U.S. patent application number 10/084635 was filed with the patent office on 2002-10-10 for high throughput screening assay systems in microscale fluidic devices.
Invention is credited to Bousse, Luc J., Kopf-Sill, Anne R., Parce, John Wallace.
Application Number | 20020146845 10/084635 |
Document ID | / |
Family ID | 24696690 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146845 |
Kind Code |
A1 |
Parce, John Wallace ; et
al. |
October 10, 2002 |
High throughput screening assay systems in microscale fluidic
devices
Abstract
The present invention provides novel microfluidic devices and
methods that are useful for performing high-throughput screening
assays. In particular, the devices and methods of the invention are
useful in screening large numbers of different compounds for their
effects on a variety of chemical, and preferably, biochemical
systems.
Inventors: |
Parce, John Wallace; (Palo
Alto, CA) ; Kopf-Sill, Anne R.; (Portola Valley,
CA) ; Bousse, Luc J.; (Menlo Park, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
Attorneys at Law
1100 New York Avenue, NW
Washington
DC
20005
US
|
Family ID: |
24696690 |
Appl. No.: |
10/084635 |
Filed: |
February 28, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10084635 |
Feb 28, 2002 |
|
|
|
09608898 |
Jun 30, 2000 |
|
|
|
09608898 |
Jun 30, 2000 |
|
|
|
09250029 |
Feb 11, 1999 |
|
|
|
09250029 |
Feb 11, 1999 |
|
|
|
08671987 |
Jun 28, 1996 |
|
|
|
5942443 |
|
|
|
|
Current U.S.
Class: |
436/514 |
Current CPC
Class: |
B01L 2200/0647 20130101;
Y10S 366/02 20130101; Y10S 436/805 20130101; B01J 19/0093 20130101;
G01N 33/5064 20130101; Y10S 436/807 20130101; B01L 2200/0605
20130101; Y10S 435/81 20130101; G01N 33/5044 20130101; B01L
2300/0877 20130101; B01L 2200/0689 20130101; G01N 33/5091 20130101;
B01L 3/502761 20130101; G01N 33/5008 20130101; B01L 2300/0816
20130101; B01L 3/502784 20130101; B01L 2400/0415 20130101; B01L
2200/0673 20130101; G01N 27/44791 20130101; G01N 35/08 20130101;
B01L 3/502753 20130101; G01N 33/5304 20130101; G01N 33/57415
20130101; B01L 2300/0864 20130101; B01L 2300/0887 20130101; Y10S
436/806 20130101; Y10S 436/809 20130101; G01N 33/502 20130101; Y10S
435/97 20130101; B01L 3/5025 20130101; G01N 33/5094 20130101; B01L
9/527 20130101; B01L 3/50273 20130101; B01L 2200/12 20130101; B01L
2300/0861 20130101; B01L 2200/0668 20130101; G01N 33/5011
20130101 |
Class at
Publication: |
436/514 |
International
Class: |
G01N 033/558 |
Claims
What is claimed is:
1. A method of screening a plurality of test compounds for an
effect on a biochemical system, comprising: providing a substrate
having at least a first surface, and at least two intersecting
channels fabricated in said first surface, at least one of said at
least two intersecting channels having at least one cross-sectional
dimension in a range from 0.1 to 500 .mu.m; flowing a first
component of a biochemical system in a first of said at least two
intersecting channels; flowing at least a first test compound from
a second channel into said first channel whereby said first test
compound contacts said first component of said biochemical system;
and detecting an effect of said at least first test compound on
said biochemical system.
2. The method of claim 1, wherein said at least first component of
a biochemical system produces a detectable signal representative of
a function of said biochemical system.
3. The method of claim 1, wherein said at least first component
further comprises an indicator compound which interacts with said
first component to produce a detectable signal representative of a
functioning of said biochemical system.
4. The method of claim 1, wherein said first component of a
biochemical system comprises an enzyme and a substrate for said
enzyme, wherein action of said enzyme on said substrate produces a
detectable signal.
5. The method of claim 1, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein at least one of said receptor or ligand has a detectable
signal associated therewith.
6. The method of claim 1, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein binding of said receptor to said ligand produces a
detectable signal.
7. The method of claim 1, wherein said at least first component of
a biochemical system is a biological barrier and said effect of
said at least first test compound is an ability of said test
compound to traverse said barrier.
8. The method of claim 7, wherein said barrier is selected from the
group consisting of an epithelial or an endothelial layer.
9. The method of claim 1, wherein said at least first component of
a biochemical system comprises cells, and said detecting step
comprises determining an effect of said test compound on said
cells.
10. The method of claim 9, wherein said cells are capable of
producing a detectable signal corresponding to a cellular function,
and said detecting step comprises detecting an effect of said test
compound on said cellular function by detecting a level of said
detectable signal.
11. The method of claim 9, wherein said detecting step comprises
detecting an effect of said test compound on viability of said
cells.
12. A method of screening a plurality of test compounds for an
effect on a biochemical system, comprising: providing a substrate
having at least a first surface, and at least two intersecting
channels fabricated in said first surface, at least one of said at
least two intersecting channels having at least one cross-sectional
dimension in a range from 0.1 to 500 .mu.m; continuously flowing a
first component of a biochemical system in a first channel of said
at least two intersecting channels; periodically introducing a
different test compound into said first channel from a second
channel of said at least two intersecting channels; and detecting
an effect of said test compound on said at least first component of
a biochemical system.
13. The method of claim 12, wherein said step of periodically
introducing comprises flowing a plurality of different test
compounds into said first channel from a second channel of said at
least two intersecting channels, each of said plurality of
different test compounds being physically isolated from each other
of said plurality of different test compounds.
14. The method of claim 12, wherein said at least first component
of a biochemical system produces a detectable signal representative
of a function of said biochemical system.
15. The method of claim 14, wherein said detecting comprises
monitoring said detectable signal from said continuously flowing
first component at a point on said first channel, said detectable
signal having a steady state intensity, and wherein said effect of
said interaction between said first component and said test
compound comprises a deviation from said steady state intensity of
said detectable signal.
16. The method of claim 14, wherein said at least first component
further comprises an indicator compound which interacts with said
first component to produce a detectable signal representative of a
functioning of said biochemical system.
17. The method of claim 16, wherein said first component of a
biochemical system comprises an enzyme and said indicator compound
comprises a substrate for said enzyme, wherein action of said
enzyme on said substrate produces a detectable signal.
18. The method of claim 14, wherein said at least first component
of a biochemical system comprises a receptor/ligand binding pair,
wherein at least one of said receptor or ligand has a detectable
signal associated therewith.
19. The method of claim 18, wherein said receptor and said ligand
flow along said first channel at different rates.
20. The method of claim 14, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein binding of said receptor to said ligand produces a
detectable signal.
21. The method of claim 12, wherein said at least first component
of a biochemical system comprises cells, and said detecting step
comprises determining an effect of said test compound on said
cells.
22. The method of claim 21, wherein said cells are capable of
producing a detectable signal corresponding to a cellular function,
and said detecting step comprises detecting an effect of said test
compound on said cellular function by detecting a level of said
detectable signal.
23. The method of claim 21, wherein said detecting step comprises
detecting an effect of said test compound on viability of said
cells.
24. A method of screening a plurality of different test compounds
for an effect on a biochemical system, comprising: providing a
substrate having at least a first surface, and a plurality of
reaction channels fabricated in said first surface, each of said
plurality of reaction channels being fluidly connected to at least
two transverse channels fabricated in said surface; introducing at
least a first component of a biochemical system into said plurality
of reaction channels; flowing a plurality of different test
compounds through at least one of said at least two transverse
channels, each of said plurality of test compounds being introduced
into said at least one transverse channels in a discrete volume;
directing each of said plurality of different test compounds into a
separate one of said plurality of reaction channels; and detecting
an effect of each of said test compounds on said at least one
component of said biochemical system.
25. The method of claim 24, wherein said at least first component
of said biochemical system produces a flowable detectable signal
representative of a function of said biochemical system.
26. The method of claim 25, wherein said detectable flowable signal
produced in each of said plurality of reaction channels is flowed
into and through said second transverse channel, each of said
detectable flowable signals produced in each of said plurality of
reaction channels being physically isolated from each other of said
detectable flowable signals, whereupon each of said detectable
flowable signals is separately detected.
27. The method of claim 25, wherein said flowable signal comprises
a soluble signal.
28. The method of claim 27, wherein said soluble signal is selected
from fluorescent or calorimetric signals.
29. The method of claim 24, wherein said at least first component
further comprises an indicator compound which interacts with said
first component to produce a detectable signal representative of a
functioning of said biochemical system.
30. The method of claim 29, wherein said first component of a
biochemical system comprises an enzyme and said indicator compound
comprises a substrate for said enzyme, wherein action of said
enzyme on said substrate produces a detectable signal.
31. The method of claim 24, wherein said at least first component
of a biochemical system comprises a receptor/ligand binding pair,
wherein at least one of said receptor or ligand has a detectable
signal associated therewith.
32. The method of claim 24, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein binding of said receptor to said ligand produces a
detectable signal.
33. The method of claim 24, wherein said at least first component
of a biochemical system comprises cells, and said detecting step
comprises determining an effect of said test compound on said
cells.
34. The method of claim 33, wherein said cells are capable of
producing a detectable signal corresponding to a cellular function,
and said detecting step comprises detecting an effect of said test
compound on said cellular function by detecting a level of said
detectable signal.
35. The method of claim 34, wherein said detecting step comprises
detecting an effect of said test compound on viability of said
cells.
36. The method of claim 24, wherein each of said plurality of
different test compounds is immobilized upon a separate bead, and
said step of directing each of said plurality of different test
compounds into a separate one of said plurality of reaction
channels comprises: lodging one of said separate beads at an
intersection of said first transverse channel and each of said
plurality of reaction channels; and controllably releasing said
test compounds from each of said separate beads into each of said
plurality of reaction channels.
