U.S. patent application number 10/359961 was filed with the patent office on 2003-07-24 for microfluidic devices, systems and methods for performing integrated reactions and separations.
This patent application is currently assigned to Caliper Technologies Corp.. Invention is credited to Chow, Andrea W., Kopf-Sill, Anne R., Parce, J. Wallace, Sundberg, Steven A..
Application Number | 20030138359 10/359961 |
Document ID | / |
Family ID | 26787598 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138359 |
Kind Code |
A1 |
Chow, Andrea W. ; et
al. |
July 24, 2003 |
Microfluidic devices, systems and methods for performing integrated
reactions and separations
Abstract
Microfluidic devices for performing integrated reaction and
separation operations. The devices comprise a planar substrate
having a first surface with an integrated channel network disposed
therein. The reaction region in the integrated microscale channel
network has a mixture of at least first and second reactants
located therein, wherein the mixture interacts to produce one or
more products. The reaction region is configured to maintain
contact between the first and second reactants contained within it.
The device also includes a separation region in the integrated
channel network, where the separation region is configured to
separate the first reactant from the product, when the first
reactant and product are flowing through the separation region.
Inventors: |
Chow, Andrea W.; (Los Altos,
CA) ; Kopf-Sill, Anne R.; (Portola Valley, CA)
; Parce, J. Wallace; (Palo Alto, CA) ; Sundberg,
Steven A.; (San Francisco, CA) |
Correspondence
Address: |
CALIPER TECHNOLOGIES CORP
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043
US
|
Assignee: |
Caliper Technologies Corp.
605 Fairchild Drive
Mountain View
CA
94043
|
Family ID: |
26787598 |
Appl. No.: |
10/359961 |
Filed: |
February 6, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10359961 |
Feb 6, 2003 |
|
|
|
09442073 |
Nov 15, 1999 |
|
|
|
6551836 |
|
|
|
|
10359961 |
Feb 6, 2003 |
|
|
|
09093489 |
Jun 8, 1998 |
|
|
|
6274089 |
|
|
|
|
60108628 |
Nov 16, 1998 |
|
|
|
Current U.S.
Class: |
422/400 ;
204/451; 204/601 |
Current CPC
Class: |
B01F 33/30 20220101;
B01L 2400/0487 20130101; B01L 2400/0418 20130101; G01N 27/44791
20130101; Y10T 436/25375 20150115; G01N 27/44743 20130101; G01N
2001/4038 20130101; B01L 3/502753 20130101; B01L 2400/0421
20130101; B01L 2200/0673 20130101; B01L 2300/087 20130101; B01L
2200/0605 20130101; B01L 3/502784 20130101; B01L 2300/0864
20130101; B01L 2400/0415 20130101; Y10T 436/117497 20150115; B01L
2300/0816 20130101; Y10T 436/2575 20150115; B01L 2300/0867
20130101; B01L 3/50273 20130101 |
Class at
Publication: |
422/101 ;
204/451; 204/601 |
International
Class: |
G01N 027/447 |
Claims
What is claimed is:
1. A microfluidic device for performing integrated reaction and
separation operations, comprising: a body structure having an
integrated microscale channel network disposed therein; a reaction
region within the integrated microscale channel network, the
reaction region having a mixture of at least first and second
reactants disposed in and flowing through the reaction region, the
mixture interacting to produce one or more products, wherein the
reaction region is configured to maintain contact between the first
and second reactants flowing therethrough; and a separation region
in the integrated channel network, the separation region in fluid
communication with the reaction region and being configured to
separate the first reactant from the one or more products flowed
therethrough.
2. The microfluidic device of claim 1, wherein the reaction region
comprises a microscale reaction channel having first and second
ends and the separation region comprises a microscale separation
channel having first and second ends.
3. The microfluidic device of claim 2, wherein the reaction channel
and the separation channel are in fluid communication and cross at
a first intersection between the first and second ends of the
reaction channel and the separation channel, respectively.
4. The microfluidic device of claim 3, further comprising an
electrokinetic material transport system operably coupled to the
first and second ends of the reaction channel and the first and
second ends of the separation channel for electrokinetically
transporting material through the reaction channel and into the
separation channel.
5. The microfluidic device of claim 4, wherein at least two of the
first and second reactants and product have different
electrophoretic mobilities under an applied electric field.
6. The microfluidic device of claim 4, wherein the reaction channel
comprises first and second fluid regions disposed therein, the
first fluid region comprising the first and second reactants and
the product, and having a first conductivity, the first fluid
region being bounded by the second fluid regions, wherein the
second fluid regions have a second conductivity that is lower than
the first conductivity.
7. The microfluidic device of claim 3, wherein the separation
channel comprises a separation inducing buffer, the separation
inducing buffer having a conductivity that is higher than the
second conductivity.
8. The microfluidic device of claim 3, wherein the separation
channel comprises a separation inducing buffer, the separation
inducing buffer having a conductivity that is lower than the first
conductivity.
9. The microfluidic device of claim 3, wherein the separation
channel comprises a separation inducing buffer, the separation
inducing buffer having a conductivity that is the same as the first
conductivity.
10. The microfluidic device of claim 3, further comprising: at
least first and second conductivity measuring electrodes disposed
in electrical contact with opposite sides of the reaction channel
adjacent to the first intersection; and a conductivity detector
operably coupled to the first and second conductivity measuring
electrodes.
11. The microfluidic device of claim 3, further comprising at least
a third reactant in the reaction region, the second and third
reactants interacting to produce the product, and wherein the first
reactant comprises a test compound.
12. The microfluidic device of claim 3, wherein the separation
channel comprises a separation medium disposed therein.
13. The microfluidic device of claim 3, further comprising: a
source of at least first reactant in fluid communication with the
reaction channel; and a source of at least second reactant in fluid
communication with the reaction channel.
14. The microfluidic device of claim 13, wherein the source of at
least first reactant comprises at least a first reactant reservoir
connected to the reaction channel via a first reactant channel, and
the source of at least second reactant comprises: a source of at
least a second reactant separate from the body structure; and an
external sample accessing capillary in fluid communication with the
reaction channel, for contacting the second reactant reservoir and
transporting a volume of the second reactant into the reaction
channel.
15. The microfluidic device of claim 13, wherein the source of at
least first reactant comprises a first reactant reservoir disposed
in the body structure and connected to the reaction channel via a
first reactant channel, and the source of second reactant comprises
a second reactant reservoir disposed in the body structure and
connected to the reaction channel via a second reactant
channel.
16. The microfluidic device of claim 3, wherein the body structure
comprises at least first and second planar substrates, a plurality
of grooves being fabricated into a first planar surface of the
first substrate, and a first planar surface of the second substrate
being mated to the first planar substrate of the first substrate
covering the plurality of grooves and defining the integrated
channel network.
17. The microfluidic device of claim 16, wherein at least one of
the first and second substrates comprise a silica-based
substrate.
18. The microfluidic device of claim 17, wherein the silica-based
substrate is selected from glass, quartz, fused silica, or
silicon.
19. The microfluidic device of claim 18, wherein the silica based
substrate comprises glass.
20. The microfluidic device of claim 16, wherein at least one of
the first and second substrates comprises a polymeric material.
21. The microfluidic device of claim 20, wherein the polymeric
material is selected from polymethylmethacrylate, polycarbonate,
polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane,
polysulfone, polystyrene, polymethylpentene, polypropylene,
polyethylene, polyvinylidine fluoride, and
acrylonitrile-butadiene-styrene copolymer.
22. The microfluidic device of claim 21, wherein the polymeric
material comprises polymethylmethacrylate.
23. The microfluidic device of claim 3, wherein channels in the
integrated channel network have at least one cross-sectional
dimension between about 0.1 and about 500 .mu.m.
24. The microfluidic device of claim 3, wherein channels in the
integrated channel network have at least one cross-sectional
dimension between about 1 and about 100 .mu.m.
25. The microfluidic device of claim 24, wherein channels in the
integrated channel network have at least one cross-sectional
dimension between about 10 and about 100 .mu.m.
26. The microfluidic device of claim 2, wherein the reaction
channel comprises alternating first and second fluid regions, the
first region having a higher ionic concentration than the second
fluid region, the reaction mixture being localized in a first fluid
region.
27. The microfluidic device of claim 2, wherein the reaction
channel and the separation channel are in fluid communication via a
connecting channel, the connecting channel intersecting the
reaction channel between the first and second ends of the reaction
channel, and intersecting the separation channel between the first
and second ends of the separation channel.
28. The microfluidic device of claim 27, further comprising an
electrokinetic material transport system operably coupled to the
first and second ends of the reaction channel and the first and
second ends of the separation channel for electrokinetically
transporting material through the reaction channel and into the
separation channel.
29. The microfluidic device of claim 28, wherein at least two of
the first and second reactants and product have different
electrophoretic mobilities under an applied electric field.
30. The microfluidic device of claim 27, wherein the connecting
channel comprises a smaller cross-sectional area than the first or
second channels.
31. The microfluidic device of claim 27, wherein the connecting
channel comprises a length less than about 1 mm.
32. The microfluidic device of claim 27, wherein the connecting
channel comprises a length less than about 0.5 mm.
33. The microfluidic device of claim 32, wherein the reaction
channel comprises first and second fluid regions disposed therein,
the first fluid region comprising the first and second reactants
and the product, and having a first conductivity, the first fluid
region being bounded by the second fluid regions, wherein the
second fluid regions have a second conductivity that is lower than
the first conductivity.
34. The microfluidic device of claim 32, wherein the separation
channel comprises a separation inducing buffer, the separation
inducing buffer having a conductivity that is higher than the
second conductivity.
35. The microfluidic device of claim 34, wherein the separation
inducing buffer comprises a conductivity that is from about 2 to
about 100 times greater than the second conductivity.
36. The microfluidic device of claim 34, wherein the separation
inducing buffer has a conductivity that is lower than the first
conductivity.
37. The microfluidic device of claim 34, wherein the separation
inducing buffer comprises a conductivity that is from about 2 to
about 100 times less than the first conductivity.
38. The microfluidic device of claim 34, wherein the separation
inducing buffer has a conductivity approximately equal to the first
conductivity.
39. The microfluidic device of claim 27, further comprising: at
least first and second conductivity measuring electrodes disposed
in electrical or capacitive contact with opposite sides of the
reaction channel adjacent to the first intersection; and a
conductivity detector operably coupled to the first and second
conductivity measuring electrodes.
40. The microfluidic device of claim 27, further comprising at
least a third reactant in the reaction channel, the second and
third reactants interacting to produce the product, and wherein the
first reactant comprises a test compound.
41. The microfluidic device of claim 27, wherein the separation
channel comprises a separation medium disposed therein.
42. The microfluidic device of claim 27, wherein the reaction
region comprises alternating first and second fluid regions, the
first region having a higher ionic concentration than the second
fluid region, the reaction mixture being localized in a first fluid
region.
43. The microfluidic device of claim 2, wherein the first end of
the reaction channel is in fluid communication with the first end
of the separation channel at a first junction, and further
comprising a buffer channel having first and second ends, the first
end of the buffer channel in fluid communication with the reaction
channel and the separation channel at the first junction, the
second end of the buffer channel being in fluid communication with
a source of separation inducing buffer.
44. The microfluidic device of claim 43, wherein the first and
second channel portions are co-linear.
45. The microfluidic device of claim 43, further comprising an
electrokinetic material transport system operably coupled to the
second ends of the reaction channel, the separation channel and the
buffer channel for electrokinetically transporting material from
the reaction region to the separation region, and for introducing
separation inducing buffer into the separation channel from the
buffer channel.
46. The microfluidic device of claim 45, wherein at least two of
the first and second reactants and product have different
electrophoretic mobilities under an applied electric field.
47. The microfluidic device of claim 43, wherein the reaction
channel comprises first and second fluid regions disposed therein,
the first fluid region comprising the first and second reactants
and the product, and having a first conductivity, the first fluid
region being bounded by the second fluid regions, wherein the
second fluid regions have a second conductivity that is lower than
the first conductivity.
48. The microfluidic device of claim 43, wherein the separation
inducing buffer has a conductivity that is greater than the second
conductivity.
49. The microfluidic device of claim 48, wherein the separation
inducing buffer has a conductivity that is from about 2 to about
100 times greater than the second conductivity.
50. The microfluidic device of claim 48, wherein the separation
inducing buffer has a conductivity that is lower than the first
conductivity.
51. The microfluidic device of claim 48, wherein the separation
inducing buffer has a conductivity that is from about 2 to about
100 times less than the first conductivity.
52. The microfluidic device of claim 48, wherein the separation
inducing buffer has a conductivity that is approximately equal to
the first conductivity.
53. The microfluidic device of claim 48, wherein the separation
inducing buffer has a conductivity that is approximately equal to
the second conductivity.
54. The microfluidic device of claim 43, further comprising at
least a third reactant in the reaction region, the second and third
reactants interacting to produce the product, and wherein the first
reactant comprises a test compound.
55. The microfluidic device of claim 43, wherein the separation
channel comprises a separation medium disposed therein.
