U.S. patent application number 11/176805 was filed with the patent office on 2005-11-03 for high throughput screening assay systems in microscale fluidic devices.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Bousse, Luc J., Kopf-Sill, Anne R., Parce, J. Wallace.
Application Number | 20050241941 11/176805 |
Document ID | / |
Family ID | 27100659 |
Filed Date | 2005-11-03 |
United States Patent
Application |
20050241941 |
Kind Code |
A1 |
Parce, J. Wallace ; et
al. |
November 3, 2005 |
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, J. Wallace; (Palo
Alto, CA) ; Kopf-Sill, Anne R.; (Portola Valley,
CA) ; Bousse, Luc J.; (Los Altos, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
27100659 |
Appl. No.: |
11/176805 |
Filed: |
July 6, 2005 |
Related U.S. Patent Documents
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Application
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11176805 |
Jul 6, 2005 |
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10637730 |
Aug 7, 2003 |
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10637730 |
Aug 7, 2003 |
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09718236 |
Nov 21, 2000 |
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6630353 |
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09718236 |
Nov 21, 2000 |
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08881696 |
Jun 24, 1997 |
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6267858 |
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08881696 |
Jun 24, 1997 |
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08671987 |
Jun 28, 1996 |
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5942443 |
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08881696 |
Jun 24, 1997 |
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08761575 |
Dec 6, 1996 |
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6046056 |
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Current U.S.
Class: |
204/450 ;
204/600 |
Current CPC
Class: |
B01J 2219/00828
20130101; B01L 2200/027 20130101; B01L 2200/0647 20130101; B01J
2219/0097 20130101; B01L 3/502784 20130101; B01L 2400/0487
20130101; Y10T 436/11 20150115; B01L 3/5025 20130101; B01J 19/0093
20130101; B01L 2300/0867 20130101; B01L 2400/0415 20130101; B01L
2400/0418 20130101; B01L 2300/0864 20130101; Y10S 436/805 20130101;
Y10T 436/117497 20150115; Y10T 436/25 20150115; B01L 2300/0887
20130101; Y10T 436/110833 20150115; G01N 33/5011 20130101; Y10T
436/113332 20150115; B01L 2200/0668 20130101; Y10S 435/817
20130101; G01N 33/5064 20130101; B01L 2300/0816 20130101; B01J
2219/00833 20130101; G01N 33/5091 20130101; B01J 2219/00952
20130101; B01L 2200/0673 20130101; B01J 2219/00853 20130101; G01N
35/08 20130101; Y10S 436/806 20130101; G01N 27/44791 20130101; B01L
3/502715 20130101; G01N 33/502 20130101; B01L 3/502761 20130101;
Y10S 435/81 20130101; B01J 2219/00831 20130101; B01L 2300/0861
20130101; G01N 33/5097 20130101; B01J 2219/00961 20130101; G01N
33/5304 20130101; B01L 3/502753 20130101; B01L 2200/0605 20130101;
B01L 2300/0877 20130101; Y10S 436/804 20130101; G01N 33/5008
20130101; G01N 33/57415 20130101; Y10S 366/02 20130101; B01L
3/50273 20130101 |
Class at
Publication: |
204/450 ;
204/600 |
International
Class: |
G01N 027/27 |
Claims
What is claimed is:
1. A microfluidic device, comprising: means for continuously
flowing interacting components of a biochemical system through a
first of at least two intersecting channels; means for flowing a
test compound from a second channel into the first channel such
that the test compound contacts at least one component of the
biochemical system; means for detecting an effect of the test
compound on interactions of the components of the biochemical
system.
2. The microfluidic device of claim 1, wherein the components of a
biochemical system comprise an enzyme and a substrate for the
enzyme, and wherein action of the enzyme on the substrate produces
a detectable signal.
3. The microfluidic device of claim 1, wherein the components of a
biochemical system comprise a receptor/ligand binding pair, wherein
at least one of the receptor and the ligand has a detectable signal
associated therewith.
4. The microfluidic device of claim 1, wherein the components of a
biochemical system comprise a receptor/ligand binding pair, wherein
binding of the receptor to the ligand produces a detectable
signal.
5. The microfluidic device of claim 1, wherein the components of a
biochemical system comprise components of a cell, and wherein the
cell is capable of producing a detectable signal corresponding to a
cellular function.
6. The microfluidic device of claim 5, wherein detecting an effect
of the test compound on interactions of the components of the cell
comprises detecting an effect of the test compound on viability of
the cell.
7. The microfluidic device of claim 1, wherein the components of
the biochemical system produce a flowable detectable signal
representative of a function of the biochemical system.
8. The microfluidic device of claim 1, wherein the detectable
effect of the test compound on interactions of the components of
the biochemical system comprises a change in a steady-state
signal.
9. The microfluidic device of claim 1, wherein at least one of the
at least two intersecting channels has a cross-sectional dimension
in a range from 0.1 .mu.m to 500 .mu.m.
10. A microfluidic device, comprising: means for continuously
flowing interacting components of a biochemical system through a
plurality of channels; means for introducing a discrete volume of
one of a plurality of test compounds into each of the plurality of
channels such that the test compound contacts at least one
component of the biochemical system; and means for detecting an
effect of each test compound on interactions of the components of
the biochemical system.
11. The microfluidic device of claim 10, wherein the components of
a biochemical system comprise an enzyme and a substrate for the
enzyme, and wherein action of the enzyme on the substrate produces
a detectable signal.
12. The microfluidic device of claim 10, wherein the components of
a biochemical system comprise a receptor/ligand binding pair,
wherein at least one of the receptor and the ligand has a
detectable signal associated therewith.
13. The microfluidic device of claim 10, wherein the components of
a biochemical system comprise a receptor/ligand binding pair,
wherein binding of the receptor to the ligand produces a detectable
signal.
14. The microfluidic device of claim 10, wherein the components of
a biochemical system comprise components of a cell, and wherein the
cell is capable of producing a detectable signal corresponding to a
cellular function.
15. The microfluidic device of claim 14, wherein detecting an
effect of the test compound on interactions of the components of
the cell comprises detecting an effect of the test compound on
viability of the cell.
16. The microfluidic device of claim 10, wherein the components of
the biochemical system produce a flowable detectable signal
representative of a function of the biochemical system.
17. The microfluidic device of claim 10, wherein the detectable
effect of the test compound on interactions of the components of
the biochemical system comprises a change in a steady-state
signal.
18. The microfluidic device of claim 10, wherein at least one of
the plurality of channels has a cross-sectional dimension in a
range from 0.1 .mu.m to 500 .mu.m.
19. A microfluidic test compound processing system, comprising:
means for holding a microfluidic device; means for continually
flowing components of a biochemical system in a first of at least
two intersecting channels within the microfluidic device; means for
introducing a test compound into the microfluidic device from an
external test compound source; means for placing the introducing
means in fluid communication with the external test compound
source; means for controlling flow and direction of the test
compound within the microfluidic device such that the test compound
contacts at least one of the continually flowing components of the
biochemical system; and means for detecting an effect of the test
compound on interactions of the components of the biochemical
system.