37. An apparatus for screening test compounds for an effect on a
biochemical system, comprising: a substrate having at least one
surface; at least two intersecting channels fabricated into said
surface of said substrate, at least one of said at least two
intersecting channels having at least one cross-sectional dimension
in the range from about 0.1 to about 500 .mu.m; a source of a
plurality different test compounds fluidly connected to a first of
said at least two intersecting channels; a source of at least one
component of said biochemical system fluidly connected to a second
of said at least two intersecting channels; a fluid direction
system for flowing said at least one component within said second
of said at least two intersecting channels and for introducing said
different test compounds from said first to said second of said at
least two intersecting channels; a cover mated with said surface;
and a detection zone in said second channel for detecting an effect
of said test compound on said biochemical system.
38. The apparatus of claim 37, wherein said fluid direction system
generates a continuous flow of said at least first component along
said second of said at least two intersecting channels, and
periodically injects a test compound from said first channel into
said second channel.
39. The apparatus of claim 37, further comprising a source of a
second component of said biochemical system, and a third channel
fabricated into said surface, said third channel fluidly connecting
at least one of said at least two intersecting channels with said
source of said second component of said biochemical system.
40. The apparatus of claim 39, wherein said fluid direction system
generates a continuous flow of a mixture of said first component
and said second component along said second of said at least two
intersecting channels, and periodically injects a test compound
from said first channel into said second channel.
41. The apparatus of claim 37, wherein said fluid direction system
continuously flows said plurality of different test compounds from
said first into said second of said at least two intersecting
channels, each of said plurality of different test compounds being
separated by a fluid spacer.
42. The apparatus of claim 37, wherein said fluid direction system
comprises: at least three electrodes, each electrode being in
electrical contact with said at least two intersecting channels on
a different side of an intersection formed by said at least two
intersecting channels; and a control system for concomitantly
applying a variable voltage at each of said electrodes, whereby
movement of said test compounds or said at least first component in
said at least two intersecting channels may be controlled.
43. The apparatus of claim 37, wherein said detection system
includes a detection window in said second channel.
44. The apparatus of claim 43, wherein said detection system is a
fluorescent detection system.
45. The apparatus of claim 37, wherein said substrate is
planar.
46. The apparatus of claim 37, wherein said substrate comprises
etched glass.
47. The apparatus of claim 37, wherein said substrate comprises
etched silicon.
48. The apparatus of claim 37, further comprising an insulating
layer disposed over said etched silicon substrate.
49. The apparatus of claim 37, wherein said substrate is a molded
polymer.
50. The apparatus of claim 37, wherein said at least one component
of a biochemical system comprises an enzyme, and a substrate which
produces a detectable signal when reacted with said enzyme.
51. The apparatus of claim 50, wherein said substrate is selected
from the group consisting of chromogenic and fluorogenic
substrates.
52. The apparatus of claim 37, wherein said at least first
component of a biochemical system comprises a receptor/ligand
binding pair, wherein at least one of said receptor or ligand has a
detectable signal associated therewith.
53. The apparatus of claim 37, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein binding of said receptor to said ligand produces a
detectable signal.
54. An apparatus for detecting an effect of a test compound on a
biochemical system, comprising: a substrate having at least one
surface; a plurality of reaction channels fabricated into said
surface; at least two transverse channels fabricated into said
surface, each of said plurality of reaction channels being fluidly
connected to a first of said at least two transverse channels at a
first point in said reaction channels, and fluidly connected to a
second of said at least two transverse channels at a second point
in said reaction channels, said at least two transverse channels
and said plurality of reaction channels each having at least one
cross-sectional dimension in the range from about 0.1 to about 500
.mu.m; a source of at least one component of said biochemical
system, said source of at least one component of said biochemical
system being fluidly connected to each of said plurality of
reaction channels; a source of test compounds fluidly connected to
said first of said at least two transverse channels; a fluid
direction system for controlling movement of said test compound and
said at least one component within said at least two transverse
channels and said plurality of reaction channels; a cover mated
with said surface; and a detection system for detecting an effect
of said test compound on said biochemical system.
55. The apparatus of claim 54, wherein said fluid control system
comprises: a plurality of individual electrodes, each in electrical
contact with each terminus of said at least two transverse
channels; and a control system for concomitantly applying a
variable voltage at each of said electrodes, whereby movement of
said test compounds or said at least first component in said at
least two transverse channels and said plurality of reaction
channels may be controlled.
56. The apparatus of claim 54, wherein each of said plurality of
reaction channels comprises a bead resting well at said first point
in said plurality of reaction channels.
57. The apparatus of claim 54, wherein said source of at least one
component of a biochemical system is fluidly connected to said
plurality of reaction channels by a third transverse channel, said
third transverse channel having at least one cross sectional
dimension in a range of from 0.1 to 500 .mu.m and being fluidly
connected to each of said plurality of reaction channels at a third
point in said reaction channels.
58. The apparatus of claim 57, wherein said third point in said
reaction channels is intermediate to said first and second points
in said reaction channels.
59. The apparatus of claim 58, further comprising a particle
retention zone in each of said plurality of reaction channels,
between said third and said second points in said plurality of
reaction channels.
60. The apparatus of claim 49, wherein said particle retention zone
comprises a particle retention matrix.
61. The apparatus of claim 49, wherein said particle retention zone
comprises a microstructural filter.
62. The apparatus of claim 54, wherein said plurality of reaction
channels comprises a plurality of parallel reaction channels
fabricated into said surface of said substrate and said at least
two transverse channels are connected at opposite ends of each of
said parallel reaction channels.
63. The apparatus of claim 54, wherein said at least two transverse
channels are fabricated on said surface of said substrate in inner
and outer concentric channels, and said plurality of reaction
channels extend radially from said inner concentric channel to said
outer concentric channel.
64. The apparatus of claim 63, wherein said detection system
comprises a detection window in said second channel.
65. The apparatus of claim 64, wherein said detection system is a
fluorescent detection system.
66. The apparatus of claim 54, wherein said substrate is
planar.
67. The apparatus of claim 54, wherein said substrate comprises
etched glass.
68. The apparatus of claim 54, wherein said substrate comprises
etched silicon.
69. The apparatus of claim 54, further comprising an insulating
layer disposed over said etched silicon substrate.
70. The apparatus of claim 54, wherein said substrate is a molded
polymer.
71. The apparatus of claim 54, wherein said at least one component
of a biochemical system comprises an enzyme, and an enzyme
substrate which produces a detectable signal when reacted with said
enzyme.
72. The apparatus of claim 71, wherein said enzyme substrate is
selected from the group consisting of chromogenic and fluorogenic
substrates.
73. The apparatus of claim 54, wherein said at least first
component of a biochemical system comprises a receptor/ligand
binding pair, wherein at least one of said receptor or ligand has a
detectable signal associated therewith.
74. The apparatus of claim 54, wherein said first component of a
biochemical system comprises a receptor/ligand binding pair,
wherein binding of said receptor to said ligand produces a
detectable signal.
Description
BACKGROUND OF THE INVENTION
[0001] There has long been a need for the ability to rapidly assay
compounds for their effects on various biological processes. For
example, enzymologists have long sought better substrates, better
inhibitors or better catalysts for enzymatic reactions. Similarly,
in the pharmaceutical industries, attention has been focused on
identifying compounds that may block, reduce, or even enhance the
interactions between biological molecules. Specifically, in
biological systems, the interaction between a receptor and its
ligand often may result, either directly or through some downstream
event, in either a deleterious or beneficial effect on that system,
and consequently, on a patient for whom treatment is sought.
Accordingly, researchers have long sought after compounds or
mixtures of compounds that can reduce, block or even enhance that
interaction.
[0002] Modern drug discovery is limited by the throughput of the
assays that are used to screen compounds that possess these
described effects. In particular, screening of the maximum number
of different compounds necessitates reducing the time and labor
requirements associated with each screen.
[0003] High throughput screening of collections of chemically
synthesized molecules and of natural products (such as microbial
fermentation broths) has thus played a central role in the search
for lead compounds for the development of new pharmacological
agents. The remarkable surge of interest in combinatorial chemistry
and the associated technologies for generating and evaluating
molecular diversity represent significant milestones in the
evolution of this paradigm of drug discovery. See Pavia et al.,
1993, Bioorg. Med. Chem. Lett. 3:387-396, incorporated herein by
reference. To date, peptide chemistry has been the principle
vehicle for exploring the utility of combinatorial methods in
ligand identification. See Jung & Beck-Sickinger, 1992, Angew.
Chem. Int. Ed. Enql. 31:367-383, incorporated herein by reference.
This may be ascribed to the availability of a large and
structurally diverse range of amino acid monomers, a relatively
generic, high-yielding solid phase coupling chemistry and the
synergy with biological approaches for generating recombinant
peptide libraries. Moreover, the potent and specific biological
activities of many low molecular weight peptides make these
molecules attractive starting points for therapeutic drug
discovery. See Hirschmann, 1991, Angew. Chem. Int. Ed. Engl.
30:1278-1301, and Wiley & Rich, 1993, Med. Res. Rev. 13:
327-384, each of which is incorporated herein by reference.
Unfavorable pharmacodynamic properties such as poor oral
bioavailability and rapid clearance in vivo have limited the more
widespread development of peptidic compounds as drugs however. This
realization has recently inspired workers to extend the concepts of
combinatorial organic synthesis beyond peptide chemistry to create
libraries of known pharmacophores like benzodiazepines (see Bunin
& Ellman, 1992, J. Amer. Chem. Soc. 114:10997-10998,
incorporated herein by reference) as well as polymeric molecules
such as oligomeric N-substituted glycines ("peptoids") and
oligocarbamates. See Simon et al., 1992, Proc. Natl. Acad. Sci. USA
89:9367-9371; Zuckermann et al., 1992, J. Amer. Chem. Soc.
114:10646-10647; and Cho et al., 1993, Science 261:1303-1305, each
of which is incorporated herein by reference.
[0004] In similar developments, much as modern combinatorial
chemistry has resulted in a dramatic increase in the number of test
compounds that may be screened, human genome research has also
uncovered large numbers of new target molecules against which the
efficacy of test compounds may be screened.
[0005] Despite the improvements achieved using parallel screening
methods and other technological advances, such as robotics and high
throughput detection systems, current screening methods still have
a number of associated problems.