56. The microfluidic device of claim 43, wherein the reaction
region comprises alternating first and second fluid regions, the
first region having a higher ionic concentration than the second
fluid region, the reaction mixture being localized in a first fluid
region.
57. A microfluidic device for performing integrated reaction and
separation operations, comprising: a body structure; a first
channel disposed in the body structure, the first channel having
disposed therein, at least first and second fluid regions, the
first fluid region having an ionic concentration higher than an
ionic concentration of the second fluid region, and the first and
second fluid regions communicating at a first fluid interface;
second and third channels disposed in the body structure, the
second channel intersecting and connecting the first and third
channels at intermediate points along a length of the first and
third channels, respectively; an electrokinetic material transport
system for applying a voltage gradient along a length of the first
channel, but not the second channel, to electrokinetically move the
first fluid interface past the intermediate point of the first
channel, and force at least a portion of the first fluid regions
through the second channel into the third channel.
58. The device of claim 57, wherein the first fluid region has a
conductivity that is from about 2 to about 200 times greater than a
conductivity of the second fluid regions.
59. The device of claim 57, wherein the first fluid region has a
conductivity that is from about 2 to about 100 times greater than a
conductivity of the second fluid regions.
60. The device of claim 57, wherein the first fluid region has a
conductivity that is from about 2 to about 50 times greater than a
conductivity of the second fluid regions.
61. The device of claim 57, wherein the first fluid region has a
conductivity that is from about 2 to about 20 times greater than a
conductivity of the second fluid regions.
62. The device of claim 57, wherein the first fluid region has a
conductivity that is from about 2 to about 10 times greater than a
conductivity of the second fluid regions.
63. The device of claim 57, wherein the first fluid region
comprises at least first and second materials.
64. The device of claim 63, wherein the first and second materials
have different electrophoretic mobilities under an applied electric
field.
65. A method of performing integrated reaction and separation
operations, comprising: providing a microfluidic device comprising
a body structure having a reaction channel and a separation channel
disposed therein, the reaction channel and separation channel being
in fluid communication; flowing at least first and second reactants
through the reaction channel in a first fluid region, the first and
second reactants interacting to form at least a first product
within the first fluid region, wherein the step of transporting
through the first channel is carried out under conditions for
maintaining the first and second reactants and products
substantially within the first fluid region; directing at least a
portion of the first fluid region to the separation channel, the
separation channel being configured to separate the product from at
least one of the first and second reactants; and transporting the
portion along the separation channel to separate the product from
at least first reactant.
66. The method of claim 65, wherein: the flowing step comprises
applying a first voltage gradient along the reaction channel to
electrokinetically move the first fluid region into the
intersection; and the directing step comprises applying a second
voltage gradient along the separation channel to direct at least a
portion of the first fluid region into the separation channel; the
separating step comprises applying a third voltage gradient along
the separation channel to separate the first reactant from the
first product.
67. The method of claim 66, wherein the conditions suitable for
maintaining the first and second reactant and product substantially
within the first fluid region comprises the first fluid region
having a first conductivity and being bounded by second fluid
regions having a second conductivity that is lower than the first
conductivity.
68. The method of claim 66, wherein the first conductivity is from
about 2 to about 100 times greater than the second
conductivity.
69. The method of claim 66, wherein the separation channel has a
separation inducing buffer disposed therein, the separation buffer
having a conductivity lower than the first conductivity.
70. The method of claim 66, wherein the separation channel has a
separation inducing buffer disposed therein, the separation
inducing buffer having a conductivity approximately equivalent to
the first conductivity.
71. The method of claim 66, wherein the product and at least one of
the first and second reactants have different electrophoretic
mobilities under an applied electric field.
72. The method of claim 65, further comprising the step of
detecting the separated product in the separation channel.
73. The method of claim 65, wherein: in the providing step, the
reaction channel and the separation channel disposed in the body
structure are in fluid communication and cross at a first
intersection; the flowing step comprises flowing the first fluid
region into the first intersection; and the directing step
comprises directing the portion of the first mixture in the
intersection into the separation channel.
74. The method of claim 73, further comprising the step of
detecting when the first fluid region is disposed in the
intersection.
75. The method of claim 74, wherein the step of detecting when the
first fluid region is disposed in the intersection comprises
detecting a change in conductivity of fluid at the
intersection.
76. The method of claim 74, wherein the first and second fluid
regions have optical characteristics that are distinguishable from
each other, and the step of detecting when the first fluid region
is disposed in the intersection comprises detecting within the
intersection, the optical characteristics indicating the presence
of the first fluid region.
77. The method of claim 74, wherein the optical characteristics
that are distinguishable from each other comprise a fluorophore or
chromophore disposed within at least one of the first or second
fluid regions.
78. The method of claim 77, wherein the optical characteristics
that are distinguishable from each other comprise a first
chromophore or fluorophore disposed in the first fluid region and a
second chromophore or fluorophore disposed in the second fluid
region, the first fluorophore or chromophore being distinguishable
from the second chromophore or fluorophore.
79. The method of claim 69, wherein: in the providing step, the
reaction channel and the separation channel are in fluid
communication via a connecting channel the connecting channel
intersecting the reaction channel at a first intersection and
intersecting the separation channel at a second intersection; the
flowing step comprises flowing the first fluid region into the
first intersection; and the directing step comprises directing at
least a portion of the first fluid region through the connecting
channel into the separation channel.
80. The method of claim 79, wherein the directing step comprises
providing a voltage gradient between the reaction channel and
separation channel to electrokinetically direct a portion of the
first fluid region from the reaction channel, through the
connecting channel and into the separation channel.
81. The method of claim 79, wherein the directing step comprises
flowing the first fluid region along the reaction channel through
the first intersection, a pressure differential present at an
interface of the first and second fluid regions forcing a portion
of the first fluid region into the connecting channel and into the
separation channel.
82. The method of claim 69, wherein: in the providing step, the
reaction channel has first and second ends, the separation channel
has first and second ends, the first end of the reaction channel
being in fluid communication with the first end of the separation
channel at a first junction, and further comprising a buffer
channel having first and second ends, the first end of the buffer
channel in fluid communication with the reaction channel and
separation channel at the first junction; the flowing step
comprises flowing the first fluid region along the reaction channel
to the first junction; and the directing step comprises directing
the portion of the first mixture in the intersection into the
separation channel.
83. The method of claim 82, wherein the directing step comprises
directing at least a portion of the first fluid region into the
separation channel while concomitantly injecting the separation
inducing buffer from the third channel into the separation
channel.
84. A method of directing fluid transport in a microscale channel
network, comprising: providing a microfluidic device having at
least first and second intersecting channels disposed therein, the
first channel being intersected by the second channel at an
intermediate point; introducing first and second fluid regions into
the first channel, wherein the first and second fluid regions are
in communication at a first fluid interface, and wherein the first
fluid region has a higher conductivity than the second fluid
region; applying an electric field across a length of the first
channel, but not across the second channel, to electroosmotically
transport the first and second fluid regions through the first
channel past the intermediate point, whereby a portion of the first
fluid is forced into the second channel.
85. A method of transporting materials in an integrated
microfluidic channel network, comprising: providing a first
microscale channel that is intersected at an intermediate point, by
a second channel; introducing first and second fluid regions
serially into the first channel, the first and second fluid regions
being in communication at a first fluid interface; applying a
motive force to the first and second fluid regions to move the
first and second fluid regions past the intermediate point, the
first and second fluid regions having different flow rates under
said motive force, the different flow rates producing a pressure
differential at the first interface, the pressure differential
resulting in a portion of the first material being injected into
the second channel.
86. The method of claim 86, wherein the motive force comprises an
electric field applied across a length of the first channel.
87. A method of performing integrated reaction and separation
operations in a microfluidic system, comprising: providing a
microfluidic device comprising a body, and a reaction channel and a
separation channel disposed therein, the reaction channel being in
fluid communication with the separation channel; transporting at
least first and second reactants through the first region, the
first and second reactants are maintained substantially together
allowing reactants to interact to form at least a first product in
the first mixture; transporting the first mixture including the
product to the second region wherein the product is separated from
at least one of the reactants; and separating the product from at
least one of the reactants.
88. A method of performing integrated reaction and separation
operations in a microfluidic system, comprising: providing a
microfluidic device having at least first and second channel
regions disposed therein, the first and second channel regions
being connected by a first connecting channel; introducing first
reactants into the first channel region, the first reactants being
contained within a first material region having a first ionic
concentration, the first region being bounded by second regions
having a second ionic concentration, the second ionic concentration
being lower than the first ionic concentration; transporting the
first and second material regions past an intersection of the first
channel region and the first connecting channel, whereby at least a
portion of the first material region is diverted through the
connecting channel and into the second channel region.
89. A method of performing integrated reaction and separation
operations in a microfluidic device, comprising: providing a
microfluidic device having a reaction channel portion and a
separation channel portion, the reaction channel portion being
fluidly connected and intersecting the separation channel portion
at a first intersection; transporting at least a first reactant
through the reaction channel portion within a first discrete fluid
region, under conditions whereby the reactant reacts to produce at
least a first product, within the first fluid region, the first
fluid region being bounded by at least a second fluid region;
detecting when the at least first fluid region reaches the first
intersection; injecting a portion of the at least first fluid
region into the separation channel; separating the product from the
at least first reactant.
90. The method of claim 89, wherein the first fluid region has a
conductivity higher than the second fluid region, and the detecting
step comprises detecting a change in conductivity in the first
intersection when the first fluid region reaches the first
intersection.
91. The method of claim 89, wherein at least one of the first and
second fluid regions comprises a marker compound, and the detecting
step comprises detecting when the marker compound is present in the
first intersection.
92. A microfluidic device for performing integrated reaction and
separation operations, comprising: a body structure having an
integrated microscale channel network disposed therein; a reaction
region within the integrated microscale channel network, the
reaction region having a mixture of at least a first reactant and a
first product disposed in and flowing through the reaction region,
wherein the reaction region is configured to maintain contact
between the first reactant and the first product flowing
therethrough; and a separation region in the integrated channel
network, the separation region in fluid communication with the
reaction region and being configured to separate the first reactant
from the first product flowed therethrough.
93. A microfluidic device for analyzing electrokinetic mobility
shifts of analytes, comprising: a body structure; a first
microfluidic channel portion having substantially no electrical
field applied across its length; a second microfluidic channel
portion having an electrical field applied across its length, the
second channel portion being fluidly connected to the first channel
portion; and a pressure source in communication with at least one
of the first channel portion and the second channel portion for
moving a material through the first channel portion into the second
channel portion.
94. The microfluidic device of claim 93, comprising first and
second electrodes in electrical contact at first and second ends of
the second channel portion, respectively, each of the first and
second electrodes being operably coupled to an electrical power
source, for applying the electric field across the length of the
second channel portion.
95. The microfluidic device of claim 93, wherein the pressure
source is a positive pressure source and is operably coupled to the
first channel portion.
96. The microfluidic device of claim 93, wherein the pressure
source comprises a negative pressure source, and is operably
coupled to the second channel portion, for drawing the analytes
from the first channel portion into the second channel portion.
97. The microfluidic device of claim 93, wherein the first channel
portion is fluidly connected to a source of first and second
analytes.
98. The microfluidic device of claim 93, wherein the first channel
portion is fluidly connected to a source of at least a third
analyte.
99. The microfluidic device of claim 98, further comprising a
capillary element extending out of the body structure, which
capillary element includes a capillary channel disposed therein,
the capillary channel being open at a first end and fluidly
connected to the first channel portion at a second end, and wherein
fluid communication between the first channel portion and the
source of at least a third analyte is provided by contacting the
open end of the capillary channel with a source of the third
analyte.
100. The microfluidic device of claim 99, wherein the first and
second electrodes are disposed in electrical contact with third and
fourth channels that are in fluid communication with the second
channel portion at the first and second ends of the second channel
portion, respectively.
101. A method of analyzing an effect of a first analyte on a second
analyte, comprising: contacting the first analyte with the second
analyte in a first microfluidic channel portion having
substantially no electric field applied across its length;
transporting at least a portion of the first analyte and second
analyte to a second channel portion that is in fluid communication
with the first channel portion and which has an electric field
applied across its length; measuring a change, if any, in an
electrokinetic mobility of the second analyte in the second channel
portion, a change in the electrokinetic mobility of the second
analyte being indicative of an effect of the first analyte on the
second analyte.
102. The method of claim 101, wherein the effect of the first
analyte on the second analyte is a binding of the first analyte to
the second analyte, which results in a change of the electrokinetic
mobility of the second analyte.
103. The method of claim 101, wherein the effect of the first
analyte on the second analyte is a cleavage effect, which results
is a change in an electrokinetic mobility of the second
analyte.
104. The method of claim 101, further comprising: contacting the
first and second analytes in the first channel with a third
analyte; and measuring a change in the electrokinetic mobility of
the second analyte in the presence of the third analyte relative to
a change in the electrokinetic mobility of the second analyte in
the absence of the third analyte.
