluidic network structure having a
desired configuration.
[0075] Step 1 as illustrated in FIG. 5a involves forming a first
layer of the structure comprising, for example, a first, lower
level of the microfluidic network. Of course, in other embodiments,
layers comprising an upper or intermediate level of the structure
can be molded before or at the same time a lower layer is molded.
In general, the order of the molding steps is not particularly
critical and the various layers of the overall structure can be
molded in any order that is desired or convenient. In the
illustrated embodiment, a lower mold master 300 is provided having
a series of topological features 302 protruding from an upper
surface 304 of the lower mold master. A second mold master 306
having a flat, featureless surface 308 facing surface 304 of mold
master 300 is provided and placed in contact with an upper surface
of topological features 302 of mold master 304. Disposed between
mold masters 304 and 306 is a layer of hardenable liquid 310, which
upon solidification forms a replica molded layer including therein
a plurality of channels, formed by topological features 302 of mold
master 304, which, channels, in preferred embodiments, pass
completely through the thickness of the entire layer of liquid 310,
upon hardening, thus forming a membrane structure comprised of the
hardened liquid.
[0076] Mold master 300, having positive, high-relief topological
features 302 formed on a surface 304 thereof comprises, in some
preferred embodiments, a substrate that has been modified, for
example, via photolithography or any suitable micromachining method
apparent to those of ordinary skill in the art. Topological
features 302 are shaped, sized, and positioned to correspond to a
desired arrangement of channels in the level of the overall
microfluidic network structure being formed by the mold master. In
one preferred embodiment, mold master 300 comprises a silicon wafer
having a surface 304 that has been via photolithography utilizing a
photomask having a pattern therewithin corresponding to a desired
pattern of topological features 302. Techniques for forming
positive relief patterns of topological features on silicon, or
other materials, utilizing photolithography and photomasks, are
well known and understood by those of ordinary skill in the art
and, for example, are described in Qin, D., et al. "Rapid
Prototyping of Complex Structures with Feature Sizes Larger Than 20
microns," Advanced Materials, 8(11):pp. 917-919 and Madou, M.,
Fundamentals of Microfabrication, CRC Press, Boca Raton, Fla.,
(1997), both incorporated herein by reference.
[0077] In a particularly preferred embodiment, mold master 300
comprises a silicon or other substrate, which has been spincoated
with one or more layers of a commercially available polymeric
photoresist material. In such preferred embodiments, topological
features 302 can be easily, conveniently, and accurately formed in
the layer(s) of photoresist forming surface 304 of substrate 300
via exposure of photoresist to radiation through a photomask and
subsequent development of the photoresist material to remove
photoresist material from the surface and regions surrounding
features 302 thus leaving behind topological features 302 in
positive relief. A variety of positive and negative photoresists
can be utilized for such purposes and are well known to those of
ordinary skill in the art.
[0078] One particularly preferred method for forming topological
features 302 on a surface of a substrate coated with one or more
layers of photoresist is described in more detail below in the
context of FIG. 8. The photomask utilized, as described above,
provides a pattern therein able to selectively block radiation
reaching the layer(s) of photoresist so that, upon development of
the layer, a pattern of topological features will be formed, which
features correspond to a desired arrangement of channels within the
replica molded layer. Such patterns can be designed with the aid of
any one of a number of commercially available computer aided design
(CAD) programs, as would be apparent to those of ordinary skill in
the art.
[0079] Mold master 306 can be comprised of the same material as
mold master 300; however, in preferred embodiments, mold master 306
is formed of an elastomeric material, for example, an elastomeric
polymer. Mold master 306 is, in preferred embodiments, formed of an
elastomeric material because the elastomeric nature of the mold
master enables an improved seal at the interface of surface 308 of
mold master 306 and the upper surfaces of topological features 302
of lower mold master 300 to be formed so as to essentially
completely exclude hardenable liquid 310 from the interface between
the topological features 302 and surface 308 of mold master 306.
This preferred ("sandwich") method enables, upon the hardening of
hardenable liquid 310, the production of a membrane comprised of
the hardened fluid having channels disposed therein which
completely traverse the entire thickness of the membrane and which
are not blocked by a thin layer of hardened liquid.
[0080] For some embodiments, it is also desirable that upper mold
master 306 be transparent in order to be able to visualize
topological features 302 during the molding process. Alternatively,
in other embodiments, upper mold master 306 can comprise a rigid,
non-elastomeric material and lower mold master 300, including
topological features 302 forming the channels of the molded
structure, can be formed of an elastomeric material. In such an
embodiment, the elastomeric mold master having positive relief
topological features disposed on its surface is preferably itself
formed as a molded replica of a pre-master having a surface
including a plurality of negative, low-relief features therein,
which form the positive relief features in the elastomeric mold
master upon creating a replica mold of the pre-master surface. In
yet other embodiments, the upper and lower mold masters of the
invention can both comprise elastomeric materials and can be formed
of the same, or different elastomeric materials. In addition,
although less preferred, upper mold master 306 can be eliminated
entirely and hardenable fluid 310 may simply be spuncast onto
surface 304 of lower mold master 300 to a thickness corresponding
to the height of topological features 302. Such method is generally
less preferred for producing molded membranes according to the
invention because it is generally desired that the uppermost and
lowermost surfaces of the membrane be as flat and smooth as
possible to enable conformal sealing and prevention of leakage upon
assembly of the layers into the overall microfluidic network
structure.
[0081] In preferred embodiments, hardenable liquid 310 is placed
upon surface 304 of lower mold master 300 in an amount sufficient
to form a layer over the region of surface 304 including
topological features 302, corresponding to the channel structure in
the layer to be formed, which layer having a thickness at least
equal to the height of topological features 302 above surface 304.
Subsequent to placing liquid 310 on surface 304, the method
involves bringing surface 308 of upper mold master 306 into contact
with the upper surface of features 302. In alternative embodiments,
a lower mold master and upper mold master can be brought into
contact prior to addition of the hardenable liquid, and the
hardenable liquid can be applied to the region between the facing
surfaces of the mold masters by adding a sufficient amount in the
region of the space between the upper mold master and lower mold
master around their periphery (e.g. periphery 312), and
subsequently allowing hardenable liquid 310 to flow into the space
surrounding the topological features of the mold master(s) via
capillary action. Such method for utilizing capillary action for
creating a molded replica structure as described in detail in
commonly owned, copending U.S. patent application Ser. No.
09/004,583 entitled "Method of Forming Articles Including
Waveguides Via Capillary Micromolding and Microtransfer Molding,"
and International Pat. Publication No. WO 97/33737, each
incorporated herein by reference.
[0082] Hardenable liquid 310 can comprise essentially any liquid
known to those of ordinary skill in the art that can be induced to
solidify or spontaneously solidifies into a solid capable of
containing and transporting fluids contemplated for use in and with
the microfluidic network structures. In preferred embodiments,
hardenable liquid 310 comprises a polymeric liquid or a liquid
polymeric precursor (i.e. a "prepolymer"). Suitable polymeric
liquids can include, for example, thermoplastic polymers, thermoset
polymers, or mixture of such polymers heated above their melting
point; or a solution of one or more polymers in a suitable solvent,
which solution forms a solid polymeric material upon removal of the
solvent, for example, by evaporation. Such polymeric materials,
which can be solidified from, for example, a melt state or by
solvent evaporation, are well known to those of ordinary skill in
the art.
[0083] In preferred embodiments, hardenable liquid 310 comprises a
liquid polymeric precursor. Where hardenable liquid 310 comprises a
prepolymeric precursor, it can be, for example, thermally
polymerized to form a solid polymeric structure via application of
heat to mold master 300 and/or mold master 306; or, in other
embodiments, can be photopolymerized if either mold master 300 or
mold master 306 is transparent to radiation of the appropriate
frequency. Curing and solidification via free-radical
polymerization can be carried out as well. These and other forms of
polymerization are known to those of ordinary skill in the art and
can be applied to the techniques of the present invention without
undue experimentation. All types of polymerization, including
cationic, anionic, copolymerization, chain copolymerization,
cross-linking, and the like can be employed, and essentially any
type of polymer or copolymer formable from a liquid precursor can
comprise hardenable liquid 310 in accordance with the invention. An
exemplary, non-limiting list of polymers that are potentially
suitable include polyurethane, polyamides, polycarbonates,
polyacetylenes and polydiacetylenes, polyphosphazenes,
polysiloxanes, polyolefins, polyesters, polyethers, poly(ether
ketones), poly(alkaline oxides), poly(ethylene terephthalate),
poly(methyl methacrylate), polystyrene, and derivatives and block,
random, radial, linear, or teleblock copolymers, cross-linkable
materials such as proteinaceous materials and/or blends of the
above. Gels are suitable where dimensionally stable enough to
maintain structural integrity upon removal from the mold masters,
as described below. Also suitable are polymers formed from
monomeric alkylacrylates, alkylmethacrylates, alpha-methylstyrene,
vinyl chloride and other halogen-containing monomers, maleic
anhydride, acrylic acid, acrylonitrile, and the like. Monomers can
be used alone, or mixtures of different monomers can be used to
form homopolymers and copolymers. The particular polymer,
copolymer, blend, or gel can be selected by those of ordinary skill
in the art using readily available information and routine testing
and experimentation so as to tailor a particular material for any
of a wide variety of potential applications.
[0084] According to some preferred embodiments of the invention,
hardenable liquid 310 comprises a fluid prepolymeric precursor
which forms an elastomeric polymer upon curing and solidification.
A variety of elastomeric polymeric materials are suitable for such
fabrications, and are also suitable for forming mold masters, for
embodiments where one or both of the mold masters is composed of an
elastomeric material. A non-limiting list of examples of such
polymers includes polymers of the general classes of silicone
polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are
characterized by the presence of a three-membered cyclic ether
group commonly referred to as an epoxy group, 1,2-epoxide, or
oxirane. For example, diglycidyl ethers of bisphenol A can be used,
in addition to compounds based on aromatic amine, triazine, and
cycloaliphatic backbones. Another example includes the well-known
Novolac polymers. Examples of silicone elastomers suitable for use
according to the invention include those formed from precursors
including the chlorosilanes such as methylchlorosilanes,
ethylchlorosilanes, and phenylchlorosilanes, and the like. A
particularly preferred silicone elastomer is polydimethylsiloxane
(PDMS). Exemplary polydimethylsiloxane polymers include those sold
under the trademark Sylgard by Dow Chemical Co., Midland, Mich.,
and particularly Sylgard 182, Sylgard 184, and Sylgard 186.
[0085] Silicone polymers, for example, PDMS, are especially
preferred for use in the invention because they have several
desirable beneficial properties simplifying fabrication of the
microfluidic network structures, described herein. First, such
materials are inexpensive, readily available, and can be solidified
from a prepolymeric liquid via curing with heat. For example, PDMSs
are typically curable by exposure of the prepolymeric liquid to
temperatures of about, for example, 65.degree. C. to about
75.degree. C. for exposure times of about, for example, 1 hour.
Second, silicone polymers, such as PDMS, are elastomeric and are
thus useful for forming certain of the mold masters used in some
embodiments of the invention. In addition, microfluidic networks
formed from elastomeric materials can have the advantage of
providing structures which are flexible and conformable to the
shape of a variety of substrates to which they may be applied, and
elastomeric networks can provide reduced resistance to fluid flow
for a given applied pressure drop, as compared to non-elastomeric
structures, and can also be more easily fabricated to include
active elements therein, for example integrated valves and pumping
elements, which elements can utilize the flexibility and elasticity
of the material for their performance.
[0086] Another distinct advantage for forming the inventive
microfluidic networks from silicone polymers, such as PDMS, is the
ability of such polymers to be oxidized, for example by exposure to
an oxygen-containing plasma such as an air plasma, so that the
oxidized structures contain at their surface chemical groups
capable of cross-linking to other oxidized silicone polymer
surfaces or to the oxidized surfaces of a variety of other
polymeric and non-polymeric materials. Thus, membranes, layers, and
other structures produced according to the invention utilizing
silicone polymers, such as PDMS, can be oxidized and essentially
irreversibly sealed to other silicone polymer surfaces, or to the
surfaces of other substrates reactive with the oxidized silicone
polymer surfaces, without the need for separate adhesives or other
sealing means. In addition, microfluidic structures formed from
oxidized silicone polymers can include channels having surfaces
formed of oxidized silicone polymer, which surfaces can be much
more hydrophilic than the surfaces of typical elastomeric polymers.
Such hydrophilic channel surfaces can thus be more easily filled
and wetted with aqueous solutions than can structures comprised of
typical, unoxidized elastomeric polymers or other hydrophobic
materials.
[0087] In addition to being irreversibly sealable to itself,
oxidized PDMS can also be sealed irreversibly to a range of
oxidized materials other than itself including, for example, glass,
silicon, silicon oxide, quartz, silicon nitride, polyethylene,
polystyrene, glassy carbon, and epoxy polymers, which have been
oxidized in a similar fashion to the PDMS surface (for example, via
exposure to an oxygen-containing plasma). Oxidation and sealing
methods useful in the context of the present invention are
described in more detail below and also in Duffy et al., Rapid
Prototyping of Microfluidic Systems and Polydimethylsiloxane,
Analytical Chemistry, Vol. 70, pages 474-480, 1998, incorporated
herein by reference.
[0088] For clarity and simplicity, the discussion below involving
the inventive methods for forming microfluidic structures according
to the invention in many instances makes specific reference to a
preferred embodiment wherein the layers comprising the structure
and/or one or more mold masters are formed from a hardenable liquid
comprising a fluid prepolymer of PDMS. It should be understood, as
the discussion above makes clear, that such reference is pure
exemplary, and a wide variety of other materials can be utilized in
place of or in addition to PDMS to achieve the various objects,
features, and benefits of the present invention, as would be
apparent to those of ordinary skill in the art.
[0089] Referring again to FIG. 5a, in Step 2, PDMS, comprising
hardenable liquid 310, is cured and solidified, for example by
application of heat to raise the temperature of the PDMS prepolymer
to between about 65.degree. C. to about 75.degree. C. for about 1
hour, as described above. In order to prevent seepage of the PDMS
between surface 308 and the upper surface of topological features
302, it is preferred to apply pressure to one or both of lower
surface 314 of mold master 300 and upper surface 316 of mold master
306. It has been found, within the context of the invention, that a
pressure of approximately between about 10-100 g/mm.sup.2
(100-1,000 kPa) or greater is generally sufficient to prevent PDMS
prepolymer from seeping between topological features 302 and
surface 308 so as to cause blockage of subsequent channels formed
within the cured membrane.