[0006] For example, screening large numbers of samples using
existing parallel screening methods have high space requirements to
accommodate the samples and equipment, e.g., robotics, etc., high
costs associated with that equipment, and high reagent requirements
necessary for performing the assays.
[0007] Additionally,, in many cases, reaction volumes must be very
small to account for the small amounts of the test compounds that
are available. Such small volumes compound errors associated with
fluid handling and measurement, e.g., evaporation. Additionally,
fluid handling equipment and methods have typically been unable to
handle these volume ranges with any acceptable level of accuracy
due in part to surface tension effects in such small volumes.
[0008] The development of systems to address these problems must
consider a variety of aspects of the assay process. Such aspects
include target and compound sources, test compound and target
handling, specific assay requirements, and data acquisition,
reduction storage and analysis. In particular, there exists a need
for high throughput screening methods and associated equipment and
devices that are capable of performing repeated, accurate assay
screens, and operating at very small volumes.
[0009] The present invention meets these and a variety of other
needs. In particular, the present invention provides novel methods
and apparatuses for performing screening assays which address and
provide meaningful solutions to these problems.
SUMMARY OF THE INVENTION
[0010] The present invention generally provides methods of
screening a plurality of test compounds for an effect on a
biochemical system. These methods typically utilize microfabricated
substrates which have at least a first surface, and at least two
intersecting channels fabricated into that first surface. At least
one of the intersecting channels will have at least one
cross-sectional dimension in a range from 0.1 to 500 .mu.m. The
methods involve flowing a first component of a biochemical system
in a first of the at least two intersecting channels. At least a
first test compound is flowed from a second channel into the first
channel whereby the test compound contacts the first component of
the biochemical system. An effect of the test compound on the
biochemical system is then detected.
[0011] In a related aspect, the method comprises continuously
flowing the first component of a biochemical system in the first
channel of the at least two intersecting channels. Different test
compounds are periodically introduced into the first channel from a
second channel. The effect, if any, of the test compound on the
biochemical system is then detected.
[0012] In an alternative aspect the methods utilize a substrate
having at least a first surface with a plurality of reaction
channels fabricated into the first surface. Each of the plurality
of reaction channels is fluidly connected to at least two
transverse channels also fabricated in the surface. The at least a
first component of a biochemical system is introduced into the
plurality of reaction channels, and a plurality of different test
compounds is flowed through at least one of the at least two
transverse channels. Further, each of the plurality of test
compounds is introduced into the transverse channel in a discrete
volume. Each of the plurality of different test compounds is
directed into a separate reaction channel and the effect of each of
test compounds on the biochemical system is then detected.
[0013] The present invention also provides apparatuses for
practicing the above methods. In one aspect, the present invention
provides an apparatus for screening test compounds for an effect on
a biochemical system. The device comprises a substrate having at
least one surface with at least two intersecting channels
fabricated into the surface. The at least two intersecting channels
have at least one cross-sectional dimension in the range from about
0.1 to about 500 .mu.m. The device also comprises a source of
different test compounds fluidly connected to a first of the at
least two intersecting channels, and a source of at least one
component of the biochemical system fluidly connected to a second
of the at least two intersecting channels. Also included are fluid
direction systems for flowing the at least one component within the
intersecting channels, and for introducing the different test
compounds from the first to the second of the intersecting
channels. The apparatus also comprises a detection zone in the
second channel for detecting an effect of said test compound on
said biochemical system.
[0014] In preferred aspects, the apparatus of the invention
includes a fluid direction system which comprises at least three
electrodes, each electrode being in electrical contact with the at
least two intersecting channels on a different side of an
intersection formed by the at least two intersecting channels. The
fluid direction system also includes a control system for
concomitantly applying a variable voltage at each of the
electrodes, whereby movement of the test compounds or the at least
first component in the at least two intersecting channels may be
controlled.
[0015] In another aspect, the present invention provides an
apparatus for detecting an effect of a test compound on a
biochemical system, comprising a substrate having at least one
surface with a plurality of reaction channels fabricated into the
surface. The apparatus also has at least two transverse channels
fabricated into the surface, wherein each of the plurality of
reaction channels is fluidly connected to a first of the at least
two transverse channels at a first point in each of the reaction
channels, and fluidly connected to a second transverse channel at a
second point in each of the reaction channels. The apparatus
further includes a source of at least one component of the
biochemical system fluidly connected to each of the reaction
channels, a source of test compounds fluidly connected to the first
of the transverse channels, and a fluid direction system for
controlling movement of the test compound and the first component
within the transverse channels and the plurality reaction channels.
As above, the apparatuses also include a detection zone in the
second transverse channel for detecting an effect of the test
compound on the biochemical system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic illustration of one embodiment of a
microlaboratory screening assay system of the present invention
which can be used in running a continuous flow assay system.
[0017] FIGS. 2A and 2B show a schematic illustration of the
apparatus shown in FIG. 1, operating in alternate assay systems.
FIG. 2A shows a system used for screening effectors of an
enzyme-substrate interaction. FIG. 2B illustrates the use of the
apparatus in screening effectors of receptor-ligand
interactions.
[0018] FIG. 3 is a schematic illustration of a "serial input
parallel reaction" microlaboratory assay system in which compounds
to be screened are serially introduced into the device but then
screened in a parallel orientation within the device.
[0019] FIGS. 4A-4F show a schematic illustration of the operation
of the device shown in FIG. 3, in screening a plurality of bead
based test compounds.
[0020] FIG. 5 shows a schematic illustration of a continuous flow
assay device incorporating a sample shunt for performing prolonged
incubation followed by a separation step.
[0021] FIG. 6A shows a schematic illustration of a serial input
parallel reaction device for use with fluid based test compounds.
FIGS. 6B and 6C show a schematic illustration of fluid flow
patterns within the device shown in FIG. 6A.
[0022] FIG. 7 shows a schematic illustration of one embodiment of
an overall assay systems which employs multiple microlaboratory
devices labeled as "LabChips" for screening test compounds.
[0023] FIG. 8 illustrates the parameters of a fluid flow system on
a small chip device for performing enzyme inhibitor screening.
[0024] FIG. 9 shows a schematic illustration of timing for
sample/spacer loading in a microfluidic device channel.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0025] I. General
[0026] The present invention provides novel microlaboratory systems
and methods that are useful for performing high-throughput
screening assays. In particular, the present invention provides
microfluidic devices and methods of using such devices that are
useful in screening large numbers of different compounds for their
effects on a variety of chemical, and preferably, biochemical
systems.
[0027] As used herein, the phrase "biochemical system" generally
refers to a chemical interaction that involves molecules of the
type generally found within living organisms.
[0028] Such interactions include the full range of catabolic and
anabolic reactions which occur in living systems including
enzymatic, binding, signalling and other reactions. Further,
biochemical systems, as defined herein, will also include model
systems which are mimetic of a particular biochemical interaction.
Examples of biochemical systems of particular interest in
practicing the present invention include, e.g., receptor-ligand
interactions, enzyme-substrate interactions, cellular signaling
pathways, transport reactions involving model barrier systems
(e.g., cells or membrane fractions) for bioavailability screening,
and a variety of other general systems. Cellular or organismal
viability or activity may also be screened using the methods and
apparatuses of the present invention, i.e., in toxicology
studies.
[0029] In order to provide methods and devices for screening
compounds for effects on biochemical systems, the present invention
generally incorporates model in vitro systems which mimic a given
biochemical system in vivo for which effector compounds are
desired. The range of systems against which compounds can be
screened and for which effector compounds are desired, is
extensive. For example, compounds may be screened for effects in
blocking, slowing or otherwise inhibiting key events associated
with biochemical systems whose effect is undesirable. For example,
test compounds may be screened for their ability to block systems
that are responsible, at least in part, for the onset of disease or
for the occurrence of particular symptoms of diseases, including,
e.g., hereditary diseases, cancer, bacterial or viral infections
and the like. Compounds which show promising results in these
screening assay methods can then be subjected to further testing to
identify effective pharmacological agents for the treatment of
disease or symptoms of a disease.
[0030] Alternatively, compounds can be screened for their ability
to stimulate, enhance or otherwise induce biochemical systems whose
function is believed to be desirable, e.g., to remedy existing
deficiencies in a patient.
[0031] Once a model system is selected, batteries of test compounds
can then be applied against these model systems. By identifying
those test compounds that have an effect on the particular
biochemical system, in vitro, one can identify potential effectors
of that system, in vivo.
[0032] In their simplest forms, the biochemical system models
employed in the methods and apparatuses of the present invention
will screen for an effect of a test compound on an interaction
between two components of a biochemical system, e.g.,
receptor-ligand interaction, enzyme-substrate interaction, and the
like. In this form, the biochemical system model will typically
include the two normally interacting components of the system for
which an effector is sought, e.g., the receptor and its ligand or
the enzyme and its substrate.
[0033] Determining whether a test compound has an effect on this
interaction then involves contacting the system with the test
compound and assaying for the functioning of the system, e.g.,
receptor-ligand binding or substrate turnover. The assayed function
is then compared to a control, e.g., the same reaction in the
absence of the test compound or in the presence of a known
effector.
[0034] Although described in terms of two-component biochemical
systems, the methods and apparatuses may also be used to screen for
effectors of much more complex systems where the result or end
product of the system is known and assayable at some level, e.g.,
enzymatic pathways, cell signaling pathways and the like.
Alternatively, the methods and apparatuses described herein may be
used to screen for compounds that interact with a single component
of a biochemical system, e.g., compounds that specifically bind to
a particular biochemical compound, e.g., a receptor, ligand,
enzyme, nucleic acid, structural macromolecule, etc.
[0035] Biochemical system models may also be embodied in whole cell
systems. For example, where one is seeking to screen test compounds
for an effect on a cellular response, whole cells may be utilized.
Modified cell systems may also be employed in the screening systems
encompassed herein. For example, chimeric reporter systems may be
employed as indicators of an effect of a test compound on a
particular biochemical system. Chimeric reporter systems typically
incorporate a heterogenous reporter system integrated into a
signaling pathway which signals the binding of a receptor to its
ligand. For example, a receptor may be fused to a heterologous
protein, e.g., an enzyme whose activity is readily assayable.