105. The method of claim 101, wherein the second analyte has a
detectable label associated with it.
106. The method of claim 105, wherein the detectable label
comprises an optically detectable label.
107. The method of claim 106, wherein the optically detectable
label comprises a fluorescent label.
108. The method of claim 101, wherein the first ad second analytes
are transported into the second microfluidic channel portion by
applying a pressure differential between the first channel portion
and the second channel portion
109. A method of analyzing an electrokinetic mobility shift in a
first analyte, comprising: flowing the first analyte through a
first microscale channel portion having substantially no electrical
field applied across it; introducing the first analyte into a
second microfluidic channel portion; applying an electric field
across a length of the second microfluidic channel portion but not
the first microfluidic channel portion; measuring an electrokinetic
mobility of the first analyte under the electric field applied in
the second channel portion.
110. The method of claim 109, wherein the first analyte comprises a
product of an interaction between at least first and second
precursor analytes, the first and second precursor analytes having
a first and second electrokinetic mobilities, respectively, and the
first analyte having a third electrokinetic mobility.
111. The method of claim 110, wherein third electrokinetic mobility
is different from at least one of the first and second
electrokinetic mobilities.
112. The method of claim 111, wherein the first precursor analyte
comprises a detectable label, the detectable label becoming part of
the first analyte when the first and second precursor analytes
interact.
113. The method of claim 112, wherein the second electrokinetic
mobility is different from the first electrokinetic mobility.
114. The method of claim 113, wherein the first and second
precursor analytes are moved from the first channel portion to the
second channel portion by applying a pressure differential between
the first and second channel portions to force the first and second
precursor analytes into the second channel portion.
115. Use of a microfluidic device for performing integrated
reaction and separation operations, the device comprising: a body
structure having an integrated microscale channel network disposed
therein; a reaction region within the integrated microscale channel
network, the reaction region having a mixture of at least first and
second reactants disposed in and flowing through the reaction
region, the mixture interacting to produce one or more products,
wherein the reaction region is configured to maintain contact
between the first and second reactants flowing therethrough; and a
separation region in the integrated channel network, the separation
region in fluid communication with the reaction region and being
configured to separate the first reactant from the one or more
products flowed therethrough.
116. Use of a microfluidic device for performing integrated
reaction and separation operations, the device comprising: a body
structure having an integrated microscale channel network disposed
therein; a reaction region within the integrated microscale channel
network, the reaction region having a mixture of at least a first
reactant and a first product disposed in and flowing through the
reaction region, wherein the reaction region is configured to
maintain contact between the first reactant and the first product
flowing therethrough; and, a separation region in the integrated
channel network, the separation region in fluid communication with
the reaction region and being configured to separate the first
reactant from the first product flowed therethrough.
117. Use of a device selected from any one of the devices of claims
1-64 and 92-100 for practicing a method selected from any one of
the methods of claims 65-91 and 101-114.
118. An assay utilizing a use set forth in any one of claims
115-117.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to U.S. Ser. No.
60/108,628, filed Nov. 16, 1998 and U.S. Ser. No. 09/093,489 filed
Jun. 8, 1998.
BACKGROUND OF THE INVENTION
[0002] In the analysis of biological and chemical systems, a number
of advantages are realized by the process of miniaturization. For
example, by miniaturizing analytical and synthetic processes, one
obtains advantages in: (1) reagent volumes, where reagents are rare
and/or expensive to produce or purchase; (2) reaction times, where
mixing or thermal modulation of reactants is a rate limiting
parameter; and (3) integration, allowing one to combine multiple
preparative and analytical/synthetic operations in a single
bench-top unit.
[0003] Despite the advantages to be obtained through miniaturized
laboratory systems, or microfluidic systems, early attempts at
developing such systems suffered from a number of problems. Of
particular note was the inability of early systems to control and
direct fluid movement through microfluidic channels and chambers in
order to mix, react and separate reaction components for analysis.
Specifically, many of the early microfluidic systems utilized
micromechanical fluid direction system, e.g., microfabricated
pumps, valves and the like, which were expensive to fabricate and
required complex control systems to be properly operated. Many of
these systems also suffered from dead volumes associated with the
mechanical elements, which prevented adequate fluid control
substantially below the microliter or 100 nanoliter range.
Pneumatic systems were also developed to move fluids through
microfluidic channels, which systems were simpler to operate.
Again, however, these systems lacked sufficient controllability to
move small, precise amounts of fluids.
[0004] Pioneering developments in controlled electrokinetic
material transport have subsequently allowed for the precise
control and manipulation of extremely small amounts of fluids and
other materials within interconnected channel structures, without
the need for mechanical valves and pumps. See Published
International Patent Application No. WO 96/04547, to Ramsey. In
brief, by concomitantly controlling electric fields in a number of
intersecting channels, one can dictate the direction of flow of
materials and/or fluids at an unvalved intersection.
[0005] These advances in material transport and direction within
microfluidic channel networks have provided the ability to perform
large numbers of different types of operations within such
networks. See, e.g., commonly owned Published International
Application No. 98/00231 to Parce et al., and Published
International Application No.98/00705, describing the use of such
systems in performing high-throughput screening operations.
[0006] Despite the wide-ranging utility and relative simplicity of
these advances, in some cases, it may be desirable to provide
simpler solutions to material transport needs within a microfluidic
system. The present invention meets these and other needs.
[0007] In particular, the present invention provides material
direction methods and systems that take advantage of certain flow
properties of the materials, in conjunction with novel structures,
to controllably direct material flow through an integrated
microfluidic channel structure.
SUMMARY OF THE INVENTION
[0008] In a first aspect, this invention provides a microfluidic
device for performing integrated reaction and separation
operations. The device comprises a body structure having an
integrated microscale channel network disposed therein. The
reaction region within the integrated microscale channel network
has a mixture of at least first and second reactants disposed in
and flowing through the reaction region, wherein the mixture
interacts to produce one or more products. The reaction region is
configured to maintain contact between the first and second
reactants flowing therethrough. The device also includes a
separation region in the integrated channel network, where the
separation region is in fluid communication with the reaction
region and is configured to separate the first reactant from the
one or more products flowing therethrough.
[0009] The invention also provides a device for performing
integrated reaction and separation operations. The device comprises
a planar substrate having a first channel disposed in the substrate
containing at least first and second fluid regions. The first fluid
region has an ionic concentration higher than an ionic
concentration of the second fluid region, and the first and second
fluid regions communicates at a first fluid interface. Second and
third channels are disposed in the substrate, the second channel
intersects and connects the first and third channels at
intermediate points along a length of the first and third channels,
respectively. The device also includes an electrokinetic material
transport system for applying a voltage gradient along a length of
the first channel, but not the second channel which
electrokinetically moves the first fluid interface past the
intermediate point of the first channel and forces at least a
portion of the first fluid regions through the second channel into
the third channel.
[0010] This invention also provides methods of performing
integrated reaction and separation operations which include
providing a microfluidic device comprising a body structure having
a reaction channel and a separation channel disposed therein, the
reaction channel and separation channel being in fluid
communication. At least first and second reactants flow through the
reaction channel in a first fluid region. The first and second
reactants interact to form at least a first product within the
first fluid region. The step of transporting through the first
channel is carried out under conditions for maintaining the first
and second reactants and products substantially within the first
fluid region. At least a portion of the first fluid region is
directed to the separation channel, which is configured to separate
the product from at least one of the first and second reactants.
The portion is then transported along the separation channel to
separate the product from at least the first reactant.
[0011] The invention also provides methods of directing fluid
transport in a microscale channel network comprising a microfluidic
device having at least first and second intersecting channels
disposed therein, the first channel being intersected by the second
channel at an intermediate point. First and second fluid regions
are introduced into the first channel, wherein the first and second
fluid regions are in communication at a first fluid interface, and
wherein the first fluid region has a higher conductivity than the
second fluid region. An electric field is applied across a length
of the first channel, but not across the second channel, to
electroosmotically transport the first and second fluid regions
through the first channel past the intermediate point, whereby a
portion of the first fluid is forced into the second channel.
[0012] The invention also provides methods of transporting
materials in an integrated microfluidic channel network comprising
a first microscale channel that is intersected at an intermediate
point by a second channel. First and second fluid regions are
introduced serially into the first channel and are in communication
at a first fluid interface. A motive force is applied to the first
and second fluid regions to move the first and second fluid regions
past the intermediate point. The first and second fluid regions
have different flow rates or inherent velocities under said motive
force. The different inherent velocities produce a pressure
differential at the first interface that results in a portion of
the first material being injected into the second channel.
[0013] The invention also provides methods of performing integrated
reaction and separation operations in a microfluidic system,
comprising a microfluidic device with a body, a reaction channel,
and a separation channel disposed therein. The reaction channel is
in fluid communication with the separation channel. At least first
and second reactants are transported through the first region. The
first and second reactants are maintained substantially together to
allow reactants to interact to form at least a first product in the
first mixture. The first mixture, including the product, is
transported to the second region wherein the product is separated
from at least one of the reactants.
[0014] The invention also provides methods of performing integrated
reaction and separation operations in a microfluidic system,
comprising a microfluidic device having at least first and second
channel regions disposed therein, the first and second channel
regions are connected by a first connecting channel. First
reactants are introduced into the first channel region, the first
reactants being contained within a first material region having a
first ionic concentration. The first region is bounded by second
regions having a second ionic concentration, the second ionic
concentration is lower than the first ionic concentration. The
first and second material regions are transported past an
intersection of the first channel region and the first connecting
channel, whereby at least a portion of the first material region is
diverted through the connecting channel and into the second channel
region.
[0015] In related aspects, the present invention also provides
microfluidic devices for analyzing electrokinetic mobility shifts
of analytes, where the device includes a body structure having a
first microfluidic channel portion disposed therein, where the
first channel portion has substantially no electrical field applied
across its length. A second microfluidic channel portion is also
included, but where the second channel portion has an electrical
field applied across its length. The second channel portion being
fluidly connected to the first channel portion. The device also
includes a pressure source in communication with at least one of
the first channel portion and the second channel portion for moving
a material through the first channel portion into the second
channel portion.
[0016] Relatedly, the present invention also provides methods of
analyzing materials using the described devices. In particular, the
methods of the invention analyze an effect of a first analyte on a
second analyte. The methods steps include contacting the first
analyte with the second analyte in a first microfluidic channel
portion having substantially no electric field applied across its
length. At least a portion of the first analyte and second analyte
is transported to a second channel portion that is in fluid
communication with the first channel portion and which has an
electric field applied across its length. A change in the
electrokinetic mobility of the second analyte, if any, is measured
in the second channel portion, where a change in the electrokinetic
mobility of the second analyte is indicative of an effect of the
first analyte on the second analyte.
[0017] Similarly provided are methods of analyzing an
electrokinetic mobility shift in a first analyte, which methods
comprise flowing the first analyte through a first microscale
channel portion having substantially no electrical field applied
across it. The first analyte is then introduced into a second
microfluidic channel portion. An electric field is then applied
across a length of the second microfluidic channel portion but not
across the length of the first microfluidic channel portion.
Finally, an electrokinetic mobility of the first analyte is
measured under the electric field applied in the second channel
portion.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 schematically illustrates an example of a
microfluidic device incorporating a layered body structure.
[0019] FIG. 2 schematically illustrates a control system for
electrokinetically moving materials within a microfluidic
device.
[0020] FIG. 3 illustrates an example of an embodiment of a
microfluidic device of the present invention for performing
integrated reaction and separation operations. FIG. 3A illustrates
the elements of the device itself, while FIGS. 3B-3C illustrate the
operation of the device in transporting, reacting and separating
reaction components within the device of FIG. 3A. FIG. 3D
illustrates an alternate configuration for the device shown in FIG.
3A, and FIG. 3E illustrates a close-up view of an intersection in a
device of the invention which incorporates conductivity measuring
capabilities at the intersection for controlling injection of
reaction mixtures into separation channels.
[0021] FIG. 4 illustrates one alternate embodiment of a
microfluidic device according to the present invention for
performing integrated reaction and separation operations. FIG. 4A
illustrates the elements of the device itself, while FIG. 4B
illustrates the operation of the device in transporting, reacting
and separating reaction components within the device of FIG.
4A.
[0022] FIG. 5 is a schematic illustration of the pressure profile
across fluid regions of differing ionic concentration when being
transported through a microscale channel by electrokinetic
forces.
[0023] FIG. 6 illustrates an alternate device for performing a
contained reaction operation followed by a separation operations in
a continuous flow mode. FIG. 6A schematically illustrates the
structure of the device itself, while FIG. 6B schematically
illustrates the operation of the device.
[0024] FIG. 7 illustrates a microfluidic device channel layout used
in performing integrated operations where the first portion of the
operation requires containment of reactants while the second
portion requires their separation.
[0025] FIG. 8 illustrates the fluorescence signal of rhodamine B
and fluorescein monitored at various locations along the main
channel during the continuous flow mode operation using the device
shown in FIG. 7.