[0090] Step 3 involves peeling the cured membrane from one or both
of mold master 300 and 306. In preferred embodiments, as discussed
above, materials are selected for mold master 300, mold master 306,
and hardenable liquid 310, which allow removal of the solidified
membrane upon solidification of the hardenable liquid without
destruction of the molded structure. In especially preferred
embodiments, because a solidified layer is typically thin and
fragile (for example, layer 318 can vary in thickness from about 20
.mu.m to about 1 mm), mold master 300 and mold master 306 are
selected or treated such that layer 318 adheres to the surface of
one of the mold masters more strongly than to the surface of the
other mold master. Such differential adhesion allows the mold
masters to be peeled apart such that the fragile molded layer 318
remains adherent to and is supported by one or the other of the
mold masters. Such differential adhesion of layer 318 can be
created by selecting materials comprising mold master 306 and
surface 304 of mold master 300 having different chemical properties
such that the non-covalent interfacial adhesion between layer 318
and surface 304 differs from that between layer 318 and surface
308. Those of ordinary skill in the art can readily determine
appropriate materials for comprising hardenable liquid 310, mold
master 300, and mold mater 306 and/or surface treatments which can
be applied to either or both of the mold masters that allow for
differences in non-covalent interfacial adhesion between layer 318
and the surfaces of the mold masters, enabling layer 318 to be
selectively removed from one of the surfaces while remaining
adherent to the other. Interfacial free energies for a wide variety
of materials are readily available to those of ordinary skill in
the art and can be utilized, along with routine screening tests,
for example measuring forces required to peel apart various
combinations of materials, by those of ordinary skill in the art to
readily select a combination of materials, without undue
experimentation, for enabling layer 318 to be selectively removed
from the surface of one mold master while remaining adherent to and
supported by the surface of the other mold master.
[0091] For example, in the illustrated embodiment, lower mold
master 300 includes an upper surface 304 comprising a negative
photopolymer (SU-8-50, Microlithography Chemical Corp., Newton,
Mass.), upper mold master 306 comprises oxidized PDMS, and
hardenable fluid 310 comprises a PDMS prepolymer. Also in the
illustrated embodiment, surfaces 308 and 304, before contact with
fluid 310 were silanized to facilitate the removal of PDMS replica
layer 318 after curing. In an exemplary embodiment, the masters
were silanized by exposing the surfaces to a chlorosilane vapor,
for example a vapor containing
tridecafluoro-1,1,2,2-tetrahydrooctal-1-trichlorosilane. PDMS
replica layer 318 adheres more strongly to silanized PDMS mold
master 306 than to silanized surface 304 of mold master 300 and
remains supported by and attached to mold master 306 upon applying
a peeling force tending to separate the two mold masters, resulting
in molded replica layer 318 remaining adherent and supported by
mold master 306, as illustrated in Step 3. In an alternative
embodiment, instead of utilizing a silanized PDMS layer for mold
master 306 in combination with silanized mold master 300, as
described above, mold master 306 can comprise a layer or sheet of a
material having a very low interfacial free energy, for example
Teflon.TM. (polytetrafluoroethylene (PTFE)). In such an embodiment,
replica molded layer 318 will tend to remain adherent to mold
master 300 upon applying a peeling force tending to separate mold
master 306 and mold master 300.
[0092] Step 4 of FIG. 5a illustrates an optional step comprising
conformally contacting molded replica layer 318, supported by mold
master 306, with a lower substrate layer 320, and, optionally,
irreversibly sealing lower surface 319 of layer 318 to the upper
surface 322 of substrate 320. In the illustrated embodiment,
substrate 320 comprises a PDMS slab having a flat upper surface
322. Both lower surface 319 of layer 318 and upper surface 322 of
substrate 320 have been oxidized, for example by exposure to an air
plasma in a plasma cleaner, as discussed above and in more detail
below, prior to bringing the surfaces into contact, so that when
brought into conformal contact, an irreversible seal spontaneously
forms between surface 319 and surface 322 providing a fluid-tight
seal at the bottom of channels 321 in layer 318. Exposure of the
PDMS surfaces to the oxygen-containing plasma is believed to cause
the formation of Si--OH groups at the surface of the PDMS, which
react with other Si--OH groups to form bridging, covalent siloxane
(Si--O--Si) bonds by a condensation reaction between the two
oxidized PDMS surfaces.
[0093] In alternative embodiments, where it is not desired to
permanently seal layer 318 to substrate 320, the surfaces may not
be oxidized so that they do not irreversibly seal to each other but
rather may simply be brought into conformal contact with each
other, which conformal contact between the two essentially flat
planar surfaces can be sufficient, for microfluidic applications
involving vacuum or low pressures, to form a fluid-tight seal.
Also, in some applications, such as microcontact surface patterning
with the inventive microfluidic networks as described in more
detail below, it may be desirable to provide a "patterning" surface
of the microfluidic network having channels therein which are not
sealed by a substrate, and which can be brought into contact with a
material surface in order to form on the surface a pattern defined
by the channels in the "patterning" surface of the microfluidic
network.
[0094] In yet other embodiments, substrate 320 can comprise a
material different from one or both of molded layer 318 and mold
master 306, for example, a material other than PDMS. In some such
embodiments, substrate 320 can comprise, for example, the surface
of a silicon wafer or microchip, or other substrate advantageous
for use in certain applications of the microfluidic network
provided according to the invention. Molded layer 318 can, as
described above, be irreversibly sealed to such alternative
substrates or may simply be placed in conformal contact without
irreversible sealing. For embodiments where it is desired to
irreversibly seal a molded replica layer 318 comprising PDMS to a
substrate 320 not comprising PDMS, it is preferred that substrate
320 be selected from the group of materials other than PDMS to
which oxidized PDMS is able to irreversibly seal (e.g., glass,
silicon, silicon oxide, quartz, silicon nitride, polyethylene,
polystyrene, epoxy polymers, and glassy carbon surfaces which have
been oxidized). For embodiments involving hardenable liquids other
than PDMS prepolymers, which form molded replica layers not able to
be sealed via the oxidation methods described above, when it is
desired to irreversibly seal such layers to each other or to a
substrate, alternative sealing means can be utilized, as would be
apparent to those of ordinary skill in the art, including, but not
limited to, the use of separate adhesives, thermal bonding, solvent
bonding, ultrasonic welding, etc.
[0095] Step 5 illustrated in FIG. 5a comprises the removal of upper
mold master 306 to expose flat, top surface 317 of molded replica
318 thus yielding a first, lower level of the overall microfluidic
network structure having a series of channels 321 disposed in a
desired pattern therein. In an alternative embodiment to the
illustrated membrane sandwich method for forming membrane layer
318, in Step 1 a molded replica can be formed by placing mold
master 300 in the bottom of a dish or other container having a
depth in excess of the height of topological features 302 and
filling the container to a level in excess of the height of
features 302 with a hardenable liquid, such as PDMS prepolymer.
Upon curing and removing the cured structured from the container
and from mold master 300, a structure similar to that obtained at
the end of Step 5 is formed, except comprising channels that do not
penetrate through the entire thickness of the molded replica layer.
Such an embodiment is described in further detail in the context of
the fabrication method illustrated in FIG. 7 below. In addition, as
illustrated in FIG. 5, to facilitate the stacking and alignment of
additional molded replica layers comprising the second, third, and
any higher levels of the microfluidic structure, lower layer 318
can be trimmed such that it is essentially uniform in thickness and
has a desired overall size and perimeter shape.
[0096] FIG. 5b illustrates the formation of a second molded replica
layer comprising the third, intermediate level of a microfluidic
network structure containing therein connecting channels as
previously described. Steps 6-8 are essentially similar to Steps
1-3 described above in the context of FIG. 5a, except that lower
mold master 330 has an upper surface 332 including thereon positive
relief topological features 334 protruding above surface 332 that
are shaped, sized, and positioned to form channels within the
molded replica structure corresponding to a desired arrangement of
connecting channels within the third, intermediate level of the
microfluidic network structure being fabricated. In addition, if
desired, additional features (not shown) can be included on the
surface 332 of mold master 330 corresponding to channels that are
disposed within (i.e., have longitudinal axis coplanar with) the
third, intermediate level of the microfluidic network structure
being formed.
[0097] Step 7 involves curing PDMS prepolymer 310 (or other
hardenable liquid) as previously described for Step 2 above, and
Step 8 involves selectively removing molded replica layer 340 from
mold master 330 while it remains supported by an adherent to upper
mold master 306, as described for Step 3 above. Optional step 9
involves removing molded replica layer 340 from upper mold master
306 and, if desired, trimming layer 340 so that it has an
essentially identical overall size and perimeter shape as layer 318
above. Step 10 involves placing molded replica layer 340 into
conformal contact with upper surface 317 of molded replica layer
318, aligning the channels 342 in molded replica layer 340 with
channels 321 in molded replica layer 318 to provide a desired
registration between the channels of the first, lower level of the
structure comprised of layer 318 and the third, intermediate level
of the structure comprised of layer 340, followed by irreversibly
sealing together layers 318 and 340. In alternative embodiments,
the alignment and sealing steps can be delayed if desired and
performed in one step for all of the layers (i.e., all three
channel-forming layers) comprising the overall structure which have
been formed and stacked upon each other (e.g. see FIG. 5c below).
In addition, for embodiments wherein upper mold master 306 is
transparent, for example for embodiments where upper mold master
comprises PDMS, and especially for embodiments including replica
layers having a large number of channels disposed completely
through the entire thickness of the membrane layer or having
channels shaped so that the molded replica membrane layer is not
free-standing when removed from a support surface (e.g., channels
comprising continuous, closed geometric shapes, spiral shaped
channels, etc.), layer 340 is preferably not removed from mold
master 306 as illustrated in Step 9, but instead, mold master 306,
with molded replica layer 340 attached thereto, is placed in
contact with upper surface 317 of molded replica layer 318 and
aligned and sealed as described in step 10 prior to removing mold
master 306, so that the molded replica layer remains attached to
and supported by a mold master during each of the manipulation
steps and is never free-standing.
[0098] Alignment of the molded replica features comprising the
channels of layers 318 and 340 can be accomplished utilizing a
microscope, such as a stereo microscope, in combination with an
alignment stage and/or micromanipulators for accurately positioning
the layers and registering the features with respect to each other.
For a preferred embodiment wherein layers 318 and 340 are comprised
of PDMS, layers 318 and 340 can be aligned and sealed to each other
by either of the preferred methods described directly below. In a
first method, layer 340 is placed upon layer 318 and carefully
aligned with respect to layer 318 to provide a desired alignment
and registration of channels by utilizing a stereo microscope and a
micromanipulator. Layers 318 and 340 are then carefully slightly
separated from each other (e.g. by a few millimeters), without
changing the registered lateral alignment of channels within the
layers, to provide a small space between surface 317 of layer 318
and surface 341 of layer 340. The aligned structure having the
layers slightly separated is then exposed to an oxygen-containing
plasma in order to oxidize surfaces 317 and 341. The layers are
then carefully brought together without altering or disturbing the
lateral alignment of the channels, so that surfaces 317 and 341
spontaneously seal to each other upon conformal contact.
[0099] In the second, especially preferred, embodiment, alignment
and sealing of the layers proceeds as follows. The upper surface
317 of layer 318 and lower surface 341 of layer 340 are oxidized
utilizing the oxygen-containing plasma exposure method described
previously, and a liquid that is essentially non-reactive with the
oxidized surfaces is placed upon layer 317 to form a continuous
layer of liquid thereupon, upon which, surface 341 of layer 340 is
placed. The liquid, in addition to being essentially non-reactive
with the oxidized surfaces of the PDMS, also preferably prevents
degradation of the active Si--OH groups present on the surfaces for
a period of time sufficiently long to enable alignment of the
surfaces with respect to each other and removal of the liquid.
After placing layer 340 onto the fluid-covered surface of layer
318, layer 340 is aligned with respect to layer 318 to yield a
desired registration and alignment of features (channels) for
forming the microfluidic network structure. The non-reactive liquid
is then removed from between the two surfaces bringing the two
surfaces into conformal contact with each other and spontaneously
sealing the two surfaces together.
[0100] A variety of liquids can potentially be utilized as the
non-reactive liquid in the context of the inventive alignment
method above described. As previously discussed, appropriate
liquids will be essentially non-reactive with the oxidized surfaces
and will preferably stabilize and delay degradation of the active
chemical groups contained within the oxidized surfaces. It has been
found, in the context of the present invention, that polar liquids,
and especially those comprising compounds including hydroxyl
moieties, are effective for use as the non-reactive liquid.
Especially preferred are water, alcohols, and mixtures thereof with
alcohols, and alcohol-water mixtures being particularly preferred,
especially those including methanol and/or trifluoroethanol. The
non-reactive liquid, in preferred embodiments, is removed from
between the oxidized surfaces of the layers via evaporation of the
liquid, and thus, in such embodiments, as the non-reactive liquid
evaporates the oxidized surfaces of the layers are simultaneously
brought together in conformal contact whereupon the surfaces react
to create an essentially irreversible seal.
[0101] While we have described above an embodiment wherein layer
340 comprising the third, intermediate layer of the structure is
aligned and sealed with respect to layer 318 comprising a first,
lower level of the structure prior to the fabrication of the molded
replica layer comprising a second, upper level of the structure, in
other embodiments, as mentioned above, the upper layer is formed
prior to sealing the lower and intermediate layers together, so
that the intermediate and upper layers can be stacked, aligned, and
sealed to the lower layer in a single step, eliminating the need to
selectively oxidize only lower surface 341 of intermediate layer
340 so as to prevent degradation of an oxidized upper surface 343
of intermediate layer 340 prior to the formation, stacking, and
alignment of the upper layer to the intermediate layer (as shown
and described in FIG. 5c below).
[0102] FIG. 5c illustrates the final steps for forming the overall
three-layer, three-level microfluidic network according to this
first fabrication method embodiment of the invention. Step 11 and
Step 12 of FIG. 5c are analogous to Steps 1 and 2 of FIG. 5a and
Steps 6 and 7 of FIG. 5b and involve sandwiching a hardenable
liquid 310, such as PDMS, between upper mold master 306 and a lower
mold master 350 having an upper surface 352 including thereon
topological features 354 in positive relief constructed and
positioned for forming channels disposed within the second, upper
level of the final overall microfluidic network structure.
Hardenable liquid 310 is cured and solidified in Step 12, as
previously described, and, in preferred embodiments, molded replica
layer 360 is preferentially separated from surface 352 of lower
mold master 350 while remaining in contact with and supported by
upper mold master 306, as previously described. Molded replica
layer 360, which comprises the second, upper level of the overall
structure, includes molded channels 362 disposed within layer 360.
Step 14 involves optionally removing molded replica membrane layer
360 from upper mold master 306, as previously described for Step 9
discussed in the context of FIG. 5b. In step 15, molded replica
layer 360, formed in Step 12 above, is stacked upon intermediate
layer 340, produced as described in the context of FIG. 5b above,
and is subsequently aligned with respect to lower layers 340 and
318 such that channels 362 are registered and arranged in a desired
alignment with respect to channels 342 of layer 340 and channels
321 of layer 318 to provide a desired overall three-dimensional
fluidic network structure. Layer 360 is preferably sealed to layer
340 by utilizing one of the aligning and sealing methods previously
described in the context of Step 10 of FIG. 5b above.