20. The microfluidic test compound processing system of claim 19,
wherein at least one of the at least two intersecting channels has
a cross-sectional dimension in a range from 0.1 .mu.m to 500 .mu.m.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/637,730, filed Aug. 7, 2003, which is a
continuation of U.S. patent application Ser. No. 09/718,236, filed
Nov. 21, 2000, which is a continuation of U.S. patent application
Ser. No. 08/881,696, filed Jun. 24, 1997, now U.S. Pat. No.
6,267,858, which is a continuation-in-part of U.S. patent
application Ser. No. 08/671,987, filed Jun. 28, 1996, now U.S. Pat.
No. 5,942,443 and U.S. patent application Ser. No. 08/761,575,
filed Dec. 06, 1996, now U.S. Pat. No. 6,046,056, all of which are
hereby incorporated herein by reference in their entirety for all
purposes. PCT Application No. PCT/US97/10894, which designates the
United States of America and is substantially identical to the
present application, was co-filed in the United States Receiving
Office on Jun. 24, 1997. This application is also incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] This application relates to apparatus and assay systems for
detecting molecular interactions. The apparatus comprise a
substrate with one or more intersecting channels and an
electroosmotic fluid movement component, or other component for
moving fluid in the channels on the substrate.
BACKGROUND OF THE INVENTION
[0003] 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. Similarly, the ability to rapidly process samples for
detection of biological molecules relevant to diagnostic or
forensic analysis is of fundamental value for, e.g., diagnostic
medicine, archaeology, anthropology, and modern criminal
investigation.
[0004] 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 a maximum number of
different compounds necessitates reducing the time and labor
requirements associated with each screen.
[0005] 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. Eng. 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
& Elhman, 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.
[0006] 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 (e.g., genes and
gene products such as proteins and RNA) against which the efficacy
of test compounds are screened.
[0007] 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. 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.
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., due to evaporation, small
dispensing errors, or the like. 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.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention 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
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
the 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 optionally 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 are
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 of reaction
channels. As above, the apparatuses also optionally 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.
[0022] FIGS. 6B and 6C show a schematic illustration of fluid flow
patterns within the device shown in FIG. 6A.
[0023] FIG. 7 shows a schematic illustration of one embodiment of
an overall assay systems which employs multiple microlaboratory
devices labeled as "LabChips.TM." for screening test compounds.
[0024] FIG. 8 is a schematic illustration of a chip layout used for
a continuous-flow assay screening system.
[0025] FIGS. 9A and 9B shows fluorescence data from a continuous
flow assay screen. FIG. 9A shows fluorescence data from a test
screen which periodically introduced a known inhibitor (IPTG) into
a .beta.-galactosidase assay system in a chip format. FIG. 9B shows
a superposition of two data segments from FIG. 9A, directly
comparing the inhibitor data with control (buffer) data.
[0026] FIG. 10 illustrates the operating parameters of a fluid flow
system on a small chip device for performing enzyme inhibitor
screening.
[0027] FIG. 11 shows a schematic illustration of timing for
sample/spacer loading in a microfluidic device channel.
[0028] FIG. 12, panels A-G schematically illustrate electrodes used
in apparatuses of the invention.
DETAILED DESCRIPTION
[0029] I. Applications for the Invention
[0030] 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 in screening
large numbers of different compounds for their effects on a variety
of chemical, and preferably, biochemical systems.
[0031] As used herein, the phrase "biochemical system" generally
refers to a chemical interaction that involves molecules of the
type generally found within living organisms. Such interactions
include the full range of catabolic and anabolic reactions which
occur in living systems including enzymatic, binding, signaling and
other reactions. Further, biochemical systems, as defined herein,
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, e.g.,
in toxicology studies. Biological materials which are assayed
include, but are not limited to, cells, cellular fractions
(membranes, cytosol preparations, etc.), agonists and antagonists
of cell membrane receptors (e.g., cell receptor-ligand interactions
such as e.g., transferring, c-kit, viral receptor ligands (e.g.,
CD4-HIV), cytokine receptors, chemokine receptors, interleukin
receptors, immunoglobulin receptors and antibodies, the cadherein
family, the integrin family, the selectin family, and the like;
see, e.g., Pigott and Power (1993) The Adhesion Molecule FactsBook
Academic Press New York and Hulme (ed) Receptor Ligand Interactions
A Practical Approach Rickwood and Hames (series editors) IRL Press
at Oxford Press NY), toxins and venoms, viral epitopes, hormones
(e.g., opiates, steroids, etc.), intracellular receptors (e.g.
which mediate the effects of various small ligands, including
steroids, thyroid hormone, retinoids and vitamin D; for reviews
see, e.g., Evans (1988) Science, 240:889-895; Ham and Parker (1989)
Curr. Opin. Cell Biol., 1:503-511; Burustein et al. (1989), Ann.
Rev. Physiol., 51:683-699; Truss and Beato (1993) Endocr. Rev.,
14:459-479), peptides, retro-inverso peptides, polymers of
.alpha.-, .beta.-, or .omega.-amino acids (D- or L-), enzymes,
enzyme substrates, cofactors, drugs, lectins, sugars, nucleic acids
(both linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies. Synthetic polymers such as
heteropolymers in which a known drug is covalently bound to any of
the above, such as polyurethanes, polyesters, polycarbonates,
polyureas, polyamides, polyethyleneimines, polyarylene sulfides,
polysiloxanes, polyimides, and polyacetates are also assayed. Other
polymers are also assayed using the systems described herein, as
would be apparent to one of skill upon review of this disclosure.
One of skill will be generally familiar with the biological
literature. For a general introduction to biological systems, see,
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods
in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al. (1989) Molecular Cloning--A Laboratory
Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor Press, NY, (Sambrook); Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (through 1997 Supplement) (Ausubel); Watson et
al. (1987) Molecular Biology of the Gene, Fourth Edition The
Benjamin/Cummings Publishing Co., Menlo Park, Calif.; Watson et al.
(1992) Recombinant DNA Second Edition Scientific American Books,
NY; Alberts et al. (1989) Molecular Biology of the Cell Second
Edition Garland Publishing, NY; Pattison (1994) Principles and
Practice of Clinical Virology; Darnell et al., (1990) Molecular
Cell Biology second edition, Scientific American Books, W. H.
Freeman and Company; Berkow (ed.) The Merck Manual of Diagnosis and
Therapy, Merck & Co., Rahway, N.J.; Harrison's Principles of
Internal Medicine, Thirteenth Edition, Isselbacher et al. (eds).
(1994) Lewin Genes, 5th Ed., Oxford University Press (1994); The
"Practical Approach" Series of Books (Rickwood and Hames (series
eds.) by IRL Press at Oxford University Press, NY; The "FactsBook
Series" of books from Academic Press, NY; Product information from
manufacturers of biological reagents and experimental equipment
also provide information useful in assaying biological systems.
Such manufacturers include, e.g., the SIGMA chemical company (Saint
Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB
Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San
Diego, Calif., and Applied Biosystems (Foster City, Calif.), as
well as many other commercial sources known to one of skill.
[0032] 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 are optionally screened for
effects in blocking, slowing or otherwise inhibiting key events
associated with biochemical systems whose effect is undesirable.