Activation of the receptor by ligand binding then activates the
heterologous protein which then allows for detection. Thus, the
surrogate reporter system produces an event or signal which is
readily detectable, thereby providing an assay for receptor/ligand
binding. Examples of such chimeric reporter systems have been
previously described in the art.
[0036] Additionally, where one is screening for bioavailability,
e.g., transport, biological barriers may be included. The term
"biological barriers" generally refers to cellular or membranous
layers within biological systems, or synthetic models thereof.
Examples of such biological barriers include the epithelial and
endothelial layers, e.g. vascular endothelia and the like.
[0037] Biological responses are often triggered and/or controlled
by the binding of a receptor to its ligand. For example,
interaction of growth factors, i.e., EGF, FGF, PDGF, etc., with
their receptors stimulates a wide variety of biological responses
including, e.g., cell proliferation and differentiation, activation
of mediating enzymes, stimulation of messenger turnover,
alterations in ion fluxes, activation of enzymes, changes in cell
shape and the alteration in genetic expression levels. Accordingly,
control of the interaction of the receptor and its ligand may offer
control of the biological responses caused by that interaction.
[0038] Accordingly, in one aspect, the present invention will be
useful in screening for compounds that affect an interaction
between a receptor molecule and its ligands. As used herein, the
term "receptor" generally refers to one member of a pair of
compounds which specifically recognize and bind to each other. The
other member of the pair is termed a "ligand." Thus, a
receptor/ligand pair may include a typical protein receptor,
usually membrane associated, and its natural ligand, e.g., another
protein or small molecule.
[0039] Receptor/ligand pairs may also include antibody/antigen
binding pairs, complementary nucleic acids, nucleic acid
associating proteins and their nucleic acid ligands. A large number
of specifically associating biochemical compounds are well known in
the art and can be utilized in practicing the present
invention.
[0040] Traditionally, methods for screening for effectors of a
receptor/ligand interaction have involved incubating a
receptor/ligand binding pair in the presence of a test compound.
The level of binding of the receptor/ligand pair is then compared
to negative and/or positive controls. Where a decrease in normal
binding is seen, the test compound is determined to be an inhibitor
of the receptor/ligand binding. Where an increase in that binding
is seen, the test compound is determined to be an enhancer or
inducer of the interaction.
[0041] In the interest of efficiency, screening assays have
typically been set up in multiwell reaction plates, e.g.,
multi-well microplates, which allow for the simultaneous, parallel
screening of large numbers of test compounds.
[0042] A similar, and perhaps overlapping, set of biochemical
systems includes the interactions between enzymes and their
substrates. The term "enzyme" as used herein, generally refers to a
protein which acts as a catalyst to induce a chemical change in
other compounds or "substrates."
[0043] Typically, effectors of an enzyme's activity toward its
substrate are screened by contacting the enzyme with a substrate in
the presence and absence of the compound to be screened and under
conditions optimal for detecting changes in the enzyme's activity.
After a set time for reaction, the mixture is assayed for the
presence of reaction products or a decrease in the amount of
substrate. The amount of substrate that has been catalyzed is them
compared to a control, i.e., enzyme contacted with substrate in the
absence of test compound or presence of a known effector. As above,
a compound that reduces the enzymes activity toward its substrate
is termed an "inhibitor," whereas a compound that accentuates that
activity is termed an "inducer."
[0044] Generally, the various screening methods encompassed by the
present invention involve the serial introduction of a plurality of
test compounds into a microfluidic device. Once injected into the
device, the test compound may be screened for effect on a
biological system using a continuous serial or parallel assay
orientation.
[0045] As used herein, the term "test compound" refers to the
collection of compounds that are to be screened for their ability
to affect a particular biochemical system. Test compounds may
include a wide variety of different compounds, including chemical
compounds, mixtures of chemical compounds, e.g., polysaccharides,
small organic or inorganic molecules, biological macromolecules,
e.g., peptides, proteins, nucleic acids, or an extract made from
biological materials such as bacteria, plants, fungi, or animal
cells or tissues, naturally occurring or synthetic compositions.
Depending upon the particular embodiment being practiced, the test
compounds may be provided, e.g., injected, free in solution, or may
be attached to a carrier, or a solid support, e.g., beads. A number
of suitable solid supports may be employed for immobilization of
the test compounds. Examples of suitable solid supports include
agarose, cellulose, dextran (commercially available as, i.e.,
Sephadex, Sepharose) carboxymethyl cellulose, polystyrene,
polyethylene glycol (PEG), filter paper, nitrocellulose, ion
exchange resins, plastic films, glass beads,
polyaminemethylvinylether maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
Additionally, for the methods and apparatuses described herein,
test compounds may be screened individually, or in groups. Group
screening is particularly useful where hit rates for effective test
compounds are expected to be low such that one would not expect
more than one positive result for a given group.
[0046] II. Assay Systems
[0047] As described above, the screening methods of the present
invention are generally carried out in microfluidic devices or
"microlaboratory systems," which allow for integration of the
elements required for performing the assay, automation, and minimal
environmental effects on the assay system, e.g., evaporation,
contamination, human error. A number of devices for carrying out
the assay methods of the invention are described in substantial
detail below. However, it will be recognized that the specific
configuration of these devices will generally vary depending upon
the type of assay and/or assay orientation desired. For example, in
some embodiments, the screening methods of the invention can be
carried out using a microfluidic device having two intersecting
channels. For more complex assays or assay orientations,
multichannel/intersection devices may be employed. The small scale,
integratability and self-contained nature of these devices allows
for virtually any assay orientation to be realized within the
context of the microlaboratory system.
[0048] A. Continuous Flow Assay Systems
[0049] In one preferred aspect, the methods and apparatuses of the
invention are used in screening test compounds using a continuous
flow assay system. Generally, the continuous flow assay system can
be readily used in screening for inhibitors or inducers of
enzymatic activity, or for agonists or antagonists of
receptor-ligand binding. In brief, the continuous flow assay system
involves the continuous flow of the particular biochemical system
along a microfabricated channel. As used herein, the term
"continuous" generally refers to an unbroken or contiguous stream
of the particular composition that is being continuously flowed.
For example, a continuous flow may include a constant fluid flow
having a set velocity, or alternatively, a fluid flow which
includes pauses in the flow rate of the overall system, such that
the pause does not otherwise interupt the flow stream. The
functioning of the system is indicated by the production of a
detectable event or signal. Typically, such detectable signals will
include chromophoric or fluorescent signals that are associated
with the functioning of the particular model system used. For
enzyme systems, such signals will generally be produced by products
of the enzyme's catalytic action, e.g., on a chromogenic or
fluorogenic substrate. For binding systems, e.g., receptor ligand
interactions, signals will typically involve the association of a
labeled ligand with the receptor, or vice versa.
[0050] In preferred aspects, the continuous system generates a
constant signal which varies only when a test compound is
introduced that affects the system. Specifically, as the system
components flow along the channel, they will produce a relatively
constant signal level at a detection zone or window of the channel.
Test compounds are periodically introduced into the channel and
mixed with the system components. Where those test compounds have
an effect on the system, it will cause a deviation from the
constant signal level at the detection window. This deviation may
then be correlated to the particular test compound screened.
[0051] One embodiment of a device for use in a serial or continuous
assay geometry is shown in FIG. 1. As shown, the overall device 100
is fabricated in a planar substrate 102. Suitable substrate
materials are generally selected based upon their compatibility
with the conditions present in the particular operation to be
performed by the device. Such conditions can include extremes of
pH, temperature, salt concentration, and application of electrical
fields. Additionally, substrate materials are also selected for
their inertness to critical components of an analysis or synthesis
to be carried out by the device.
[0052] Examples of useful substrate materials include, e.g., glass,
quartz and silicon as well as polymeric substrates, e.g. plastics.
In the case of conductive or semi-conductive substrates, it will
generally be desirable to include an insulating layer on the
substrate. This is particularly important where the device
incorporates electrical elements, e.g., electrical fluid direction
systems, sensors and the like. In the case of polymeric substrates,
the substrate materials may be rigid, semi-rigid, or non-rigid,
opaque, semi-opaque or transparent, depending upon the use for
which they are intended. For example, devices which include an
optical or visual detection element, will generally be fabricated,
at least in part, from transparent materials to allow, or at least,
facilitate that detection. Alternatively, transparent windows of,
e.g., glass or quartz, may be incorporated into the device for
these types detection elements. Additionally, the polymeric
materials may have linear or branched backbones, and may be
crosslinked or non-crosslinked. Examples of particularly preferred
polymeric materials include, e.g., polydimethylsiloxanes (PDMS),
polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone,
polycarbonate and the like.
[0053] The device shown in FIG. 1 includes a series of channels
110, 112, and optional reagent channel 114, fabricated into the
surface of the substrate. At least one of these channels will
typically have very small cross sectional dimensions, e.g., in the
range of from about 0.1 .mu.m to about 500 .mu.m. Preferably the
cross-sectional dimensions of the channels will be in the range of
from about 0.1 to about 200 .mu.m and more preferably in the range
of from about 0.1 to about 100 .mu.m. In particularly preferred
aspects, each of the channels will have at least one
cross-sectional dimension in the range of from about 0.1 .mu.m to
about 100 .mu.m. Although generally shown as straight channels, it
will be appreciated that in order to maximize the use of space on a
substrate, serpentine, saw tooth or other channel geometries, to
incorporate effectively longer channels in shorter distances.
[0054] Manufacturing of these microscale elements into the surface
of the substrates may generally be carried out by any number of
microfabrication techniques that are well known in the art. For
example, lithographic techniques may be employed in fabricating,
e.g., glass, quartz or silicon substrates, using methods well known
in the semiconductor manufacturing industries such as
photolithographic etching, plasma etching or wet chemical etching.
Alternatively, micromachining methods such as laser drilling,
micromilling and the like may be employed. Similarly, for polymeric
substrates, well known manufacturing techniques may also be used.
These techniques include injection molding or stamp molding methods
where large numbers of substrates may be produced using, e.g.,
rolling stamps to produce large sheets of microscale substrates or
polymer microcasting techniques where the substrate is polymerized
within a micromachined mold.
[0055] The devices will typically include an additional planar
element which overlays the channeled substrate enclosing and
fluidly sealing the various channels to form conduits. Attaching
the planar cover element may be achieved by a variety of means,
including, e.g., thermal bonding, adhesives or, in the case of
certain substrates, e.g., glass, or semi-rigid and non-rigid
polymeric substrates, a natural adhesion between the two
components. The planar cover element may additionally be provided
with access ports and/or reservoirs for introducing the various
fluid elements needed for a particular screen.
[0056] The device shown in FIG. 1 also includes reservoirs 104, 106
and 108, disposed and fluidly connected at the ends of the channels
110 and 114. As shown, sample channel 112, is used to introduce the
plurality of different test compounds into the device. As such,
this channel will generally be fluidly connected to a source of
large numbers of separate test compounds that will be individually
introduced into the sample channel 112 and subsequently into
channel 110.
[0057] The introduction of large numbers of individual, discrete
volumes of test compounds into the sample may be carried out by a
number of methods. For example, micropipettors may be used to
introduce the test compounds into the device. In preferred aspects,
an electropipettor may be used which is fluidly connected to sample
channel 112. An example of such an electropipettor is described in,
e.g., U.S. patent application Ser. No. ______, filed ______
(Attorney Docket No. 017646-000500) the disclosure of which is
hereby incorporated herein by reference in its entirety for all
purposes. Generally, this electropipettor utilizes electroosmotic
fluid direction as described herein, to alternately sample a number
of test compounds and spacer compounds. The pipettor then delivers
individual, physically isolated sample or test compound volumes, in
series, into the sample channel for subsequent manipulation within
the device. Individual samples are typically separated by a slug of
low ionic strength spacer fluid. These low ionic strength spacers
have higher voltage drop over the length of the plug, thereby
driving the electrokinetic pumping. On either side of the sample
plug, which is typically in higher ionic strength solution, are
fluid plugs referred to as guard plugs or bands at the interface of
the sample plug. These guard bands typically comprise a high ionic
strength solution to prevent migration of the sample elements into
the spacer fluid band, resulting in electrophoretic bias. The use
of such guard bands is described in greater detail in U.S. patent
application Ser. No. ______, filed ______, (Attorney Docket No.
017646-000500) which is incorporated herein by reference.
[0058] Alternatively, the sample channel 112 may be individually
fluidly connected to a plurality of separate reservoirs via
separate channels. The separate reservoirs each contain a separate
test compound with additional reservoirs being provided for
appropriate spacer compounds. The test compounds and/or spacer
compounds are then transported from the various reservoirs into the
sample channels using appropriate fluid direction schemes. In
either case, it generally is desirable to separate the discrete
sample volumes, or test compounds, with an appropriate spacer
buffer.
[0059] As shown, the device also includes a detection window or
zone 116 at which a signal from the biochemical system may be
monitored. This detection window typically will include a
transparent cover allowing visual or optical observation and
detection of the assay results, e.g., observation of a colorometric
or fluorometric response.
[0060] In particularly preferred aspects, monitoring of the signals
at the detection window is achieved using an optical detection
system. For example, fluorescence based signals are typically
monitored using, e.g., laser activated fluorescence detection
systems which employ a laser light source at an appropriate
wavelength for activating the fluorescent indicator within the
system. Fluorescence is then detected using an appropriate detector
element, e.g., a photomultiplier tube (PMT). Similarly, for screens
employing colorometric signals, spectrophotometric detection
systems may be employed which direct a light source at the sample
and provide a measurement of absorbance or transmissivity of the
sample.
[0061] In alternative aspects, the detection system may comprise a
non-optical detectors or sensors for detecting a particular
characteristic of the system disposed within detection window 116.
Such sensors may include temperature, conductivity, potentiometric
(pH, ions), amperometric (for compounds that may be oxidized or
reduced, e.g., O.sub.2, H.sub.2O.sub.2, I.sub.2, oidzable/reducible
organic compounds, and the like).
[0062] In operation, a fluid first component of a biological
system, e.g., a receptor or enzyme, is placed in reservoir 104. The
first component is flowed through main channel 110, past the
detection window, 116, and toward waste reservoir 108. A second
component of the biochemical system, e.g., a ligand or substrate,
is concurrently flowed into the main channel 110 from the side
channel 114, whereupon the first and second components mix and are
able to interact. Deposition of these elements within the device
may be carried out in a number of ways. For example, the enzyme and
substrate, or receptor and ligand solutions can be introduced into
the device through sealable access ports in the planar cover.
Alternatively, these components may be added to their respective
reservoirs during manufacture of the device. In the case of such
pre-added components, it may be desirable to provide these
components in a stabilized form to allow for prolonged shelf-life
of the device. For example, the enzyme/substrate or receptor/ligand
components may be provided within the device in lyophilized form.
Prior to use, these components may be easily reconstituted by
introducing a buffer solution into the reservoirs. Alternatively,
the components may be lyophilized with appropriate buffering salts,
whereby simple water addition is all that is required for
reconstitution.
[0063] As noted above, the interaction of the first and second
components is typically accompanied by a detectable signal. For
example, in those embodiments where the first component is an
enzyme and the second a substrate, the substrate may be a
chromogenic or fluorogenic substrate which produces an optically
detectable signal when the enzyme acts upon the substrate. In the
case where the first component is a receptor and the second is a
ligand, either the ligand or the receptor may bear a detectable
signal. In either event, the mixture and flow rate of compounds
will typically remain constant such that the flow of the mixture of
the first and second components past the detection window 116 will
produce a steady-state signal. By "steady state signal" is
generally meant a signal that has a regular, predictable signal
intensity proile. As such, the steady-state signal may include
signals having a constant signal intensity, or alternatively, a
signal with a regular periodic intensity, against which variations
in the normal signal profile may be measured. This latter signal
may be generated in cases where fluid flow is periodically
interrupted for, e.g., loading additional test compounds, as
described in the description of the continuous flow systems.
Although the signal produced in the above-described enzymatic
system will vary along the length of the channel, i.e., increasing
with time of exposure as the enzyme converts the fluorogenic
substrate to the fluorescent product, the signal at any specific
point along the channel will remain constant, given a constant flow
rate.
[0064] From sample channel 112, test compounds may be periodically
or serially introduced into the main channel 110 and into the
stream of first and second components. Where these test compounds
have an effect on the interaction of the first and second elements,
it will produce a deviation in the signal detected at the detection
window. As noted above, typically, the various different test
compounds to be injected through channel 112 will be separated by a
spacer fluid to allow differentiation of the effects, or lack of
effects, from one test compound to another. In those embodiments
where electroosmotic fluid direction systems are employed, the
spacer fluids may also function to reduce any electrophoretic bias
that can occur within the test sample. The use of these spacer
fluids as well as the general elimination of electrophoretic bias
within the sample or test compound plugs is substantially described
in U.S. patent application Ser No. ______, filed ______ (Attorney
Docket No. 017646-000500) previously incorporated herein by
reference.
[0065] By way of example, a steady, continuous flow of enzyme and
fluorogenic substrate through main channel 110 will produce a
constant fluorescent signal at the detection window 116. Where a
test compound inhibits the enzyme, it will produce a momentary but
detectable drop in the level of signal at the detection window. The
timing of the drop in signal can then be correlated with a
particular test compound based upon a known injection to detection
time-frame. Specifically, the time required for an injected
compound to produce an observed effect can be readily determined
using positive controls.
[0066] For receptor/ligand systems, a similar variation in the
steady state signal may also be observed. Specifically, the
receptor and its fluorescent ligand can be made to have different
flow rates along the channel. This can be accomplished by
incorporating size exclusion matrices within the channel, or, in
the case of electroosmotic methods, altering the relative
electrophoretic mobility of the two compounds so that the receptor
flows more rapidly down the channel. Again, this may be
accomplished through the use of size exclusion matrices, or through
the use of different surface charges in the channel which will
result in differential flow rates of charge-varied compounds. Where
a test compound binds to the receptor, it will result in a dark
pulse in the fluorescent signal followed by a brighter pulse.
Without being bound to a particular theory of operation, it is
believed that the steady state signal is a result of both free
fluorescent ligand, and fluorescent ligand bound to the receptor.
The bound ligand is traveling at the same flow rate as the receptor
while the unbound ligand is traveling more slowly. Where the test
compound inhibits the receptor-ligand interaction, the receptor
will not `bring along` the fluorescent ligand, thereby diluting the
fluorescent ligand in the direction of flow, and leaving an excess
of free fluorescent ligand behind. This results in a temporary
reduction in the steady-state signal, followed by a temporary
increase in fluorescence. Alternatively, schemes similar to those
employed for the enzymatic system may be employed, where there is a
signal that reflects the interaction of the receptor with its
ligand. For example, pH indicators which indicate pH effects of
receptor-ligand binding may be incorporated into the device along
with the biochemical system, i.e., in the form of encapsulated
cells, whereby slight pH changes resulting from binding can be
detected. See Weaver, et al., Bio/Technology (1988) 6:1084-1089.
Additionally, one can monitor activation of enzymes resulting from
receptor ligand binding, e.g., activation of kinases, or detect
conformational changes in such enzymes upon activation, e.g.,
through incorporation of a fluorophore which is actuivated or
quenched by the conformational change to the enzyme upon
activation.
[0067] Flowing and direction of fluids within the microscale
fluidic devices may be carried out by a variety of methods. For
example, the devices may include integrated microfluidic
structures, such as micropumps and microvalves, or external
elements, e.g., pumps and switching valves, for the pumping and
direction of the various fluids through the device. Examples of
microfluidic structures are described in, e.g., U.S. Pat. Nos.
5,271,724, 5,277,556, 5,171,132, and 5,375,979. See also, Published
U.K. Patent Application No. 2 248 891 and Published European Patent
Application No. 568 902.