[0103] As previously mentioned, in some preferred embodiments,
layer 340 is aligned with respect to layer 318 and layer 360 is
aligned with respect to layer 340 and the layers are sealed
together in a single step after alignment, which step, for such
embodiments, can take place at Step 15 of FIG. 5c. In such
embodiments, layer 340 would not be irreversibly sealed to layer
318 prior to the addition of layer 360 to the stack and alignment
of layer 360 with respect to layer 340 and 318. In such
embodiments, wherein layers 340 and 360 are both aligned and sealed
in a single step, the alignment and sealing methods utilized can be
essentially the same as those previously described for aligning and
sealing layer 340 to layer 318 in the context of Step 10 of FIG.
5b. In addition, in some embodiments where it is desired to
irreversibly seal together some portions of the surfaces of the
layers of the structures while leaving non-irreversibly sealed
other portions, such portions which are not desired to be
irreversibly sealed can be coated with a protective coating (e.g.
petroleum jelly) prior to oxidation in order to prevent oxidation
of that portion of the surface so that it will not irreversibly
seal to other oxidized surfaces upon contact.
[0104] Also provided, according to the invention, is a method for
self-aligning layers 318, 340, and 360 with respect to each other
to provide a desired alignment and registration of the channels
within each of the layers, without the need for manual alignment
with the aid of a microscope and/or micromanipulator. The
self-alignment method provided according to the invention can be
utilized for the embodiments described above wherein the layers are
oxidized and separated from each other by a layer of liquid during
alignment of the layers. Details of this self-alignment method are
described below in the context of FIG. 6 and rely on the
interaction between the surface tension of the liquid between the
layers and specific alignment features provided within the layers
being aligned.
[0105] Microfluidic network structure 370 obtained at the
conclusion of Step 15 of FIG. 5c can comprise, for some
embodiments, a complete structure, useful, for example, for
applications wherein it is desired that channels 362 in layer 360
remain uncovered and exposed to the surroundings. For example, one
particular embodiment utilizing a microfluidic network structure
similar in configuration to structure 370 involves utilizing the
microfluidic network structure as a stamping template for
selectively applying a fluid to a material surface to create a
pattern on the material's surface corresponding to the pattern of
channels 362 in layer 360. In such embodiments, surface 364 of
layer 360 comprises a stamping surface, which is placeable in
contact with a material surface for forming a pattern thereon, and
microfluidic network structure 370 comprises a three-dimensional
microfluidic stamp. Specific uses and patterns producible by such
microfluidic stamps are described in greater detail below.
[0106] For other embodiments where it is desired to form a
microfluidic network structure having an enclosed network of
channels, optional Step 16 of FIG. 5c involves contacting upper
surface 364 of layer 360 with an upper substrate layer 380 to form
enclosed microfluidic network structure 390. In some preferred
embodiments, where layers 318, 360, and 364 comprise PDMS, upper
substrate layer 380 is also comprised of PDMS and is irreversibly
sealed to surface 364 via the self-sealing method utilizing
oxidation of the PDMS surfaces with an oxygen-containing plasma
described in detail above. In alternative embodiments, however,
upper substrate layer 380 may simply be placed in conformal contact
with upper layer 364 and not irreversibly sealed thereto. In
addition, upper substrate 380, in some embodiments, is not formed
of the same material (e.g., PDMS) as layers 318, 360, and 364 of
the structure. Upper substrate 380 can be essentially any of the
materials mentioned previously for comprising substrate layer 320
previously described above in the context of FIG. 5a or any other
substrate which can contact surface 364 conformally.
[0107] In order to provide fluid communication between channels
contained within layers 318, 360, and 364 of structure 390 and the
surrounding environment, lower substrate layer 320 and/or upper
substrate layer 380 can include, formed therein, inlet/outlet
conduits 392 providing fluid communication between the channels of
the structure and the external environment. Conduits 392 can be
formed within substrate layer by a variety of machining and/or
molding methods, as would be apparent to those of ordinary skill in
the art. In one embodiment, the conduits 392 in substrate 320,
comprising PDMS, are formed by carefully boring into layer 320 with
a small diameter syringe needle. In other embodiments, substrate
layer 392 can itself comprise a replica molded structure with
conduits 392 corresponding to and formed by topological features
present on a surface of a mold master utilized to form substrate
layer 320. In addition, as would be apparent to those of ordinary
skill in the art, other features can be machined within, or molded
within one or both of substrate layers 320 and 380 to provide
various desired structures and functions for particular
applications. For example, upper substrate layer 380 as shown
includes traversing therethrough a small diameter channel 394,
having a characteristic cross sectional dimension on the order of a
few microns to a few tens of microns, which conduit 394 serves the
function of providing a relief valve to prevent over pressure of
the channels contained within the structure defined by layers 318,
340, and 360.
[0108] FIGS. 6a-6c illustrate one method for self-aligning various
layers of the microfluidic network structures with respect to each
other provided by the invention. The self-alignment method outlined
in FIGS. 6a-6c can be utilized for embodiments involving the
alignment and sealing methods discussed above involving disposing
layers of the structure separated from each other by a layer of
liquid disposed therebetween. Such a method is useful, for example,
for aligning layers 340 and 318 with respect to each other and
layers 360 and 340 with respect to each other in the previously
described microfluidic network fabrication method. In addition, the
self-alignment method described in FIGS. 6a-6c can also be utilized
for performing self-alignment in the context of the methods
described below in FIG. 7 and FIG. 10.
[0109] One embodiment for implementing the self-aligning method
provided according to the invention is illustrated in FIG. 6a. FIG.
6a shows a first layer 400 and a second layer 402 including therein
replica molded features 404 and 406 respectively, comprising, for
example, channels disposed within each of the layers, which
channels are desired to be registered and aligned with respect to
each other in a certain way. In the illustrated embodiment, a
plurality of self-alignment elements 408 are formed at selected,
predetermined locations within layer 400 and layer 402. In the
illustrated embodiment, self-alignment features 408 comprise
vertically disposed channels traversing, in some preferred
embodiments, essentially completely through layers 400 and 402 such
that upon bringing layer 402 into conformal contact with layer 400
upper surface 410 of layer 402 is in fluid communication with lower
surface 412 of layer 400 through vertically disposed channels
formed by the alignment of the self-alignment elements contained
within layers 400 and 402 respectively. In other embodiments, one
or more of the alignment elements may not completely traverse the
layer in which it is disposed, but may instead comprise an
indentation, bump, or other feature within or on the surface of the
layer.
[0110] In order to effect proper self-alignment, it is important
that layers 400 and 402 be essentially identical in size and
perimetric shape, when viewed in the x-y plane along the negative
z-axis direction as illustrated, such that the perimeter of layers
402 and 400 essentially identically overlap when the layers are
brought together into properly aligned conformal contact.
Optionally, in other embodiments, proper self-alignment can also be
effected if, instead of being essentially identical in size and
perimetric shape, one of the layers is much larger than the other
so that the meniscus of liquid formed around the edge of the
smaller layer does not change appreciably in total surface area
with small movements of the two layers with respect to each
other.
[0111] Self-alignment elements 408, in preferred embodiments, are
formed within layers 400 and 402, during the replica molding
process for forming the layers, by topological features provided
within the mold masters utilized for molding. Such topological
features can be positioned and located within the mold master
surface at selected, strategic positions with respect to features
within the mold master surface for forming channels 404 and 406
through use of a CAD computer program, such as described above for
designing the overall layout of the topological features for
forming the various channels within the replica molded layer
structures. Topological features forming self-alignment elements
408 are positioned with respect to topological features forming
channel structures 404 and 406 so that when layer 400 and layer 402
are superimposed such that alignment holes 408 are precisely
aligned with respect to each other, channel features 404 and 406
are also oriented with respect to each other in a desired
registered alignment.
[0112] FIG. 6b and FIG. 6c illustrate the manner by which alignment
holes 408 interact with a fluid layer 412 disposed between layers
400 and 402 to effect self-alignment. When self-alignment holes 408
and features 404 and 406 are properly aligned with respect to each
other, as shown in FIG. 6b, the layers are arranged in an
equilibrium position in which the interfacial area 414 between
fluid layer 412 and the surrounding gaseous environment is
minimized and there are no net capillary forces, due to the surface
tension of fluid layer 412, tending to change the position of layer
400 or layer 402 with respect to each other.
[0113] By contrast, when features 404, 406, and self-alignment
holes 408 are misaligned with respect to each other, as illustrated
in FIG. 6c, the interfacial area 414 between fluid layer 412 and
the surrounding gaseous atmosphere is increased with respect to the
interfacial surface area when the system is in its equilibrium
position as shown in FIG. 6b above, and there will be a net
resulting capillary force in the direction shown by arrow 416, due
to surface tension effects of fluid layer 412, tending to bring the
system into the equilibrium position illustrated in FIG. 6b.
[0114] In alternative embodiments, an essentially identical
self-aligning effect as illustrated in FIGS. 6a-6c can be achieved
without the need for forming self-alignment holes or features, such
as 408, in the layers which are to be self-aligned with respect to
each other. In such alternative embodiments, the layers can be
formed without self-alignment holes, such as 408, but instead be
formed or trimmed to have perimeter shapes, which are essentially
identical to each other, so that the layers when stacked upon one
another with a fluid layer therebetween, as illustrated in FIGS. 6b
and 6c, will have a minimum free energy equilibrium position
defined by an essentially precise and exact overlay of the
essentially identical perimetric shapes of the two layers. The
features comprising channels within the layers are, in such
embodiments, strategically positioned with respect to the
peripheral border of the layers, so that, when the layers are
aligned in the above-described minimum energy, no net capillary
force equilibrium position, the perimeters of the layers are
precisely superimposed upon each other and the features comprising
the channels within the layers are also similarly aligned with
respect to each other in a desired registration. FIG. 6d
illustrates one contemplated embodiment of a perimetric shape for
enabling the above-described self-alignment of various layers of
the structure without the need for alignment holes.
[0115] The above-described self-alignment techniques are able to
self-align a stack of as many individual layers as is desired,
essentially simultaneously and in parallel. The self-alignment
technique described herein is also capable of self-aligning
elements with respect to each other within a margin of error of
approximately +/-10 .mu.m or less, providing sufficient alignment
precision for most of the channel sizes and configurations
contemplated for the structures provided according to the invention
(e.g., channel structures having a cross-sectional dimension
ranging from about 20 .mu.m to about 500 .mu.m). The alignment
precision obtainable by the above-described self-alignment
technique is typically comparable or better than that obtainable
via manual alignment techniques utilizing a stereomicroscope and
conventional micromanipulation equipment.
[0116] The above-described self-alignment techniques are especially
well suited for embodiments involving alignment of oxidized PDMS
layers utilizing the above-described alignment/sealing method using
a non-reactive liquid disposed between and able to wet the oxidized
PDMS layers. However, those of ordinary skill in the art will
readily realized that the above-described self-alignment technique
can be utilized for aligning layers comprised of essentially any of
the suitable materials for forming the microfluidic system
discussed above and can be utilized for self-aligning layers that
are not reactive with respect to each other and do not become
essentially irreversibly sealed to each other upon contact but,
instead, are simply aligned in conformal, non-sealing contact with
each other. Those of ordinary skill in the art can readily select
appropriate liquids having desired surface-wetting properties (for
use in the self-aligning technique when utilizing the technique to
self align surfaces comprised of materials other than oxidized
PDMS) using no more than known, published surface wetting
properties for various liquids on various surfaces or routine
screening tests not requiring undue experimentation. In addition,
while the above-described self-alignment technique has been
exemplified in the context of aligning two replica molded layers of
the overall microfluidic structure with respect to each other. In
other embodiments, the technique can be utilized to align a replica
molded layer comprising one or more levels of the microfluidic
structure to the surface of a substrate, for example a silicon
microchip, or the like. Utilization of the self-aligning method for
aligning a layer of the microfluidic network to a substrate
surface, for example a surface of a silicon microchip, may be
important for applications where the microfluidic network is
utilized as an on-chip sensor, detector, analyzer, etc.
[0117] FIG. 7 illustrates an alternative embodiment for fabricating
a microfluidic network structure according to the invention. Unlike
the method previously described in the context of FIGS. 5a-5c, the
fabrication method described in FIG. 7 involves the formation, by
replica molding, alignment, and assembly of only two, as opposed to
three, discrete layers forming the three levels of the overall
microfluidic network structure.
[0118] As described above in the context of FIGS. 5a-5c, the method
outlined in FIG. 7 can potentially utilize a wide variety of
hardenable liquids for forming the replica molded components of the
microfluidic network structure. Such hardenable liquids were
described previously in the context of FIGS. 5a-5c. As previously,
in preferred embodiments, the replica molded structure is formed of
a polymeric material, more preferably an elastomeric material, and
most preferably a transparent elastomeric material. In a
particularly preferred embodiment illustrated and exemplified in
FIG. 7, the replica molded structures are formed of PDMS.
[0119] In Step 1 of the method illustrated in FIG. 7, a mold master
500 having a surface 502 including a series of topological features
504 thereon protruding from the surface in positive relief is
formed in a manner essentially equivalent to that described for
forming mold master 300 of FIG. 5a. Topological features 504 are
shaped, sized, and laid out on surface 502 in a pattern
predetermined to form a desired arrangement of channels disposed in
the upper, third level of the overall microfluidic network
structure. Mold master 502 is then placed in the bottom of a petri
dish or other container having a depth exceeding the height of the
upper surfaces of topological features 504 on surface 502.
[0120] In Step 2, a hardenable liquid is added to the container
containing master 500 in an amount sufficient to completely cover
and submerge topological features 504. As discussed in FIG. 5a
above, surface 502 of mold master 500, in preferred embodiments, is
treated with a release agent, for example a silanizing agent, to
permit release of the replica molded structure from the surface
without undue damage or distortion of the replica molded structure.
Also in Step 2, as described above in the context of FIGS. 5a-5c,
the hardenable liquid, for example a PDMS prepolymer solution, is
cured and solidified to form a solid molded replica 510 of surface
502 of mold master 500. Molded replica 510 is removed from surface
502 after curing as illustrated in Step 2. In the illustrated
embodiment, molded replica 510 comprises a PDMS slab which can, as
illustrated, be trimmed to a desired overall size and perimetric
shape. Molded replica 510 includes therein, but not completely
extending therethrough, a series of indentations 512 in lower
surface 514 corresponding to topological features 504 of mold
master 500. Indentations 512 form channels disposed within the
third, upper level of the overall microfluidic network to be
fabricated.
[0121] Steps 3 and 4 of the method illustrated in FIG. 7 comprise
the formation of a replica molded membrane layer including therein
both channels disposed in the first, lower level of the overall
microfluidic network structure and connecting channels of the
third, intermediate level of the overall microfluidic network
structure forming fluidic connections between the channels disposed
in the first, lower level and the second, upper level of the
structure. The molded replica membrane layer, having two levels of
features formed therein, is formed by a membrane sandwich
fabrication method (Steps 3 and 4) similar to the method previously
described in the context of FIGS. 5a-5c, except that mold master
520 includes a surface 522 having formed thereon a plurality of
topological features 524 in positive relief protruding from surface
522, that include features, for example feature 526, that are
two-level topological features, which are characterized by a first
portion 528 having a first height with respect to a region of
surface 522 adjacent to feature 526 and a second portion 530, which
is integrally connected to the first portion, having a second
height with respect to surface 522 adjacent feature 526, which
second height is greater than the height of first portion 528.
