High throughput screening assay systems in microscale fluidic devices

What is claimed is:

1. An apparatus for screening test compounds for an effect on a flowing biochemical system, comprising:

a substrate having at least one surface;

at least two intersecting channels fabricated into said surface of said substrate, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range from about 0.1 to about 500 .mu.m;

a source of a plurality of different test compounds fluidly connected to a first of said at least two intersecting channels;

a source of at least one component of said biochemical system fluidly connected to a second of said at least two intersecting channels;

a fluid direction system for flowing said at least one component within said second of said at least two intersecting channels and for flowing said different test compounds from said first to said second of said at least two intersecting channels;

a cover mated with said surface; and

a detection zone in said second channel for detecting an effect of said test compound on said flowing biochemical system.

2. The apparatus of claim 1, wherein said fluid direction system generates a continuous flow of said at least one component along said second of said at least two intersecting channels, and periodically injects a test compound from said first channel into said second channel.

3. The apparatus of claim 1, further comprising a source of a second component of said biochemical system, and a third channel fabricated into said surface, said third channel fluidly connecting at least one of said at least two intersecting channels with said source of said second component of said biochemical system.

4. The apparatus of claim 3, wherein said fluid direction system generates a continuous flow of a mixture of said at least one component and said second component along said second of said at least two intersecting channels, and periodically ejects a test compound from said first channel into said second channel.

5. The apparatus of claim 1, wherein said fluid direction system continuously flows said plurality of different test compounds from said first into said second of said at least two intersecting channels, each of said plurality of different test compounds being separated by a fluid spacer.

6. The apparatus of claim 1, wherein said fluid direction system comprises:

at least tree electrodes, each electrode being in electrical contact with said at least two intersecting channels on a different side of an intersection formed by said at least two intersecting channels; and

a control system for concomitantly applying a variable voltage at each of said electrodes, whereby movement of said test compounds or said at least one component in said at least two intersecting channels may be controlled.

7. The apparatus of claim 1, wherein said detection system includes a detection window in said second channel.

8. The apparatus of claim 7, wherein said detection system is a fluorescent detection system.

9. The apparatus of claim 1, wherein said substrate is planar.

10. The apparatus of claim 1, wherein said substrate comprises etched glass.

11. The apparatus of claim 1, wherein said substrate comprises etched silicon.

12. The apparatus of claim 1, further comprising an insulating layer disposed over said etched silicon substrate.

13. The apparatus of claim 1, wherein said substrate is a molded polymer.

14. The apparatus of claim 1, wherein said at least one component of a biochemical system comprises an enzyme, and an enzyme substrate which produces a detectable signal when reacted with said enzyme.

15. The apparatus of claim 14, wherein said enzyme substrate is selected from the group consisting of chromogenic and fluorogenic enzyme substrates.

16. The apparatus of claim 1, wherein said at least one component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.

17. The apparatus of claim 1, wherein said at least one component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.

18. An apparatus for detecting an effect of a test compound on a flowing biochemical system, comprising:

a substrate having at least one surface;

a plurality of reaction channels fabricated into said surface;

at least two transverse channels fabricated into said surface, each of said plurality of reaction channels being fluidly connected to a first of said at least two transverse channels at a first point in said reaction channels, and fluidly connected to a second of said at least two transverse channels at a second point in said reaction channels, said at least two transverse channels and said plurality of reaction channels each having at least one cross-sectional dimension in the range from about 0.1 to about 500 .mu.m;

a source of at least one component of said biochemical system, said source of at least one component of said biochemical system being fluidly connected to each of said plurality of reaction channels;

a source of test compounds fluidly connected to said first of said at least two transverse channels;

a fluid direction system for controlling movement of said test compound and said at least one component within said at least two transverse channels and said plurality of reaction channels;

a cover mated with said surface; and

a detection system for detecting an effect of said test compound on said flowing biochemical system.

19. The apparatus of claim 18, wherein said fluid control system comprises:

a plurality of individual electrodes, each in electrical contact with each terminus of said at least two transverse channels; and

a control system for concomitantly applying a variable voltage at each of said electrodes, whereby movement of said test compounds or said at least first component in said at least two transverse channels and said plurality of reaction channels may be controlled.

20. The apparatus of claim 18, wherein each of said plurality of reaction channels comprises a bead resting well at said first point in said plurality of reaction channels.