[0068] Although microfabricated fluid pumping and valving systems
may be readily employed in the devices of the invention, the cost
and complexity associated with their manufacture and operation can
generally prohibit their use in mass-produced disposable devices as
are envisioned by the present invention. For that reason, in
particularly preferred aspects, the devices of the invention will
typically include an electroosmotic fluid direction system. Such
fluid direction systems combine the elegance a fluid direction
system devoid of moving parts, with an ease of manufacturing, fluid
control and disposability. Examples of particularly preferred
electroosmotic fluid direction systems include, e.g., those
described in International Patent Application No. WO 96/04547 to
Ramsey et al., which is incorporated herein by reference in its
entirety for all purposes.
[0069] In brief, these fluidic control systems typically include
electrodes disposed within the reservoirs that are placed in fluid
connection with the plurality of intersecting channels fabricated
into the surface of the substrate. The materials stored in the
reservoirs are transported through the channel system delivering
appropriate volumes of the various materials to one or more regions
on the substrate in order to carry out a desired screening
assay.
[0070] Fluid transport and direction is accomplished through
electroosmosis or electrokinesis. In brief, when an appropriate
fluid is placed in a channel or other fluid conduit having
functional groups present at the surface, those groups can ionize.
For example, where the surface of the channel includes hydroxyl
functional groups at the surface, protons can leave the surface of
the channel and enter the fluid. Under such conditions, the surface
will possess a net negative charge, whereas the fluid will possess
an excess of protons or positive charge, particularly localized
near the interface between the channel surface and the fluid. By
applying an electric field along the length of the channel, cations
will flow toward the negative electrode. Movement of the positively
charged species in the fluid pulls the solvent with them. The
steady state velocity of this fluid movement is generally given by
the equation: 1 v = E 4
[0071] where v is the solvent velocity, .epsilon. is the dielectric
constant of the fluid, .xi. is the zeta potential of the surface, E
is the electric field strength, and .eta. is the solvent viscosity.
Thus, as can be easily seen from this equation, the solvent
velocity is directly proportional to the surface potential.
[0072] To provide appropriate electric fields, the system generally
includes a voltage controller that is capable of applying
selectable voltage levels, simultaneously, to each of the
reservoirs, including ground. Such a voltage controller can be
implemented using multiple voltage dividers and multiple relays to
obtain the selectable voltage levels. Alternatively, multiple,
independent voltage sources may be used. The voltage controller is
electrically connected to each of the reservoirs via an electrode
positioned or fabricated within each of the plurality of
reservoirs.
[0073] Incorporating this electroosmotic fluid direction system
into the device shown in FIG. 1 involves incorporation of an
electrode within each of the reservoirs 104, 106 and 108, and at
the terminus of sample channel 112 or at the terminus of any fluid
channels connected thereto, whereby the electrode is in electrical
contact with the fluid disposed in the respective reservoir or
channel. Substrate materials are also selected to produce channels
having a desired surface charge. In the case of glass substrates,
the etched channels will possess a net negative charge resulting
from the ionized hydroxyls naturally present at the surface.
Alternatively, surface modifications may be employed to provide an
appropriate surface charge, e.g., coatings, derivatization, e.g.,
silanation, or impregnation of the surface to provide appropriately
charged groups on the surface. Examples of such treatments are
described in, e.g., Provisional Patent Application Ser. No. ______,
filed Apr. 16, 1996 (Attorney Docket No. 017646-002600) which is
hereby incorporated herein by reference in its entirety for all
purposes.
[0074] Modulating voltages are then concomitantly applied to the
various reservoirs to affect a desired fluid flow characteristic,
e.g., continuous flow of receptor/enzyme, ligand/substrate toward
the waste reservoir with the periodic introduction of test
compounds. Particularly, modulation of the voltages applied at the
various reservoirs can move and direct fluid flow through the
interconnected channel structure of the device in a controlled
manner to effect the fluid flow for the desired screeening assay
and apparatus.
[0075] FIG. 2A shows a schematic illustration of fluid direction
during a typical assay screen. Specifically, shown is the injection
of a test compound into a continuous stream of an
enzyme-fluorogenic substrate mixture. As shown in FIG. 2A, and with
reference to FIG. 1, a continuous stream of enzyme is flowed from
reservoir 104, along main channel 110. Test compounds 120,
separated by appropriate fluid spacers 121 are introduced from
sample channel 112 into main channel 110. Once introduced into the
main channel, the test compounds will interact with the flowing
enzyme stream. The mixed enzyme/test compound plugs are then flowed
along main channel 110 past the intersection with channel 114. A
continuous stream of fluorogenic or chromogenic substrate which is
contained in reservoir 106, is introduced into sample channel 110,
whereupon it contacts and mixes with the continuous stream of
enzyme, including the discrete portions (or "plugs") of the stream
which include the test compounds 122. Action of the enzyme upon the
substrate will produce an increasing level of the fluorescent or
chromatic signal. This increasing signal is indicated by the
increasing shading within the main channel as it approaches the
detection window. This signal trend will also occur within those
test compound plugs which have no effect on the enzyme/substrate
interaction, e.g., test compound 126. Where a test compound does
have an effect on the interaction of the enzyme and the substrate,
a variation will appear in the signal produced. For example,
assuming a fluorogenic substrate, a test compound which inhibits
the interaction of the enzyme with its substrate will result in
less fluorescent product being produced within that plug. This will
result in a non-fluorescent, or detectably less fluorescent, plug
within the flowing stream as it passes detection window 116. For
example, as shown, test compound 128, a putative inhibitor of the
enzyme-substrate interaction, shows detectably lower flourescence
than the surrounding stream. This is indicated by a lack of shading
of test compound plug 128.
[0076] A detector adjacent to the detection window monitors the
level of fluorescent signal being produced by the enzyme's activity
on the fluorogenic or chromogenic substrate. This signal remains at
a relatively constant level for those test compounds which have no
effect on the enzyme-substrate interaction. When an inhibitory
compound is screened, however, it will produce a momentary drop in
the fluorescent signal representing the reduced or inhibited enzyme
activity toward the substrate. Conversely, inducer compounds upon
screening, will produce a momentary increase in the fluorescent
signal, corresponding to the increased enzyme activity toward the
substrate.
[0077] FIG. 2B provides a similar schematic illustration of a
screen for effectors of a receptor-ligand interaction. As in FIG.
2A, a continuous stream of receptor is flowed from reservoir 104
through main channel 110. Test compounds 150 separated by
appropriate spacer fluids 121 are introduced into the main channel
110 from sample channel 112, and a continuous stream of fluorescent
ligand from reservoir 106 is introduced from side channel 114.
Fluorescence is indicated by shading within the channel. As in FIG.
2A, the continuous stream of fluorescent ligand and receptor past
the detection window 116 will provide a constant signal intensity.
The portions of the stream containing the test compounds which have
no effect on the receptor-ligand interaction will provide the same
or similar level of fluorescence as the rest of the surrounding
stream, e.g., test compound 152. However, the presence of test
compounds which possess antagonistic or inhibitory activity toward
the receptor-ligand interaction will result in lower levels of that
interaction in those portions of the stream where those compounds
are located, e.g., test compound 154. Further, differential flow
rates for the receptor bound fluorescent ligand and free
fluorescent ligand will result in a detectable drop in the level of
fluorescence which corresponds to the dilution of the fluorescence
resulting from unbound, faster moving receptor. The drop in
fluorescence is then followed by an increase in fluorescence 156
which corresponds to an accumulation of the slower moving, unbound
fluorescent ligand.
[0078] In some cases, it may be desirable to provide an additional
channel for shunting off or extracting the reaction mixture slug
from the running buffer and/or spacer compounds. This may be the
case where one wishes to keep the reaction elements contained
within the sample plug during the reaction, while allowing these
elements to be separated during a data aquisition stage. As
described previously, one can keep the various elements of the
reaction together in the sample plug that is moving through the
reaction channel by incorporating appropriate spacer fluids between
samples. Such spacer fluids are generally selected to retain the
samples within their original slugs, i.e., not allowing smearing of
the sample into the spacer fluid, even during prolonged reaction
periods. However, this goal can be at odds with those assays which
are based upon the separation of elements of the assay, e.g.,
ligand-receptor assays described above, or where a reaction product
must be separated in a capillary.
[0079] A schematic illustration of one embodiment of a device 500
for performing this sample shunting or extraction is shown in FIG.
5. As shown, the samples or test compounds 504 are introduced to
the device or chip via the sample channel 512. Again, these are
typically introduced via an appropriate sample injection device
506, e.g., a capillary pipettor. The ionic strength and lengths of
the spacer solution plugs 502 and guard band plugs 508 are selected
such that those samples with the highest electrophoretic mobility
will not migrate through the spacer fluid/guard bands in the length
of time that it takes the sample to travel down the reaction
channel.
[0080] Assuming a receptor ligand assay system, test compounds pass
into the device 500 and into reaction channel 510, where they are
first combined with the receptor. The test compound/receptor are
flowed along the reaction channel in the incubation zone 510a.
Following this initial incubation, the test compound/receptor mix
is combined with a labelled ligand (e.g., fluorescent ligand)
whereupon this mixture flows along the second incubation region
510b of reaction channel 510. The lengths of the incubation regions
and the flow rates of the system (determined by the potentials
applied at each of the reservoirs 514, 516, 518, 520, 522, and at
the terminus of sample channel 512) determine the time of
incubation of the receptor with the fluorescent ligand and test
compound. The ionic strengths of the solutions containing the
receptors and fluorescent ligands, as well as the flow rates of
material from the reservoirs housing these elements into the sample
channel are selected so as to not interfere with the spacer
fluid/guard bands.
[0081] The isolated sample plugs containing receptor, fluorescent
ligand and test compound are flowed along the reaction channel 510
by the application of potentials at, e.g., reservoirs 514, 516, 518
and at the terminus of sample channel 512. Potentials are also
applied at reservoirs 520 and 522, at the opposite ends of
separation channel 524, to match the potentials at the two ends of
the transfer channel, so that the net flow across the transfer
channel is zero. As the sample plug passes the intersection of
reaction channel 510 and transfer channel 526, the potentials are
allowed to float at reservoirs 518 and 522, whereupon the
potentials applied at reservoirs 514, 516, 520, and at the terminus
of sample channel 512, result in the sample plug being shunted
through transfer channel 526 and into separation channel 524. Once
in the separation channel, the original potentials are reapplied to
all of the reservoirs to stop the net fluid flow through transfer
channel 526. The diversion of the sample plugs can then be repeated
with each subsequent sample plug. Within the separation channel,
the sample plug may be exposed to different conditions than those
of the reaction channel. For example, a different flow rate may be
used, capillary treatments may allow for separation of
differentially charged or different sized species, and the like. In
a preferred aspect, samples are shunted into the separation channel
to place the samples into a capillary filled with high ionic
strength buffer, i.e., to remove the low ionic strength spacer,
thereby allowing separation of the various sample components
outside the confines of the original sample plug. For example, in
the case of the above-described receptor/ligand screen, the
receptor/ligand complex may have a different electrophoretic
mobility from the ligand alone, in the transfer channel, thereby
allowing more pronounced separation of the complex from the ligand,
and its subsequent detection.
[0082] Such modifications have a wide variety of uses, particularly
where it may be desirable to separate reaction products following
reaction, e.g., in cleavage reactions, fragmentation reactions, PCR
reactions, and the like.
[0083] B. Serial in Parallel Assay Systems
[0084] More complex systems can also be produced within the scope
of the present invention. For example, a schematic illustration of
one alternate embodiment employing a "serial input parallel
reaction" geometry is shown in FIG. 3. As shown, the device 300
again includes a planar substrate 302 as described previously.
Fabricated into the surface of the substrate 302 are a series of
parallel reaction channels 312-324. Also shown are three transverse
channels fluidly connected to each of these parallel reaction
channels. The three transverse channels include a sample injection
channel 304, an optional seeding channel 306 and a collection
channel 308. Again, the substrate and channels are generally
fabricated utilizing the materials and to the dimensions generally
described above. Although shown and described in terms of a series
of parallel channels, the reaction channels may also be fabricated
in a variety of different orientations. For example, rather than
providing a series of parallel channels fluidly connected to a
single transverse channel, the channels may be fabricated
connecting to and extending radially outward from a central
reservoir, or may be arranged in some other non-parallel fashion.
Additionally, although shown with three transverse channels, it
will be recognized that fewer transverse channels may be used
where, e.g., the biochemical system components are predisposed
within the device. Similarly, where desired, more transverse
channels may be used to introduce further elements into a given
assay screen. Accordingly, the serial-in- parallel devices of the
present invention will typically include at least two and
preferably three, four, five or more transverse channels.
Similarly, although shown with 7 reaction channels, it will be
readily appreciated that the microscale devices of the present
invention will be capable of comprising more than 7 channels,
depending upon the needs of the particular screen. In preferred
aspects, the devices will include from 10 to about 500 reaction
channels, and more preferably, from 20 to about 200 reaction
channels.
[0085] This device may be particularly useful for screening test
compounds serially injected into the device, but employing a
parallel assay geometry, once the samples are introduced into the
device, to allow for increased throughput.
[0086] In operation, test compounds are serially introduced into
the device, separated as described above, and flowed along the
transverse sample injection channel 304 until the separate test
compounds are adjacent the intersection of the sample channel 304
with the parallel reaction channels 310-324. As shown in FIGS.
4A-4F, the test compounds may be provided immobilized on individual
beads. In those cases where the test compounds are immobilized on
beads, the parallel channels may be optionally fabricated to
include bead resting wells 326-338 at the intersection of the
reaction channels with the sample injection channel 304. Arrows 340
indicate the net fluid flow during this type of sample/bead
injection. As individual beads settle into a resting well, fluid
flow through that particular channel will be generally restricted.
The next bead in the series following the unrestricted fluid flow,
then flows to the next available resting well to settle in
place.
[0087] Once in position adjacent to the intersection of the
parallel reaction channel and the sample injection channel, the
test compound is directed into its respective reaction channel by
redirecting fluid flows down those channels. Again, in those
instances where the test compound is immobilized on a bead, the
immobilization will typically be via a cleavable linker group,
e.g., a photolabile, acid or base labile linker group. Accordingly,
the test compound will typically need to be released from the bead,
e.g., by exposure to a releasing agent such as light, acid, base or
the like prior to flowing the test compound down the reaction
channel.
[0088] Within the parallel channel, the test compound will be
contacted with the biochemical system for which an effector
compound is being sought. As shown, the first component of the
biochemical system is placed into the reaction channels using a
similar technique to that described for the test compounds. In
particular, the particular biochemical system is typically
introduced via one or more transverse seeding channels 306. Arrows
342 illustrate the direction of fluid flow within the seeding
channel 306. The biochemical system may be solution based, e.g., a
continuously flowing enzyme/substrate or receptor ligand mixture
like that described above, or as shown in FIGS. 4A-4F, may be a
whole cell or bead based system, e.g., beads which have
enzyme/substrate systems immobilized thereon.
[0089] In those instances where the biochemical system is
incorporated in a particle, e.g., a cell or bead, the parallel
channel may include a particle retention zone 344. Typically, such
retention zones will include a particle sieving or filtration
matrix, e.g., a porous gel or microstructure which retains
particulate material but allows the free flow of fluids. Examples
of microstructures for this filtration include, e.g., those
described in U.S. Pat. No. 5,304,487, which is hereby incorporated
by reference in its entirety for all purposes. As with the
continuous system, fluid direction within the more complex systems
may be generally controlled using microfabricated fluid direction
structures, e.g., pumps and valves. However, as the systems grow
more complex, such systems become largely unmanageable.
Accordingly, electroosmotic systems, as described above, are
generally preferred for controlling fluid in these more complex
systems. Typically, such systems will incorporate electrodes within
reservoirs disposed at the termini of the various transverse
channels to control fluid flow thorough the device. In some
aspects, it may be desirable to include electrodes at the termini
of all the various channels. This generally provides for more
direct control, but also grows less managable as systems grow more
complex. In order to utilize fewer electrodes and thus reduce the
potential complexity, it may often be desireable in parallel
systems, e.g., where two fluids are desired to move at similar
rates in parallel channels, to adjust the geometries of the various
flow channels. In particular, as channel length increases,
resistance along that channel will also increase. As such, flow
lengths between electrodes should be designed to be substantially
the same regardless of the parallel path chosen. This will
generally prevent the generation of transverse electrical fields
and thus promote equal flow in all parallel channels. To accomplish
substantially the same resistance between the electrodes, one can
alter the geometry of the channel structure to provide for the same
channel length, and thus, the channel resistance, regardless of the
path travelled. Alternatively, resistance of channels may be
adjusted by varying the cross-sectional dimensions of the paths,
thereby creating uniform resistance levels regardless of the path
taken.
[0090] As the test compounds are drawn through their respective
parallel reaction channels, they will contact the biochemical
system in question. As described above, the particular biochemical
system will typically include a flowable indicator system which
indicates the relative functioning of that system, e.g., a soluble
indicator such as chromogenic or fluorogenic substrate, labelled
ligand, or the like, or a particle based signal, such as a
precipitate or bead bound signalling group. The flowable indicator
is then flowed through the respective parallel channel and into the
collection channel 308 whereupon the signals from each of the
parallel channels are flowed, in series, past the detection window,
116.
[0091] FIGS. 4A-4F, with reference to FIG. 3, show a schematic
illustration of the progression of the injection of test compounds
and biochemical system components into the "serial input parallel
reaction" device, exposure of the system to the test compounds, and
flowing of the resulting signal out of the parallel reaction
channels and past the detection window. In particular, FIG. 4A
shows the introduction of test compounds immobilized on beads 346
through sample injection channel 304. Similarly, the biochemical
system components 348 are introduced into the reaction channels
312-324 through seeding channel 306. Although shown as being
introduced into the device along with the test compounds, as
described above, the components of the model system to be screened
may be incorporated into the reaction channels during manufacture.
Again, such components may be provided in liquid form or in
lyophilized form for increased shelf life of the particular
screening device.
[0092] As shown, the biochemical system components are embodied in
a cellular or particle based system, however, fluid components may
also be used as described herein. As the particulate components
flow into the reaction channels, they may be retained upon an
optional particle retaining matrix 344, as described above.
[0093] FIG. 4B illustrates the release of test compounds from the
beads 346 by exposing the beads to a releasing agent. As shown, the
beads are exposed to light from an appropriate light source 352,
e.g., which is able to produce light in a wavelength sufficient to
photolyze the linker group, thereby releasing compounds that are
coupled to their respective beads via a photolabile linker
group.
[0094] In FIG. 4C, the released test compounds are flowed into and
along the parallel reaction channels as shown by arrows 354 until
they contact the biochemical system components. The biochemical
system components 348 are then allowed to perform their function,
e.g., enzymatic reaction, receptor/ligand interaction, and the
like, in the presence of the test compounds. Where the various
components of the biochemical system are immobilized on a solid
support, release of the components from their supports can provide
the initiating event for the system. A soluble signal 356 which
corresponds to the functioning of the biochemical system is then
generated (FIG. 4D). As described previously, a variation in the
level of signal produced is an indication that the particular test
compound is an effector of the particular biochemical system. This
is illustrated by the lighter shading of signal 358.
[0095] In FIGS. 4E and 4F, the soluble signal is then flowed out of
reactions channels 312-324 into the detection channel 308, and
along the detection channel past the detection window 116.
[0096] Again, a detection system as described above, located
adjacent the detection window will monitor the signal levels. In
some embodiments, the beads which bore the test compounds may be
recovered to identify the test compounds which were present
thereon. This is typically accomplished by incorporation of a
tagging group during the synthesis of the test compound on the
bead. As shown, spent bead 360, i.e., from which a test compound
has been released, may be transported out of the channel structure
through port 362 for identification of the test compound that had
been coupled to it. Such identification may be accomplished outside
of the device by directing the bead to a fraction collector,
whereupon the test compounds present on the beads may be
identified, either through identification of a tagging group, or
through identification of residual compounds. Incorporation of
tagging groups in combinatorial chemistry methods has been
previously described using encrypted nucleotide sequences or
chlorinated/fluorinated aromatic compounds as tagging groups. See,
e.g., Published PCT Application No. WO 95/12608. Alternatively, the
beads may be transported to a separate assay system within the
device itself whereupon the identification may be carried out.
[0097] FIG. 6A shows an alternate embodiment of a "serial input
parallel reaction" device which can be used for fluid based as
opposed to bead based systems. As shown the device 600 generally
incorporates at least two transverse channels as were shown in
FIGS. 3 and 4, namely, sample injection channel 604 and detection
channel 606. These transverse channels are interconnected by the
series of parallel channels 612-620 which connect sample channel
604 to detection channel 606.
[0098] The device shown also includes an additional set of channels
for directing the flow of fluid test compounds into the reaction
channels. In particular, an additional transverse pumping channel
634 is fluidly connected to sample channel 604 via a series of
parallel pumping channels 636-646. The pumping channel includes
reservoirs 650 and 652 at its termini. The intersections of
parallel channels 636-646 are staggered from the intersections of
parallel channels 612-620 with sample channel 604, e.g., half way
between. Similarly, transverse pumping channel 608 is connected to
detection channel 606 via parallel pumping channels 622-632. Again,
the intersections of parallel pumping channels 622-632 with
detection channel 606 are staggered from the intersections of
reaction channels 612-620 with the detection channel 606.
[0099] A schematic illustration of the operation of this system is
shown in FIGS. 6B-6C. As shown, a series of test compounds,
physically isolated from each other, are introduced into sample
channel 604 using the methods described previously. For
electrokinetic systems, potentials are applied at the terminus of
sample channel 604, as well as reservoir 648. Potentials are also
applied at reservoirs 650:652, 654:656, and 658:660. This results
in a fluid flow along the transverse channels 634, 604, 606 and
608, as illustrated by the arrows, and a zero net flow through the
parallel channel arrays interconnecting these transverse channels,
as shown in FIG. 6B. Once the test compound slugs are aligned with
parallel reaction channels 612-620, connecting sample channel 604
to detection channel 606, as shown by the shaded areas in FIG. 6B,
flow is stopped in all transverse directions by removing the
potentials applied to the reservoirs at the ends of these channels.
As described above, the geometry of the channels can be varied to
maximize the use of space on the substrate. For example, where the
sample channel is straight, the distance between reaction channels
(and thus, the number of parallel reactions that can be carried out
in a size limited substrate) is dictated by the distance between
sample plugs. These restrictions, however, can be eliminated
through the inclusion of altered channel geometries. For example,
in some aspects, the length of a spacer/guard band plug can be
accomodated by a serpentine, square-wave, saw tooth or other
reciprocating channel geometry. This allows packing a maximum
number of reaction channels onto the limited area of the substrate
surface.
[0100] Once aligned with the parallel reaction channels, the sample
is then moved into the parallel reaction channels 612-620 by
applying a first potential to reservoirs 650 and 652, while
applying a second potential to reservoirs 658 and 660, whereby
fluid flow through parallel pumping channels 636-646 forces the
test compounds into parallel reaction channels 612-620, as shown in
FIG. 6C. During this process, no potential is applied at reservoirs
648, 654, 656, or the terminus of sample channel 604. Parallel
channels 636-646 and 622-632 are generally adjusted in length such
that the total channel length, and thus the level of resistance,
from reservoirs 650 and 652 to channel 604 and from reservoirs 658
and 660 to channel 606, for any path taken will be the same.
Resistance can generally be adjusted by adjusting channel length or
width. For example, channels can be lengthened by including folding
or serpentine geometries. Although not shown as such, to accomplish
this same channel length, channels 636 and 646 would be the longest
and 640 and 642 the shortest, to create symetric flow, thereby
forcing the samples into the channels. As can be seen, during
flowing of the samples through channels 612-620, the resistance
within these channels will be the same, as the individual channel
length is the same.
[0101] Following the reaction to be screened, the sample
plug/signal element is moved into detection channel 606 by applying
a potential from reservoirs 650 and 652 to reservoirs 658 and 660,
while the potentials at the remaining reservoirs are allowed to
float. The sample plugs/signal are then serially moved past the
detection window/detector 662 by applying potentials to reservoirs
654 and 656, while applying appropriate potentials at the termini
of the other transverse channels to prevent any flow along the
various parallel channels.
[0102] Although generally described in terms of screening assays
for identification of compounds which affect a particular
interaction, based upon the present disclosure, it will be readily
appreciated that the above described microlaboratory systems may
also be used to screen for compounds which specifically interact
with a component of a biochemical system without necessarily
affecting an interaction between that component and another element
of the biochemical system. Such compounds typically include binding
compounds which may generally be used in, e.g., diagnostic and
therapeutic applications as targeting groups for therapeutics or
marker groups, i.e. radionuclides, dyes and the like. For example,
these systems may be used to screen test compounds for the ability
to bind to a given component of a biochemical system.
[0103] III. Microlaboratory System
[0104] Although generally described in terms of individual discrete
devices, for ease of operation, the systems described will
typically be a part of a larger system which can monitor and
control the functioning of the devices, either on an individual
basis, or in parallel, multi-device screens. An example of such a
system is shown in FIGS. 7.
[0105] As shown in FIG. 7, the system may include a test compound
processing system 700. The system shown includes a platform 702
which can hold a number of separate assay chips or devices 704. As
shown, each chip includes a number of discrete assay channels 706,
each having a separate interface 708, e.g., pipettor, for
introducing test compounds into the device. These interfaces are
used to sip test compounds into the device, separated by sipping
spacer fluid and guard band fluids, into the device. In the system
shown, the interfaces of the chip are inserted through an opening
710 in the bottom of the platform 702, which is capable of being
raised and lowered to place the interfaces in contact with test
compounds or wash/spacer/guard band fluids, which are contained in,
e.g., multiwell micro plates 711, positioned below the platform,
e.g., on a conveyor system 712. In operation, multiwell plates
containing large numbers of different test compounds are stacked
714 at one end of the conveyor system. The plates are placed upon
the conveyor separated by appropriate buffer reservoirs 716 and
718, which may be filled by buffer system 720. The plates are
stepped down the conveyor and the test compounds are sampled into
the chips, interspersed by appropriate spacer fluids. After loading
the test compounds into the chips, the multiwell plates are then
collected or stacked 722 at the opposite end of the system. The
overall control system includes a number of individual
microlaboratory systems or devices, e.g., as shown in FIG. 7. Each
device is connected to a computer system which is appropriately
programmed to control fluid flow and direction within the various
chips, and to monitor, record and analyze data resulting from the
screening assays that are performed by the various devices. The
devices will typically be connected to the computer through an
intermediate adapter module which provides an interface between the
computer and the individual devices for implementing operational
instructions from the computer to the devices, and for reporting
data from the devices to the computer. For example, the adapter
will generally include appropriate connections to corresponding
elements on each device, e.g., electrical leads connected to the
reservoir based electrodes that are used for electroosmotic fluid
flow, power inputs and data outputs for detection systems, either
electrical or fiberoptic, and data relays for other sensor elements
incorporated into the devices. The adapter device may also provide
environmental control over the individual devices where such
control is necessary, e.g., maintaining the individual devices at
optimal temperatures for performing the particular screening
assays.
[0106] As shown, each device is also equipped with appropriate
fluid interfaces, e.g., micropipettors, for introducing test
compounds into the individual devices. The devices may be readily
attached to robotic systems which allow test compounds to be
sampled from a number of multiwell plates that are moved along a
conveyor system. Intervening spacer fluids can also be introduced
via a spacer solution reservoir.
EXAMPLES
[0107] An assay screen is performed to identify inhibitors of an
enzymatic reaction. A schematic of the chip to be used is shown in
FIG. 8. The chip has a reaction channel 5 cm in length which
includes a 1 cm incubation zone and a 4 cm reaction zone. The
reservoir at the beginning of the sample channel is filled with
enzyme solution and the side reservoir is filled with the
fluorogenic substrate. Each of the enzyme and substrate are diluted
to provide for a steady state signal in the linear signal range for
the assay system, at the detector. Potentials are applied at each
of the reservoirs (sample source, enzyme, substrate and waste) to
achieve an applied field of 200 V/cm. This applied field produces a
flow rate of 2 mm/second. During passage of a given sample through
the chip, there will generally be a diffusive broadening of the
sample. For example, in the case of a small molecule sample, e.g.,
1 mM benzoic acid diffusive broadening of approximately 0.38 mm and
an electrophoretic shift of 0.4 mm is seen.
[0108] Test compound plugs in 150 mM NaCl are introduced into the
sample channel separated by guard bands of 150 mM NaCl and spacer
plugs of 5 mM borate buffer. Once introduced into the sample
channel shown, sample requires 12 seconds to travel the length of
the sample channel and reach the incubation zone of the reaction
channel. This is a result of the flow rate of 2 mm/sec, allowing
for 1 second for moving the sample pipettor from the sample to the
spacer compounds. Allowing for these interuptions, the net flow
rate is 0.68 mm/sec. Another 12 seconds is required for the enzyme
test compound mixture to travel through the incubation zone to the
intersection with the substrate channel where substrate is
continuously flowing into the reaction zone of the reaction
channel. Each test compound then requires 48 seconds to travel the
length of the reaction zone and past the fluorescence detector. A
schematic of timing for sample/spacer loading is shown in FIG. 9.
The top panel shows the sample/spacer/guard band distribution
within a channel, whereas the lower panel shows the timing required
for loading the channel. As shown, the schematic includes the
loading (sipping) of high salt (HS) guard band ("A"), moving the
pipettor to the sample ("B"), sipping the sample ("C"), moving the
pipettor to the high salt guard band solution ("D") sipping the
high salt ("E"), moving the pipettor to the low salt (LS) spacer
fluid ("F"), sipping the low salt spacer ("G") and returning to the
high salt guard band ("H"). The process is then repeated for each
additional test compound.
[0109] A constant base fluorescent signal is established at the
detector in the absence of test compounds. Upon introduction of the
test compounds, a decrease in fluorescence is seen which, based
upon time delays corresponds to a specific individual test
compound. This test compound is tentatively identified as an
inhibitor of the enzyme, and further testing is conducted to
confirm this and quantitate the efficacy of this inhibitor.
[0110] 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 true scope of the invention. All publications and patent
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication or patent document were so individually
denoted.
* * * * *