[0122] The term "integrally connected," as used herein in the
context of describing two-level topological features of mold
masters, refers to such features having at least a first portion
and a second portion, the second portion having a height or depth
with respect to the surface of the mold master adjacent the feature
different from the first portion, wherein the first and second
portion comprise two different regions of a continuous structure or
comprise two discrete structures each having at least one surface
in direct contact with at least one surface of the other. By
providing such two-level topological features on mold master 520,
the illustrated method allows simultaneous formation and alignment
of channels disposed within two levels of the overall microfluidic
network structure. Thus, by forming two levels of the overall
structure within a single layer in a single replica molding step,
the present method eliminates the need to align two discrete layers
comprising the first, lower level of the structure and the third,
intermediate level of the structure with respect to each other
after formation of the molded replica layers. Thus, as described
below, the present method requires only a single alignment step for
assembling the molded replica layers into the overall microfluidic
network structure.
[0123] A variety of photolithography and micromachining methods
known to those of ordinary skill in the art, which are capable of
forming features on a surface having multiple heights or depths
with respect to the surface, can potentially be utilized in the
context of the present invention for forming the two-level
topological features 526 of mold master 520. A particularly
preferred embodiment for forming mold master 520 involves an
inventive method for forming two-level topological features in
photoresist, and is described in more detail below in the context
of FIG. 8.
[0124] After formation of mold master 520, a layer of hardenable
liquid, for example PDMS, is placed upon surface 522 of mold master
520 and covered with an upper mold master 540, having a lower
surface 542 that is essentially flat and featureless, so that
surface 542 is in conformal contact with the uppermost surfaces of
the two-level topological features 526 on surface 522 of mold
master 520. As previously described in the context of FIGS. 5a-5c,
mold master 540 can comprise a variety of materials including, for
example, an elastomeric polymer slab, for example formed of PDMS, a
polymeric sheet, a flat silicon wafer, etc. In preferred
embodiments, as previously discussed, it is desirable that the
interfacial adhesion strength between surface 522 of mold master
520 and the hardened molded replica differ from the interfacial
surface adhesion between surface 542 of mold master 540 and the
hardened liquid comprising the molded replica. In the illustrated
embodiment, surface 522 of mold master 520 comprises a silanized
polymeric negative photoresist layer and mold master 540 comprises
a Teflon.TM. (PTFE) sheet.
[0125] In Step 4, pressure is uniformly applied to surface 544 of
upper mold master 540 and surface 546 of lower mold master 520 to
enable the upper surfaces 548 of topological features 526 to make
sealing contact with surface 542 of mold master 540 during the
hardening and curing process forming the replica molded membrane
layer. In Step 4, the hardenable liquid, for example PDMS
prepolymer, is cured to form a two-level replica molded membrane
550. Two-level replica molded membrane 550 includes a plurality of
first channels 552, disposed within a lower surface 554 of the
membrane, comprising channels disposed within the first level of
the overall microfluidic network structure, and also includes
vertically oriented connecting channels 554 that completely
penetrate the thickness of the membrane and interconnect lower
surface 554 and upper surface 556 of the membrane, forming the
connecting channels disposed within the third, intermediate level
of the overall microfluidic network structure. Channels 552
comprise replica molded features corresponding to first portions
528 of topological features 526 of mold master 520 and connecting
channels 555 comprise replica molded features corresponding to
second portions 530 of two-level topological features 526 of mold
master 520.
[0126] In the illustrated embodiment, the PDMS membrane comprising
molded replica layer 550 is separated from the mold masters by
first peeling PTFE sheet 540 from the upper surface 556 of the
membrane and subsequently peeling the membrane from upper surface
522 of mold master 520. In other embodiments, molded replica 550
can remain in contact with upper surface 522 of mold master 520
during the subsequent, and below described, aligning and sealing
steps, in order to support membrane 550 and prevent distortion or
destruction of the molded features therein. It should be
understood, that for more complex structures, additional replica
molded membranes such as 550 can be stacked upon each other in the
assembly of the microfluidic network structure to yield structures
having more than three levels of interconnected microfluidic
channels.
[0127] In the final step of fabrication, Step 5, replica molded
slab 510 and replica molded membrane 550 are aligned with respect
to each other to yield the desired microfluidic network structure,
brought into conformal contact with each other, and optionally
sealed together by methods previously described above in the
context of FIGS. 5a-5c to yield the final microfluidic network
structure 560. As previously described, the structure 560 can
include inlet conduits 562 and outlet conduits 564 for each of the
non-interconnected fluid flow paths disposed within the structure,
or other interconnections between the flow paths within the
structure and the external environment as required or desired for a
particular application. In the illustrated embodiment, microfluidic
network structure 560 includes three non-fluidically interconnected
fluid flow paths therein. The first flow path 561 has an inlet and
outlet in the foreground and is shaded light gray; the second flow
path 563 has an inlet and outlet that are centrally disposed shaded
in black; and the third flow path 565 has an inlet and outlet in
the background and is shaded dark gray.
[0128] In addition, lowermost surface 554 of structure 560 includes
therein a pattern indentations corresponding to the channels of the
first, lower level of the microfluidic network structure formed
within the bottom surface 554 of the replica molded membrane 550.
Thus, microfluidic network structure 560 is useful for embodiments
wherein the microfluidic network structure is utilized as a surface
patterning stamp for depositing materials onto a material surface
in a pattern corresponding to the channels disposed within surface
554, or otherwise creating a patterned surface with a pattern
corresponding to the pattern of the channels disposed within
surface 554. In alternative embodiments, surface 554 can be placed
in conformal contact with, and optionally sealed to, a solid PDMS
slab, or other substrate or surface, to form an enclosed
microfluidic network structure, as described previously in the
context of FIGS. 5a-5c.
[0129] FIG. 8 illustrates a preferred method for preparing mold
masters that have a surface including thereon one or more two-level
topological features. While the illustrated method is useful for
forming two-level topological features in layers of either negative
or positive photoresist materials, in the embodiment illustrated, a
negative photoresist material (e.g., SU8-50) is utilized as an
example. In addition, while, in the illustrated embodiment,
two-level topological features comprising positive, high-relief
features protruding from the surface of the mold master are
fabricated, it should be understood that the method is also well
suited to produce two-level topological features comprising
negative, low-relief features characterized by indentations,
grooves, or channels within the surface of the mold master. Any
variations in the below described technique for producing two-level
positive, high-relief features in negative photoresist that are
required in order to produce two-level features in positive
photoresist and/or to produce two-level features comprising
negative, low-relief features involve only simple extensions of the
below-described method that would be apparent to those of ordinary
skill in the art.
[0130] In Step 1 of the method illustrated in FIG. 8, a silicon
wafer 600, or other suitable substrate, is coated with a layer of
photoresist 602, by a conventional spin-coating technique or other
suitable coating technique known to those of ordinary skill in the
art. Layer 602 is spin-coated to a depth corresponding to the
desired depth of the deepest feature to be formed on the first
level of the mold master (e.g. a depth corresponding to the deepest
channel to be disposed in the level of the microfluidic channel
structure to be replica molded by the first level of the mold
master. The thickness of layer 602 will typically range from about
20 .mu.m to about 500 .mu.m, and can, in some embodiments, be as
thick as about 1 mm.
[0131] In Step 2, the photoresist is "soft baked" by being exposed
to an elevated temperature for a short period of time to drive off
solvent used in the spin-coating process For example, for SU8-50
negative photoresist, the coated substrate is exposed to a
temperature of about 95-105.degree. C. for a period of several
minutes. In Step 3, a first photomask 604 including thereon a
pattern 606 corresponding to features 626 of the first level of the
mold master is placed in contact with negative photoresist layer
602. As would be apparent to those of ordinary skill in the art, a
wide variety of photomasks can be utilized according to the present
inventive method; however, in a preferred embodiment illustrated,
photomask 604 comprises a high resolution transparency film having
a pattern printed thereon. Designs for the channel system printed
upon the high resolution transparency are preferably generated with
a CAD computer program. In the illustrated embodiment, a
high-resolution (e.g., 3000-5000 dpi) transparency, which acts as
photomask 604, is produced by a commercial printer from the CAD
program design file. In the illustrated embodiment, essentially the
entirety of photomask 604 is rendered opaque to the radiation used
to expose the photoresist by a layer of toner, and the fluidic
channel-forming features to be formed on the surface of negative
photoresist 602 correspond to transparent regions 606 of the
photomask surface.
[0132] In addition to regions 606 corresponding to features in the
mold master for forming fluidic channels within the molded replica
structure formed with the mold master, photomask 604 also includes
peripheral transparent regions 608, which correspond to topological
features for forming alignment tracks useful for aligning the mold
masters with respect to each other in certain methods for forming
microfluidic structures as described in more detail below in FIGS.
9a and 9b.
[0133] In Step 4, upper surface 603 of photoresist layer 602 is
exposed to radiation, for example ultraviolet (UV) radiation of a
frequency and intensity selected to cross-link exposed areas of the
negative photoresist, through the transparent regions of the
printed pattern of photomask 604. In Step 5, after exposure to
cross-linking radiation, the first photomask 604 is removed from
the surface, the photoresist is hard-baked (e.g. at about
95-105.degree. C. for several minutes) and a second layer of
photoresist is spin-coated on top of surface 603 of photoresist
602. The second layer of photoresist is spin-coated to a thickness
sufficient for forming features in the mold master corresponding to
the connecting channels disposed within the third, intermediate
level of the replica molded microfluidic network structure formed
with the mold master. Typically, the thickness of the second level
of photoresist will range from about 20 .mu.m to about 1 mm. Wafer
600, now containing a first, exposed layer of photoresist and a
second layer of unexposed photoresist can then be subject to
another soft-baked procedure to drive off solvent from the
unexposed layer of photoresist, similarly as described in Step 2
above.
[0134] As illustrated in Step 5 of FIG. 8, regions of the first
layer of photoresist that were exposed to the radiation (e.g.,
regions 610 and 612) typically exhibit a change in the degree of
transparency and/or refractive index of the photoresist, thus
rendering them visible through the upper layer of newly spin-cast,
unexposed photoresist. This visibility allows a second photomask to
be easily aligned with respect to the first exposed pattern by
using a standard photomask aligner. In other embodiments,
especially where the exposed pattern may not be visually apparent,
visible alignment features or elements can be included on the
surface of wafer 600 to enable alignment of the second photomask to
achieve a desired two-level pattern, as would be apparent to those
of ordinary skill in the art.
[0135] In Step 6, a second photomask 614 including thereon printed
patterns 616, corresponding the second level portions of the
two-level topological features of the mold master, which form the
connecting channels in the intermediate level of the replica molded
microfluidic network structure formed with the mold master, and
618, corresponding to a second level of the optional alignment
tracks. It should be understood, that while, in the illustrated
embodiment, features 606 corresponding to topological features for
forming channels disposed in the first level of the microfluidic
network structure comprise linear features, in other embodiments,
features 606 can be non-linear, thus forming curved topological
features resulting in non-linear, curved channels within the first
level of the microfluidic structure. Similarly, any of the
previously described structures and methods for forming channels
disposed within a particular level of microfluidic network
structure can include channels that are non-linear and curved
within the plane or curved surface defining the level of the
microfluidic network structure in which the channels are disposed
in addition to, or instead of, the straight channels previously
illustrated.
[0136] Printed pattern 616, creating topological features for
forming channels within the microfluidic network structure can
also, in some embodiments, include features parallel and contiguous
with regions 610 formed within the first layer of photoresist and
corresponding to printed pattern 606, such that some of the
topological features produced on the surface of the mold master by
the illustrated method include features that form channels having a
longitudinal axis parallel to the first level of the replica molded
microfluidic network structure formed with the mold master, and
which have an overall depth within the replica molded microfluidic
network structure formed with the mold master, which is equal to
the combined depth of the first level and the third, intermediate
level of the structure (i.e., for forming replica molded
microfluidic network structures having deep channels that are
disposed within both the first level and the third, intermediate
level of the microfluidic network structure).
[0137] Photomask 614 is aligned in Step 6 with respect to exposed
pattern 610 and the second, unexposed layer of photoresist is
exposed, in Step 7, to the cross-linking radiation through
photomask 614. Following exposure, mask 614 is removed from the top
layer of photoresist, and the photoresist is hard-baked as
described above. If desired, the above-mentioned steps can be
repeated with additional layers of photoresist and additional
photomasks to produce more than two levels of topological features
on the surface of wafer 600. After the desired number of layers of
photoresist have been coated onto wafer 600 and exposed to
cross-linking radiation as described above, the relief pattern is
developed in Step 8 by exposing the photoresist to a developing
agent that dissolves and removes non-cross-linked photoresist
material leaving behind a mold master 620 having a surface 622
including thereon a pattern of two-level high relief features 624
having a first portion 626 with a first height above surface 622
and a second portion 628 having a second height above surface 622,
which is greater than height 626. First portion 626 of the
topological features forms the channels disposed within the first
level of the replica molded microfluidic network structure formed
with mold master 620, and second portion 628 of the topological
features forms the connecting channels traversing the third,
intermediate level of a microfluidic network structure replica
molded using mold master 620.
[0138] Also formed on surface 622 of mold master 620 by the
above-outlined process are alignment tracks 630 having a height
corresponding to the height of the second portion 628 of
topological features 624. While, in the illustrated embodiment, the
second layer of photoresist was spin-coated onto a first layer of
exposed photoresist before developing the first layer, in an
alternative embodiment, the first layer of photoresist can be
developed before spin-coating the second layer of photoresist if
desired. Solvents useful for developing the unexposed portions of
the photoresist are selected based on the particular photoresist
material employed. Such developing agents are well known to those
of ordinary skill in the art and are typically specified by the
commercial manufacturers of many of the photoresists useful for
performing the methods of the invention. For example, for the
illustrated embodiment utilizing SU8-50 negative photoresist,
uncross-linked photoresist can be removed during development by
exposing the photoresist to propylene glycol methyl ether acetate.
Two-level mold master 620, subsequent to formation as described
above, is preferably coated with a release agent, for example by
silanizing the surface, in order to facilitate removal of a molded
replica from the surface of the mold master.
[0139] FIGS. 9a and 9b illustrate the steps of a third embodiment
of the method according to the invention for fabricating a
three-dimensional microfluidic network structure. The method
illustrated in FIGS. 9a and 9b comprises a membrane sandwich
technique similar to that previously described in Steps 3 and 4 of
the method illustrated in FIG. 7, except that instead of forming a
replica molded membrane layer between a bottom master including
two-level topological features and a top mold master having an
essentially flat, planar surface, as was illustrated in the method
of FIG. 7, in the method according to FIGS. 9a and 9b, a replica
molded membrane layer is formed between two mold masters, both
including topological features thereon and at least one including
at least one two-level topological feature thereon, thus yielding a
replica molded membrane including therein a microfluidic network
structure containing all three of the above-discussed levels. In
some embodiments, both the upper and lower mold masters utilized
for forming the three-level replica molded membrane layer according
to the embodiment of FIGS. 9a and 9b can comprise mold masters, for
example similar to mold masters 500 and 520 shown in FIG. 7.