[0026] FIG. 9 illustrates the fluorescence signal of rhodamine B
and Fluorescein monitored at various locations along the main
channel and separation channel during the injection mode operation
using the device shown in FIG. 7.
[0027] FIG. 10 schematically illustrates alternate devices for
carrying out integrated reaction and separation operations. FIG.
10a illustrates an integrated colinear channel for performing
reactions and separations under pressure and electrokinetic flow,
while FIG. 10b illustrates an alternate device which includes a
separate but connected channel in which electrokinetic separations
are carried out.
DETAILED DESCRIPTION OF THE INVENTION
[0028] I. General
[0029] A. Desirability for Integration
[0030] In chemical and biochemical analyses, a number of useful
analytical operations require processes that include two or more
operational steps. For example, many operations require that a
sample material undergo some preparative reaction(s) prior to the
ultimate analytical operation. Alternatively, some analytical
operations require multiple different process steps in the ultimate
analytical operation. As a specific example, a large number of
operations require a reaction step and a separation step, which
depending upon the analytical operation, may be in either order.
Such operations are easily carried out where one is operating at
the bench scale, e.g., utilizing reagent volumes well in excess of
5 or 10 .mu.l, permitting the use of conventional fluid handling
equipment and technology.
[0031] However, when operating in the microfluidic range, e.g., on
the submicroliter to nanoliter level, conventional fluid handling
technologies fail. Specifically, conventional fluidic systems,
e.g., pipettors, tubing, pumps, valves, injectors, and the like,
are incapable of transporting, dispensing and/or measuring reagent
volumes in the submicroliter, nanoliter or picoliter range. While
microfluidic technology provides potential avenues for addressing
many of these issues, early proposals in microfluidics lacked the
specific control to optimize such systems. For example, a great
deal of microfluidic technology to date has been developed using
mechanical fluid and material transport systems, e.g.,
microfabricated pumps and valves, pneumatic or hydraulic systems,
acoustic systems, and the like. These technologies all suffer from
problems of inaccurate fluid control, as well as excessive volume
requirements, e.g., in pump and valve dead volumes. Failing in this
regard, such systems are largely inadequate for performing multiple
integrated operations on microfluidic scale fluid or reagent
volumes.
[0032] The present invention, on the other hand, provides
microfluidic systems that have precise fluidic control at the
submicroliter, nanoliter and even picoliter range. Such control
permits the ready integration of multiple operations within a
single microfluidic device, and more particularly, the integration
of a reaction operation and a separation operation, within a single
device. Further, microfluidic systems of the present invention,
that incorporate such control also offer advantages of
automatability, low cost and high or ultra-high-throughput.
[0033] In a particular aspect, the microfluidic devices and systems
of the invention include microscale or microfluidic channel
networks that comprise a reaction region and a separation region.
These two regions are connected to allow the controlled movement of
material from one region to the other. As noted above, this is made
simpler by precise control of material transport within the channel
network. In particularly preferred aspects, material transport is
carried out using a controlled electrokinetic material transport
system. In alternate preferred aspects, combined pressure-based and
electrokinetic transport systems are used.
[0034] As used herein, the term "microfluidic" generally refers to
one or more fluid passages, chambers or conduits which have at
least one internal cross-sectional dimension, e.g., depth, width,
length, diameter, etc., that is less than 500 .mu.m, and typically
between about 0.1 .mu.m and about 500 .mu.m. In the devices of the
present invention, the microscale channels or chambers preferably
have at least one cross-sectional dimension between about 0.1 .mu.m
and 200 .mu.m, more preferably between about 0.1 .mu.m and 100
.mu.m, and often between about 1 .mu.m and 20 .mu.m. Accordingly,
the microfluidic devices or systems prepared in accordance with the
present invention typically include at least one microscale
channel, usually at least two intersecting microscale channels, and
often, three or more intersecting channels disposed within a single
body structure. Channel intersections may exist in a number of
formats, including cross intersections, "T" intersections, or any
number of other structures whereby two channels are in fluid
communication.
[0035] The microfluidic devices of the present invention typically
employ a body structure that has the integrated microfluidic
channel network disposed therein. In preferred aspects, the body
structure of the microfluidic devices described herein typically
comprises an aggregation of two or more separate layers which when
appropriately mated or joined together, form the microfluidic
device of the invention, e.g., containing the channels and/or
chambers described herein. Typically, the microfluidic devices
described herein will comprise a top portion, a bottom portion, and
an interior portion, wherein the interior portion substantially
defines the channels and chambers of the device.
[0036] FIG. 1 illustrates a general example of a two-layer body
structure 10, for a microfluidic device. In preferred aspects, the
bottom portion of the device 12 comprises a solid substrate that is
substantially planar in structure, and which has at least one
substantially flat upper surface 14. A variety of substrate
materials may be employed as the bottom portion. Typically, because
the devices are microfabricated, substrate materials will be
selected based upon their compatibility with known microfabrication
techniques, e.g., photolithography, wet chemical etching, laser
ablation, air abrasion techniques, injection molding, embossing,
and other techniques. The substrate materials are also generally
selected for their compatibility with the full range of conditions
to which the microfluidic devices may be exposed, including
extremes of pH, temperature, salt concentration, and application of
electric fields. Accordingly, in some preferred aspects, the
substrate material may include materials normally associated with
the semiconductor industry in which such microfabrication
techniques are regularly employed, including, e.g., silica based
substrates, such as glass, quartz, silicon or polysilicon, as well
as other substrate materials, such as gallium arsenide and the
like. In the case of semiconductive materials, it will often be
desirable to provide an insulating coating or layer, e.g., silicon
oxide, over the substrate material, and particularly in those
applications where electric fields are to be applied to the device
or its contents.
[0037] In additional preferred aspects, the substrate materials
will comprise polymeric materials, e.g., plastics, such as
polymethylmethacrylate (PMMA), polycarbonate,
polytetrafluoroethylene (TEFLON.TM.), polyvinylchloride (PVC),
polydimethylsiloxane (PDMS), polysulfone, polystyrene,
polymethylpentene, polypropylene, polyethylene, polyvinylidine
fluoride, ABS (acrylonitrile-butadiene-styrene copolymer), and the
like. Such polymeric substrates are readily manufactured using
available microfabrication techniques, as described above, or from
microfabricated masters, using well known molding techniques, such
as injection molding, embossing or stamping, or by polymerizing the
polymeric precursor material within the mold (See U.S. Pat. No.
5,512,131). Such polymeric substrate materials are preferred for
their ease of manufacture, low cost and disposability, as well as
their general inertness to most extreme reaction conditions. Again,
these polymeric materials may include treated surfaces, e.g.,
derivatized or coated surfaces, to enhance their utility in the
microfluidic system, e.g., provide enhanced fluid direction, e.g.,
as described in U.S. patent application Ser. No. 08/843,212, filed
Apr. 14, 1997 (Attorney Docket No. 17646-002610), and which is
incorporated herein by reference in its entirety for all
purposes.
[0038] The channels and/or chambers of the microfluidic devices are
typically fabricated into the upper surface of the bottom substrate
or portion 12, as microscale grooves or indentations 16, using the
above described microfabrication techniques. The top portion or
substrate 18 also comprises a first planar surface 20, and a second
surface 22 opposite the first planar surface 20. In the
microfluidic devices prepared in accordance with the methods
described herein, the top portion also includes a plurality of
apertures, holes or ports 24 disposed therethrough, e.g., from the
first planar surface 20 to the second surface 22 opposite the first
planar surface.
[0039] The first planar surface 20 of the top substrate 18 is then
mated, e.g., placed into contact with, and bonded to the planar
surface 14 of the bottom substrate 12, covering and sealing the
grooves and/or indentations 16 in the surface of the bottom
substrate, to form the channels and/or chambers (i.e., the interior
portion) of the device at the interface of these two components.
The holes 24 in the top portion of the device are oriented such
that they are in communication with at least one of the channels
and/or chambers formed in the interior portion of the device from
the grooves or indentations in the bottom substrate. In the
completed device, these holes function as reservoirs for
facilitating fluid or material introduction into the channels or
chambers of the interior portion of the device, as well as
providing ports at which electrodes may be placed into contact with
fluids within the device, allowing application of electric fields
along the channels of the device to control and direct fluid
transport within the device.
[0040] In many embodiments, the microfluidic devices will include
an optical detection window disposed across one or more channels
and/or chambers of the device. Optical detection windows are
typically transparent such that they are capable of transmitting an
optical signal from the channel/chamber over which they are
disposed. Optical detection windows may merely be a region of a
transparent cover layer, e.g., where the cover layer is glass or
quartz, or a transparent polymer material, e.g., PMMA,
polycarbonate, etc. Alternatively, where opaque substrates are used
in manufacturing the devices, transparent detection windows
fabricated from the above materials may be separately manufactured
into the device.
[0041] These devices may be used in a variety of applications,
including, e.g., the performance of high throughput screening
assays in drug discovery, immunoassays, diagnostics, genetic
analysis, and the like. As such, the devices described herein, will
often include multiple sample introduction ports or reservoirs, for
the parallel or serial introduction and analysis of multiple
samples. In preferred aspects, however, these devices are coupled
to a sample introduction port, e.g., a pipettor, which serially
introduces multiple samples into the device for analysis. Examples
of such sample introduction systems are described in e.g.,
Published International Patent Application Nos. WO 98/00231 and
98/00707, each of which is hereby incorporated by reference in its
entirety for all purposes.
[0042] As described above, the devices and systems of the present
invention preferably employ electrokinetic transport systems for
manipulating fluids and other materials within the microfluidic
channel networks. As used herein, "electrokinetic material
transport systems" include systems which transport and direct
materials within an interconnected channel and/or chamber
containing structure, through the application of electrical fields
to the materials, thereby causing material movement through and
among the channel and/or chambers, i.e., positively charged species
will generally be attracted to the negative electrode, while
negative ions will be attracted to the positive electrode.
[0043] Such electrokinetic material transport and direction systems
include those systems that rely upon the electrophoretic mobility
of charged species within the electric field applied to the
structure. Such systems are more particularly referred to as
electrophoretic material transport systems. Other electrokinetic
material direction and transport systems rely upon the
electroosmotic flow of fluid and material within a channel or
chamber structure which results from the application of an electric
field across such structures. In brief, when a fluid is placed into
a channel which has a surface bearing charged functional groups,
e.g., hydroxyl groups in etched glass channels or glass
microcapillaries, those groups can ionize. In the case of hydroxyl
functional groups, this ionization, e.g., at neutral pH, results in
the release of protons from the surface and into the fluid,
creating a concentration of protons at near the fluid/surface
interface, or a positively charged sheath surrounding the bulk
fluid in the channel. Application of a voltage gradient across the
length of the channel, will cause the proton sheath to move in the
direction of the voltage drop, i.e., toward the negative electrode.
Although described as electrophoretic or electroosmotic, the
material transport systems used in conjunction with the present
invention often rely upon a combination of electrophoretic and
electroosmotic transporting forces to move materials.
[0044] "Controlled electrokinetic material transport and
direction," as used herein, refers to electrokinetic systems as
described above, which employ active control of the voltages
applied at multiple, i.e., more than two, electrodes. Rephrased,
such controlled electrokinetic systems concomitantly regulate
voltage gradients applied across at least two intersecting
channels. Controlled electrokinetic material transport is described
in Published PCT Application No. WO 96/04547, to Ramsey, which is
incorporated herein by reference in its entirety for all purposes.
In particular, the preferred microfluidic devices and systems
described herein, include a body structure which includes at least
two intersecting channels or fluid conduits, e.g., interconnected,
enclosed chambers, which channels include at least three
unintersected termini. The intersection of two channels refers to a
point at which two or more channels are in fluid communication with
each other, and encompasses "T" intersections, cross intersections,
"wagon wheel" intersections of multiple channels, or any other
channel geometry where two or more channels are in such fluid
communication. An unintersected terminus of a channel is a point at
which a channel terminates not as a result of that channel's
intersection with another channel, e.g., a "T" intersection. In
preferred aspects, the devices will include at least three
intersecting channels having at least four unintersected termini.
In a basic cross channel structure, where a single horizontal
channel is intersected and crossed by a single vertical channel,
controlled electrokinetic material transport operates to
controllably direct material flow through the intersection, by
providing constraining flows from the other channels at the
intersection. For example, assuming one was desirous of
transporting a first material through the horizontal channel, e.g.,
from left to right, across the intersection with the vertical
channel. Simple electrokinetic material flow of this material
across the intersection could be accomplished by applying a voltage
gradient across the length of the horizontal channel, i.e.,
applying a first voltage to the left terminus of this channel, and
a second, lower voltage to the right terminus of this channel, or
by allowing the right terminus to float (applying no voltage).
However, this type of material flow through the intersection would
result in a substantial amount of diffusion at the intersection,
resulting from both the natural diffusive properties of the
material being transported in the medium used, as well as
convective effects at the intersection.