21. The apparatus of claim 18, wherein said source of at least one component of a biochemical system is fluidly connected to said plurality of reaction channels by a third transverse channel, said third transverse channel having at least one cross sectional dimension in a range of from 0.1 to 500 .mu.m and being fluidly connected to each of said plurality of reaction channels at a third point in said reaction channels.

22. The apparatus of claim 21, wherein said third point in said reaction channels is intermediate to said first and second points in said reaction channels.

23. The apparatus of claim 22, further comprising a particle retention zone in each of said plurality of reaction channels, between said third and said second points in said plurality of reaction channels.

24. The apparatus of claim 23, wherein said particle retention zone comprises a particle retention matrix.

25. The apparatus of claim 23, wherein said particle retention zone comprises a microstructural filter.

26. The apparatus of claim 18, wherein said plurality of reaction channels comprises a plurality of parallel reaction channels fabricated into said surface of said substrate and said at least two transverse channels are connected at opposite ends of each of said parallel reaction channels.

27. The apparatus of claim 18, wherein said at least two transverse channels are fabricated on said surface of said substrate in inner and outer concentric channels, respectively, and said plurality of reaction channels extend radially from said inner concentric channel to said outer concentric channel.

28. The apparatus of claim 27, wherein said detection system comprises a detection window in said second channel.

29. The apparatus of claim 28, wherein said detection system is a fluorescent detection system.

30. The apparatus of claim 18, wherein said substrate is planar.

31. The apparatus of claim 18, wherein said substrate comprises etched glass.

32. The apparatus of claim 18, wherein said substrate comprises etched silicon.

33. The apparatus of claim 18, further comprising an insulating layer disposed over said etched silicon substrate.

34. The apparatus of claim 18, wherein said substrate is a molded polymer.

35. The apparatus of claim 18, wherein said at least one component of a biochemical system comprises an enzyme, and an enzyme substrate which produces a detectable signal when reacted with said enzyme.

36. The apparatus of claim 35, wherein said enzyme substrate is selected from the group consisting of chromogenic and fluorogenic substrates.

37. The apparatus of claim 18, wherein said at least one component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.

38. The apparatus of claim 18, wherein said at least one component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.

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BACKGROUND OF THE INVENTION

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.

Modern drug discovery is limited by the throughput of the assays that are used to screen compounds that possess these described effects. In particular, screening of the maximum number of different compounds necessitates reducing the time and labor requirements associated with each screen.

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. Engl. 31: 367-383, incorporated herein by reference. This may be ascribed to the availability of a large and structurally diverse range of amino acid monomers, a relatively generic, high-yielding solid phase coupling chemistry and the synergy with biological approaches for generating recombinant peptide libraries. Moreover, the potent and specific biological activities of many low molecular weight peptides make these molecules attractive starting points for therapeutic drug discovery. See Hirschmann, 1991, Angew. Chem. Int. Ed. Engl. 30: 1278-1301, and Wiley & Rich, 1993, Med. Res. Rev. 13: 327-384, each of which is incorporated herein by reference. Unfavorable pharmacodynamic properties such as poor oral bioavailability and rapid clearance in vivo have limited the more widespread development of peptidic compounds as drugs however. This realization has recently inspired workers to extend the concepts of combinatorial organic synthesis beyond peptide chemistry to create libraries of known pharmacophores like benzodiazepines (see Bunin & Ellman, 1992, J. Amer. Chem. Soc. 114: 10997-10998, incorporated herein by reference) as well as polymeric molecules such as oligomeric N-substituted glycines ("peptoids") and oligocarbamates. See Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371; Zuckermann et al., 1992, J. Amer. Chem. Soc. 114: 10646-10647; and Cho et al., 1993, Science 261:1303-1305, each of which is incorporated herein by reference.

In similar developments, much as modern combinatorial chemistry has resulted in a dramatic increase in the number of test compounds that may be screened, human genome research has also uncovered large numbers of new target molecules against which the efficacy of test compounds may be screened.

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., evaporation. Additionally, fluid handling equipment and methods have typically been unable to handle these volume ranges with any acceptable level of accuracy due in part to surface tension effects in such small volumes.

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.

The present invention meets these and a variety of other needs. In particular, the present invention provides novel methods and apparatuses for performing screening assays which address and provide meaningful solutions to these problems.

SUMMARY OF THE INVENTION

The present invention generally provides methods of screening a plurality of test compounds for an effect on a biochemical system. These methods typically utilize microfabricated substrates which have at least a first surface, and at least two intersecting channels fabricated into that first surface. At least one of the intersecting channels will have at least one cross-sectional dimension in a range from 0.1 to 500 .mu.m. The methods involve flowing a first component of a biochemical system in a first of the at least two intersecting channels. At least a first test compound is flowed from a second channel into the first channel whereby the test compound contacts the first component of the biochemical system. An effect of the test compound on the biochemical system is then detected.

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.

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.

The present invention also provides apparatuses for practicing the above methods. In one aspect, the present invention provides an apparatus for screening test compounds for an effect on a biochemical system. The device comprises a substrate having at least one surface with at least two intersecting channels fabricated into the surface. The at least two intersecting channels have at least one cross-sectional dimension in the range from about 0.1 to about 500 .mu.m. The device also comprises a source of different test compounds fluidly connected to a first of the at least two intersecting channels, and a source of at least one component of the biochemical system fluidly connected to a second of the at least two intersecting channels. Also included are fluid direction systems for flowing the at least one component within the intersecting channels, and for introducing the different test compounds from the first to the second of the intersecting channels. The apparatus also comprises a detection zone in the second channel for detecting an effect of said test compound on said biochemical system.

In preferred aspects, the apparatus of the invention includes a fluid direction system which comprises at least three electrodes, each electrode being in electrical contact with the at least two intersecting channels on a different side of an intersection formed by the at least two intersecting channels. The fluid direction system also includes a control system for concomitantly applying a variable voltage at each of the electrodes, whereby movement of the test compounds or the at least first component in the at least two intersecting channels may be controlled.

In another aspect, the present invention provides an apparatus for detecting an effect of a test compound on a biochemical system, comprising a substrate having at least one surface with a plurality of reaction channels fabricated into the surface. The apparatus also has at least two transverse channels fabricated into the surface, wherein each of the plurality of reaction channels is fluidly connected to a first of the at least two transverse channels at a first point in each of the reaction channels, and fluidly connected to a second transverse channel at a second point in each of the reaction channels. The apparatus further includes a source of at least one component of the biochemical system fluidly connected to each of the reaction channels, a source of test compounds fluidly connected to the first of the transverse channels, and a fluid direction system for controlling movement of the test compound and the first component within the transverse channels and the plurality reaction channels. As above, the apparatuses also include a detection zone in the second transverse channel for detecting an effect of the test compound on the biochemical system.

BRIEF DESCRIPTION OF THE DRAWINGS

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.

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.

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.

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.

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.

FIG. 6A shows a schematic illustration of a serial input parallel reaction device for use with fluid based test compounds.

FIGS. 6B and 6C show a schematic illustration of fluid flow patterns within the device shown in FIG. 6A.

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.

FIG. 8 is a schematic illustration of a chip layout used for a continuous-flow assay screening system.

FIG. 9 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.

FIG. 10 illustrates the operating parameters of a fluid flow system on a small chip device for performing enzyme inhibitor screening.

FIG. 11 shows a schematic illustration of timing for sample/spacer loading in a microfluidic device channel.

DESCRIPTION OF THE PREFERRED EMBODIMENT

I. General 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.

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, signalling and other reactions. Further, biochemical systems, as defined herein, will also include model systems which are mimetic of a particular biochemical interaction. Examples of biochemical systems of particular interest in practicing the present invention include, e.g., receptor-ligand interactions, enzyme-substrate interactions, cellular signaling pathways, transport reactions involving model barrier systems (e.g., cells or membrane fractions) for bioavailability screening, and a variety of other general systems. Cellular or organismal viability or activity may also be screened using the methods and apparatuses of the present invention, i.e., in toxicology studies.

In order to provide methods and devices for screening compounds for effects on biochemical systems, the present invention generally incorporates model in vitro systems which mimic a given biochemical system in vivo for which effector compounds are desired. The range of systems against which compounds can be screened and for which effector compounds are desired, is extensive. For example, compounds may be screened for effects in blocking, slowing or otherwise inhibiting key events associated with biochemical systems whose effect is undesirable. For example, test compounds may be screened for their ability to block systems that are responsible, at least in part, for the onset of disease or for the occurrence of particular symptoms of diseases, including, e.g., hereditary diseases, cancer, bacterial or viral infections and the like. Compounds which show promising results in these screening assay methods can then be subjected to further testing to identify effective pharmacological agents for the treatment of disease or symptoms of a disease.

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.

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.

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.

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 sam