However, as previously discussed, it is desirable for at least one
of the mold masters to be formed of an elastomeric material to
improve sealing contact between portions of the surfaces of the
mold masters that are in contact during the replica molding process
so as to prevent undesirable leakage of hardenable liquid into such
regions of contact. Therefore, in preferred embodiments, the upper
mold master and/or lower mold master are formed from an elastomeric
material having a surface with topological features thereon.
[0140] In some particularly preferred embodiments, elastomeric mold
masters are formed using a replica molding procedure, similar to
that used to form the various layers of the microfluidic structure,
to form topological features on the elastomeric mold master that
are formed during replica molding from topological features on a
pre-master prepared by photolithography or micromachining. The
method illustrated in FIGS. 9a and 9b correspond to such a
preferred embodiment. In the illustrated embodiment, the top mold
master, as well as the replica molded membrane layer, are formed
from an elastomeric material comprising PDMS. As referred to and
discussed extensively above, PDMS, while being preferred for
forming many of the structures and mold masters according to the
invention, comprises only one example of a material formable from a
hardenable liquid useful for forming the mold masters and
microfluidic networks according to the invention. A wide variety of
alternative materials and hardenable liquids have been previously
discussed in the context of the methods illustrated in FIGS. 5 and
7, and such materials, or other materials apparent to those of
ordinary skill in the art, can be substituted for PDMS in the
method illustrated in FIGS. 9a and 9b below.
[0141] FIG. 9a illustrates one preferred method for forming an
elastomeric top mold master for use in forming a three-level
replica molded membrane layer. In Step 1, a pre-master mold is
fabricated by forming topological features on a surface of a
substrate 700, for example as previously illustrated in the context
of FIG. 8. Since, in the illustrated embodiment, it is desired that
the topological features formed in the replica molded top mold
master comprise positive, high-level relief features protruding
from the surface of the mold master, the topological features
formed on surface 702 of substrate 700 comprise negative, low-level
relief features characterized by grooves or channels 704, 706 seen
more clearly in the cross-sectional view. In the illustrated
embodiment, pre-master mold 700 is fabricated using a two-level
photolithography technique similar to that described in FIG. 8.
Topological features 706 have a greater depth than topological
features 704 and essentially traverse the entire thickness of
photoresist layer 708. In the illustrated embodiment, topological
features 706 correspond to and form topological features in the
replica molded elastomeric mold master which are alignment tracks,
whose function is explained in more detail below. Topological
features 704 correspond to and form topological features in the
replica molded mold master which are responsible for forming
channels ultimately disposed in the second, upper level of the
replica molded three-level membrane layer. It should be understood
that in alternative embodiments, one or more of topological
features 704 can comprise two-level topological features having a
first portion with a first depth with respect to surface 702 and a
second portion with a second, greater depth (e.g. corresponding to
the depth of topological features 706) with respect to surface 702.
For such embodiments, a replica molded top mold master would
include two-level topological features in positive relief for
forming channels disposed in the second, upper level of the replica
molded membrane as well as connecting channels traversing the
membrane. In such embodiments, the lower mold master can include
channel-forming topological features having a single, uniform
height or can include channel-forming topological features that are
also two-level topological features.
[0142] In Step 2, pre-master mold 700 is placed into the bottom of
container 712. The container is then filled with a hardenable
liquid, such as PDMS prepolymer, to a level at least covering upper
surface 702 of pre-master mold 700. Subsequently, the hardenable
liquid is cured or solidified, as previously discussed, and, in
Step 3, is removed from the pre-master mold, optionally trimmed,
and treated with a release agent, for example by silanization or
oxidation followed by silanization. The resulting structure 720
comprises a replica molded mold master including a surface 722
having disposed thereon topological features 724 at a first height
with respect to surface 722 and corresponding to topological
features 704 of pre-master 700, and topological features 726 having
a second, greater height with respect to surface 722 and
corresponding to topological features 706 on pre-master 700.
Topological features 724 comprise channel-forming features and
topological features 726 comprise alignment tracks.
[0143] FIG. 9b illustrates steps for forming the replica molded
three-level membrane layer with the upper mold master 720 produced
according to the steps outlined in FIG. 9a above and a lower mold
master 620 produced according to the method outlined previously in
FIG. 8. In Step 4, a quantity of hardenable liquid 310, for example
PDMS prepolymer, is placed in contact with upper surface 622 of
lower mold master 620 in an amount sufficient to form a layer
having a thickness at least equal to the height of topological
features 628 and 630. Upper mold master 720 is then brought into
contact with lower mold master 620 in Step 5 and is manually
manipulated until topological features 726 comprising alignment
tracks in the upper mold master mate and interdigitate with
topological features 630 comprising alignment tracks in the lower
mold master. Upon mating and interdigitating of alignment tracks
726 and 630, the alignment and relative position of channel-forming
topological features 724 of the upper mold master and
channel-forming topological features 624 of the lower mold master
is such that they are properly positioned and aligned with respect
to each other to form the desired three-dimensional microfluidic
network channel structure within the replica molded membrane layer.
The interface between the upper mold master 720 and lower mold
master 620 during the replica molding process in Step 5 is seen
more clearly in the cross-sectional view. The cross-sectional view
illustrates that, upon proper alignment, alignment tracks 726 of
upper mold master 720 mate and interdigitate with alignment tracks
630 in lower mold master 620. In addition, the cross-sectional view
also clearly illustrates the conformal, sealing contact made
between channel-forming feature 725 in upper mold master 720 and
the upper surface of second portions 628 of the topological
features on the surface of the lower mold master.
[0144] In Step 6, hardenable liquid 310, for example PDMS
prepolymer, is cured, as previously described and upper mold master
720 is peeled away from lower mold master 620. In the illustrated
embodiment, where upper mold master 720 comprises silanized PDMS,
lower mold master 620 has an upper surface 622 comprising polymeric
photoresist and hardenable liquid 310 comprises PDMS prepolymer,
the replica molded PDMS membrane layer 730 formed upon curing will
adhere more strongly to surface 722 of upper mold master 720 than
to surface 622 of lower mold master 620 and, upon peeling away of
upper mold master 720, will remain adhered to and supported by
upper mold master 720, thus preventing damage to the membrane.
[0145] Replica molded membrane layer 730 includes therein channels
732 disposed within lower surface 734 of membrane 730, formed by
first portion 626 of topological features 624 of lower mold master
620; upper channels 736 disposed within upper surface 738 of the
membrane, formed by topological features 724 of the upper mold
master; and connecting channels 740 traversing the membrane and
interconnecting surface 734 and surface 738, which interconnecting
channels are formed by second portions 628 of two-level topological
features 624 of lower mold master 620. Thus, in the presently
described method, a single replica molded layer is formed that
includes therein all three levels required to form a
three-dimensional microfluidic network structures according to the
invention. In addition, because of the provision of alignment
tracks 726 and 630, the entire three-dimensional network structure
was formed without the need for performing an alignment of features
or channels requiring the use of a microscope or micromanipulator.
Because the present method does not require visual alignment of
features or channels, it can be especially useful for forming
microfluidic membrane structures from materials that are opaque to
visible light.
[0146] When, as illustrated, the three-level membrane is formed by
utilizing one mold master formed via a photolithographic or
micromachining technique (e.g. mold master 620) together with an
elastomeric mold master (e.g. 720), which is formed by replica
molding a pre-master mold formed via a photolithographic or
micromachining technique (e.g. pre-master 700), if the hardenable
liquid utilized to form the replica molded mold master (e.g. as
illustrated in Step 2 of FIG. 9a) has a tendency to shrink during
hardening, this shrinkage should be taken into account when sizing
and positioning the topological features of the pre-master, so that
topological features of the replica molded mold master will
properly match those of the other mold master to yield the desired
alignment of channels. For example, when PDMS is used to form one
mold master, it has been found that the size and relative spacing
of the features in the pre-master should be increased by about
0.66% over that desired for the final PDMS mold master in order to
account for shrinkage of the mold master during curing of the
pre-polymer.
[0147] Replica molded polymeric membrane 730 can be removed from
upper mold master 720 and can be utilized as a stand-alone
structure or can be stacked with other such structures to form more
complex networks. Optionally, and as shown in Step 7, before
removal from upper mold master 720, lower surface 734 of membrane
730 can be brought into conformal contact with a lower substrate
layer 750, for example, a flat piece of PDMS, silicon wafer,
microchip, or other substrate, and can optionally be sealed thereto
as previously described. Substrate layers, instead of having flat
smooth surfaces as illustrated, can, in other embodiments, include
topological features thereon that are matable with topological
features on the surface of the replica molded membrane, for
example, alignment tracks 739, so that, upon interdigitation of the
matable topological features on the substrate layer and one or more
topological features on the surface of the replica molded membrane,
the membrane is aligned and oriented in a desired configuration
with respect to the substrate.
[0148] After contacting the membrane with the substrate layer and,
optionally, essentially irreversibly sealing the membrane to the
substrate layer, upper mold master 720 can then be removed from
upper surface 738 of membrane 730 as illustrated in step 8. The
resulting microfluidic network structure 760 can be utilized as
shown or after trimming away the regions of the membrane including
alignment tracks 739. Structure 760 is useful, for example, as a
microfluidic membrane stamp for patterning a material surface, the
stamping surface comprising upper surface 738 of membrane 730,
which has channels 736 disposed therein. Structure 760 is also
useful for embodiments wherein the microfluidic network structure
is utilized as a mold in which to form three-dimensional networks
of materials having a structure corresponding to the channel
structure in membrane 730, as described in more detail below.
[0149] For embodiments where it is desired to provide an enclosed
series of microfluidic channels, upper surface 738 of membrane 730
is subsequently placed in conformal contact with and, optionally
sealed to, an upper substrate layer 770. Upper substrate layer 770
can comprise a slab of PDMS or other substrate layer desirable for
a particular application, as previously discussed. Also, as
previously discussed, inlet and outlet conduits can be formed
within either or both of substrate layers 770 and 750 in order to
interconnect the fluid flow paths of the microfluidic channel
structure to the external environment.
[0150] FIG. 9c illustrates a modification of the embodiment for
fabricating the three-dimensional microfluidic structure, as
illustrated in FIGS. 9a and 9b. In the modification illustrated in
FIG. 9C, the upper and lower mold masters utilized for forming the
three-level replica molded membrane layer each include two-level
topological features thereon for forming the connecting channels
traversing the replica-molded membrane.
[0151] The two-level features of the upper and lower mold masters
that form the connecting channels through the membrane are
configured to have complementary, mateable shapes, such that when
the mold masters are placed together during the replica molding
step (e.g., step 5 as illustrated in FIG. 9b), the mateable,
channel-forming topological features on the upper and lower mold
masters will mate/interdigitate with each other, for example, as
shown in FIG. 9c(iv). Providing such mateable, connecting
channel-forming features can reduce any tendency of the hardenable
liquid for forming the replica molded membrane to be incompletely
excluded from the regions forming the connecting channels during
the molding process, which incomplete exclusion can lead to the
formation of an undesirable, thin layer of hardened polymer
occluding the connecting channels after molding. In the modified
embodiment illustrated, wherein the two-level topological features
of the upper and lower mold master that form the connecting
channels are configured with shapes that are mateable with each
other, the hardenable liquid can be more effectively and thoroughly
excluded from the region molding the connecting channels, thus
effectively eliminating any tendency to form a thin film of
hardened material occluding the connecting channels upon formation
of the membrane.
[0152] In addition, the mateable, connecting channel-forming
two-level topological features can also serve a purpose similar to
that of the alignment tracks discussed above. Namely, upon mating
or interdigitation of the mateable, connecting channel-forming
features of the upper and lower mold master, the alignment and
relative position of the various other channel-forming topological
features of the upper and lower mold master will be properly
positioned and aligned with respect to each other to form the
desired three-dimensional microfluidic network channel structure
within the replica molded membrane layer. Also, relative motion
between the upper and lower mold masters, leading to misalignment,
during the replica molding step can be reduced or eliminated.
Accordingly, although the alignment track-forming features are
illustrated in the modified embodiment shown in FIG. 9c, because
the mateably-shaped connecting channel-forming topological features
of the upper and lower mold master can perform essentially the same
function and fulfil essentially the same purpose, in some
embodiments utilizing the modified mold masters, the alignment
track-forming features could be eliminated.
[0153] FIG. 9c(i) and (ii) illustrate a modified pre-master mold
781 for forming the replica molded upper mold master 782 that
includes the two-level connecting channel-forming topological
features configured to mate/interdigitate with complementary
connecting channel-forming features in the lower mold master.
Pre-master mold 781 can be fabricated by forming topological
features on surface 702, for example as previously illustrated in
the context of FIGS. 8 and 9a. As previously described in the
context of FIG. 9a, since it is desired that the topological
features formed in the replica molded top mold master comprised
positive, high-level relief features protruding from the surface of
the mold master, the topological features formed on surface 702
comprise negative, low-level relief features characterized by
grooves or channels 704, 706, and 783. In the illustrated
embodiment, pre-master mold 781 is fabricated using a two-level
forming photolithography technique similar to that described above
in FIG. 8.
[0154] One-level channel-forming feature 704, and two-level
alignment track forming feature 706 are essentially identical to
those previously described in the context of FIG. 9a. In contrast
to the embodiment illustrated previously in FIG. 9a, however,
pre-master mold 781 includes a topological feature 784
corresponding to and forming a topological feature in the replica
molded mold master, which is responsible for forming a channel
ultimately disposed in the second, upper-level of the replica
molded three-level membrane layer, which feature 784 includes, and
is bounded by, topological features 783, which are configured to
form connecting channel-forming features in the replica molded
upper mold master that will have a shape that is mateable to
complementary connecting channel-forming features in the lower mold
master. Topological feature 783, shown in cross-section, comprises
an outer ring 785 in two-level negative relief surrounding a
central post 786, the ring and post together forming a
"donut"-shaped two-level annulus.
[0155] FIG. 9c(ii) illustrates the resulting upper mold master
formed by replica molding pre-master 781, as discussed previously
in the context of FIG. 9a, Step 2. The resulting structure 782
comprises a replica molded mold master including a surface 722
having disposed thereon topological features 724 and 787 at a first
height 791 with respect to surface 722 corresponding to topological
features 704 and 784, respectively, of pre-master 781, and
topological features 726 having a second, greater height 794 with
respect to surface 722 and corresponding to topological features
706 on pre-master 781. Upper mold master 782 also includes
topological features 788, corresponding to topological features 783
of pre-master 781, features 788 including a central hole region
790, in which the molded material comprising the mold master
extends to a position at the first height 791 with respect to
surface 722, and an outer peripheral ring 789 having a second,
greater height 794 with respect to surface 722. Topological
features 788 comprise two-level, connecting channel-forming
features, having a shape that is mateable to corresponding features
on the lower mold master.