For example, test compounds are optionally 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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. Typically, such assays involve the measurement of a
parameter of the biochemical system. By "parameter of the
biochemical system" is meant some measurable evidence of the
system's functioning, e.g., the presence or absence of a labeled
group or a change in molecular weight (e.g., in binding reactions,
transport screens), the presence or absence of a reaction product
or substrate (in substrate turnover measurements), or an alteration
in electrophoretic mobility (typically detected by a change in
elution time of a labeled compound).
[0037] 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 are
optionally 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.
[0038] 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 are optionally
utilized. Modified cell systems may also be employed in the
screening systems encompassed herein. For example, chimeric
reporter systems are optionally 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 is
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.
[0039] Additionally, where one is screening for bioavailability,
e.g., transport, biological barriers are optionally 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.
[0040] 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 epidermal growth actor,
FGF fibroblast growth factor, PDGF platelet derived growth factor,
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.
[0041] 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. Receptoriligand 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.
[0042] 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.
[0043] In the interest of efficiency, screening assays have
typically been set up in multiweli reaction plates, e.g.,
multi-well microplates, which allow for the simultaneous, parallel
screening of large numbers of test compounds.
[0044] 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."
[0045] 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."
[0046] 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 is screened for effect on a biological
system using a continuous serial or parallel assay orientation.
[0047] 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 are provided, e.g., injected, free in solution, or are
optionally attached to a carrier, or a solid support, e.g., beads.
A number of suitable solid supports are 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 are 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. Alternatively,
such group screening is used where the effects of different test
compounds are differentially detected in a single system, e.g.,
through electrophoretic separation of the effects, or differential
labeling which enables separate detection.
[0048] Test compounds are commercially available, or derived from
any of a variety of biological sources apparent to one of skill and
as described, supra. In one aspect, a tissue homogenate or blood
sample from a patient is tested in the assay systems of the
invention. For example, in one aspect, blood is tested for the
presence or activity of a biologically relevant molecule. For
example, the presence and activity level of an enzyme are detected
by supplying and enzyme substrate to the biological sample and
detecting the formation of a product using an assay systems of the
invention. Similarly, the presence of infectious pathogens
(viruses, bacteria, fungi, or the like) or cancerous tumors can be
tested by monitoring binding of a labeled ligand to the pathogen or
tumor cells, or a component of the pathogen or tumor such as a
protein, cell membrane, cell extract or the like, or alternatively,
by monitoring the presence of an antibody against the pathogen or
tumor in the patient's blood. For example, the binding of an
antibody from a patient's blood to a viral protein such as an HIV
protein is a common test for monitoring patient exposure to the
virus. Many assays for detecting pathogen infection are well known,
and are adapted to the assay systems of the present invention.
[0049] Biological samples are derived from patients using well
known techniques such as venipuncture or tissue biopsy. Where the
biological material is derived from non-human animals, such as
commercially relevant livestock, blood and tissue samples are
conveniently obtained from livestock processing plants. Similarly,
plant material used in the assays of the invention are conveniently
derived from agricultural or horticultural sources. Alternatively,
a biological sample can be from a cell or blood bank where tissue
and/or blood are stored, or from an in vitro source such as a
culture of cells. Techniques and methods for establishing a culture
of cells for use as a source for biological materials are well
known to those of skill in the art. Freshney Culture of Animal
Cells, a Manual of Basic Technique, Third Edition Wiley-Liss, New
York (1994) provides a general introduction to cell culture.
[0050] II. Assay Systems
[0051] 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, or the like. 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 are optionally
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.
[0052] A. Electrokinetic Material Transport
[0053] In preferred aspects, the devices, methods and systems
described herein, employ electrokinetic material transport systems,
and preferably, controlled electrokinetic material transport
systems. As used herein, "electrokinetic material transport
systems" include systems which transport and direct materials
within an interconnected channel and/or chamber containing
structure, through the application of electrical fields to the
materials, thereby causing material movement through and among the
channel and/or chambers, i.e., cations will move toward the
negative electrode, while anions will move toward the positive
electrode.
[0054] Such electrokinetic material transport and direction systems
include those systems that rely upon the electrophoretic mobility
of charged species within the electric field applied to the
structure. Such systems are more particularly referred to as
electrophoretic material transport systems. Other electrolinetic
material direction and transport systems rely upon the
electroosmotic flow of fluid and material within a channel or
chamber structure which results from the application of an electric
field across such structures. In brief, when a fluid is placed into
a channel which has a surface bearing charged functional groups,
e.g., hydroxyl groups in etched glass channels or glass
microcapillaries, those groups can ionize. In the case of hydroxyl
functional groups, this ionization, e.g., at neutral pH, results in
the release of protons from the surface and into the fluid,
creating a concentration of protons at near the fluid/surface
interface, or a positively charged sheath surrounding the bulk
fluid in the channel. Application of a voltage gradient across the
length of the channel, will cause the proton sheath, as well as the
fluid it surrounds, to move in the direction of the voltage drop,
i.e., toward the negative electrode.
[0055] "Controlled electrokinetic material transport and
direction," as used herein, refers to electrokinetic systems as
described above, which employ active control of the voltages
applied at multiple, i.e., more than two, electrodes. Rephrased,
such controlled electrokinetic systems concomitantly regulate
voltage gradients applied across at least two intersecting
channels. Controlled electrolinetic material transport is described
in Published PCT Application No. WO 96/04547, to Ramsey, which is
incorporated herein by reference in its entirety for all purposes.
In particular, the preferred microfluidic devices and systems
described herein, include a body structure which includes at least
two intersecting channels or fluid conduits, e.g., interconnected,
enclosed chambers, which channels include at least three
unintersected termini. The intersection of two channels refers to a
point at which two or more channels are in fluid communication with
each other, and encompasses "T" intersections, cross intersections,
"wagon wheel" intersections of multiple channels, or any other
channel geometry where two or more channels are in such fluid
communication. An unintersected terminus of a channel is a point at
which a channel terminates not as a result of that channel's
intersection with another channel, e.g., a "T" intersection. In
preferred aspects, the devices will include at least three
intersecting channels having at least four unintersected termini.
In a basic cross channel structure, where a single horizontal
channel is intersected and crossed by a single vertical channel,
controlled electrokinetic material transport operates to
controllably direct material flow through the intersection, by
providing constraining flows from the other channels at the
intersection. For example, assuming one was desirous of
transporting a first material through the horizontal channel, e.g.,
from left to right, across the intersection with the vertical
channel. Simple electrokinetic material flow of this material
across the intersection could be accomplished by applying a voltage
gradient across the length of the horizontal channel, i.e.,
applying a first voltage to the left terminus of this channel, and
a second, lower voltage to the right terminus of this channel, or
by allowing the right terminus to float (applying no voltage).
However, this type of material flow through the intersection would
result in a substantial amount of diffusion at the intersection,
resulting from both the natural difflusive properties of the
material being transported in the medium used, as well as
convective effects at the intersection.