[0045] In controlled electrokinetic material transport, the
material being transported across the intersection is constrained
by low level flow from the side channels, e.g., the top and bottom
channels. This is accomplished by applying a slight voltage
gradient along the path of material flow, e.g., from the top or
bottom termini of the vertical channel, toward the right terminus.
The result is a "pinching" of the material flow at the
intersection, which prevents the diffusion of the material into the
vertical channel. The pinched volume of material at the
intersection may then be injected into the vertical channel by
applying a voltage gradient across the length of the vertical
channel, i.e., from the top terminus to the bottom terminus. In
order to avoid any bleeding over of material from the horizontal
channel during this injection, a low level of flow is directed back
into the side channels, resulting in a "pull back" of the material
from the intersection.
[0046] In addition to pinched injection schemes, controlled
electrokinetic material transport is readily utilized to create
virtual valves which include no mechanical or moving parts.
Specifically, with reference to the cross intersection described
above, flow of material from one channel segment to another, e.g.,
the left arm to the right arm of the horizontal channel, can be
efficiently regulated, stopped and reinitiated, by a controlled
flow from the vertical channel, e.g., from the bottom arm to the
top arm of the vertical channel. Specifically, in the `off` mode,
the material is transported from the left arm, through the
intersection and into the top arm by applying a voltage gradient
across the left and top termini. A constraining flow is directed
from the bottom arm to the top arm by applying a similar voltage
gradient along this path (from the bottom terminus to the top
terminus). Metered amounts of material are then dispensed from the
left arm into the right arm of the horizontal channel by switching
the applied voltage gradient from left to top, to left to right.
The amount of time and the voltage gradient applied dictates the
amount of material that will be dispensed in this manner.
[0047] A schematic illustration of a system 30 for carrying out
analytical operations within a microfluidic device using controlled
electrokinetic material transport is illustrated in FIG. 2. As
shown, the microfluidic device 10, is connected to an electrical
controller 34 via a series of electrical leads/electrodes 32. The
electrodes are disposed in the reservoirs that are disposed at the
termini of the channels in the channel network within the device
10. The electrical controller typically includes a power supply, as
well as appropriate circuitry for regulation of voltage and/or
currents applied to each of the electrical leads/electrodes 32 to
control material transport, as described above. One example of such
a power supply is that described in commonly owned Published
International Patent Application No. WO 98/00707. The system shown,
also includes a computer 36, which includes appropriate software or
other programming for instructing the electrical controller to
apply appropriate voltage/current profiles to the various
reservoirs or channel termini in order to achieve a desired
material movement within the device, e.g., for a given operation.
In addition to instructing the electrical controller, the computer
also receives data from the controller relating to the electrical
parameters within the device, e.g., applied current/voltage,
resistance, etc., as well as receiving data from the detector 38.
For example, in typical applications, the detector 38 is an
optical, e.g., fluorescence detector, which detects relative
fluorescence levels within the device and reports the data to the
computer 36 for storage and subsequent analysis. The detector is
generally disposed adjacent a detection window that is disposed in
the device, e.g., a translucent or transparent region of the device
10. Accordingly, the computer is typically programmed to instruct
the operation of the system, as well as receive, store and analyze
the data generated by the system.
[0048] Although described for the purposes of illustration with
respect to a four way, cross intersection, these controlled
electrokinetic material transport systems can be readily adapted
for more complex interconnected channel networks, e.g., arrays of
interconnected parallel channels.
[0049] In alternate aspects, the present invention provides
microfluidic devices, systems and methods of using them, for
performing reaction and separation operations within an integrated
microfluidic channel network, that utilize different material
direction and transport means in order to ensure reactants in the
reaction channel portion are maintained together, while reactants
are allowed to separate within the separation channel portion.
[0050] As described above, the integrated device typically includes
at least a first channel portion that is configured so as to
maintain reactants that are flowing through it, together. In the
context of the present embodiment, this is typically accomplished
by driving the flow of the reactants through the first channel
portion using a pressure-based flow system. By using pressure-based
flow, different reactants do not suffer from biasing effects of
differential electrophoretic mobilities, as is true under purely
electrokinetic material transport systems. In operation, first and
second analytes that are to be kept in contact are flowed along the
first channel portion, or reaction channel, where that channel
portion has substantially no applied electric field disposed across
it. The absence of an electric field avoids the electrophoretic
biasing problem noted above. A second microfluidic channel portion,
in fluid communication with the first channel portion is then used
to perform the separation operation. In particular, at least a
portion of the reactants that are flowing through the first channel
portion are introduced into the second channel portion. The second
channel portion has an electric field applied across its length, in
order to promote the electrophoretic separation of reactants.
Application of an electric field is generally carried out as
described herein, e.g., via electrodes disposed in electrical
communication with the termini of the second channel portion,
either directly, or via connecting channels. Typically, the
materials flowing through the second channel portion have a net
flow in one direction, e.g., toward the detection zone, as a result
of one or both of electroosmotic flow and/or pressure based flow
from the first channel. As a result, even species with
electrophoretic mobilities opposite to the desired direction of
flow, e.g., away from the detection zone in the second channel
portion, will still have a net flow in that direction, and thereby
permit their detection.
[0051] In those instances where the interaction of the first and
second analytes has an electrophoretic mobility altering effect on
one or both of the analytes, e.g., resulting in a product that has
an electrophoretic mobility different from one or more of the
original analytes, the applied electric field within the second
channel portion will result in a separation of the product from the
original analytes. The product is then detected, allowing a
quantitative determination of the interaction of the analytes.
[0052] An exemplary assay that is carried out according to the
methods of the present invention is a nonfluorogenic phosphatase
assay which employs a phosphorylated fluorescent substrate that is
dephosphorylated by a phosphatase enzyme to yield a more negatively
charged fluorescent product. Thus, the action of the phosphatase on
the phosphorylated substrate has a mobility altering effect on the
dephosphorylated product. In the systems described herein, the
assay is carried out by flowing the phosphatase enzyme and
fluorescent phosphorylated substrate through the first channel
portion by applying a pressure differential across the first
channel portion, to force or draw the reactants through the
channel. Because there is no electric field applied across the
length of the channel, there is nothing to cause the separation of
the dephosphorylated product from the phosphorylated substrate. The
mixture of product, substrate and enzyme is then directed into the
second channel portion which has, or is capable of having an
electric field applied across its length. When subjected to the
electric field, the dephosphorylated fluorescent product has a
substantially different mobility within the second channel portion
than the phosphorylated fluorescent substrate. As these two
fluorescent components are physically separated, they are
therefore, separately detectable. The production of the separately
detectable species, e.g., substrate and product, is indicative that
the enzyme has acted on the substrate. Assuming then that one
wanted to screen a variety of materials to determine whether those
materials had an effect on the phosphatase activity, it would
merely require introducing those materials into the first reaction
channel, one at a time, as a third reactant contacting the
phosphatase enzyme and substrate. One would again measure the
relative amount of fluorescent product produced, and compare it to
a control reaction, e.g., where no effector of that interaction was
present.
[0053] The reaction mixture is optionally introduced into the
second channel portion as discrete aliquots or plugs, which are
then separated to yield two separate peaks of the detected label,
or as a continuous flow of the reaction mixture which produces a
constant label signal which is interrupted when an effector of the
desired interaction is introduced. Such continuous flow assay
formats are described in great detail in Published International
Patent Application No. WO98/00231, which is incorporated herein by
reference. In brief, variations in the mobility of the labeled
portion of the reaction mixture in discrete regions, e.g., regions
where effectors (inhibitors/enhancers) are introduced, results in
an accumulation or depletion of the labeled product either before
or after the particular reaction region. This is due to the change
in amount of product within those regions resulting from the
presence of, e.g., an inhibitor, which is then made detectable by
the differential mobility of product and substrate.
[0054] B. Specific Assay Examples
[0055] As noted above, a number of useful analytical operations
require processes that include two or more operational steps. For
example, a number of analytical assays require the performance of a
reaction step followed by a separation step. This is typically the
case where the activity that is sought to be detected in the assay
does not itself produce a change in the level of a detectable
signal, such as the production or depletion of a colored,
radioactive or fluorescent species, e.g., product or substrate, an
alteration in detectable solution characteristics, e.g.. pH,
conductivity, etc. or the like. In such cases, it is often
necessary to be able to separate reactants from products in order
to then distinguish between these components and determine their
relative quantities.
[0056] Specific examples of analytical operations that do not
produce an alteration in the level of detectable signal in a
mixture of reactants and products are those assays referred to as
"non-fluorogenic" or "non-chromogenic" assays. In particular, for a
number of assay types, reagents are available that will produce a
colored or fluorescent signal in response to a particular activity.
For example, for a number of enzymes, fluorogenic or chromogenic
substrates are commercially available. In the case of fluorogenic
substrates, the substrate can be either non-fluorescent or have a
low level of fluorescence as a substrate. Alternatively, the
substrate may be fluorescent while the product is non-fluorescent
or detectably less fluorescent than the substrate. However, upon
reaction with the enzyme of interest, a fluorescent product is
produced (or the fluorescent substrate is consumed). By measuring
the amount of fluorescence produced or consumed, one can determine
the relative activity of the enzyme.
[0057] Other examples of fluorogenic reactants include, e.g.,
nucleic acid or molecular beacons. These molecular beacons include
a fluorophore/quencher pair, at different ends of a
self-complementary nucleic acid sequence or at different ends of
two complementary probes. In its native state, autohybridization of
the probe or probes places the fluorophore adjacent to the
quencher, thereby quenching the fluorescent signal. However, under
denaturing conditions, or when the beacon is hybridized to a
complementary nucleic acid sequence, the fluorophore is separated
from its quencher, and a fluorescent signal is detectable.
[0058] In the case of non-fluorogenic assays, however, reagents
often are not available that will produce an altered fluorescence
following the reaction of interest, i.e., there is no change in
fluorescent quantum efficiency of the product from the substrate,
or between the free and bound (or complexed) reactants. Thus, while
a substrate may bear a detectable label, the products of the action
of an enzyme on that substrate will bear the same label and be
present in the same mixture, and are therefore not separately
detectable without, for example, a subsequent separation step. The
same is true, for example, where a ligand bears a detectable label,
and is contacted with a receptor of interest in a mixture. The free
ligand bears the same label as the ligand/receptor complex, and is
therefore generally indistinguishable from the bound or complexed
ligand/receptor in typical fluorescent intensity detection systems,
without at least a subsequent separation step.
[0059] Despite these difficulties however, many reactions do result
in changes in other properties of the reactants/products. For
example, in many cases, a reaction will produce a change in charge
and/or size of the reactants and/or products. As noted previously,
because reactants and products of these non-fluorogenic assays
cannot be distinguished from each other with respect to
fluorescence intensity or spectrum, when present in a mixture of
the two, it is generally necessary to separate them prior to
detection.
[0060] As in bench scale operations, it is these changes in
reactant characteristics that are exploited in separating the
reactants and products in the microfluidic devices of the present
invention. Specifically, the devices and systems of the present
invention that are used in performing such non-fluorogenic assays,
comprise an interconnected microfluidic channel structure that
includes a reaction region and a separation region. In particularly
preferred aspects, the devices include a channel portion in which
reactants are maintained together, in order to allow the reaction
to progress. Following the reaction, the unreacted reactants and
the products are moved to a separation channel or channel portion,
where separation of the reactants and products is carried out,
followed by detection of the desired component, typically the
product.
[0061] In addition to non-fluorogenic enzyme assays, a number of
other assays are non-fluorogenic or non-chromogenic. For example,
with the possible exception of assays that utilize a molecular
beacon, e.g., certain nucleic acid binding assays, most binding
assays are non-fluorogenic or non-chromogenic. In particular, the
bound or complexed components of the assay do not change in the
amount or spectrum of fluorescence over that of the free
components. Thus, in a mixture the bound and free components are
typically indistinguishable. Again, such assays typically utilize a
separation step to first separate, then identify the relative
levels of bound and free components. In most cases, such assays are
carried out by tethering one member of the binding pair, e.g., the
receptor or ligand, or one strand of complementary nucleic acids.
The other binding member that bears a fluorescent label is then
contacted with the tethered member, and the labeled material that
does not bind is washed away, leaving the bound fluorescent, or
otherwise labeled material to be detected. This is one of the basic
principles behind the development of molecular array technologies.
See, e.g., U.S. Pat. No., 5,143,854, to Pirrung et al.
Alternatively, such assays would require the separation of bound
and free components using, e.g., a chromatographic step.
[0062] The devices and systems of the invention are equally
applicable to such binding assays, and utilize the same principles
as outlined above. In particular, bound complexes often have
different charges, sizes or charge:mass ratios from their separate
reactant components. These differences are exploited, as described
above, to separate the reactants, e.g., unbound labeled ligand and
unbound receptors, from the products, e.g., complexed labeled
ligand and receptor. The separated components are then separately
detected, whereby their relative concentrations are determined.
[0063] Although described in terms of reactions that employ two or
more reactants followed by separation of reactants and the
products, it will be apparent that the methods and devices of the
invention are readily employed in separating a product from the
reactant in a single reactant reaction, e.g., where product is
formed from the single reactant, e.g., a spontaneous reaction
(degradation, association, aggregation, etc.), as a result of a
thermal or photo-induced reaction (photolysis etc.).