[0156] The lower mold master 792, illustrated in FIG. 9c(iii) is
substantially similar to lower mold master 620 illustrated and
discussed previously in the context of FIGS. 8 and 9b; however, the
second portions (e.g., portions 628 as illustrated in FIG. 9b) of
two-level topological features 626 of FIG. 9b, which are now called
out by figure label 793, are somewhat smaller in diameter than
those illustrated in FIG. 9b, and are sized and positioned to mate
and interdigitate with holes 790 of interconnecting channel-forming
topological features 788 of upper mold master 782, when the mold
masters are brought together and alligned for forming the
three-level microfluidic membrane as illustrated in FIG.
9c(iv).
[0157] It should be understood that while, in the illustrated
embodiment, the shape of the matable connecting channel-forming
topological features of the upper mold master comprises a circular,
donut-shape annulus, and that of the lower mold master connecting
channel-forming topological features comprises a post, in other
embodiments, this configuration could be reversed such that the
annulus-shaped features are present on the lower mold master and
the posts are present on the upper mold master. In addition, in
other embodiments, upper mold master 782, as discussed previously,
need not be a replica molded elastomeric structure, but instead
could comprise a mold master formed in photoresist, or other
material, for example similar to lower mold master 792, which could
be formed by, for example a micro-machining technique or, more
preferably, as previously discussed in the context of FIG. 8.
[0158] It should also be understood that while the mateable,
interconnecting channel-forming features illustrated in the present
embodiment comprise a circular cylindrical post-annulus
arrangement, in other embodiments, the interdigitating, mateable
shapes of the interconnecting channel-forming features of the upper
and lower mold masters could be selected from an extremely wide
variety of suitably mateable shapes. For example, instead of a
circular post mating with an annulus having a circular
centrally-disposed bore therein, a variety of alternative
cylindrical shapes could instead be utilized, for example squares,
triangles, rectangles, n-sided polygons, ovals, etc. Alternatively,
mateable configurations other than a post-annulus configuration, as
illustrated, could be employed. For example, one of the mold
masters could include interconnecting channel-forming features
including a slot element that is mateable with a corresponding
groove element in the interconnecting channel-forming features of
the other mold master, or, alternatively, one mold master could
provide interconnecting channel-forming features including a half
cylinder-shaped element with the other mold master also providing
interconnecting channel-forming features including half
cylinder-shaped elements, which half cylinders-shaped elements of
the first and second mold masters to mate together to together form
cylindrical interconnecting channel-forming features. Those of
ordinary skill in the art will readily envision a wide variety of
such mateable shapes and configurations suitable for use in the
present context and providing substantially equivalent function and
performance as described above. Each of such alternative
configurations is deemed to be an equivalent structure falling
within the scope of the present invention.
[0159] FIG. 10 illustrates a method for forming the five-level
microfluidic network structure, shown previously in FIG. 4a,
comprising a coiled network of interconnected channels forming a
first fluid flow path surrounding a straight channel forming a
second fluid flow path. The method in FIG. 10 is based upon the
methods previously described in FIGS. 8, 9a, and 9b discussed
above. In the method shown in FIG. 10, two separate molded replica
membrane layers are formed, which are subsequently aligned with
respect to each other and sealed together to form the final,
overall, five-level coiled network structure 220. The first molded
replica membrane layer 800 comprises three levels of the overall
structure and a second molded replica membrane layer 810 comprises
the remaining two levels of the overall microfluidic network
structure. Molded replica layer 800 comprising three levels is
formed by the membrane sandwich method previously discussed in the
context of FIG. 9b and utilizing a lower mold master 802 having
formed thereon a plurality of two-level topological features 804
having first portions 806, forming channels 807 disposed within the
first, lowermost level of the overall microfluidic network
structure, and second portions 808 forming connecting channels
traversing the level adjacent to and positioned immediately above
the lowermost level of the microfluidic network structure upon
replica molding.
[0160] Upper mold master 812 is preferably a replica molded
elastomeric material (e.g. like mold master 720) and includes a
bottom surface 814 having a plurality of single-level topological
features 816 protruding therefrom including a centrally disposed
feature 818, forming the straight channel 819 disposed on the
second, upper level of membrane layer 800, and a plurality of
features 820, aligned with second portions 808 of topological
features 804 of lower mold master 802, forming a continuation of
connecting channels 821 through the second, upper level of replica
molded layer 800 upon replica molding. Molded replica layer 810,
comprising the two uppermost levels of the overall structure, is
formed by the same membrane sandwich method utilizing lower mold
master 802 and an upper mold master 830, which comprises a flat
slab of preferably elastomeric material. Two-level topological
features 804, having first portions 806 and second portions 808,
form a series of channels 832 disposed within lower surface 834 of
molded replica layer 810 and form connecting channels 833
traversing the thickness of molded replica layer 810, upon replica
molding of layer 810.
[0161] In order to complete the assembly and form the overall
coiled microfluidic network structure 220, molded replica layer 810
is rotated 180.degree. in the direction of arrow 836, stacked on
top of molded replica layer 800, aligned so that the replica molded
channels are registered to form the desired coiled channel network
structure, brought into conformal contact with, and optionally
sealed to molded replica layer 800. Optionally, surface 834 of
molded replica layer 810 and/or surface 809 of molded replica layer
800 can be brought into conformal contact with, and optionally
sealed to, a substrate layer (e.g., 838, 839) prior to or
subsequent to stacking, aligning, and, optionally, sealing layers
800 and 810 to each other. If desired, excess material comprising
layers 800 and 810 can be trimmed from the structure as illustrated
in the final step of FIG. 10. The resulting structure 220 includes
the coiled, two fluid flow path microfluidic network previously
described in detail in the context of FIG. 4a above.
[0162] In addition to being useful as fluid flow directing networks
for applications requiring fluid management in very small scale
devices, for example, in micro total analysis systems (.mu.TAS),
the microfluidic network structures provided according to the
invention are also useful for a variety of other uses. For example,
microfluidic channel systems fabricated according to the invention
can be used to fabricate a variety of microstructures having
three-dimensional structures corresponding to a three-dimensional
network of channels within a microfluidic network structure. Such
microstructures can be formed by filling the channel network of the
microfluidic systems with a hardenable liquid, solidifying the
hardenable liquid within the network channels, and, optionally,
removing the surrounding microfluidic network structure to yield a
free-standing microstructure comprised of the solidified hardenable
liquid. The hardenable liquid utilized for form microstructures
that are replica molded within the inventive microfluidic network
systems can comprise essentially any of the hardenable liquids
described above as being useful for forming the microfluidic
network structures themselves. The hardenable liquids chosen to
form the replica molded microstructures should be chemically
compatible with the microfluidic network structure and, for
embodiments where it is desired to selectively remove a surrounding
microfluidic network structure, should be resistant, once hardened,
to whatever treatment is required to dissolve or otherwise remove
the surrounding microfluidic network structure. In one particular
illustrative example, a microfluidic network structure produced
according to the invention and composed of PDMS can be filled with
an epoxy prepolymer, so that the epoxy prepolymer essentially
completely fills the microfluidic channel structure of the
microfluidic network. The epoxy prepolymer can then be cured, for
example by exposure to ultraviolet light through the surrounding
PDMS microfluidic channel structure, in order to cure the epoxy
prepolymer and form a solid microstructure within the channels. The
surrounding PDMS microfluidic network can then be dissolved, for
example with tetrabutylammonium fluoride (1.0 M in tetrahydrofuran)
leaving behind a free-standing microstructure, comprised of epoxy
polymer, having a three-dimensional structure corresponding to the
three-dimensional network of channels in the PDMS microfluidic
channel structure.
[0163] In another illustrative application for certain microfluidic
channel structures provided by the invention, the microfluidic
channel structure is used as a three-dimensional microfluidic
applicator or "stamp" for forming a pattern on a material surface
corresponding to a pattern of channels disposed in one level of the
microfluidic network structure. The "stamping surface" of such
structures includes disposed therein a series of channels forming
indentations, which channels can deliver material to a substrate
surface in contact with the "stamping surface" in order to form a
pattern thereon corresponding to the pattern of channels in the
stamping surface. Examples of structures discussed previously
having "stamping surfaces" are microfluidic channel structure 560
illustrated in FIG. 7 having a stamping surface 554, and
microfluidic channel structure 760 illustrated in FIG. 9b having a
stamping surface 738.
[0164] The method for patterning a material surface with a
microfluidic network structure provided according to the invention
comprises contacting a stamping surface of the microfluidic network
structure with a material surface to be stamped, and, while
maintaining the stamping surface in contact with the material
surface being stamped, at least partially filing one or more flow
paths of the microfluidic channel structure with a fluid so that at
least a portion of the fluid contacts the material surface.
Subsequently, if desired, the stamping surface can be removed from
the material surface, yielding a pattern on the material surface,
according to the pattern of channels disposed within the stamping
surface, formed by contact of the material surface with the
fluid.
[0165] One example of such a stamped pattern is illustrated in FIG.
11. The microfluidic stamp utilized for forming the pattern in FIG.
11 was previously illustrated in FIG. 1a. In forming the pattern in
FIG. 11, microfluidic network 100 (FIG. 1a) is formed so that lower
surface 134 is configured as a stamping surface, with the channels
disposed therein comprising indentations within the surface exposed
to the external environment. For embodiments wherein the
microfluidic network structures are utilized as stamps/applicators,
it is especially preferred that the microfluidic network structures
be formed of an elastomeric material, so that the stamping surface
of the stamp is able to make a fluid-tight conformal seal with a
wide variety of shapes and textures of material surfaces.
[0166] The microfluidic stamps provided according to the invention
can be utilized to form patterns on material surfaces comprised of
an extremely wide variety of materials, as would be apparent to
those of ordinary skill in the art. The structures provided
according to the invention, when used as stamps, can be utilized,
for example: to form patterned self-assembled monolayers (SAMs) on
material surfaces; to form patterns of inorganic materials on
surfaces; to form patterns of organic and/or biological materials
on surfaces; to form patterns on surfaces via contacting the
surfaces with a material that chemically reacts with and/or
degrades/etches the material surface; etc. Essentially any material
able to be printed via conventional microcontact printing
techniques can be patterned onto a surface using the inventive
microfluidic stamping structures provided by the invention. A
variety of such materials and applications is described in detail
in U.S. Pat. Nos. 5,512,131; 5,620,850; 5,776,748; 5,900,160;
5,951,881; and 5,976,826, each of which is incorporated herein by
reference.
[0167] The microfluidic stamping structures provided according to
the invention have several advantages over traditional
two-dimensional microfluidic stamps. For example, the microfluidic
stamping structures provided according to the invention have the
ability to simultaneously form a plurality of patterns onto a
material surface, each of which patterns is comprised of a
different material or "ink". In general, the number of different
patterns and materials which can be patterned onto a material
surface simultaneously by the stamps provided according to the
invention is equal to the number of independent, non-fluidically
interconnected fluid flow paths disposed within the microfluidic
stamping structure.
[0168] In order to form multiple patterns with different "inks"
utilizing traditional two-dimensional microcontact printing stamps,
individual stamps each having a separate pattern thereon must be
utilized, with each stamp being inked with a different fluid, and
with each pattern being carefully overlaid upon the previous
pattern and aligned thereto. By utilizing the three-dimensional
microfluidic channel structures provided according to the
invention, the inventive stamps are able to form, simultaneously,
essentially any desired number of arbitrarily complex patterns on a
material surface using a single stamp in a single stamping
step.
[0169] For example, referring again to FIG. 11, the microfluidic
channel system of FIG. 1a having a stamping surface 134 is able to
simultaneously form an overall pattern on material surface 900
corresponding to seven discrete subpatterns (A-G), each subpattern
corresponding to channels disposed within stamping surface 134 of
one of the seven fluid flow paths (102, 104, 106, 108, 110, 112,
114) of the microfluidic channel system shown in FIG. 1a. As
illustrated, each of subpatterns A-G includes discrete pattern
features (902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922,
and 924) which are non-continuous, and which are non-intersecting
with each other. In general, the microfluidic stamps provided
according to the invention are capable of forming patterns
comprised of discrete regions, wherein the discrete regions are
non-continuous with each other, and wherein discrete regions
corresponding to and formed by channels within the stamping surface
of the structure corresponding to two different non-fluidically
interconnected fluid flow paths are non-intersecting with each
other.
[0170] In the illustrated pattern shown in FIG. 11, it is possible
to pattern up to seven different materials ("inks") onto material
surface 900 simultaneously using microfluidic stamp 100 by filling
each of the separate flow paths of the microfluidic network with a
different fluid after contacting stamping surface 134 with material
surface 900. For example, patterned regions labeled "A" in FIG. 11
can comprise a first patterned material, regions labeled "B" can
comprise a second patterned material, regions labeled "C" can
comprise a third patterned material, regions labeled "D" can
comprise a fourth patterned material, regions labeled "E" can
comprise a fifth patterned material, regions labeled "F" can
comprise a sixth patterned material, and regions labeled "G" can
comprise a seventh patterned material. The overall pattern that
results on material surface 900 corresponds to each of the seven
individual subpatterns (A-G) formed by contact of material surface
900 with the particular fluids contained within each of the
individual flow paths forming subpatterns A-G.
[0171] In some embodiments, regions of stamping surfaces disposed
between channel indentations that make conformal contact with the
material surface being stamped can also, if desired, be coated with
another material or, "ink". In such embodiments, in addition to
forming patterns corresponding to the channel structures in the
stamping surface as described above, the regions surrounding,
contiguous with, and separating the patterns formed by the channel
structures ("printing regions") can also contain a deposited
material, carried by the printing regions, which material is
printed on the material surface upon conformal contact of the
"printing regions" of the stamping surface with the material
surface. The above technique enables an operator to essentially
simultaneously perform a conventional microcontact printing step
and a step of depositing material in a predetermined pattern on the
material surface via the channels disposed in the stamping surface
of the microfluidic stamp.
[0172] Because it is possible to create arbitrarily complex
patterns comprising a large number of patterned regions containing
different patterned materials, the stamps provided according to the
invention potentially have an extremely wide range of use for a
wide variety of applications. For example, in one preferred
application, the inventive stamps can be utilized to pattern cells
and/or proteins onto surfaces. For example, proteins can be
selectively patterned onto a surface which are adhesive to cells,
non-adhesive to cells, or selectively adhesive to certain cells
while non-adhesive to other cells. By forming patterns with such
proteins, complex patterns of one cell type or a variety of cell
types can be selectively patterned onto surfaces for various
applications, for example, for forming biosensors or performing
drug screening tests. With the microfluidic stamps provided
according to the invention it is possible, in principle, to pattern
a large number, for example in excess of 200 or 300, different cell
types, each separated from each other and arranged in a patterned
array format. Such patterning can be accomplished, according to the
invention, by, for example, selectively patterning proteins onto a
surface adherent to particular cell types followed by contact of
the patterned material surface with one or more cell suspensions,
or by selectively patterning a plurality of different cell types
onto a surface directly using a microfluidic stamp and filling
particular fluid flow paths within the stamp with suspensions
containing a discrete cell type or mixture of cell types desired to
be patterned onto the surface. The ability to form patterns
comprising arrays of regions, with each region including a
particular cell type or mixture of cell types, can enable the
creation of material surfaces for use in biosensors or drug
screening devices having cells patterned thereon that can be easily
and readily identified by their spatial locations on the
surface.