[0056] In controlled electrokinetic material trasort, the material
being transported across the intersection is constrained by low
level flow from the side channels, e.g., the top and bottom
channels. This is accomplished by applying a slight voltage
gradient along the path of material flow, e.g., from the top or
bottom termini of the vertical channel, toward the right terminus.
The result is a "pinching" of the material flow at the
intersection, which prevents the diffusion of the material into the
vertical channel. The pinched volume of material at the
intersection may then be injected into the vertical channel by
applying a voltage gradient across the length of the vertical
channel, i.e., from the top terminus to the bottom terminus. In
order to avoid any bleeding over of material from the horizontal
channel during this injection, a low level of flow is directed back
into the side channels, resulting in a "pull back" of the material
from the intersection.
[0057] In addition to pinched injection schemes, controlled
electrokinetic material transport is readily utilized to create
virtual valves which include no mechanical or moving parts.
Specifically, with reference to the cross intersection described
above, flow of material from one channel segment to another, e.g.,
the left arm to the right arm of the horizontal channel, can be
efficiently regulated, stopped and reinitiated, by a controlled
flow from the vertical channel, e.g., from the bottom arm to the
top arm of the vertical channel. Specifically, in the `off` mode,
the material is transported from the left arm, through the
intersection and into the top arm by applying a voltage gradient
across the left and top termini. A constraining flow is directed
from the bottom arm to the top arm by applying a similar voltage
gradient along this path (from the bottom terminus to the top
terminus). Metered amounts of material are then dispensed from the
left arm into the right arm of the horizontal channel by switching
the applied voltage gradient from left to top, to left to right.
The amount of time and the voltage gradient applied dictates the
amount of material that will be dispensed in this manner. Although
described for the purposes of illustration with respect to a four
way, cross intersection, these controlled electrolinetic material
transport systems can be readily adapted for more complex
interconnected channel networks, e.g., arrays of interconnected
parallel channels.
[0058] B. Continuous Flow Assay Systems
[0059] 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 interrupt the flow stream. The
functioning of the system is indicated by the production of a
detectable event or signal. In one preferred embodiment, such
detectable signals include optically detectable 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.
[0060] A wide variety of other detectable signals and labels can
also be used in the assays and apparatuses of the invention. In
addition to the chromogenic and fluorogenic labels described above,
radioactive decay, electron density, changes in pH, solvent
viscosity, temperature and salt concentration are also conveniently
measured.
[0061] More generally, labels are commonly detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. For example, useful nucleic acid labels include
32P, 35S, fluorescent dyes, electron-dense reagents, enzymes (e.g.,
as commonly used in an ELISA), biotin, dioxigenin, or haptens and
proteins for which antisera or monoclonal antibodies are available.
A wide variety of labels suitable for labeling biological
components are known and are reported extensively in both the
scientific and patent literature, and are generally applicable to
the present invention for the labeling of biological components.
Suitable labels include radionucleotides, enzymes, substrates,
cofactors, inhibitors, fluorescent moieties, chemiluminiescent
moieties, magnetic particles, and the like. Labeling agents
optionally include e.g., monoclonal antibodies, polyclonal
antibodies, proteins, or other polymers such as affinity matrices,
carbohydrates or lipids. Detection proceeds by any of a variety of
known methods, including spectrophotometric or optical tracking of
radioactive or fluorescent markers, or other methods which track a
molecule based upon size, charge or affinity. A detectable moiety
can be of any material having a detectable physical or chemical
property. Such detectable labels have been well-developed in the
field of gel electrophoresis, column chromatograpy, solid
substrates, spectroscopic techniques, and the like, and in general,
labels useful in such methods can be applied to the present
invention. Thus, a label is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical thermal, or chemical means. Useful labels in
the present invention include fluorescent dyes (e.g., fluorescein
isothiocyanate, Texas red, rhodamine, and the like), radiolabels
(e.g., 3H, 125I, 35S, 14C, 32P or 33P), enzymes (e.g., LacZ, CAT,
horse radish peroxidase, alkaline phosphatase and others, commonly
used as detectable enzymes, either as marker products or as in an
EUISA), nucleic acid intercalators (e.g., ethidium bromide) and
colorimetric labels such as colloidal gold or colored glass or
plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
[0062] Fluorescent labels are particularly preferred labels.
Preferred labels are typically characterized by one or more of the
following: high sensitivity, high stability, low background, low
environmental sensitivity and high specificity in labeling.
[0063] Fluorescent moieties, which are incorporated into the labels
of the invention, are generally are known, including 1- and
2-amironaphthalene, p,p'-diaminostilbenes, pyrenes, quaternary
phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone
imines, anthracenes, oxacarbocyanine, merocyanine,
3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl
benzene, 1,2-benzophenazin, retinol, bis-3-aminopyridiniuim salts,
hellebrigenin, tetracycline, sterophenol,
benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen,
7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin,
porphyrins, triarylmethanes and flavin. Individual fluorescent
compounds which have functionalities for linking to an element
desirably detected in an apparatus or assay of the invention, or
which can be modified to incorporate such functionalities include,
e.g., dansyl chloride; fluoresceins such as
3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;
N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl
2-amino-6-sulfonatonaphthalene; 4-acetamido-4-isothiocyanato-sti-
lbene-2,2'-disulfonic acid; pyrene-3-sulfonic acid;
2-toluidinonaphthalene6-sulfonate;
N-phenyl-N-methyl-2-aminoaphthalene-6-- sulfonate; ethidim bromide;
stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl
phosphatidylethanolamine; N,N'-dioctadecyl oxacarbocyanine:
N,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)stearate;
d-3-aminodesoxy-equilenin; 12-(9'-anthroyl)stearate;
2-methylanthracene; 9-vinylanracene;
2,2'(vinylene-p-phenylene)bisbenzoxazole;
p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene;
6-dimethylamino-1,2-benzoph- enazin; retinol;
bis(3'-aminopyridinium) 1,10-decandiyl diiodide;
sulfonaphthylhydrazone of hellibrienin; chlorotetracycline;
N-(7-dimethylamino4-methyl-2-oxo-3-chromenyl)maleinide;
N-(p-(2-benzimidazolyl)-phenyl)maleimide;
N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin;
4-chloro-7-nitro-2,1,3-benzooxadiazole- ; merocyanine 540;
resorufm; rose bengal; and 2,4-diphenyl-3(2H)-furanone. Many
fluorescent tags are commercially available from SIGMA chemical
company (Saint Louis, Mo.), Molecular Probes, R&D systems
(Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway,
N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes
Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research,
Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
and Applied Biosystems (Foster City, Calif.) as well as other
commercial sources known to one of skill.
[0064] Desirably, fluorescent labels absorb light above about 300
nm, preferably about 350 nm, and more preferably above about 400
nm, usually emitting at wavelengths greater than about 10 nm higher
than the wavelength of the light absorbed. It should be noted that
the absorption and emission characteristics of the bound label may
differ from the unbound label. Therefore, when referring to the
various wavelength ranges and characteristics of the labels, it is
intended to indicate the labels as employed and not the label which
is unconjugated and characterized in an arbitrary solvent.
[0065] Fluorescent labels are one preferred class of detectable
labels, in part because by irradiating a fluorescent label with
light, one can obtain a plurality of emissions. Thus, a single
label can provide for a plurality of measurable events. Detectable
signal may also be provided by chemiluminescent and bioluminescent
sources. Chemiluminescent sources include a compound which becomes
electronically excited by a chemical reaction and may then emit
light which serves as the detectible signal or donates energy to a
fluorescent acceptor. A diverse number of families of compounds
have been found to provide chemiluminescence under a variety or
conditions. One family of compounds is
2,3-dihydro-1,4-phthalazinedione. The most popular compound is
luminol, which is a 5-amino compound. Other members of the family
include the 5-amino-6,7,8-trimethoxy-and the dimethylamino[ca]benz
analog. These compounds can be made to luminesce with alkaline
hydrogen peroxide or calcium hypochlorite and base. Another family
of compounds is the 2,4,5-triphenylimidazoles, with lophine as the
common name for the parent product. Chemiluminescent analogs
include para-dimethylamino and-methoxy substituents.
Chemiluminescence may also be obtained with oxalates, usually
oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g.,
hydrogen peroxide, under basic conditions. Other useful
chemiluminescent compounds are also known and available, including
--N-alkyl acridimim esters (basic H.sub.2O.sub.2) and dioxetanes.
Alternatively, luciferins may be used in conjunction with
luciferase or lucigenins to provide bioluminescence.
[0066] The label is coupled directly or indirectly to a molecule to
be detected (a product, substrate, enzyme, or the like) according
to methods well known in the art. As indicated above, a wide
variety of labels are used, with the choice of label depending on
the sensitivity required, ease of conjugation of the compound,
stability requirements, available instrumentation, and disposal
provisions. Non radioactive labels are often attached by indirect
means. Generally, a ligand molecule (e.g., biotin) is covalently
bound to a polymer. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. A number of
ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with labeled, anti-ligands. Alternatively,
any haptenic or antigenic compound can be used in combination with
an antibody. Labels can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
Means of detecting labels are well known to those of skill in the
art. Thus, for example, where the label is a radioactive label,
means for detection include a scintillation counter or photographic
film as in autoradiography. Where the label is a fluorescent label,
it may be detected by exciting the fluorochrome with the
appropriate wavelength of light and detecting the resulting
fluorescence, e.g., by microscopy, visual inspection, via
photographic film, by the use of electronic detectors such as
digital cameras, charge coupled devices (CCDs) or photomultipliers
and phototubes, and the like. Fluorescent labels and detection
techniques, particularly microscopy and spectroscopy are preferred.
Similarly, enzymatic labels are detected by providing appropriate
substrates for the enzyme and detecting the resulting reaction
product. Finally, simple calorimetric labels are often detected
simply by observing the color associated with the label. For
example, conjugated gold often appears pink, while various
conjugated beads appear the color of the bead.
[0067] 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.
[0068] 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.
[0069] 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 material and
fluid direction systems, sensors and the like. In the case of
polymeric substrates, the substrate materials are optionally 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, are
optionally incorporated into the device for these types detection
elements. Additionally, the polymeric materials may have linear or
branched backbones, and are optionally crosslinked or
non-crosslinked. Examples of particularly preferred polymeric
materials include, e.g., polydimethylsiloxanes (PDMS),
polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone,
polycarbonate and the like.
[0070] 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.
[0071] 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 are optionally 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 are optionally 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
are optionally 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.
[0072] 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 is 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.
[0073] 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.
[0074] The introduction of large numbers of individual, discrete
volumes of test compounds into the sample is carried out by a
number of methods. For example, micropipettors are optionally used
to introduce the test compounds into the device. In preferred
aspects, an electropipettor is 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. 08/671,986,
filed Jun. 28, 1996, now U.S. Pat. No. 5,779,868, 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, or "subject materials," and
spacer compounds. The pipettor then delivers individual, physically
isolated sample or test compound volumes in subject material
regions, in series, into the sample channel for subsequent
manipulation within the device. Individual samples are typically
separated by a spacer region of low ionic strength spacer fluid.
These low ionic strength spacer regions have higher voltage drop
over their length than do the higher ionic strength subject
material or test compound regions, thereby driving the
electrokinetic pumping. On either side of the test compound or
subject material region, which is typically in higher ionic
strength solution, are fluid regions referred to as first spacer
regions (also referred to as "guard bands"), that contact the
interface of the subject material regions. These first spacer
regions typically comprise a high ionic strength solution to
prevent migration of the sample elements into the lower ionic
strength fluid regions, or second spacer region, which would result
in electrophoretic bias. The use of such first and second spacer
regions is described in greater detail in U.S. patent application
Ser. No. 08/671,986, filed Jun. 28, 1996, now U.S. Pat. No.
5,779,868, which is incorporated herein by reference.
[0075] Alternatively, the sample channel 112 is optionally
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 material
direction schemes. In either case, it generally is desirable to
separate the discrete sample volumes, or test compounds, with
appropriate spacer regions.
[0076] As shown, the device also includes a detection window or
zone 116 at which a signal from the biochemical system is
optionally 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.
[0077] 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 which direct a light source at the sample are optionally
used, providing a measurement of absorbance or transmissivity of
the sample.
[0078] In alternative aspects, the detection system may comprise
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 are oxidized or
reduced, e.g., O.sub.2, H.sub.2O.sub.2, I.sub.2,
oxidizable/reducible organic compounds, and the like).
[0079] In operation, a flowable first component of a biological
system, e.g., a fluid comprising a receptor or enzyme, is placed in
reservoir 104. This 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 is 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 open or sealable access ports in the planar
cover. Alternatively, these components are optionally added to
their respective reservoirs during manufacture of the device. In
the case of such pre-added components, it is 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 are optionally provided within the
device in lyophilized form. Prior to use, these components are
easily reconstituted by introducing a buffer solution into the
reservoirs. Alternatively, the components are lyophilized with
appropriate buffering salts, whereby simple water addition is all
that is required for reconstitution.
[0080] 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 is 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 optionally includes 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 profile. 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 is measured. This latter signal is
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.
[0081] From sample channel 112, test compounds is periodically or
serially introduced into the main channel 110 and into the stream
of first and second components as fluid regions containing the test
compound, also referred to as the "subject material regions." 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 corresponding to the subject
material region. As noted above, typically, the various different
test compounds to be injected through channel 112 will be separated
by a first and even second spacer fluid regions 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 fluid regions may
also function to reduce any electrophoretic bias that can occur
within the test sample. The use of these spacer regions to drive
the electroosmotic flow of fluids, as well as in the general
elimination of electrophoretic bias within the sample or test
compound or subject material regions is substantially described in
U.S. patent application Ser. No. 08/671,986, filed Jun. 28, 1996,
now U.S. Pat. No. 5,779,868, previously incorporated herein by
reference.
[0082] 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, introduction of a test compound,
i.e., in a subject material region, will produce a momentary but
detectable drop in the level of signal at the detection window
corresponding with that subject material region. 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.
[0083] 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 is 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 is 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 is 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 activated or quenched by the conformational
change to the enzyme upon activation.
[0084] Flowing and direction of fluids within the microscale
fluidic devices is 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.
[0085] Although microfabricated fluid pumping and valving systems
are 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 of 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 U.S. Pat. No. 5,858,195 to Ramsey et al., which is
incorporated herein by reference in its entirety for all
purposes.
[0086] 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.
[0087] Fluid and materials transport and direction is accomplished
through electroosmosis or electrokinesis. In brief, when an
appropriate material, typically comprising a 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
[0088] where v is the solvent velocity, .epsilon. is the dielectric
constant of the fluid, .zeta. 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.
[0089] 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 are optionally 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.
[0090] 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 hydrolysis naturally present at the surface.
Alternatively, surface modifications are optionally 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., U.S. Pat. No. 5,885,470,
which is hereby incorporated herein by reference in its entirety
for all purposes.
[0091] In brief, 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 and salt
concentration. Additionally, substrate materials are also selected
for their inertness to critical components of an analysis or
synthesis to be carried out by the device. Polymeric 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 a transparent polymeric material to facilitate that
detection. Alternatively, transparent windows of, e.g. glass or
quartz, may be incorporated into the device for these detection
elements. Additionally, the polymeric materials may have linear or
branched backbones, and may be crosslinked or non-crosslinked.
Examples of polymeric materials include, e.g., Acrylics, especially
PMMAs (polymethylmethacrylates); exemplar acrylics include e.g.,
Acrylite M-30 or Acrylite L40 available from CYRO Industries,
Rockaway, N.J., or PLEXIGLAS VS UVT available from Autohaas North
America; polycarbonates (e.g., Makrolon CD-2005 available from The
Plastics and Rubber division of Mobay Corporation (Pittsburg, Pa.)
or Bayer Corporation, or LEXAN OQ 1020L or LEXAN OQ 1020, both
available from GE Plastics) polydimethylsiloxanes (PDMS),
polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone,
polycarbonate and the like. Optical, mechanical, thermal,
electrical, and chemical resistance properties for many plastics
are well known (and are generally available from the manufacturer),
or can easily be determined by standard assays.
[0092] As described herein, the electrokinetic fluid control
systems employed in the devices of the present invention generally
utilize a substrate having charged functional groups at its
surface, such as the hydroxyl groups present on glass surfaces. As
described, devices of the present invention can also employ plastic
or other polymeric substrates. In general, these substrate
materials have hydrophobic surfaces. As a result, use of
electrokinetic fluid control systems in devices utilizing polymeric
substrates used in the present invention typically employs
modification of the surfaces of the substrate that are in contact
with fluids.
[0093] Surface modification of polymeric substrates may take on a
variety of different forms. For example, surfaces may be coated
with an appropriately charged material. For example, surfactants
with charged groups and hydrophobic tails are desirable coating
materials. In short, the hydrophobic tails will localize to the
hydrophobic surface of the substrate, thereby presenting the
charged head group at the fluid layer.
[0094] In one embodiment, preparation of a charged surface on the
substrate involves the exposure of the surface to be modified,
e.g., the channels and/or reaction chambers, to an appropriate
solvent which partially dissolves or softens the surface of the
polymeric substrate. A detergent is then contacted with the
partially dissolved surface. The hydrophobic portion of the
detergent molecules will associate with the partially dissolve
polymer. The solvent is then washed from the surface, e.g., using
water, whereupon the polymer surface hardens with the detergent
embedded into the surface, presenting the charged head group to the
fluid interface.
[0095] In alternative aspects, polymeric materials, such as
polydimethylsiloxane, may be modified by plasma irradiation. In
particular, plasma irradiation of PDMS oxidizes the methyl groups,
liberating the carbons and leaving hydroxyl groups in their place,
effectively creating a glass-like surface on the polymeric
material, with its associated hydroxyl functional groups.
[0096] The polymeric substrate may be rigid, semi-rigid, nonrigid
or a combination of rigid and nonrigid elements, depending upon the
particular application for which the device is to be used. In one
embodiment, a substrate is made up of at least one softer, flexible
substrate element and at least one harder, more rigid substrate
element, one of which includes the channels and chambers
manufactured into its surface. Upon mating the two substrates, the
inclusion of the soft element allows formation of an effective
fluid seal for the channels and chambers, obviating the need and
problems associated with gluing or melting more rigid plastic
components together.
[0097] A number of additional elements are added to the polymeric
substrate to provide for the electrokinetic fluid control systems.
These elements may be added either during the substrate formation
process, i.e., during the molding or stamping steps, or they may be
added during a separate, subsequent step. These elements typically
include electrodes for the application of voltages to the various
fluid reservoirs, and in some embodiments, voltage sensors at the
various channel intersections to monitor the voltage applied.
[0098] Electrodes may be incorporated as a portion of the molding
process. In particular, the electrodes may be patterned within the
mold so that upon introduction of the polymeric material into the
mold, the electrodes will be appropriately placed. Alternatively,
the electrodes and other elements may be added after the substrate
is formed, using well known microfabrication methods, e.g.,
sputtering or controlled vapor deposition methods followed by
chemical etching.
[0099] Whether polymeric or other substrates are used, modulating
voltages are 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 screening assay and apparatus.
[0100] FIG. 2A shows a schematic illustration of fluid direction
during a typical assay screen. Specifically, shown is the injection
of a test compound (in a subject material region) 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 spacer regions 121, e.g., low ionic
strength spacer regions, 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 regions 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 subject material regions 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 or subject
material regions 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 subject
material region. This will result in a non-fluorescent, or
detectably less fluorescent region within the flowing stream as it
passes detection window 116, which corresponds to the subject
material region. For example, as shown, a subject material region
including a test compound 128, which is a putative inhibitor of the
enzyme-substrate interaction, shows detectably lower fluorescence
than the surrounding stream. This is indicated by a lack of shading
of subject material region 128.
[0101] 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, produce a momentary increase in the fluorescent signal,
corresponding to the increased enzyme activity toward the
substrate.
[0102] 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 or subject material
regions 150 separated by appropriate spacer fluid regions 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 subject material regions
in 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 or subject material region 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 or
subject material region 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.
[0103] In some embodiments, it is desirable to provide an
additional channel for shunting off or extracting the subject
material region reaction mixture from the running buffer and/or
spacer regions. This may be the case where one wishes to keep the
reaction elements contained within the a discrete fluid region
during the reaction, while allowing these elements to be separated
during a data acquisition stage. As described previously, one can
keep the various elements of the reaction together in the subject
material region that is moving through the reaction channel by
incorporating appropriate spacer fluid regions between samples.
Such spacer fluid regions are generally selected to retain the
samples within their original subject material regions, i.e., not
allowing smearing of the sample into the spacer regions, 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. Thus, it may be desirable to remove those elements which
prevented such separation during the initial portions of the fluid
direction.
[0104] A schematic illustration of one embodiment of a device 500
for performing this sample or subject material shunting or
extraction is shown in FIG. 5. As shown, the subject materials or
test compounds 504 are introduced to the device or chip via the
sample channel 512. Again, these are typically introduced via an
appropriate injection device 506, e.g., a capillary pipettor. The
ionic strength and lengths of the first spacer regions 508 and
second spacer regions 502 are selected such that those samples with
the highest electrophoretic mobility will not migrate through the
first spacer regions 508 into the second spacer regions 502 in the
length of time that it takes the sample to travel down the reaction
channel.
[0105] 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, in
the form of the subject material regions, are flowed along the
reaction channel in the incubation zone 510a. Following this
initial incubation, the test compound/receptor mix is combined with
a labeled 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 first and second spacer regions.
[0106] The isolated subject material regions 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 subject material region 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 subject
material region 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 subject material can then be repeated with each subsequent
subject material region. Within the separation channel, the subject
material region is 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, the subject material is shunted into the
separation channel to place the subject material into a capillary
filled with high ionic strength buffer, i.e., to remove the low
ionic strength spacer regions, thereby allowing separation of the
various sample components outside the confines of the original
subject material region. 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.
[0107] Such modifications have a wide variety of uses, particularly
where it is desirable to separate reaction products following
reaction, e.g., in cleavage reactions, fragmentation reactions, PCR
reactions, and the like.
[0108] C. Serial in Parallel Assay Systems
[0109] 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 are optionally fabricated
connecting to and extending radially outward from a central
reservoir, or are optionally arranged in some other non-parallel
fashion. Additionally, although shown with three transverse
channels, it will be recognized that fewer transverse channels are
used where, e.g., the biochemical system components are predisposed
within the device. Similarly, where desired, more transverse
channels are optionally 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.
[0110] 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.
[0111] In operation, test compounds in discrete subject material
regions, are serially introduced into the device, separated as
described above, and flowed along the transverse sample injection
channel 304 until the separate subject material regions 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 are optionally provided immobilized on individual
beads. In those cases where the test compounds are immobilized on
beads, the parallel channels are 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.
[0112] 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.
[0113] 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 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 are optionally 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.
[0114] 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 is desirable to include electrodes at the termini of
all the various channels. This generally provides for more direct
control, but also grows less manageable as systems grow more
complex. In order to utilize fewer electrodes and thus reduce the
potential complexity, it may often be desirable 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 traveled. Alternatively, resistance of channels are optionally
adjusted by varying the cross-sectional dimensions of the paths,
thereby creating uniform resistance levels regardless of the path
taken.
[0115] 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, labeled
ligand, or the like, or a particle based signal, such as a
precipitate or bead bound signaling 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.
[0116] FIGS. 4A4F, 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
are optionally incorporated into the reaction channels during
manufacture. Again, such components are optionally provided in
liquid form or in lyophilized form for increased shelf life of the
particular screening device.
[0117] 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 are optionally retained upon
an optional particle retaining matrix 344, as described above.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 are
optionally 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, is optionally transported out of the channel
structure through port 362 for identification of the test compound
that had been coupled to it. Such identification are optionally
accomplished outside of the device by directing the bead to a
fraction collector, whereupon the test compounds present on the
beads are optionally 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 are optionally transported to a separate
assay system within the device itself whereupon the identification
is carried out.
[0122] 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.
[0123] 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.
[0124] 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 in separate subject material
regions, are introduced into sample channel 604 using the methods
described previously. For electroosmotic 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 subject
material regions containing the test compounds 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
subject material regions. These restrictions, however, can be
eliminated through the inclusion of altered channel geometries. For
example, in some aspects, the length of a first and second spacer
regions can be accommodated 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.
[0125] Once aligned with the parallel reaction channels, the
sample, or subject material, 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 subject material 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 symmetric 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.
[0126] Following the reaction to be screened, the subject material
region/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 subject material regions/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.
[0127] Although shown with channels which intersect at right
angles, it will be appreciated that other geometries are also
appropriate for serial input parallel reactions. For example, U.S.
application Ser. No. 08,835,101, filed Apr. 4, 1997, describes
advantages to parabolic geometries and channels which vary in width
for control of fluid flow. In brief, fluid flow in electroosmotic
systems is controlled by and therefore related to current flow
between electrodes. Resistance in the fluid channels varies as a
function of path length and width, and thus, different length
channels have different resistances. If this differential in
resistance is not corrected for, it results in the creation of
transverse electrical fields which can inhibit the ability of the
devices to direct fluid flow to particular regions. The current,
and thus the fluid flow, follows the path of least resistance,
e.g., the shortest path. While this problem of transverse
electrical fields is alleviated through the use of separate
electrical systems, i.e., separate electrodes, at the termini of
each and every parallel channel, production of devices
incorporating all of these electrodes, and control systems for
controlling the electrical potential applied at each of these
electrodes can be complex, particularly where one is dealing with
hundreds to thousands of parallel channels in a single small scale
device, e.g., 1-2 cm.sup.2. Accordingly, the present invention
provides microfluidic devices for affecting serial to parallel
conversion, by ensuring that current flow through each of a
plurality of parallel channels is at an appropriate level to ensure
a desired flow pattern through those channels or channel networks.
A number of methods and substrate/channel designs for accomplishing
these goals are appropriate.
[0128] In one example of parabolic geometry for the channels in an
apparatus of the invention, the substrate includes a main channel.
A series of parallel channels terminate in a main channel. The
opposite termini of these parallel channels are connected to
parabolic channels. Electrodes are disposed at the termini of these
parabolic channels. The current flow in each of the parallel
channels is maintained constant or equivalent, by adjusting the
length of the parallel channels, resulting in a parabolic channel
structure connecting each of the parallel channels to its
respective electrodes. The voltage drop within the parabolic
channel between the parallel channels is maintained constant by
adjusting the channel width to accommodate variations in the
channel current resulting from the parallel current paths created
by these parallel channels. The parabolic design of the channels,
in combination with their tapering structures, results in the
resistance along all of the parallel channels being equal,
resulting in an equal fluid flow, regardless of the path chosen.
Generally, determining the dimensions of channels to ensure that
the resistances among the channels are controlled as desired, may
be carried out by well known methods, and generally depends upon
factors such as the make-up of the fluids being moved through the
substrates.
[0129] 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 are optionally used to screen test compounds for the
ability to bind to a given component of a biochemical system.
[0130] II. Microlaboratory System
[0131] 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 FIG. 7.
[0132] 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
first and second spacer 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/first spacer fluids/second spacer 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 members 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 fluid
regions. 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.
[0133] 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 readily be
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 fluid regions can also be
introduced via a spacer solution reservoir.
[0134] III. Fluid Electrode Interface to Prevent Degdation of
Chemical Species in a Microchip
[0135] When pumping fluids or other materials electroosmotically or
electrophoretically through an apparatus of the invention, chemical
species in the fluid can be degraded if high voltages or currents
are applied, or if voltages are applied for a long period of time.
Designs which retard movement of chemical species from the
electrode to a channel entrance or retard the movement of chemical
species to the electrode improve performance of chemical assays by
reducing unwanted degradation of chemical species within the
sample. These designs are particularly preferred in assay systems
where voltages are applied for long periods, e.g., several hours to
several days.
[0136] Electrode designs which reduce degradation of chemical
species in the assays of the invention are illustrated by
consideration of FIG. 12, panels A-G. The designs retard the moving
of chemical species from the electrode to the channel entrance or
retard the movement of chemical species to the electrode improve
performance of chemical assays. FIG. 12A shows a typical electrode
design, in which electrode 1211 is partially submerged in reservoir
1215 fluidly connected to fluid channel 1217.
[0137] In comparison, FIG. 12B utilizes a salt bridge between
electrode with frit 1219 and fluid reservoir 1221 fluidly connected
to fluid channel 1223.
[0138] FIG. 12C reduces degradation of chemical species by
providing electrode 1225 submersed in first fluid reservoir 1227
fluidly connected to second fluid reservoir 1229 by large channel
1231 which limits diffusion, but has a low electroosmotic flow.
[0139] FIG. 12D provides a similar two part reservoir, in which
electrode 1235 is submersed in first fluid reservoir 1237 fluidly
connected to second fluid reservoir 1241 by small channel 1243
which is treated to reduce or eliminate electroosmotic flow.
[0140] FIG. 12E provides another similar two part reservoir, in
which electrode 1245 is submersed in first fluid reservoir 1247
fluidly connected to second fluid reservoir 1251 by channel 1253.
Channel 1253 is filled with a material such as gel, Agar, glass
beads or other matrix material for reducing electroosmotic
flow.
[0141] FIG. 12F provides a variant two part reservoir system, in
which electrode 1255 is submersed in first fluid reservoir 1257
fluidly connected to second fluid reservoir 1259 by channel 1261.
The fluid level in second fluid reservoir 1259 is higher than the
fluid level in first fluid reservoir 1257, which forces fluid
towards electrode 1255.
[0142] FIG. 12G provides a second variant two part reservoir, in
which electrode 1265 is submersed in first fluid reservoir 1267
fluidly connected to second fluid reservoir 1269 by channel 1271.
The diameter on first fluid reservoir 1267 is small enough that
capillary forces draw fluid into first fluid reservoir 1267.
[0143] Modifications can be made to the method and apparatus as
hereinbefore described without departing from the spirit or scope
of the invention as claimed, and the invention can be put to a
number of different uses, including:
[0144] The use of a microfluidic system containing at least a first
substrate having a first channel and a second channel intersecting
said first channel, at least one of said channels having at least
one cross-sectional dimension in a range from 0.1 to 500 .mu.m, in
order to test the effect of each of a plurality of test compounds
on a biochemical system.
[0145] The use of a microfluidic system as hereinbefore described,
wherein said biochemical system flows through one of said channels
substantially continuously, enabling sequential testing of said
plurality of test compounds.
[0146] The use of a microfluidic system as hereinbefore described,
wherein the provision of a plurality of reaction channels in said
first substrate enables parallel exposure of a plurality of test
compounds to at least one biochemical system.
[0147] The use of a microfluidic system as hereinbefore described,
wherein each test compound is physically isolated from adjacent
test compounds.
[0148] The use of a substrate carrying intersecting channels in
screening test materials for effect on a biochemical system by
flowing said test materials and biochemical system together using
said channels.
[0149] The use of a substrate as hereinbefore described, wherein at
least one of said channels has at least one cross-sectional
dimension of range 0.1 to 500 .mu.m.
[0150] An assay utilizing a use of any one of the microfluidic
systems or substrates hereinbefore described.
[0151] The invention provides, inter alia, 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. Apparatus as
hereinbefore described, having 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 and an assay
apparatus including an apparatus as hereinbefore described are also
provided.
EXAMPLES
[0152] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which can be changed
or modified to yield essentially similar results.
Example 1
[0153] Enzyme Inhibitor Screen
[0154] The efficacy of performing an enzyme inhibition assay screen
was demonstrated in a planar chip format. A 6-port planar chip was
employed having the layout shown in FIG. 8. The numbers adjacent
the channels represent the lengths of each channel in millimeters.
Two voltage states were applied to the ports of the chip. The first
state (State 1) resulted in flowing of enzyme with buffer from the
top buffer well into the main channel. The second voltage state
(State 2) resulted in the interruption of the flow of buffer from
the top well, and the introduction of inhibitor from the inhibitor
well, into the main channel along with the enzyme. A control
experiment was also run in which buffer was placed into the
inhibitor well.
[0155] Applied voltages at each port for each of the two applied
voltage states were as follows:
1 State 1 State 2 Top Buffer Well (I) 1831 1498 Inhibitor Well(II)
1498 1900 Enzyme Well (III) 1891 1891 Substrate Well (IV) 1442 1442
Bottom Buffer Well (V) 1442 1442 Detect./Waste Well (VI) 0 0
[0156] To demonstrate the efficacy of the system, an assay was
designed to screen inhibitors of .beta.-galactosidase using the
following enzyme/substrate/inhibitor reagents:
[0157] Enzyme: .beta.-Galactosidase (180 U/ml in 50 mM Tris/300
.mu.g/ml BSA
[0158] Substrate: Fluorescein-digalactoside (FDG) 400 .mu.M
[0159] Inhibitor: IPTG, 200 mM
[0160] Buffer: 20 mM Tris, pH 8.5
[0161] Enzyme and substrate were continually pumped through the
main channel from their respective ports under both voltage states.
Inhibitor or Buffer were delivered into the main channel
alternately from their respective wells by alternating between
voltage state 1 and voltage state 2. When no inhibitor was present
at the detection end of the main channel, a base line level of
fluorescent product was produced. Upon introduction of inhibitor,
the fluorescent signal was greatly reduced, indicating inhibition
of the enzyme/substrate interaction. Fluorescent data obtained from
the alternating delivery of inhibitor and buffer into the main
channel is shown in FIG. 9A. FIG. 9B a superposition of the two
data segments from FIG. 9A, directly comparing the inhibitor data
with control (buffer) data. The control shows only a minor
fluctuation in the fluorescent signal that apparently resulted from
a dilution of the enzyme substrate mixture, whereas the inhibitor
screen shows a substantial reduction in the fluorescent signal,
indicating clear inhibition.
Example 2
[0162] Screening of Multiple Test Compounds
[0163] An assay screen is performed to identify inhibitors of an
enzymatic reaction. A schematic of the chip to be used is shown in
FIG. 10. 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.
[0164] Subject material regions containing test compounds in 150 mM
NaCl are introduced into the sample channel separated by first
spacer regions of 150 mM NaCl and second spacer regions of 5 mM
borate buffer. Once introduced into the sample channel shown, the
subject material region 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 interruptions, 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 subject material region containing the test compounds
then requires 48 seconds to travel the length of the reaction zone
and past the fluorescence detector. A schematic of timing for
subject material region/spacer region loading is shown in FIG. 11.
The top panel shows the subject material/first spacer region/second
spacer region 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)
first spacer fluid ("A"), moving the pipettor to the sample or
subject material ("B"), sipping the sample or subject material
("C"), moving the pipettor to the high salt first spacer fluid
("D") sipping the first spacer fluid ("E"), moving the pipettor to
the low salt (LS) or second spacer fluid ("F"), sipping the second
spacer fluid ("G") and returning to the first spacer fluid ("H").
The process is then repeated for each additional test compound.
[0165] 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 similar to that
shown in FIGS. 9A and 9B, 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.
[0166] While tie 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.
* * * * *