[0064] Related methods are also described in PCT/US98/11969
(WO98/56956), and are incorporated herein by reference.
[0065] C. Devices, Systems and Methods
[0066] Integration of multiple different operations within a single
microfluidic device can create a number of difficulties. For
example, as noted above, there are a number of difficulties
associated with accurately transporting microscale fluid volumes
within integrated channel structures. However, even more problems
arise where different operations to be performed within the
microscale channels have markedly differing, and even conflicting
goals. For example, in a number of analytical operations, in the
reaction portion of the overall operation it is generally desirable
to maintain all of the reactants in contact with one another, to
ensure that the reaction will proceed. For the separation portion
of the operation, however, it is generally necessary to separate
those very same reactants from one another, and/or from their
products.
[0067] As used herein, the terms "reactant" and "product" are not
intended to denote any specific type of interaction, but are
generally used to refer to an interaction between two or more
chemical, biochemical or biological species, which interaction
includes, chemical, biochemical, electrical, physical or other
types of interactions. Some specific nonlimiting examples of
reactants and their respective products include, e.g.,
complementary single stranded nucleic acids and their double
stranded products, ligands and receptors, and the complexes formed
therefrom, enzymes and substrates, and the products produced
therefrom, cells and cell affectors and products of such
interactions, e.g., agglutinated cells, secreted cellular products,
cells with activated incorporated reporter systems, etc.
[0068] In its simplest embodiment, the operations carried out using
the devices and systems of the invention are performed by providing
a first channel into which the various reactants are introduced as
a continuous mixture. After the reaction has been allowed to occur,
a portion of the mixture is then aliquoted into a separate channel
region in which separation of the reaction components occurs.
Separation typically involves a chromatographic or electrophoretic
separation of these components in the separation channel. The
separated components are then detected at a detection window in the
separation channel. Although described in terms of mixtures of
reactants, it will be readily appreciated that the present
invention is useful in performing integrated reaction and
separation operations where a single reactant is introduced into
the system. For example, photolyzable compounds that are first
photolyzed, then separated, fall within the scope of "reactants" as
defined herein. Similarly, heat labile compounds that dissociate
(e.g., double stranded nucleic acids), degrade, or hydrolyze under
elevated temperatures also fall within this scope.
[0069] FIG. 3A schematically illustrates a microfluidic device for
performing these integrated operations from a top and end view.
FIGS. 3B and 3C illustrate the use of the device of FIG. 3A in an
"injection mode," e.g., where reaction mixtures are injected into a
connected channel. As shown in FIG. 3A, the device 300, includes a
substrate 302 that includes a reaction channel 304 that connects a
first reactant source and a waste reservoir 308. As shown, the
first reactant source is shown as an inlet 306 from an external
sample accessing capillary 306a, e.g., an electropipettor (See WO
98/00705). A second reactant reservoir 310 is fluidly connected to
the reaction channel 304 via channel 312. A third reactant
reservoir 314 is connected to the reaction channel 304 via channel
316. Separation channel 318 intersects and crosses the reaction
channel 304 at a first intersection 320, and connects separation
buffer reservoir 322 and waste reservoir 324. In operation, the
first reactant is introduced into the reaction channel through the
external sample accessing capillary 306a. The second reactant is
flowed into the reaction channel from second reactant reservoir 310
via channel 312, whereupon it is mixed with the first reactant. An
optional third reactant is introduced into reaction channel 304
from reservoir 314 via channel 316. The reaction mixture is flowed
through the reaction channel 304 past the first intersection 320
and toward the waste reservoir 308.
[0070] A portion of this reaction mixture at the intersection 320
is then injected into the separation channel 318, which includes an
appropriate buffer, medium or matrix for separating the components
of the mixture. Typically, the separation medium is selected to
permit the electrophoretic separation of the components of the
reaction mixture, e.g., reactants and products. Generally, the
separation medium is selected to substantially reduce the relative
level of electroosmotic flow of fluid within the separation
channel, leaving electrophoresis as the primary force in moving the
materials, and through which differentiation of those materials is
achieved. In most cases, it is sufficient that the separation
medium comprises a buffer that includes an ionic strength that is
sufficiently high, such that electrophoretic differentiation of
species is allowed to occur in the channel, e.g., before
electroosmotic flow transports the material into the waste
reservoir. In some cases however, e.g., in the separation of larger
macromolecules, electrophoretic differentiation of species is
enhanced by the incorporation of a sieving component within the
separation medium, e.g., a polymer matrix component. Examples of
separation media incorporating such matrices have been widely
described for use in capillary electrophoresis applications. See,
U.S. Pat. Nos. 5,264,101 to Demorest, and U.S. Pat. No. 5,110,424
to Chin. Typically, sieving matrices are polymer solutions selected
from, e.g., agarose, cellulose, polyacrylamide polymers, e.g.,
cross-linked or non-crosslinked polyacrylamide,
polymethylacrylamide, polydimethylacrylamide, and the like. Useful
separation matrices also include other types of chromatographic
media, e.g., ion exchange matrices, hydrophobic interaction
matrices, affinity matrices, gel exclusion matrices, and the like.
Similarly, the types of separations performed in the separation
channel can be varied to include a number of different separation
types, e.g., micellar electrokinetic chromatography, isoelectric
focusing chromatography, counter-current electrophoresis, and the
like. In such cases, the products and reactants from which they are
to be separated have different partitioning coefficients (vs.
different electrophoretic mobilities) in the separation
channel.
[0071] The portion of the reaction mixture that is injected into
the separation channel is then transported along the separation
channel allowing the components of the mixture to separate. These
components are then detected at a detection window 326 at a point
along the separation channel.
[0072] While the device and methods described above are useful for
performing integrated reaction and separation operations, the
throughput of the method as described, is somewhat limited. In
particular, in the method described, only a single reaction is
carried out in the reaction channel 304 at a time. After the
separation of the reaction components has been carried out in the
separation channel 318, new reaction components are introduced into
the reaction channel for additional assays.
[0073] An alternate aspect of the present invention utilizes the
same basic injection mode concept and device structure as that
described with reference to FIG. 3A, and is illustrated in FIGS. 3B
and 3C. This alternate aspect is designed to be utilized in
conjunction with high-throughput screening assay methods and
systems that utilize controlled electrokinetic material transport
systems to serially introduce large numbers of compounds into a
microfluidic channel in which a continuous flow assay is carried
out. See, commonly assigned published International Application No.
98/00231, which is incorporated herein by reference in its
entirety. In carrying out these high-throughput assays, one or more
reactants are continuously flowed into the reaction channel 304
from reservoirs 310 and 314, as shown by arrows 330, 332 and 334.
The compound materials (an additional set of reactants) are
introduced from sampling capillary 306a, and are generally
maintained together within discrete plugs 336 of material, to
prevent smearing of one compound into the next which might result
from electrophoretic movement of differently charged materials
within the compound plug. These discrete plugs are then contacted
with a continuously flowing stream of one or more additional
reactants, e.g., enzyme and/or substrate, or members of specific
binding pairs.
[0074] Maintaining the cohesiveness of the discrete
compound/reactant plugs 336 (referred to as "reaction material
plugs") in these flowing systems, and thus allowing them to react,
is typically accomplished by providing the compound in a relatively
high ionic strength buffer ("high salt buffer" or "high
conductivity buffer"), and spacing the compound plugs with regions
of low ionic strength buffer 338 ("low salt buffer" or "low
conductivity buffer"). Because most of the voltage drop occurs
across the low conductivity buffer regions rather than the high
conductivity reaction material plugs, the material is
electroosmotically flowed through the system before there can be
extensive electrophoretic biasing of the materials in the compound
plug 336. In order to subsequently separate the reactants and
products resulting from the assay, as is often necessary in
non-fluorogenic assays, the containing influence of the high salt
plugs/low salt spacer regions must generally be overcome or
"spoiled."
[0075] In accordance with the method described above, and with
reference to FIG. 3C the containing influence of the high
conductivity material plug 336/low conductivity spacer region 338,
is overcome or spoiled by injecting a portion 340 of the high
conductivity reaction material plug 336 into the separation channel
318 that is also filled with a high conductivity buffer, as the
plug 336 moves past the intersection of the reaction channel and
separation channel. As noted above, because the separation channel
is filled with a high conductivity buffer, the electrokinetic
mobility of materials within the channel resulting from the
electrophoretic mobility of the components of the reaction material
relative to the electroosmotic movement of the fluid is
accentuated.
[0076] As the reaction material plug is transported past the
intersection 320 of the reaction channel 304 and the separation
channel 318, it is injected into the separation channel 318 by
switching the flow through the separation channel, as shown by
arrow 342. This is generally carried out by first slowing or
halting flow of the reaction material plug through the reaction
channel 304 while that plug 336 traverses the intersection 320.
Flow is then directed through the separation channel to inject the
portion of the plug that is in the intersection, into the
separation channel 318. Controlling flow streams are also
optionally provided at the intersection 320 during the reaction,
injection and separation modes, e.g., pinching flow, pull-back
flow, etc., as described above and in published International
Application No. 96/04547, previously incorporated herein by
reference.
[0077] While this method is very effective, and is also applicable
to high throughput systems, there is a measure of complexity
associated with monitoring the progress of the reaction material
plugs through the reaction channel and timing the injection of
material into the separation channel. In one aspect, the passage of
reaction material plugs through the intersection 320 is carried out
by measuring the conductivity through the intersection, e.g.,
between reservoirs 322 and 324. In particular, because the reaction
materials are contained in high ionic concentration plugs, their
passage through the intersection will result in an increase in
conductivity through the intersection and through the channel
between reservoirs 322 and 324. Measurement of conductivity between
reservoirs 322 and 324 is generally carried out using either a low
level of direct current, or using an alternating current, so as not
to disturb the electrokinetic flow of materials in the integrated
channel network. Further, because electrokinetic transport is used,
electrodes for measuring the conductivity through channels are
already in place in the wells or reservoirs of the device.
Alternatively, smaller channels are provided which intersect the
reaction channel on each side, just upstream of the injection point
or intersection, as shown in FIG. 3E. Specifically, channels 352
and 354 are provided just upstream of intersection 320, and include
electrodes 356 and 358 in electrical contact with the unintersected
termini of these channels. As used herein, the term "electrical
contact" is intended to encompass electrodes that are physically in
contact with, e.g., the fluid such that electrons pass from the
surface of the electrode into the fluid, as well as electrodes that
are capable of producing field effects within the medium with which
they are in electrical contact, e.g., electrodes that are in
capacitive contact or ionic contact with the fluid. These
electrodes are then coupled with an appropriate conductivity
detector 360 for measuring the conductivity of the fluid between
the electrodes, e.g., in the reaction channel 304, as it flows into
the intersection, which flow is indicated by arrow 362.
Conductivity is then measured across these channels to identify
when the reaction material plug is approaching the intersection.
This conductivity measurement is then used to trigger injection of
a portion of the reaction material plug into the separation channel
318. Typically, each of these additional channels includes a
reservoir at its terminus distal to the reaction channel, and
conductivity is measured via electrodes disposed in these
reservoirs. Alternatively, the two detection channels could be
provided slightly staggered so that the distance between the
channels along the length of the reaction channel is small enough
to be spanned by a single reaction material plug. The electrodes
disposed at the termini of these channels are then used to sense
the voltage difference between the intersection of each of the two
channels and the reaction channel, e.g., along the length of the
reaction channel. When a high conductivity reaction material plug
spans the distance between the two channels, the voltage difference
will be less, due to the higher conductivity of the fluid between
them.
[0078] Another preferred method of addressing this issue is
described with reference to FIG. 3D. In particular, as shown, the
device has a similar layout to that of the device shown in FIG. 3A.
However, in this aspect, the separation channel portion is channel
portion 350, which is colinear with the reaction channel portion
304, channel portion 318a functions as a waste/gating channel, and
the detection window 336a is disposed over channel portion 350.
This method of transporting the material from the reaction channel
region 304 to the separation channel region 350 is referred to as a
"continuous flow mode" or "gated injection mode."
[0079] In operation, the reaction material plugs are directed along
the reaction channel portion 304 through intersection 320, and into
waste channel 318a, toward reservoir 324, e.g., using an
electrokinetic gated flow. During operation of the device, the
resistance level between reservoirs 322 and 324 is monitored. As a
reaction material plug enters waste channel 318a, the increase in
conductivity resulting from the higher ionic concentration of the
high salt reaction material plug is used to trigger a gated
injection of a portion of that plug into the separation channel
350. Specifically, upon sensing a predetermined level of
conductivity increase, a computer linked with the electrical
controller aspect of the overall system, directs a switching of the
applied currents to produce the gated flow profile described above,
for a short period, e.g., typically less than 1 second. By gating
flow of the reaction material plugs into waste channel 318a,
conductivity changes between reservoirs 322 and 324 are more
pronounced as the length of the plug occupies a greater percentage
of the channel across which the conductivity is being measured. As
a result, one can more effectively identify meaningful conductivity
changes and thereby determine when the reaction material plugs
enter the intersection/injection point. Specifically, when using
this latter method, one is measuring conductivity changes resulting
from the length of the material plug, as opposed to measuring the
changes resulting from the width of the plug, e.g., as it passes
through an intersection across which conductivity is measured, as
described with reference to FIGS. 3B-3C, above. Again, as described
with reference to FIG. 3E above, auxiliary channels and reservoirs
may be used to measure conductivity changes across different
portions of a channel or intersecting channels, e.g., one
conductivity sensing electrode may be placed in contact with the
reaction channel, e.g., via a side channel, upstream of the
intersection while another is placed downstream of the
intresection.