[0173] Proteins can also be deposited, using the inventive
microfluidic stamps, that tend to prevent or inhibit cell adhesion
to a material surface. Such proteins are well known to those of
ordinary skill in the art and include for example bovine serum
albumin (BSA). In addition, proteins can be patterned according to
the invention that tend to promote cell adhesion to the material
surface. Such proteins include, for example, fibrinogen, collagen,
laminin, integrins, antibodies, antigens, cell receptor proteins,
cell receptor antagonists, and mixtures of the above.
[0174] As described above, the microfluidic stamping structures
provided according to the invention, can be utilized to deposit a
patterned layer of cells on a material surface. Cells which can be
patterned on material surface comprise essentially the entire range
of biological cells including, but not limited to, bacterial cells,
algae, ameba, fungal cells, cells from multi-cellular plants, and
cells from multi-cellular animals. In some preferred embodiments,
the cells comprise animal cells, and in some such embodiments
comprise mammalian cells, such as human cells.
[0175] In one preferred embodiment, the mammalian cells comprise
anchorage dependent cells, which can attach and spread onto
material surfaces. In one preferred embodiment, the microfluidic
network stamping stamp provided according to the invention is
placed with its stamping surface in conformal contact with the
material surface to be patterned with a plurality of cells, and,
after filling one or more fluid flow paths of the microfluidic
stamp with one or more suspensions of cells and before removing the
stamp from the material surface, the cells are allowed to incubate
within the channel structure of the microfluidic stamp for a period
of time sufficient to allow the cells to attach and spread onto the
material surface. In such an embodiment, the shape or pattern of
channels can be specifically designed to have a predetermined
architecture or pattern selected to simulate a desired tissue
micro-architecture in order to study the relationship between cell
shape and/or position and cell function.
[0176] In other embodiments, two or more different cell types can
be patterned onto a material surface, as described above, and,
subsequent to removing the microfluidic stamp, can be allowed to
grow upon the surface and spread such that cells of the two or more
different cells types spread together and come into contact on the
surface after a period of time has elapsed. Such a patterning and
incubation method can be useful as part of an in vitro assay, which
is able to determine and/or study interactions between different
cell types. For example, such method can form part of an in vitro
assay able to determine an angiogenic potential of a particular
type of tumor cell. In one particular application contemplated, two
different cell types comprising capillary endothelial cells and
tumor cells are patterned onto a material surface and allowed to
grow and spread upon the surface after patterning, as described
above, in order to simulate and study angiogenesis during tumor
formation. In vivo, tumor cells tend to attract and direct the
growth of capillary endothelial cells to form new blood vessels to
supply nutrients and oxygen for tumor growth. By forming a defined
pattern of capillary endothelial cells and tumor cells utilizing
the microfluidic stamps provided according to the invention, it can
be possible to enable assays able to study the differential and
competitive attraction of capillary endothelial cells to different
tumor cell lines. This technique, enabled by the present invention,
can lead to the development of a simple, standardized, and
quantitative in vitro assay for comparing the angiogenic potential
of different tumor cells.
[0177] In addition, as discussed above, the present microfluidic
network stamps enable two or more different cell types to be
patterned onto a material surface in a wide variety of patterns of
arbitrary complexity and in a predetermined arrangement, which
arrangement can be selected to simulate a distinct
micro-architecture defined by the topological relationship between
the different cell types patterned on the surface. The ability to
pattern and selectively deposit different cell types in
well-defined patterned structures, enabled by the present
invention, can enable assays designed to study the functional
significance of tissue architecture at the resolution of individual
cells, and can enable assays designed to study the molecular
interactions between different cell types that underlie processes
such as embryonic morphogenesis, formation of the blood-brain
barrier, and tumor angiogenesis.
[0178] The function and advantage of these and other embodiments of
the present invention will be more fully understood from the
examples below. The following examples are intended to illustrate
the benefits of the present invention, but do not exemplify the
full scope of the invention.
EXAMPLE 1
Fabrication of a Mold Master by Multi-Level Photolithography
[0179] A mold master of photoresist on silicon having two levels of
features in positive, high relief (i.e., protruding above the
surface of the silicon wafer) was fabricated using the two-level
photolithography technique outlined in FIG. 8. Designs for the
channel systems for the first and second levels were generated with
a CAD computer program (Free-Hand 8.0, MacroMedia, San Francisco,
Calif.). High resolution (3386 dpi) transparencies were made by
printing with a commercial printer (Linotype, Hercules Computer
Technology, Inc., Freemont, Calif.) from the CAD computer files.
Two transparencies were produced, the first comprising the
photomask for producing features in the first level of the mold
master and the second comprising photomask for producing the
features in the second level of the mold master.
[0180] Negative photoresist (SU8-50, Microlithography Chemical
Corp., Newton, Mass.) was spin-coated (at about 5,000 rpm for 20
sec) on a silicon wafer to a depth of about 50 .mu.m and soft-baked
at about 105.degree. C. for about 5 min to drive off solvent from
the spin-cast photoresist. The first transparency was then used as
a photomask and the photoresist was exposed to UV radiation for
about 45 sec (wavelength of spectral lines about: 365 nanometers,
406 nanometers, and 436 nanometers at an intensity of about 10
mW/cm.sup.2).
[0181] Without developing the uncrosslinked photoresist, a second
layer of photoresist was spin-cast to a depth of about 100 .mu.m on
top of the first layer. The second transparency comprising the
second photomask was aligned to the exposed features of the
photoresist of the first layer using a Karl Suss mask aligner and
exposed to the UV radiation for about 1 min. The silicon wafer
containing the exposed photoresist layers was then hard-baked for
about 5 min. at about 105.degree. C. The second photomask contained
the pattern corresponding to the interconnecting channels that
would eventually link channels of the first, lower level formed by
the features exposed through the first photomask, and channels of
the upper levels of the replica molded structure ultimately molded
with the mold master. As illustrated in FIG. 8, each of the
photomasks also included a pattern for forming alignment tracks
surrounding the channel system.
[0182] Both layers of photoresist were developed at the same time
to remove uncrosslinked photoresist with propylene glycol methyl
ether acetate. The resulting bottom master included tall alignment
features and channel features comprising two-level topological
features in positive relief. The surface of the bottom mold master
including the topological features was then silanized by placing
the mold master in a vacuum chamber with a few drops of
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United
Chemical Technologies, Inc., Bristol, Pa.) for about 2 hours.
Silanization of the master facilitates the removal of a PDMS
replica after molding.
EXAMPLE 2
Fabrication of a Three-Dimensional Microfluidic Network Including a
System of Channels in a "Basketweave" Configuration
[0183] In the following example, the method outlined in FIGS. 9a
and 9b was utilized to produce a microfluidic network structure
including a channel pattern therein having a basketweave structure
similar to that illustrated in FIG. 1a. First, a bottom master was
produced as described above in Example 1 having disposed thereon
two-level topological features for forming channels within the
molded replica arranged similarly to those shown schematically in
FIG. 12a by bottom master 1000. The second step of the process
comprised formation of a top master including features for forming
channels in the uppermost level of the replica molded membrane. A
similar schematic arrangement of features for producing the
channels, and the way in which the channels of the upper mold
master and lower mold master fit together to mold the overall
structure, is also illustrated in FIG. 12a, making specific
reference to upper mold master schematic 1002.
[0184] The top mold master was made by first fabricating a
two-level structure in photoresist on silicon comprising a
pre-master by a method similar to that discussed above in Example
1. The pre-master contained features in negative, low-relief (i.e.,
comprising indentations below the level of the bulk surface) so
that replica molding the upper mold master with the pre-master
produced features in positive, high-relief on the upper mold
master, as shown schematically in FIG. 12a and as shown and
discussed earlier in the context of FIG. 9a. The topological
features of the pre-master corresponding to the channel system
extended to a level below the surface of the photoresist, but did
not traverse it completely; these features were all on one level.
Alignment tracks (not shown in FIG. 12a) that were shaped and
positioned to form alignment tracks in the replica molded top mold
master that fit between alignment tracks on the bottom master (not
shown in FIG. 12a) during replica molding of the microfluidic
membrane with the mold masters were fabricated in deeper, negative
relief and went all the way through the photoresist to the silicon
wafer. The pre-master was then silanized as described above in
Example 1. The pre-master was then covered with PDMS prepolymer
(Sylgard 184.TM. silicone elastomer with about a 1:10 ratio of
curing agent to elastomeric silicone polymer) and cured at about
75.degree. C. for about 1 hour. The PDMS replica, comprising a top
mold master, was then peeled from the pre-master, trimmed, and
oxidized in a plasma cleaner (PDC-23G, Harrick, Ossining, N.Y.) for
1 min, and then was silanized by placing it in a vacuum chamber
with a few drops of
tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United
Chemical Technologies, Inc., Bristol, Pa.) for about 8 hours.
[0185] The upper mold master was then placed facedown on top of the
surface of the bottom mold master including topological features,
with a drop of PDMS prepolymer in between. The features of the
masers were aligned quickly and without magnification by manually
sliding the top master over the prepolymer and bottom master until
its tall alignment tracks slipped between the tall alignment tracks
of the bottom master. Utilizing PDMS for the top master enabled
visual observation of the features of the masters and made
alignment straightforward. A microscope was not necessary because
the alignment tracks were macroscopic. In addition to facilitating
the alignment of the segments of the channel system quickly and
without magnification, the alignment tracks also balanced the top
master and prevented the registered masters from shifting in
position in response to physical disturbances or the application of
pressure during molding.
[0186] A pressure of about 100 g/mm.sup.2 (1000 kPa) was then
applied to the top master so that prepolymer did not seep between
features that were in contact, and the PDMS was heated to about
75.degree. C. and cured in place for about 1 hour. In addition, two
flat pieces of PDMS comprising an upper and lower substrate layer
were formed by casting the PDMS prepolymer against a flat,
silanized silicon wafer and curing, as described above. To transfer
the membrane, the membrane and top master were peeled off as a
single unit from the bottom master; the surface of the membrane and
the flat pieces of PDMS were oxidized in an air plasma for 1 min,
as described above; and the oxidized surfaces were then brought
together immediately. The oxidized PDMS surface remains reactive
for a few minutes after plasma treatment. Reactivity of the surface
can be prolonged by covering the surface, if desired, with a
hydrophilic liquid such as water, methanol, trifluoroethanol, or
mixture thereof. A protected surface will still seal more than 30
min after oxidation.
[0187] After contacting the membrane with the bottom PDMS slab, the
top master was peeled off, and the top surface of the membrane was
sealed to the second oxidized flat slab to enclose the channel
system. The entire structure was then trimmed to a convenient size.
The resulting structure included a microfluidic network
incorporating eight channels in the x-direction and eight in the
y-direction, each having a width of about 100 .mu.m and a height of
about 70 .mu.m, and each alternating between crossing over and
under channels oriented perpendicular to themselves. The entire
structure had a total area in the x-y plane of about 30 mm.sup.2
and contained 64 crossovers.
[0188] FIG. 12b is a photocopy of an optical photomicrograph
showing an en face phase contrast image of the structure as viewed
in the negative z-axis direction. The optical micrograph
illustrated in FIG. 12b was taken of the replica molded membrane
alone prior to sealing the membrane between the upper and lower
PDMS substrate layers. The optical photomicrograph clearly shows
the basketweave microfluidic channel structure and the crossover
points of the channels, appearing as intersections in photographed
the x-y plane.
[0189] After enclosing the membrane between an upper and lower PDMS
support layer as described above, flow paths extending in the y
direction were filled with a solution of fluorescein and flow paths
extending in the x direction were filled with a solution of
Meldola's Blue Dye. FIG. 12c is a photocopy of a photomicrograph of
the microfluidic channel system filled as described above, with the
observer viewing the system en face in the negative z-axis
direction. FIG. 12c shows, without ambiguity, which channels cross
over and which cross under each other, and also demonstrates that
the channels do not intersect, as would be evidenced by mixed
colors at any point.
EXAMPLE 3
Fabrication of Microstructures by Replica Molding With a
Microfluidic Network Structure
[0190] A microfluidic membrane including a three-level channel
system in a basketweave pattern was produced as described in
Example 2. The microfluidic membrane was placed upon a flat PDMS
slab so that the upper surface of the PDMS slab and the lower
surface of the membrane were in conformal contact but were not
irreversibly sealed to each other. The upper surface of the
membrane was left open to the atmosphere. An epoxy prepolymer
(EP-TEK, Epoxy Technology, Billerica, Mass.) was then placed at the
channel openings and allowed to fill the channel structure by
capillary action. After approximately 1 hour standing at ambient
pressure, the epoxy had degassed and filled the channels
completely. The filled channels were then exposed to UV light (as
described above in Example 1) for about 20 min through the PDMS.
The surrounding PDMS microfluidic membrane was then dissolved in
tetrabutylammonium fluoride (1.0 M in tetrahydrofuran). FIG. 12d is
a photocopy of a scanning electron photomicrograph of the resulting
microstructure produced by the cured epoxy polymer.
EXAMPLE 4
Fabrication of a Microfluidic Network Structure Including a Coiled
Fluid Flow Path Surrounding a Straight Channel
[0191] To demonstrate the capability of stacking, registering, and
sealing membranes to each other to make structures having more than
three levels of channels, a structure was fabricated including a
straight channel surrounded by a coiled fluid flow path comprising
a series of interconnected channels. The flow path comprising the
straight channel was separated from the channels comprising the
coiled flow path by a thin, about 65-100 .mu.m, PDMS layer.
Examples of microfluidic systems that benefit from such a
configuration include heat exchange elements or countercurrent
extraction system taking advantage of the diffusion of small
molecules across the PDMS layer separating the straight channel and
the coiled fluid flow path. Multi-layer fabrication techniques such
as the one in the current example also have utility for devices for
sorting and binding particles, and for complex channel network
systems that have specific size constraints.
[0192] The method used for producing the five-level channel system
by stacking and aligning two replica molded multi-level membranes
was illustrated above in FIG. 10. Referring to FIG. 10, first,
bottom master 802 was fabricated as described above in Example 1.
Upper mold masters 820 and 830 were fabricated as described in
Example 2. Replica molded membranes 800 and 810 were fabricated of
cured PDMS prepolymer, also as described above in Example 2. Bottom
master 802 was removed from each of the membranes and flat slabs of
PDMS were sealed in their place, as described above in Example 2.
The top masters were then peeled off and the two membranes were
aligned face-to-face on the stages of micromanipulators. This
orientation required that the two-level membrane 810 be flipped
over. The membranes were brought together and aligned, and were
then backed apart by about 3 to about 5 mm without disturbing the
previous alignment. The separated membranes were then oxidized in
an air plasma, as described above, and then brought into conformal
contact.