[0080] Although described in terms of detecting changes in
conductivity, a number of methods can be used to detect when the
reaction material plug is present in or near the intersection. For
example, marker compounds may be provided within either the
reaction material region or the spacer regions. These compounds,
and thus the presence or absence of a reaction material plug or
region then can be detected at or near the injection intersection
to signal a change in the flow profile from reaction to injection
mode, e.g., injecting the reaction material into the separation
channel portion. Such marker compounds optionally include optically
detectable labels, e.g., fluorescent, chemiluminescent,
calorimetric, or colloidal materials. The marker compounds are
typically detected by virtue of a different detectable group than
that used to detect the results of the reaction of interest. For
example, where the reaction of interest results in a fluorescent
product that must be separated from a fluorescent reactant prior to
detection, the marker compound typically includes either a
non-fluorescent compound, e.g., colored, colloidal etc., or a
fluorescent compound that has a excitation and/or emission maximum
that is different from the product and/or reactant. In the latter
case, the detection system for detecting the marker compound is
typically configured to detect the marker compound without
interference from the fluorescence of the product/reactant
label.
[0081] In preferred aspects, these marker compounds are neutral
(have no net charge) at the operating pH of the system, so that
they are not electrophoretically biased during transport within
their discrete regions. Except as described above, these optically
detectable marker compounds are typically detected using a similar
or identical detection system used to detect the separated elements
of the reaction of interest, e.g., a fluorescent microscope
incorporating a PMT or photodiode, or the like.
[0082] FIG. 4A schematically illustrates an alternative mechanism
for overcoming the influence of these high salt plug/low salt
spacer regions within the separation region or channel of the
device using another version of the injection mode. As shown, the
device 400, includes a substrate 402, having a reaction channel 404
disposed therein. As shown, the reaction channel 404 is in
communication at one end with the inlet from a pipettor capillary
406 (shown from a top view). The pipettor 406 is capable of
accessing and introducing large numbers of different sample
materials into the analysis channel 404. The analysis channel is in
communication at the other end, with a waste reservoir 408.
Reservoirs 410 and 414 typically include the different reactants
needed for carrying out the reaction operation for the device and
are connected to reaction channel 404 via channels 412 and 416,
respectively. Separation channel 418 is located adjacent to
analysis channel 404, and connecting channel 420 links the two
channels at an intermediate point in both channels. Separation
channel 418 links separation buffer reservoir 422 and waste
reservoir 424. A detection window 426 is also provided within
separation channel 418, through which separated sample components
may be detected.
[0083] In one mode, the device shown in FIG. 4 is capable of taking
advantage of certain flow characteristics of fluids under
electrokinetic transport. In particular, in electrokinetically
moving different fluid regions that have different electroosmotic
flow rates, pressure gradients are created within the fluid
regions. In particular, electroosmotic fluid flow within a
microscale channel is driven by the amount of voltage drop across a
fluid region. Thus, low ionic strength, e.g., low conductance, high
resistivity, fluid regions have higher electroosmotic ("EO") flow
rates, because these regions drop a larger amount of voltage. In
contrast, higher ionic strength fluids, e.g., higher conductance
materials, drop less voltage, and thus have lower EO flow
rates.
[0084] Where a system includes different fluid regions having
different ionic strengths, these different flow rates result in
pressure differentials at or near the interface of the two fluid
regions. Specifically, where a first fluid of higher ionic
strength, e.g., a sample material, is being pushed by a second
fluid region of lower ionic strength, the trailing end of the first
fluid region is at a higher pressure from the force of the second
fluid region. Where the first fluid region is following the second
fluid region, the pulling effect of the second fluid region results
in a lower pressure region at the leading edge of the first fluid
region. A channel that includes alternating high and low ionic
strength fluid regions, will also include alternating high and low
pressure areas at or near the interfaces of the different regions.
FIG. 5 schematically illustrates the pressure gradients existing in
a channel having such different ionic strength regions. These
pressure effects were described and a method for overcoming them
set fort in commonly owned published International Application No.
WO 98/00705, incorporated herein by reference in its entirety. In
brief, in order to prevent perturbations resulting from these
pressure effects at channel intersections, the channel intersecting
the main channel is typically made shallower, as the pressure
effects drop off to the third power with decreasing channel depth,
whereas electroosmotic pumping is only reduced linearly with
channel depth. See Published International Application No. WO
98/00705.
[0085] The operation of the device shown in FIG. 4A is described
below, with reference to FIGS. 4A and 4B in the performance of a
high-throughput screening assay, which screens for affectors of a
reaction of two reactants, e.g., inhibitors or enhancers of enzyme
activity, inhibitors or enhancers of ligand receptor binding, or
any other specific binding pair. In brief, the reactants are
maintained in a relatively low ionic strength buffer, and are
placed into the first reactant reservoir 410, and the second
reactant reservoir 414. Each of these reactants is then
electrokinetically transported through the reaction channel 404
toward waste reservoir 408 in a continuous stream, as indicated by
arrows 430, 432 and 434. This electrokinetic transport is carried
out, as described above, by applying appropriate voltage gradients
between: (1) the first reactant reservoir and the waste reservoir;
and (2) the second reactant reservoir and the waste reservoir.
[0086] Periodically, a plug of material 436 that includes a
compound which is to be screened for an effect on the reaction of
the two reactants is introduced into the reaction channel by way of
the external sample accessing capillary 406 shown from an end view.
The capillary 406 is integrated with the reaction channel 404. In
particularly preferred aspects, this external sample accessing
capillary 406 is an electropipettor as described in published
International Patent Application No. WO 98/00705.
[0087] As described above, these plugs 436 of compound material are
in a relatively high ionic strength buffer solution, and are
introduced with spacer regions 438 of relatively low ionic strength
buffer. The higher ionic strength compound plugs typically approach
physiological ionic strength levels, and are preferably from about
2 to about 200 times the conductivity of the low ionic strength
buffer, in some cases, from about 2 to about 100 times the
conductivity of the low ionic strength buffer, and more preferably,
from about 2 to about 50 times the conductivity of the low ionic
strength buffer, and in many cases from about 2 to about 20 or even
10 times the conductivity of the low ionic strength buffer.
Typically, the high ionic strength buffer has a conductivity from
about 2 mS to about 20 mS, while the low ionic strength buffer has
a conductivity of from about 0.1 mS to about 5 mS, provided the low
ionic strength buffer has a lower conductivity than the higher
ionic strength buffer.
[0088] As the plugs of material 436 are transported along the
reaction channel, the two reactants are allowed to react in the
presence of the compound that is to be screened, within the plug
436, and in the absence of the compound to be screened, outside of
the plug 436, e.g., within spacer region 438. As the reaction
material plug 436 moves past the intersection of reaction channel
404 and connecting channel 420, the pressure wave caused by the
differential flow rates of the high ionic strength plugs and low
ionic strength spacer regions causes a small portion of the
material plug, or "aliquot," 440 to be injected into the connecting
channel 420.
[0089] As shown in FIG. 5, the pressure wave caused by the
interface of the high salt and low salt regions is reciprocated at
the opposite interface of the next compound plug. As such, it is
important to transport the aliquot 440 through the connecting
channel 420 into the separation channel 418 and away from the
intersection of these channels, before it is sucked back into the
reaction channel 404. This is generally accomplished by providing
the connecting channel with appropriate dimensions to permit the
aliquot to progress entirely through the connecting channel and
into the separation channel. Typically, the connecting channel will
be less than 1 mm in length, preferably less than 0.5 mm in length,
more preferably, less than 0.2 mm in length, and generally, less
than about half the width of the reaction channel, e.g., typically
from about 5 to about 100 .mu.m. Additionally, to prevent refluxing
of the aliquot into the reaction channel, flow is typically
maintained within the separation channel to move the aliquot 440
away from the intersection of connecting channel 420 and separation
channel 418, which flow is indicated by arrows 442. This same
injection process is repeated for each compound plug that is
serially introduced into the reaction channel. The effects of the
pressure wave at the intersection, and thus the size of the
injected plug can be adjusted by varying the depth of the
connecting channel at the intersection, as described above. For
example, smaller injections are achieved by making the connecting
channel shallower than the reaction channel.
[0090] The separation buffer within separation channel 418 is
selected so as to permit separation of the components within the
aliquot of reaction material. For example, whereas the materials in
the reaction channel are contained in a high salt plug to prevent
electrophoresis, the separation channel typically includes a high
salt buffer solution, which then allows the electrophoretic
separation of the components, e.g., by diluting the low salt
regions and their effects on material movement in the channels,
e.g., increased electroosmotic flow as compared to the
electrophoretic effects on the components of the reaction material.
Of course, in some cases, a high salt buffer is used in order to
create a more uniform conductivity throughout the separation
channel, allowing separation of components in the aliquot of
reaction material before the material is electroosmotically
transported out of the separation channel.
[0091] As described, in alternate or additional aspects, the
separation channel includes a separation matrix, or sieving
polymer, to assist in the separation of the components of the
reaction material aliquot.
[0092] Once the reaction material is injected into the separation
channel 418 it is transported through the separation channel and
separated into its component elements. Typically, the flow of
material within the separation channel is directed by
electrokinetic means. Specifically, a voltage gradient is typically
applied between separation buffer reservoir 422 and waste reservoir
424, causing the flow of material through the separation channel.
In addition, the voltage gradient within the separation channel
418, is typically applied at a level whereby there is no current
flow through the connecting channel 420, or only sufficient current
to prevent leakage through the connecting channel during
non-injection periods. This prevents the formation of any
transverse currents between the separation channel and the reaction
channel, which might disturb controlled material flow. Once
separated, the components of the reaction material are then
transported past a detection window 426 which has an appropriate
detector, e.g., a fluorescence scanner, microscope or imaging
system, disposed adjacent to it.
[0093] Optionally, the device illustrated in FIG. 4 employs active
material transport, e.g., electrokinetic transport, to inject a
portion 440 of the reaction material plug 436 into the separation
channel 418. In particular, the reaction material plug 436 is
electrokinetically transported along the reaction channel 404, as
described above. Once the reaction material plug 436 reaches the
intersection of the reaction channel 404 and the connecting channel
420, the electrical potentials at the various reservoirs of the
device are switched to cause current flow, and thus, flow of a
portion of the reaction material, through the connecting channel,
into the separation channel 418. The portion 440 of the reaction
material plug is then electrokinetically transported through
separation channel 418 by virtue of current flow between the
reservoirs 422 and 424. The current through the separation channel
is adjusted to match the current flowing through the reaction
channel 404, so that no transverse currents are set up through the
connecting channel. This active electrokinetic injection, as well
as the more passive pressure differential injection described
above, provide advantages over other injection modes of integrated
reaction an separation, by permitting the reaction and separation
channels to operate at the same time. Specifically, transport of
material along the reaction channel does not need to be arrested
during the separation process, and vice versa.
[0094] A simpler embodiment of the present invention and
particularly a microfluidic device for carrying it out, is
illustrated in FIG. 6. In this embodiment, the containing influence
of the high salt plugs in the reaction region or channel of the
device, as described above, is overcome or spoiled by introducing a
stream of separation inducing buffer into the system at the
junction between the reaction and separation regions. As used
herein, the term "separation inducing buffer" refers to a buffer in
which molecular species may be readily separated under appropriate
conditions. Such buffers can include pH altering buffers, sieving
buffers, varied conductivity buffers, buffers comprising separation
inducing components, e.g., drag enhancing or altering compounds
that bind to the macromolecular species to create differential
separability, and the like. For example, in the systems of the
present invention, the separation inducing buffer generally refers
to either a high salt or low salt buffer introduced into the system
at the junction point between the reaction and separation regions.
The introduction of high salt or low salt buffer lessens the
conductivity difference between the reaction material plug
(typically in high salt buffer) and the spacer region (typically in
low salt buffer), by diluting out or spoiling the differential
electrophoretic/electroosmotic forces among the different regions.