[0193] FIG. 13 shows a photocopy of an optical photomicrograph of
the resulting channel system as viewed en face along the negative
z-axis direction. Prior to the photomicrograph being taken, the two
fluid flow paths of the system were filled with a fluorescein
solution, as described in Example 2, to aid visualization of the
channel system.
EXAMPLE 5
Fabrication of a Microfluidic Stamp and Etching of a Si/SiO.sub.2
Surface and Visualization of the Etched Surface Using Optical
Interference Colors
[0194] For the present example, a three-dimensional microfluidic
stamp was produced according to the method outlined in FIG. 7.
Referring to FIG. 7, two-level lower mold master 520 was prepared
as previously described in Example 1 and one-level mold master 500
was prepared also as described in Example 1, except utilizing only
a single layer of photoresist and a single photomask to produce
only one level of topological features. The top PDMS slab 510 was
fabricated by placing mold master 500 in a container with surface
502 facing up, covering the mold master with PDMS prepolymer,
curing the PDMS prepolymer, as described above in Example 2, and
removing and trimming the molded replica to form PDMS slab 510.
[0195] PDMS membrane 550 was fabricated by sandwiching a drop of
PDMS prepolymer between master 520 and a PTFE sheet. Pressure of
between about 10 and about 50 kPa was applied tending to force the
PTFE sheet and mold master 520 together. The PDMS prepolymer was
then cured, as described in Example 2. After curing, PTFE sheet 540
was peeled away, leaving the membrane remaining attached to mold
master 520 by van der Waals interactions.
[0196] To align and seal the PDMS slab to the PDMS membrane a
micromanipulator stage was used. The slab and membrane were mounted
on the micromanipulator stage so that surface 514 was facing
surface 556. The surfaces were brought into close proximity and
aligned. After alignment, the surfaces were backed away from each
other by a few millimeters using the micromanipulator. The entire
alignment stage was then placed in a plasma cleaner (Anatech, Model
SP100 Plasma System, Springfield, Va.) and oxidized for about 40
sec in an oxygen plasma. The power level of the plasma cleaner was
about 60 watts and the oxygen pressure was about 0.2 Torr. Sealing
of the two layers was accomplished by removing the assembly from
the plasma cleaner and immediately bringing the two aligned and
oxidized PDMS surfaces into contact.
[0197] FIG. 14a illustrates schematically the channel system
disposed in the upper level 1010 of the microfluidic stamp and the
lower level 1012 of the microfluidic stamp, which lower level
having a lower surface 554 comprising the stamping surface. Surface
554 was brought into conformal contact with material surface 1014
of substrate 1016. FIG. 14b is a schematic diagram illustrating the
layout and interconnectivity of the three-level channel system
within microfluidic stamp 560 and the configuration of each of the
three non-fluidically interconnected fluid flow paths 561, 563, and
565.
[0198] To create the etched pattern on surface 1014 shown in FIG.
14c, surface 554 of the microfluidic stamp was brought into
conformal contact with surface 1014 (comprising a Si/SiO.sub.2
surface) and gentle pressure was applied to the stamp. Three
aqueous solutions containing three different concentrations of
hydrofluoric acid (10%, 5%, and 3% hydrofluoric acid, buffered at
about pH 5 with a 6:1 ratio of NH.sub.4F/HF) were allowed to flow
(.about.1 cm/sec), with each solution confined to one of the
non-fluidically interconnected flow paths in the structure. Each of
the channels in the structure had a cross-sectional area, measured
in a plane perpendicular to the channel's longitudinal axis, of
about 500 .mu.m.sup.2. Where the hydrofluoric acid solutions came
into contact with the surface, they etched away the SiO.sub.2. The
rate of etching of SiO.sub.2 for 10% hydrofluoric acid is about 20
nm/min. The lower concentrations etched at a rate proportionally
less than the most concentrated solution. The hydrofluoric acid
solutions were flowed through the channels for a period of about 26
min before removing the stamp from the surface and visualizing the
pattern.
[0199] The optical interference color of an SiO.sub.2 layer is very
sensitive to the thickness of the layer; a difference of about 30
nm, for example, can change the color from, for example, light
green to blue. Thus, patterns etched to different depths within
surface 1014 appear as different colors. Referring to FIG. 14c,
patterned features 1018, corresponding to fluid flow path 561,
which contained the 10% hydrofluoric acid solution, were etched
into surface 1014 to a depth of about 520 nm and appear green.
Etched patterned features 1020, corresponding to fluid flow path
565, which contained the 5% hydrofluoric acid solution, were etched
into surface 1014 to a depth of about 390 nm and appear yellow.
Patterned features 1022, corresponding to fluid flow path 563,
which contained the 3% hydrofluoric acid solution, were etched into
surface 1014 to a depth of about 70 nm and appear brown.
EXAMPLE 6
Patterned Deposition of Proteins Onto a Surface Using a
Three-Dimensional Microfluidic Stamp
[0200] A microfluidic stamp having a stamping surface with spirally
arranged channels therein was produced by a method similar to that
described above in Example 5. The microfluidic stamp had a
microfluidic channel system shown schematically in FIG. 15a. The
stamp included two non-fluidically interconnected fluid flow paths
1030 and 1032. The channels of fluid flow paths 1030 and 1032 are
disposed in the stamping surface of the microfluidic stamp in a
nested spiral arrangement as illustrated in FIG. 15a.
[0201] The stamping surface of the microfluidic stamp was placed in
conformal contact with a polystyrene surface of a petri dish.
Spiral flow paths 1030 was then filled with a FITC-labeled bovine
serum albumin (BSA) solution having a labeled BSA concentration of
1 mg/ml in phosphate buffer (pH 7.4). Fluid flow path 1032 was
filled with a FITC-labeled fibrinogen solution containing 0.1 mg/ml
labeled fibrinogen in phosphate buffer (pH 7.4). The proteins were
allowed to absorb onto the polystyrene surface for about 45 min.
The channels were then flushed thoroughly with phosphate buffer;
the stamp was peeled off; and the surfaces were observed en face
with fluorescence microscopy.
[0202] FIG. 15b is a photocopy of a photomicrograph taken of the
surface of the petri dish as viewed utilizing fluorescence
microscopy. Spiral pattern 1034 comprises a layer of deposited
labeled BSA and spiral pattern 1036 comprises a layer of deposited
labeled fibrinogen. Spiral pattern 1034 is brighter and more
fluorescent because the concentration of BSA used was about 10
times higher than the concentration of fluorescently labeled
fibrinogen.
EXAMPLE 7
Patterned Deposition of Cells Onto Surfaces Using Two Different
Microfluidic Systems
[0203] Cell cultures: Bovine adrenal capillary endothelial cells
(BCEs) were cultured as described in J. Folkman, C. C.
Haudenschild, B. R. Zetter, Proc. Natl. Acad. Sci. USA, Vol. 76,
pp. 5217-5221, 1982. In brief, BCEs were grown in low glucose DMEM
cell culture medium supplemented with 10% calf serum and 2 ng/ml
basic fibroblast growth factor (bFGF), and kept in a 10% CO.sub.2
atmosphere. Human bladder cancer cells (ECVs) from the ECV304 cell
line were cultured in DMEM supplemented with 10% fetal bovine serum
(FBS) and kept in a 5% CO.sub.2 atmosphere. Cells from both cell
types were labeled fluorescently before harvest at 37.degree. C. in
the CO.sub.2 incubator. BCEs were incubated with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
(DiI)-conjugated acetylated low-density lipoprotein at 4 .mu.g/ml,
which is actively taken up by BCEs and stored in endosomal granula.
ECV304 cells were incubated with 5 .mu.M 5-chloromethylfluorescein
diacetate (CMFDA), which reacts with intracellular glutathione.
Before patterning, cells were washed with PBS, dissociated from the
culture plates to which they were attached during culture with
typsin/EDTA, washed with DMEM, and resuspended in the respective
culture media at a density of about 10.sup.6 cells/ml. For
culturing patterned cells (both BCEs and ECVs) after removal of the
PDMS stamp, DMEM supplemented with 5% calf serum, 5% FBS, and 2
ng/ml bFGF was used, and the cells were kept in a 10% C0.sub.2
atmosphere.
[0204] Patterning: To form the first pattern of deposited cells, a
microfluidic stamp having the channel network structure illustrated
schematically in FIG. 16a was fabricated by a method similar to
that described above in Example 5. A stamping surface of the
microfluidic stamp included disposed therein channels comprising a
concentric square pattern. The microfluidic stamp included three
non-fluidically interconnected fluid flow paths 1040, 1042, and
1044, fluid flow path 1040 in fluid communication with outermost
concentric square pattern 1041, fluid flow path 1042 in fluid
communication with the intermediate concentric square pattern 1043,
and fluid flow path 1044 in fluid communication with the innermost
concentric square pattern 1045.
[0205] Before use, the PDMS microfluidic stamp was autoclaved at
about 121.degree. C. for about 20 min, and the walls of the
channels were coated with BSA by filling the channels with a 0
mg/ml BSA solution in pH 7.4 phosphate buffer for about 1 hour
before removing the solution and flushing with BSA-free phosphate
buffer. The stamping surface was then brought into conformal
contact with the surface of a polystyrene tissue culture dish.
Suspensions of cells (at a concentration of about 5.times.10.sup.6
cells/ml) were introduced into the three fluid flow paths and were
allowed to sediment and attach to the surface of the tissue culture
dish. The cells used were BCEs and an ECV cell line (ECV-304).
Before being deposited, the BCEs were labeled with DiI-conjugated
acetylated low-density lipoprotein, which was actively taken up by
the BCEs and stored in their endosomal granula, and the ECVs with
CMFDA, which reacted with their intracellular glutathione. The BCE
cell solutions were introduced into fluid flow paths 1040 and 1044,
and the ECV cell solution was introduced into fluid flow path 1042.
After introducing the cell suspension into the fluid flow paths of
the microfluidic stamp, the cells were cultured for about 24 hours
with the microfluidic stamp in place on the tissue culture dish
surface, so as to form a confluent layer of cells on the surface of
the tissue culture dish. After culture, the microfluidic stamp was
removed from the surface, and the surface, having cells attached
thereto, was immersed in tissue culture media, as previously
described.
[0206] FIG. 16b is a photocopy of a photomicrograph of surface of
the petri dish as observed by fluorescence microscopy. The
deposited BCE cells are attached to the surface in the outermost
concentric square pattern 1046 and the innermost concentric square
pattern 1048. Such cells, when viewed with the fluorescence
microscope appear red in color. The ECV cells are deposited on the
surface in concentric square pattern 1050 and fluoresce green when
viewed with the fluorescence microscope. FIGS. 16c and 16d are
photocopies of photomicrographs of the patterned surface as viewed
with phase-contrast microscopy, illustrating the morphology and
arrangement of the cells within each of the patterns on the
surface.
[0207] FIGS. 17a and 17b show the results of a similar cell
patterning experiment wherein two types of cells were deposited in
a chessboard-like pattern. The chessboard-like pattern was designed
as a demonstration of the potential of the microfluidic stamping
system and method of the invention to deposit multiple cell types
in an array format appropriate for a biosensor or drug screening
applications. In such an array, the responding cells could be
identified by their spatial location.
[0208] A microfluidic stamp having fluid flow paths shown
schematically in FIG. 17a was prepared by a method similar to that
described above in Example 5. The microfluidic stamp included eight
non-fluidically interconnected independent flow paths 1060, 1062,
1064, 1066, 1068, 1070, 1072, and 1074. Each of the flow paths is
in fluid communication with two square channels disposed in the
stamping surface of the microfluidic stamp. For example, fluid flow
path 1060 is in fluid communication with square channels 1076 and
1078 disposed within the stamping surface of the microfluidic
stamp.
[0209] A chessboard pattern of cells is shown in FIG. 17b, which is
a photocopy of a fluorescence photomicrograph. The patterned
surface was produced using the same procedures used for patterning
the concentric square pattern of FIGS. 16b-16d. The two cell types
used, BCEs and ECVs, were fluorescently labeled, as described
above, before being deposited onto the surface of a tissue culture
plate. Solutions of fluorescently labeled ECV cells were used to
fill fluid flow paths 1060, 1062, 1064, and 1066, and solutions of
fluorescently labeled BCE cells were used to fill fluid flow paths
1068, 1070, 1072, and 1074. The cells were cultured with the stamp
in place on the surface for 42 hours until a confluent layer of
cells were formed on the surface of the tissue culture plate. The
fluorescence photomicrograph (a photocopy of which is shown in FIG.
17b) was taken with the PDMS microfluidic stamp still in place on
the tissue culture plate surface in order to show the overlaying
weaving channel structures. The color of each of the confluent
layers of cells as viewed by fluorescence microscopy, is indicated
on the figure above each square pattern feature. The blurred red
spots 1080, 1082 and the blurred green spot 1084 comprise cells
located in the channel structure of the top level of the
microfluidic stamp above the focal plane of the microscope.
[0210] After removing the microfluidic stamp from the surface of
the tissue culture plate, the surface was placed in tissue culture
medium, as previously described, and cultured, as previously
described, to allow the two cell types to grow and spread together.
FIG. 17c shows a portion of the image of FIG. 17b illustrating a
patterned feature comprising green deposited ECV cells and red
deposited BCE cells. The two regions containing cells are separated
by an intermediate region of the tissue culture plate surface (set
off by dotted white lines), which is free of cells. FIG. 17d shows
a photocopy of a fluorescence photomicrograph taken of the
identical region of the tissue culture plate surface taken 20 hours
after removal of the stamp and subsequent culture of the plate.
FIGS. 17c and 17d are registered, and the dotted intermediate
region of FIG. 17d comprises the region in FIG. 17c that was
initially cell free. As can be seen, after 20 hours of culture
subsequent to removal of the microfluidic stamp, both cell types
have divided, grown, and spread together within the region that was
initially cell free. FIG. 17e shows the same region as shown FIG.
17d, also after 20 hours of culture subsequent to removing the
stamp, except as viewed with phase contrast light microscopy.
[0211] While several embodiments of the invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and structures
for performing the functions and/or obtaining the results or
advantages described herein, and each of such variations or
modifications is deemed to be within the scope of the present
invention. More generally, those skilled in the art would readily
appreciate that all parameters, dimensions, materials, and
configurations (list modified as appropriate) described herein are
meant to be exemplary and that actual parameters, dimensions,
materials, and configurations will depend upon specific
applications for which the teachings of the present invention are
used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically
described. The present invention is directed to each individual
feature, system, material and/or method described herein. In
addition, any combination of two or more such features, systems,
materials and/or methods, provided that such features, systems,
materials and/or methods are not mutually inconsistent, is included
within the scope of the present invention. In the claims, all
transitional phrases or phrases of inclusion, such as "comprising,"
"including," "carrying," "having," "containing," and the like are
to be understood to be open-ended, i.e. to mean "including but not
limited to." Only the transitional phrases or phrases of inclusion
"consisting of" and "consisting essentially of" are to be
interpreted as closed or semi-closed phrases, respectively, as set
forth in MPEP section 2111.03.
* * * * *