This dilution or spoiling allows electrophoretic separation of the
materials in the plug, as described above. This method is referred
to as a "continuous flow mode" because the reaction material plugs
are continuously flowing along a colinear channel, without being
redirected into an intersecting channel. Typically, the separation
inducing buffer will be either: (1) a high salt buffer having a
conductivity that is greater than the conductivity of the low salt
buffer regions, e.g., from about 2 to about 200 times greater,
preferably from about 2 to about 100 times greater, more
preferably, from about 2 to about 50 times greater, and still more
preferably, from about 2 to about 20 times greater, and often from
about 2 to about 10 times greater than the conductivity of the low
salt buffer regions; or (2) a low salt buffer having a conductivity
that is lower than the first conductivity by the same factors
described above. Of course, implied in these ranges are separation
inducing buffers that have conductivity that is substantially
approximately equivalent to either of the high salt fluid regions
or low salt fluid regions.
[0095] As shown in FIG. 6A, the device 600 is disposed in a planar
substrate 602, and includes a reaction channel region 604 and a
separation channel region 606. The reaction and separation channels
are in communication at a junction point 610. Waste reservoir 608
is disposed at the terminus of the separation channel region 606.
Also intersecting these channels at the junction point 610, is an
additional channel 612 which delivers high conductivity buffer from
reservoir 614 into the separation channel region. As with the
device described above, reactants are delivered into the junction
point 610 for reaction channel region 604 and separation channel
region 606, from first and second reactant reservoirs 616 and 618
via channels 620 and 622. Compounds that are to be screened for
effects on the reaction of the reactants are typically introduced
using an appropriate external sample accessing capillary or
pipettor 624, e.g. an electropipettor.
[0096] In operation, the reactants are transported from their
respective reservoirs 616 and 618 and along the reaction channel
region 604 in a continuous flow stream, as indicated by arrows 630,
632 and 634. Periodic plugs of compounds to be screened 636 in high
salt buffer are also flowed along the reaction channel, the
reaction mixture of the first and second reactants and the test
compound being contained within the high salt plug 636 and adjacent
low salt regions. As the plug of material 636 is transported past
the junction point 610, a stream of higher conductivity buffer,
indicated by arrow 638, continuously mixes with the reaction
mixture plug and adjacent low ionic strength regions changing the
relative field strengths across the high and low ionic strength
regions, e.g., the voltage drop across the lower ionic strength
regions is decreased. This change in field strengths allows
differentially charged material components within the reaction
mixture plug 636 to be separated into their component species 640
and 642, based upon differences in the electrophoretic mobility of
those components, as they move along the separation channel region
606. It should be noted that in accordance with the present
invention, a lower salt buffer could also function as a "spoiling
buffer" to bring the relative ionic strengths of the different
material regions closer together, and expose the entire length of
the channel to similar voltage gradients, e.g., including the
components of the reaction mixtures.
[0097] Examples of a device and system for performing integrated
reaction/separation operations using a combination of pressure flow
and electrokinetic transport are schematically illustrated in FIGS.
10a and 10b.
[0098] As shown in FIG. 10a, the device 1000 includes a body
structure 1002 which includes a first channel portion 1004 that is
fluidly connected to a second channel portion 1006. The first
channel portion is also fluidly connected to sources of reactants
1008 and 1010, via channels 1012 and 1014, respectively. The first
channel portion is also shown in fluid connection with an external
capillary element (not shown) via port 1016. As shown, the second
channel portion 1006 is fluidly connected to ports/reservoirs 1018,
1020, and 1022 via channel portions 1024, 1026 and 1028,
respectively. As shown, the device 1000 also includes a detection
window 1030 disposed across the second channel portion 1006.
[0099] In operation, first and second analytes, e.g., enzyme and
substrate, ligand and receptor, etc., are introduced into the first
channel portion 1004, from reservoirs 1008 and 1010, via channels
1012 and 1014, respectively. The first and second analytes are
moved into the first channel portion by applying an appropriate
pressure differential between the reservoirs and the first channel.
In the device shown, this is optionally accomplished by applying a
vacuum to reservoir 1022, which is translated into the first
channel portion 1004 by channels 1028 and 1006. A third analyte is
introduced into the first channel portion 1004 through the
capillary element (not shown) via inlet port 1016. Again, the
vacuum applied to the system functions to draw material that is
placed into contact with the open end of the capillary element.
Specifically, the capillary element is dipped into a source of at
least a third reactant whereby the vacuum sips the reactant into
the capillary channel and into channel portion 1004. The first,
second and optionally third reactants are permitted to react as
they move along the first channel portion 1004 toward the
intersection with channel portion 1006. As no electric field is
applied across this channel portion 1004, no electrophoretic
separation of the reactants and/or their products will occur.
[0100] Once the reaction mixture moves into channel portion 1006,
it is subjected to an electric field to promote electrophoretic
separation of the species therein. The electric field is typically
applied across channel portion 1006 by placing electrodes into
contact with fluid that is disposed in reservoirs 1020 and 1018,
creating an electric field between the reservoirs and across
channels 1024, 1006, and 1026. As the reaction components
separated, the separation is detected at detection window 1030,
typically as a fluorescent signal, or deviation from a steady state
fluorescent signal.
[0101] An alternate device construction for carrying out the same
assay methods is illustrated in FIG. 10b. Components of the device
shown in FIG. 10b that are the same as those shown in FIG. 10a are
referenced with the same reference numerals. As shown, the device
1000 includes a first channel portion 1004 that is fluidly
connected to at least first and second reactant sources, e.g.,
reservoirs 1008 and 1010, and includes the optional inlet port 1016
fluidly coupled to an external capillary element (not shown). The
first channel portion is fluidly coupled to a vacuum port/reservoir
1032. An additional channel 1034 intersects and crosses the first
channel portion 1004 and is fluidly connected to reservoir/port
1036.
[0102] As with the device illustrated in FIG. 10a, a second channel
portion 1038 is used to perform the separation operation. The
separation channel portion connects reservoirs 1040 and 1042, and
is fluidly connected to channel portion 1004 via channel 1034.
[0103] In operation, the reaction mixture, as described with
reference to FIG. 10a, is drawn into the first channel portion by
applying a vacuum to reservoir/port 1032. The reaction mixture then
moves across the intersection of channel portion 1004 and channel
1034. A portion of the reaction mixture at this intersection is
then injected into the second channel portion 1038. Injection of
the reaction mixture from the first channel portion 1004 into the
second channel portion is preferably accomplished by applying an
electrical filed across channel 1034, e.g., between reservoir/port
1036 and 1042 or 1040. Once a plug of the reaction mixture is
introduced into the second channel portion, application of an
electric field across the second channel portion 1036, e.g.,
between reservoirs 1042 and 1040, then causes the electrophoretic
separation of the different reaction components, thereby allowing
their detection at detection window 1030. One of the advantages
this latter channel structure offers over that shown in FIG. 10a is
the ability to inject discrete plugs of reaction mixture into the
separation channel. In particular, only a small volume of reaction
material is injected into the second channel portion for
separation. However, this adds complexity when performing higher
throughput assays, which are typically simpler in a continuous flow
system, e.g., as shown in FIG. 10a.
[0104] The invention is further described with reference to the
following nonlimiting examples.
EXAMPLES
[0105] The following examples demonstrate the efficacy of the
methods and devices of the present invention in performing
integrated containment or reaction and separation operations. For
these examples, a microfluidic device having the channel geometry
shown in FIG. 7 was used. In these experiments, a low salt buffer
containing 50 mM HEPES at pH 7.5, and a high salt buffer containing
50 mM HEPES+100 mM NaCl at pH 7.5 were prepared. A second high salt
buffer ("ultra high salt buffer"), containing 50 mM HEPES+200 mM
NaCl at pH 7.5, was prepared and used as the "spoiling buffer" in
the continuous flow mode. A neutral dye, Rhodamine B, and an
anionic dye, Fluorescein, were placed in the high salt buffer in
well 3 of the device shown in FIG. 7, and used as markers to track
electrophoretic containment and separation in all experiments,
because these dyes have different electrophoretic mobilities.
Example 1
[0106] Continuous Flow Mode Reaction/Separation
[0107] In the continuous flow mode, e.g., as described above with
reference to FIG. 7, above, the buffer wells of the device shown in
FIG. 7 were loaded as follows: low salt buffer was loaded in wells
1 and 4, high salt buffer with dyes was loaded in well 3, high salt
buffer was loaded in well 6, and ultra high salt buffer was loaded
in wells 2 and 8. The following voltages and currents were applied
to the listed wells, to direct movement of the materials through
the device using an eight channel current based electrical
controller which included a series of pin electrodes inserted into
the wells:
1 1 2 3 4 5 6 7 8 Time(s) Flow Profile 500 V 10 .mu.A 0 .mu.A 0.5
.mu.A 0 V 0 .mu.A 0 V 10 .mu.A 20 Fill channel w/low salt 500 V 0
.mu.A 0 .mu.A -7 .mu.A 0 V 10 .mu.A 0 V 0 .mu.A 4 Create guard
bands 500 V 0 .mu.A 10 .mu.A -7 .mu.A 0 V 0 .mu.A 0 V 0 .mu.A 1
Inject sample 500 V 10 .mu.A 0 .mu.A 0.5 .mu.A 0 V 0 .mu.A 0 V 10
.mu.A 10 Move sample down channel/separate
[0108] To monitor the degree of containment and separation of dyes,
the location of the detection point was varied along the channel
path of dye flow, and the plotted signals for each detection point
are provided in the panels of FIG. 8. This series of plots clearly
indicate that the dyes are contained in the high-low salt format
before the injection point (Panel A). The containment is
successfully disrupted, e.g., the containing influence is overcome,
upon the addition of the spoiling buffer into the main channel,
leading to separation of dyes downstream (Panels B, C, D and
E).
Example 2
[0109] Injection Mode
[0110] In the injection/separation flow mode, the wells were loaded
as follows: low salt buffer in wells 1 and 4, high salt buffer with
dyes in well 3, high salt buffer in wells 6, 2, and 8. Controlling
currents and voltages were applied as follows:
2 1 2 3 4 5 6 7 8 Time(s) Flow Profile 500 V 0 .mu.A 0 .mu.A 3
.mu.A 0 V 0 .mu.A 0 V 0 .mu.A 10 Fill channel w/low 500 V 0 .mu.A
-.5 .mu.A -7 .mu.A 0 V 10 .mu.A 0 V 0 .mu.A 4 Create guard bands
500 V 0 .mu.A 10 .mu.A -7 .mu.A 0 V 0 .mu.A 0 V 0 .mu.A 2 Inject
sample 500 V 0 .mu.A 0 .mu.A 3 .mu.A 0 V 0 .mu.A 0 V 0 .mu.A 2.8
Move sample down main channel 0 .mu.A 10 .mu.A 0 .mu.A 0 .mu.A 0 V
0 .mu.A 0 V 100 V 0.5 Cross inject sample into second channel 500 V
0 .mu.A 0 .mu.A 3 .mu.A 0 V 0 .mu.A 0 V 0 .mu.A 10 Clear main
channel -.5 .mu.A 10 .mu.A 0 .mu.A 0 .mu.A 0 V 0 .mu.A O V 100 V 60
Move sample down separation channel
[0111] The location of the detection point along the main and
separation channels again was varied to monitor the degree of
containment of the two dyes. FIG. 9 summarizes the results of the
dye signals graphically. Once again, the dyes were clearly
contained in the high-low salt format before the injection point,
(panels A and B) and were cleanly separated by electrophoresis in
the separation channel (panels C and D).
[0112] In summary, these experimental results demonstrated the
feasibility of both the continuous flow and stop flow approaches
for integrating electrophoretic containment and electrophoretic
separation in the same microfluidic device.
[0113] The discussion above is generally applicable to the aspects
and embodiments of the invention described in the claims.
[0114] Moreover, modifications can be made to the methods apparatus
and systems described herein without departing from the spirit and
scope of the invention as claimed, and the invention can be put to
a number of different uses including the following.
[0115] The use of a microfluidic integrated system or device for
performing any of the methods and assays set forth herein,
particularly the use of the devices and integrated systems set
forth herein for performing any of the assays or methods set forth
herein.
[0116] The use of any microfluidic system or device as described
herein for performing integrated reaction and separation
operations, mobility shift operations, or any other operation set
forth herein, e.g., for analysis of one or more analytes, as set
forth herein.
[0117] Use of an assay or method utilizing a feature or operational
property of any one of the microfluidic systems or devices
described herein, e.g., for practicing any method or assay set
forth herein.
[0118] Use of kits comprising any device, device element, or
instruction set, e.g., for practicing any method or assay set forth
herein, or for facilitating practice of any method or use of any
device or system set forth herein, including maintenance kits for
maintaining the devices or systems herein in an appropriate
condition to practice the methods and assays set forth herein.
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one
skilled in the art from a reading of this disclosure that various
changes in form and detail can be made without departing from the
scope of the invention. For example, all the techniques and
apparatus described above may be used in various combinations which
will be apparent upon complete review of the foregoing disclosure
and following claims. All publications and patent applications
listed herein and the references cited within those documents are
hereby incorporated herein by reference to the same extent as if
each individual publication or patent application was specifically
and individually indicated to be incorporated by reference.
Although the present invention has been described in some detail by
way of illustrations and examples for purposes of clarity and
understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *