KR20000022329A - High throughput screening assay systems in microscale fluidic devices - Google Patents

Abstract

PURPOSE: Micro-fluidic devices and methods are provided to screen large numbers of different compounds for their effects on a variety or chemical and biochemical systems. CONSTITUTION: Two series channels(110,112) and channel(114) for reagent are equipped. Each channel has at least one cross-sectional dimension in the range from about 0.1 to about 500 micrometer. Containers(104,106,108) are located at the end of channels(110,114). A sample channel is used to test compounds into the device. Biochemical signals from the system are monitored at detection window or detection area(116). The detection window observes and examines visually and optically.

Description

High Throughput Screening Assay System in Unscaled Fluid Devices

There is a long need in the art for the ability to rapidly assay compounds for their effects on various biological processes. For example, enzymaticians have long searched for better substrates, better inhibitors or better catalysts for enzymatic reactions. Similarly, the pharmaceutical industry has attracted interest in identifying compounds that can inhibit, reduce or even enhance interactions between biological molecules. In particular, for biological systems, the interaction of a receptor with its ligand can often produce one of the deleterious or beneficial effects on the system, either directly or by some downstream event, resulting in patients in need of treatment. Thus, researchers have long investigated the compounds or mixtures of compounds that can reduce, inhibit or even enhance this interaction. Similarly, the ability to quickly process a sample for the detection of biological molecules involved in diagnostic or forensic analysis is of significant value, for example in diagnostic medicine, archeology, anthropology and modern criminal investigations.

Modern drug discovery is limited by the throughput of the assays used to screen for compounds having these described effects. In particular, the screening of the maximum number of different compounds needs to reduce the time and effort required associated with each screening.

High throughput screening of collections of chemical synthetic molecules and natural products (eg, fermentation broths) plays a pivotal role in investigating lead compounds for the development of new agents. The marked rise in interest in combinatorial chemistry and related technologies for generating and evaluating molecular diversity represents a significant milestone in the development of this model of drug discovery. Pavia et al., 1993, Bioorg. Med. Chem. Lett. 3: 387-396, incorporated herein by reference]. To date, peptide chemistry has been a fundamental means for investigating the usefulness of combinatorial methods in ligand identification. Jung & Beck-Sickinger, 1992, Angew, Chem. Int. Ed. Engl. 31: 367-383, incorporated herein by reference. This may be due to the utility of large, structurally diverse ranges of amino acid monomers, relatively general high yield solid phase coupling chemistry and synergy with biological methods for generating recombinant peptide libraries. Moreover, the effective and specific biological activity of many low molecular weight peptides makes these molecules an attractive starting point for therapeutic drug discovery. 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. However, the wider development of peptide compounds as medicaments has been limited due to undesirable pharmacokinetic properties such as poor oral bioavailability and rapid in vivo clearance. This realization has recently led experimenters to extend the concept of combinatorial organic synthesis beyond peptide chemistry to benzodiazepines (Bunin & Ellman, 1992, J. Amer. Chem. Soc. 114: 10997-10998, herein incorporated by reference). Libraries of known drug specific molecule groups, such as those incorporated by reference, as well as oligomeric N-substituted glycine ("peptoids") and oligocarbamate, have been encouraged to produce ; 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, many new target molecules (eg, proteins and Genes and gene products such as RNA) have been found.

Despite the improvements achieved using parallel screening methods such as robotics and high throughput detection systems and other technological advances, current screening methods still have a number of related problems. For example, screening multiple samples using existing parallel screening methods requires large space requirements to accommodate samples and equipment, such as robotics, the high costs associated with this equipment, and the large amount of reagents required to perform the assay. This is necessary. In addition, in many cases, the reaction volume should be low because of the small amount of test compounds available. These small volumes add to errors associated with fluid handling and measurement, such as errors due to evaporation, formulations, and the like. In addition, fluid handling equipment and methods have typically been unable to handle these volume ranges with acceptable levels of accuracy due in part to the effect of surface tension at such small volumes.

Developing a system to solve these problems requires consideration of aspects of various testing methods. These aspects include target and compound sources, test compound and target handling, specific assay requirements, and data acquisition, clearance storage, and analysis. In particular, there is a need for high throughput screening methods and related equipment and devices that can perform repeat and accurate assay screening and can be operated in very low volumes.

The present invention fulfills these needs and various other needs. In particular, the present invention provides novel methods and apparatus for performing screening assays that present important solutions to these problems.

Summary of the Invention

The present invention provides a method for screening a plurality of test compounds for effects on biochemical systems. Typically these methods utilize a microfabricated substrate having at least one first surface and at least two cross channels within the first surface. One or more crossover channels will have one or more cross-sectional dimensions of 0.1-500 μm. The method of the present invention comprises flowing the first component of the biochemical system into a first channel of two or more cross channels. At least the first test compound flows from the second channel to the first channel, thereby contacting the test compound with the first component of the biochemical system. Thereafter, the effect of the test compound on the biochemical system is detected.

In a related aspect, the methods of the present invention include those that continuously flow a first component of a biochemical system into two or more crossover channels. Different test compounds are introduced periodically from the second channel into the first channel. Thereafter, the effect of the test compound on the biochemical system is detected (if effective).

In another aspect, the method of the present invention utilizes a substrate having a first surface having a plurality of reaction channels formed therein. Each of the plurality of reaction channels is also fluidly connected to two or more transverse channels formed in the surface. At least a first component of the biochemical system is introduced into the plurality of reaction channels, and the plurality of different test compounds are flowed through one or more of the two or more transverse channels. In addition, each different test compound is introduced into the transverse channel in discrete volumes. After each of a number of different test compounds is delivered in separate reaction channels, the effect of each test compound on the biochemical system is detected.

The invention also provides an apparatus for carrying out the method. In one aspect, the invention provides an apparatus for screening test compounds for effects on biochemical systems. The apparatus of the present invention includes a substrate having one or more surfaces with two or more crossing channels formed therein. The two or more crossover channels have one or more cross sectional dimensions of about 0.1 to about 500 μm. In addition, the device of the present invention provides a source of different test compounds fluidly connected to a first channel of at least two cross channels and a source of one or more components of a biochemical system fluidly connected to a second channel of at least two cross channels. Include. Fluid delivery systems for flowing one or more components into the cross channel and fluid delivery systems for introducing different test compounds from the first channel of the cross channel to the second channel. In addition, the device of the present invention optionally includes a detection region in the second channel to detect the effect of the test compound on the biochemical system.

In a preferred aspect, the device of the present invention comprises a fluid delivery system comprising at least three electrodes, each electrode being in electrical contact with at least two crossing channels at different sides of the intersection formed by the at least two crossing channels. The fluid delivery system also includes a control system for simultaneously applying a variable voltage to each electrode, whereby the movement of at least the first component in the test compound or two or more cross channels is controlled.

In another aspect, the invention provides an apparatus for detecting the effect of a test compound on a biochemical system, comprising a substrate having one or more surfaces with a plurality of reaction channels formed therein. In addition, the device of the present invention has two or more transverse channels formed in the surface, each of the plurality of reaction channels being fluidly connected to a first channel of the two or more transverse channels at a first point in each reaction channel, It is fluidly connected to the second transverse channel at a second point in each reaction channel. The apparatus of the present invention provides a source of test compound fluidly connected to each reaction channel, a source of test compound fluidly connected to a first channel of the transverse channel, and a test compound and a first within the transverse channel and the plurality of reaction channels. It further includes a fluid delivery system for controlling the movement of the component. As noted above, the device of the present invention also optionally comprises a detection zone in the second transverse channel to detect the effect of the test compound on the biochemical system.

The present invention relates to apparatus and assay systems for detecting molecular interactions. The apparatus of the present invention includes a substrate having one or more cross channels and an electroosmotic fluid transfer component, or other component for moving fluid within a channel on the substrate.

1 schematically depicts one embodiment of a microexperimental screening assay system of the present invention that may be used to implement a continuous flow assay system.

2A and 2B are schematic illustrations of the apparatus shown in FIG. 1 operating in an alternate calibration system. FIG. 2A shows a system used to screen effectors of enzyme-substrate interactions, and FIG. 2B shows a device used to screen for receptor-ligand interactions.

FIG. 3 schematically illustrates a “serial injection parallel reaction” microassay assay system wherein a compound to be screened is introduced in series in a device, but then screened in parallel orientation in the device.

4A-4F schematically illustrate the operation of the apparatus shown in FIG. 3 in the screening of multiple bead based test compounds.

FIG. 5 schematically illustrates a continuous flow assay device that includes a sample shunt for performing a separation step after a long incubation.

6A schematically illustrates a series of injection parallel reaction apparatus for use with a fluid base test compound.

FIG. 7 schematically illustrates one embodiment of a complete assay system using multiple microexperimental devices labeled “LabChips ^™ ” to screen test compounds.

8 schematically illustrates the chip arrangement used in a continuous flow assay screening system.

9 shows fluorescent data from a continuous flow assay screen. 9A shows fluorescence data from test screens in which a known inhibitor (IPTG) was periodically introduced into the β-galactosidase assay system in chip format, and FIG. 9B directly compares inhibitor data with control (buffer) data. The superposition of the two data portions from FIG. 9A is shown.

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

FIG. 11 schematically illustrates timing for sample / spacer loading in microfluidic device channels.

Panels A to G of FIG. 12 schematically illustrate electrodes used in the apparatus of the present invention.

1. Field of Application of the Invention

The present invention provides novel microexperimental systems and methods useful for performing high throughput screening assays. In particular, the present invention provides methods of using such devices for screening microfluidic devices and many different compounds for their effects on various chemicals, preferably biochemical systems.

As used herein, the term “biochemical system” generally refers to chemical interactions involving molecules of the type commonly found in living organisms. Such interactions include the full range of catabolic and assimilation reactions occurring in the survival system, including enzyme binding, signaling and other reactions. In addition, the biochemical systems defined herein also include model systems that mimic certain biochemical interactions. Examples of biochemical systems of particular interest in practicing the present invention include model barrier systems (eg, cells or membranes) for receptor-ligand interactions, enzyme-substrate interactions, cell signaling pathways, bioavailability screenings. Transport reactions, and various other general systems. Cell or organism viability or activity can also be screened using the methods and devices of the invention, for example in toxicology experiments. Biological materials to be assayed include cells, cell fractions (membrane, cytosol preparations, etc.), agonists and antagonists of cell membrane receptors (eg, cell receptor-ligand interactions, eg transferrin, c-kit, viral receptor ligands (eg For example, CD4-HIV), cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, cadherein family, intergrin family, selectin family and the like; see 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 venom, viral epitopes, hormones (e.g., Mediates the effects of various small ligands, including opiates, steroids, etc., intracellular receptors (eg, steroids, thyroid hormones, retinoids, and vitamin D). See Evans (1988) Science, 240: 889-895; Ham and Parker (1989) Curr. Opin. Cell Biol., 1: 503-511; Burnstein et al, (1989), Ann, Rev. Physiol., 51: 683-699; Truss and Beato (1993) Endocr. Rev., 14: 459-479), peptides, retro-inverso peptides, α-, β-, or ω- amino acids (D- or L-) Polymers, enzymes, enzyme substrates, cofactors, agents, lectins, sugars, nucleic acids (both linear and spherical polymer sequences), oligosaccharides, proteins, phospholipids and antibodies. Synthetic polymers, such as heteropolymers, in which known agents are covalently bonded to one of the above listed materials, for example polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, poly Mid and polyacetates are also assayed. In addition, other polymers are assayed using the systems described herein, which will be apparent to those skilled in the art having read this specification. Those skilled in the art will generally be familiar with the biological literature. For an overview of biological systems, see the following references: Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (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 Geene 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, CA; Watson et al. (1992) Recombinant DNA Second Edition Scientific American Books, NY; Alberts et al., (1990) 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 Mannual of Diagnosis and Therapy, Merck & Co., Rahway, NJ; 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,; from the manufacturer of biological reagents and laboratory equipment The product information of also provides useful information for assaying biological systems, such as, for example, SIGMA chemical company (Saint Louis, MO), R & D Systems (Minneapolis, MN), Pharmacia Pharmacia LKB Biotechnology (Piscataway, NJ), CloneTech Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., Gibco BRL Life Technologies, Inc. (Gaithersberg, MD) , Flu Fluka Chemica-Biochemica Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA) and Applied Biosystems (Foster City, CA) As well as many other commercial sources known to those skilled in the art.

To provide a method and apparatus for screening the effects of a compound on a biochemical system, the present invention generally includes an in vitro model system in which an effector compound is desired to mimic a given in vivo biochemical system. Compounds can be screened and the range of systems in which effector compounds are preferred is wide. For example, compounds are optionally screened for the effect of blocking, delaying or inhibiting important events associated with biochemical systems in which the effect is undesirable. For example, test compounds are screened for the ability to block systems that are at least partially responsible for the development of diseases or the development of certain symptoms, including genetic diseases, cancer, bacterial or viral infections, and the like. Subsequently, further tests may be conducted on the compounds that show promising results in these screening assay methods to identify agents that are effective in treating the disease or symptom of the disease.

Alternatively, the compounds can be screened for their ability to stimulate, augment or induce biochemical systems that have a function deemed desirable, for example, to treat existing defects in a patient.

Once the model system is selected, a series of test compounds can then be applied to these model systems. By identifying these test compounds that have an effect on specific biochemical systems in vitro, potential effectors of such systems in vivo can be identified.

In their simplest form, the biochemical system model used in the methods and apparatus of the present invention is a test compound for the interaction between two components of the biochemical system, for example receptor-ligand interaction, enzyme-substrate interaction, etc. Will be screened for the effect. In this form, the biochemical system model will typically include two normal interacting components of the system to which the effector has been irradiated, for example a receptor and its ligand or enzyme and its substrate.

Determining whether a test compound has an effect on this interaction includes contacting the system with the test compound and assaying for the function of the system, eg, receptor-ligand binding or substrate turnover. Thereafter, the assay function is compared with the same reaction in the control, for example in the absence of test compound or in the presence of known effectors. Typically, this assay involves the determination of the parameters of a biochemical system. "Parameter of a biochemical system" means some measurable evidence of the function of the system, such as the presence or absence of a labeling group or a change in molecular weight (eg, in a binding reaction, transport screen), the presence of a reaction product or substrate Absence (in substrate turnover measurements) or change in electrophoretic mobility (typically detected by a change in elution time of the labeling compound).

Although described for two-component biochemical systems, the methods and apparatus of the present invention can also be used to screen effectors of even more complex systems, and the output or end product of the system is known and several levels, for example enzymes. Assays in pathways, cell signaling pathways, and the like. Alternatively, the methods and devices described herein may be specifically directed to compounds that optionally interact with a single component of a biochemical system, such as certain biochemical compounds such as receptors, ligands, enzymes, nucleic acids, structural macromolecules, and the like. Used to screen for compound to bind.

In addition, biochemical system models can be embodied in the entire cell line. For example, when screening test compounds for effects on cellular responses, whole cells are optionally used. In addition, modified cell systems can be used in the screening systems included herein. For example, a chimeric reporter system is optionally used as an indicator of the effect of a test compound on a particular biochemical system. Chimeric reporter systems typically include a heterogeneous reporter system that is integrated into a signaling pathway that signals the binding of a receptor to its ligand. For example, the receptor is melted with a heterologous protein, for example an enzyme that can be easily assayed for activity. Activation of the receptor by ligand binding then activates the heterologous protein that is subsequently detected. As such, the surrogate reporter system provides an assay for receptor / ligand binding by generating a result or signal that can be easily detected. Examples of such chimeric receptor systems have been described in the prior art.

In addition, biological barriers are optionally included at the point of screening for bioavailability, eg transport. The term “biological barrier” generally relates to a cell or membrane layer, or a synthetic model thereof, in a biological system. Examples of biological barriers include epithelial and endothelial layers such as the vascular endothelial layer and the like.

Biological responses are often initiated and / or modulated by the binding of a receptor to its ligand. For example, interactions with growth factors, such as EGF, FDF, PDGF, and their receptors may, for example, be used for cell proliferation and fractionation, activation of mediatoring enzymes, stimulation of messenger exchange, modification of ion flux, activation of enzymes, cellular It stimulates a variety of biological responses, including changes in shape and changes in the level of genetic expression. Thus, modulating the interaction of a receptor with its ligand can modulate the biological response caused by this interaction.

Thus, in one aspect, the present invention will be useful in screening for compounds that affect the interaction between receptor molecules and their ligands. As used herein, the term “receptor” generally relates to a component of one of a pair of compounds that specifically recognizes and binds to each other. The other of the pair of components is referred to as "ligand". As such, receptor / ligand pairs may include universal protein receptors, generally membrane related receptors, and their natural ligands, such as another protein or small molecule. Receptor / ligand pairs may also include antibody / antigen binding pairs, complementary nucleic acids, nucleic acid related proteins, and nucleic acid ligands thereof. Many biochemical compounds that are specifically bound are well known in the art and can be used in the practice of the present invention.

Typically, methods for screening effectors of receptor / ligand interactions include incubating receptor / ligand binding pairs in the presence of test compounds. After incubation the binding level of the receptor / ligand pairs is compared with negative and / or positive controls. If normal binding appears to be reduced, the test compound is determined to be an inhibitor of receptor / ligand binding. If binding is seen to be increased, the test compound is determined to be an enhancer or inducer of interaction.

For valuable efficiencies, screening assays commonly performed on multi-well reaction plates, for example multi-well pipetteplates, where concurrency is considered, are comparable to the screening of multiple test compounds.

Similar and overlapping biochemical systems induce interactions between enzymes and their substrates. As used herein, the term “enzyme” generally relates to a protein that acts as a catalyst to induce chemical changes in other compounds or “substrates”.

Typically, effectors of enzymatic activity to the substrate of the enzyme are screened by contacting the enzyme with the substrate in the presence and absence of the compound to be screened and under optimal conditions for detecting changes in the enzyme activity. After the reaction set time, the mixture is assayed for the presence of the reaction product or the reduction of the amount of substrate. The amount of catalyzed substrate is compared to the control, ie, the enzyme contacted with the substrate in the absence of the test compound or in the presence of a known effector. As above, compounds that reduce the activity of the enzyme on the substrate of the enzyme are referred to as "inhibitors", while compounds that make the activity prominent are referred to as "derivatives".

In general, various screening methods encompassed by the present invention include the continuous introduction of multiple test compounds into a microfluidic device. Once introduced into the device, the test compounds are screened for action on biological systems using continuous series or parallel assay orientation.

As used herein, the term “test compound” relates to a compound that is screened for its ability to affect a particular biochemical system. Test compounds may be derived from chemical compounds, mixtures of chemical compounds, for example polysaccharides, small organic or inorganic molecules, biological macromolecules such as peptides, proteins, nucleic acids, or bacteria, plants, fungi, or animal cells or tissues. It can include a variety of different compounds including prepared extracts, natural or synthetic compositions. According to the specific embodiment carried out, the test compound is provided, for example, injected in free form in a solution, or optionally attached to a carrier, or a solid support, for example beads. Many suitable solid supports are used for immobilization of test compounds. Examples of suitable solid supports include agarose, cellulose, dextran (ie, commercially available as Sephadex, Sepharose), carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins , Plastic films, glass beads, polyaminemethylvinylether maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylon, silk and the like. In addition, in the methods and devices described herein, the test compounds are screened individually or in-flight. Group screening is particularly useful when it is expected to have a low hit rate for an effective test compound and has a positive result except for only one for a given group. Alternatively, the group screening is used when the results of different test compounds are differentially detected in a single system, for example via differential labeling to enable separate detection, or electrophoretic separation of the results.

Test compounds are commercially available or derived from any of a variety of biological sources described above and apparent to those skilled in the art. In one aspect, tissue equivalents or blood samples from the patient are tested with the assay system of the present invention. For example, in one aspect, blood is tested for the presence or activity of biologically related molecules. For example, the presence and activity level of an enzyme is detected by feeding the enzyme substrate to a biological sample and detecting the formation of the product using the assay system of the present invention. Similarly, in one aspect, the presence of infectious pathogens (viruses, bacteria, fungi, etc.) or cancerous tumors may be characterized by the binding of labeled ligands to pathogens or components of the pathogen or tumor, such as tumor cells, proteins, membranes, cell extracts, and the like. Or alternatively, by measuring the presence of antibodies to pathogens or tumors in the blood of the patient. For example, measuring the binding of antibodies from a patient's blood to a viral protein, such as an HIV protein, is a common test for measuring patient exposure to viruses. Many assays for pathogen infections are well known and can be applied to the assay system of the present invention.

Biological samples are derived from patients using well known techniques such as venipuncture or tissue biopsy. If biological material is derived from non-human animals, such as commercially relevant livestock, blood and tissue samples are conveniently obtained from the livestock treatment plant. Similarly, the plant material used in the assays of the present invention is conveniently derived from a crop or horticultural crop source. Alternatively, the biological sample may be supplied from a cell or blood bank in which tissue and / or blood is stored, or from an ex vivo source such as a cell culture. Techniques and methods for establishing a culture of cells for use as a source of biological material are well known to those skilled in the art. The following documents provide general induction methods for cell cultures (Freshney Culture of Animal Cells, a Manual of Basic Technique, Third Edition Wiley-Liss, New York (1994)).

II. Black system

As described above, the screening methods of the present invention are generally carried out in microfluidic devices or "microscopic experimental systems", which perform assays, automation and environmental impacts on the assay system, eg loss, contamination. Consider integrating the elements necessary to minimize human error. Numerous devices for carrying out the assay methods of the present invention are described in the detailed description below. However, it will be appreciated that the specific arrangement of such devices will generally vary depending on the assay type and / or desired assay orientation. For example, in some embodiments, the screening method of the present invention can be performed using a microfluidic device having two crossing channels. For more complex assays or assay orientations, multichannel / crossover devices are optionally used. The low integration and self-contained nature of this device ultimately takes into account any assay arrangements to be realized within the environment of microexperimental systems.

A. Electrokinetic Material Transport

In a preferred aspect, the devices, methods and systems described herein use an electrokinetic material delivery system, preferably a controlled galvanic material delivery system. As used herein, a "dynamic material delivery system" refers to the transport of materials between them through channels and / or chambers, i. While the cations are transported to the cathode, they include a system that causes the transport of material that the anions transport to the anode.

The electrokinetic material transport and induction system includes a system that depends on the electrophoretic transport of charged material in an electric field applied to the structure. The system is particularly named as the electrophoretic material transport system. Other electrokinetic material induction and delivery systems rely on the electroosmotic flow of fluids and materials within channels or chambers when an electric field is applied against the structure. Briefly stated, when fluid enters a channel having a surface with charged functional groups, for example hydroxyl groups, in an etched glass channel or glass microcapillary, the groups can be ionized. In the case of hydroxyl functional groups, such ionization at neutral pH, for example, releases protons from the surface and into the fluid, produces a concentration of protons near the fluid / surface interface, or positively surrounds a large amount of fluid in the channel. Produces a charged sheath. Application of the voltage gradient to the length of the channel will cause the proton second, as well as the fluid it surrounds, to carry in the direction of the voltage drop, ie towards the cathode.

As used herein, “adjusted electrokinetic material transport and induction” relates to an electrokinetic system that utilizes vigorous regulation of voltage applied to multiple, ie, more than two, electrodes, as described above. In other words, the regulated electrokinetic system simultaneously adjusts the voltage gradient applied for two or more crossover channels. Controlled electrokinetic material transport is described in Ramsey's PCT application WO 96/04547, which is incorporated herein by reference. In particular, the preferred microfluidic devices and systems described herein include a body structure comprising two or more cross channels or fluid conduits, eg, closed chambers connected to one another, the channels comprising three or more non-crossed ends. . An intersection point of two channels refers to a point at which two or more channels are in fluid communication with each other, and a "T" intersection, a cross, a "wagon wheel" intersection of multiple channels, or another channel structure in which two or more channels are in fluid communication with each other. It includes. The non-crossed end of the channel is the point where the channel does not cross as a result of channel crossing with another channel, eg, a "T" crossing. In a preferred aspect, the device will comprise three or more crossing channels with four or more non-crossing ends. In a basic cross channel structure, when a single horizontal channel is crossed and crossed by a single vertical channel, the regulated electrokinetic material transport regulates the material transport flow through the intersection by providing forced flow from another channel at the intersection. Works to guide enemies. For example, the first material through the horizontal channel is directed in the desired transport direction, for example from left to right with respect to the intersection with the vertical channel. Simple electrokinetic material transport of the material to the intersection is achieved by applying a voltage gradient over the length of the horizontal channel, i.e. applying a first voltage at the left end of the channel and applying a lower second voltage at the right end of the channel, or , By floating the right end (no voltage applied). However, with this type of mass flow through the cross section, a significant amount of diffusion occurs at the cross section, which results from the convective effect at the cross section, in addition to the natural diffusion properties of the material carried in the medium used.

In controlled electrokinetic material delivery, the material carried relative to the intersection is suppressed to low levels of flow from the side channels, for example the upper and lower channels. This is done by applying a weak voltage gradient along the material flow path, for example from the upper or lower end of the vertical channel towards the right end. As a result, the material flow at the crossover portion "stops", which impedes the diffusion of the material into the vertical channel. The pinched volume of material at the intersection can then be injected into the vertical channel by applying a voltage gradient over the vertical channel length, ie from the top end to the bottom end. In order to avoid release of material from the horizontal channel during this injection process, a low level of flow is directed back into the lateral channel such that "reversal" of material from the intersection occurs.

In addition to the interrupted infusion schedule, controlled electrokinetic material delivery is readily utilized to form a virtual valve that contains no mechanical or moving parts at all. Specifically, with respect to the cross-crossing portions described above, the flow of material from one channel portion to another, for example from the left branch of the horizontal channel to the right branch, is a controlled flow from the vertical channel, eg For example, it can be effectively regulated, interrupted and restarted by flow from the lower branch to the upper branch from the vertical channel. Specifically, in the "off" mode, the material is transported from the left branch into the upper branch through the intersection by applying a voltage gradient to the left and upper ends. Inhibitory flow is induced from the lower branch to the upper branch by applying a similar voltage gradient along the path (lower to upper end). The metered amount of material is then distributed from the left branch to the right branch of the horizontal channel by switching the applied voltage gradient from left to top and from left to right. The amount of voltage gradient and time applied defines the amount of material dispersed in this manner. Although described in four ways for illustrative purposes, these controlled electrokinetic material delivery systems of cross-crossing can be readily applied to more complex interconnected channel networks, for example, arrays of interconnected parallel channels.

B. Continuous Flow Assay System

In one preferred system, the methods and apparatus of the present invention are used to screen test compounds using a continuous flow assay system. In general, continuous flow assay systems can be readily used for screening for inhibitors or inducers of enzymatic activity, or for agonists or antagonists of receptor-ligand binding. In brief, a continuous flow assay system includes a continuous flow of a particular biochemical system along a microfabrication channel. As used herein, the term "continuous" generally refers to an unbroken or continuous stream of a particular composition that flows continuously. For example, a continuous flow may include a constant fluid flow with a collection of velocities, or alternatively a fluid flow that temporarily stops at the flow velocity of the entire system and does not otherwise interfere with the flow stream. The action of the stream is indicated by the generation of an event or signal that can be detected. In one preferred embodiment, the detectable signal comprises any detectable pigment production or fluorescence signal related to the action of the particular model system used. In an enzymatic system, the signal will generally be generated by the product of the catalytic action of the enzyme, for example on a pigment generating or fluorescence generating substrate. In binding systems, such as receptor ligand interactions, the signal typically involves the relationship of the labeled ligand with the receptor, or vice versa.

Various other detectable signals and labels can also be used in the assays and devices of the present invention. In addition to the pigment production and fluorescence generating signals described above, radioactive material decay, electron density, pH change, solvent viscosity, temperature and salt concentration are also suitably measured.

More generally, the label can typically be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, or other chemical means. For example, useful nucleic acid labels include 32P, 35S, fluorescent dyes, electron dense reactants, enzymes (eg, those commonly used in ELISAs), biotin, dioxygenin, or hapten and antiserum or monoclonal antibodies. Contains proteins that can be used. Various labels suitable for labeling biological components are known and widely reported in both the scientific and patent literature and can be generally applied to the present invention for labeling biological components. Suitable labels also include radiation nucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Labeling agents optionally include, for example, monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity mattresses, carbohydrates or lipids. Detection is performed by various known methods including spectrophotometric or optical detection of radioactive or fluorescent markers, or other methods of detecting molecules based on size, charge or affinity. The detectable moiety can be any substance having detectable physical or chemical properties. Such detectable labels are widely developed in the fields of gel electrophoresis, column chromatography, solid substrates, spectroscopy techniques and the like, and in general, labels useful in the method can be applied to the present invention. As such, the label is a composition that can be detected by spectroscopy, photochemistry, biochemistry, immunochemistry, electrical, optical thermal or chemical means. Labels useful in the present invention include fluorescent dyes (eg, fluorescent isothiocyanates, Texas red, rhodamine, etc.), radioactive labels (eg, 3H, 125I, 35S, 14C, 32P or 33P), enzymes (eg For example, LacZ, CAT, horseradish peroxidase, alkaline phosphatase and other substances used as detectable enzymes or used as marker products or in ELISAs, nucleic acid inserts (e.g., ethidium bromide), And colorimetric labels such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads.

Fluorescent labels are particularly preferred labels. Preferred labels are commonly characterized by one or more of high sensitivity, high stability, low background value, low environmental sensitivity and high specificity in labeling.

Commonly known fluorescent moieties that can be incorporated into the labels of the invention include 1- and 2-aminonaphthalene, p, p'-diaminobenzophenone imine, anthracene, oxacarbocyanine, merocyanine, 3- Aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol, bis-3-aminopyridinium salt, helebrigenin, tetracycline, ste Includes phenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene, 7-hydroxycoumarin, phenoxazine, carlisylate, stropantidine, porphyrin, triarylmethane and flavin do. Individual fluorescent compounds which have a functional group for binding to an enzyme which is preferably detected in the device or assay of the present invention or which can be modified to incorporate the functional group include monosyl chloride; Fluorescein, such as 3,6-dihydroxy-9-phenylxyl hydrochloride; Rod amine isothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene; N-phenyl 2-amino-6-sulfonatonaphthalene; 4-acetamido-4-isothiocyanato-stilben-2,2'-disulfonic acid, pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl-N-methyl-2-aminonaphthalene-6-sulfonate; Ethidium bromide; Stebrin; Mauromin-0,2- (9'-antroyl) palmitate; Single phosphatidylethanolamine; N, N'-dioctadecyl oxcarbocyanine; N, N'-dihexyl oxcarbocyanine; Merocyanine, 4- (3'-pyrenyl) stearate; d-3-aminodecoxy-equilenin; 12- (9'-antroyl) stearate; 2-methylanthracene; 9-vinylanthracene; 2,2 '-(vinylene-p-phenylene) bisbenzoxazole; p-bis (2- (4-methyl-5-phenyl-oxazolyl)) benzene; 6-dimethylamino-1,2-benzophenazine; Retinol; Bis (3'-aminopyrididium); 1,10-decanediyl diyodide; Sulfonaphthylhydrazone of helibrienin; Chlorotetracycline; N- (7-dimethylamino-4-methyl-2-oxo-3-chromenyl) maleimide; N- (p- (2-benzimidazolyl) -phenyl) maleimide; N- (4-fluoranthyl) maleimide; Bis (homovanylic acid); Resazarin; 4-chloro-7-nitro-2,1,3-benzooxadiazole; Merocyanine 540; Resorphin; Rose Bengal; And 2,4-diphenyl-3 (2H) -furanone. Several fluorescent tags are available from SIGMA chemical company (St. Louis, MO), Molecular Probes, R & D systems (Minneapolis, MN), Pharmacia LKB Biotechnology (New Jersey) Fitzkata Way), CloneTech Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corporation, Chem Genes Corp., Aldrich Chemical Company, Maryland Gasersburg), Fluka Chemica-Biochemika Analytica (Bucks, Switzerland), and Applied Biosystems (Foster City, CA), as well as other known to those skilled in the art. It is marketed by the company.

Preferably, the fluorescent label absorbs light at least about 300 nm, preferably at about 350 nm, more preferably at about 400 nm, and emits at a wavelength of at least about 10 nm higher than the wavelength of the absorbed light. It should be noted that the absorption and release characteristics of the bound label may differ from the unbound label. Therefore, when referring to the characteristics of the label and the various wavelength ranges, the label used is indicated and does not represent a label that is characterized and not conjugated in any solvent.

Fluorescent labels are a preferred class of detectable labels because the inventors can obtain multiple emissions by irradiating the fluorescent labels with light. Thus, a single label can be provided for measurable events. Detectable signals can also be provided by chemiluminescent and bioluminescent sources. Chemiluminescent sources include compounds that can be electrically excited by a chemical reaction and then emit light that acts as a detectable signal or provides energy to the fluorescent receptor. Various classes of compounds have been found to provide chemiluminescence under changes or conditions. One class of compounds is 2,3-dihydro-1,4-phthalazinedione. The most representative compound is luminol, a 5-amino compound. Other components of this class include 5-amino-6,7,8-trimethoxy- and dimethylamino [ca] benzene analogs. These compounds can emit light using alkaline hydrogen peroxide or calcium hypochlorite and bases. Another class of compounds is 2,4,5-triphenylimidazole, the common name for its parent product is lophin. Chemiluminescent analogues include p-dimethylamino and p-methoxy substituents. Chemiluminescence can be obtained under oxalates, generally oxalyl active esters such as p-nitrophenyl and peroxides such as hydrogen peroxide under basic conditions. Other useful chemiluminescent compounds are known and can be purchased, including -N-alkyl acridinium esters (basic H ₂ O ₂ ) and dioxetane. Alternatively, luciferin may be used in combination with luciferase or lucigenin to provide bioluminescence.

Labels are coupled directly or indirectly to the molecule to be detected (product, substrate, enzyme, etc.) according to methods well known in the art. As noted above, a wide range of labels are used with the required sensitivity, ease of conjugation of the compound, stability requirements, selection of available equipment and disposal regulations. Non-radioactive labels are often attached by indirect means. Generally, ligand molecules (eg biotin) are covalently bound to the polymer. The ligand is then bound to an anti-ligand (eg streptavidin) molecule that is inherently detectable or covalently bound to the signal system, such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Many ligands and anti ligands can be used. If the ligand has a natural anti ligand, such as biotin, thyroxine, and cortisol, it can be used with labeled anti ligand. Alternatively, any haptenic or antigenic compound can be used in combination with the antibody. The label can be conjugated directly to the signal generating compound, for example by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will mainly be hydrolases, in particular phosphatase, esterases and glycosidases, or oxidoreductases, in particular peroxidases. Fluorescent compounds include fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferon and the like. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinedione such as luminol. Means for detecting the label are well known to those skilled in the art. As such, for example, where the label is a radiolabel, the detection means comprises a scintillation counter or photographic film as in radiographic photography. If the label is a fluorescent label, the fluorescent dye is excited with light of a suitable wavelength and the resulting fluorescence is for example microscopy, visual irradiation, and electrons such as digital cameras, charge coupling devices (CCDs) or photomultipliers and phototubes. The detector can be detected by detecting by means of a photographic film by use. Fluorescent labeling and detection techniques, in particular microscopy and spectroscopy, are preferred. Similarly, enzyme labels are detected by providing a substrate suitable for the enzyme and detecting the resulting reaction product. As a result, simple colorimetric labels are often detected by simply observing the color associated with the label. For example, conjugated gold often shows pink and various conjugated beads show the color of the beads.

In a preferred embodiment, the continuous system generates a constant signal that changes only when a test compound is introduced that affects the system. In particular, when system components flow along a channel, the components will produce a relatively constant signal level in the window or detection region of the channel. If these test compounds affect the system, they will cause deviations from a constant signal level in the detection window. This deviation can then be correlated to the particular test compound being screened.

One embodiment of an apparatus for use in a series or continuous assay structure is shown in FIG. 1. As shown, the entire apparatus 100 is fabricated on a planar substrate 102. Suitable substrate materials are generally selected based on their compatibility with the conditions present in the particular operation to be performed by the device. Such conditions may include both extremes of pH, temperature, salt concentration, and application of an electric field. In addition, the base materials are chosen for their inertness to important components of the assay or synthesis that are to be performed by the device.

Examples of useful substrate materials are polymeric substrates such as plastics, as well as glass, quartz and silicone. In the case of a conductive or semiconducting substrate, it is generally preferred to include an insulating layer on the substrate. This is particularly important where the device employs electrical elements, for example electrical materials and fluid induction systems, sensors and the like. In the case of polymeric substrates, the substrate materials are optionally hard, semi-rigid, opaque, semi-opaque or transparent, depending on their intended use. For example, an apparatus comprising an optical or visible detection element will typically be fabricated, at least in part, from a transparent material that allows or at least facilitates detection. Alternatively, transparent windows, for example glass or quartz, are optionally incorporated into the device for these types of detection elements. In addition, the polymeric material may have a straight or branched backbone, optionally crosslinked or uncrosslinked. Examples of particularly preferred polymeric materials include polydimethylsiloxane (PDMS), polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone, polycarbonate and the like.

The apparatus shown in FIG. 1 includes a series of channels 110, 112 and optional reagent channels 114 fabricated into the surface of the substrate. One or more of these channels will typically have very small cross sectional dimensions, for example, in the range from about 0.1 μm to about 500 μm. Preferably, the cross sectional dimension of the channel will range from about 0.1 to about 200 μm and more preferably from about 0.1 to about 100 μm. In one particularly preferred aspect, each channel will have one or more cross-sectional dimensions in the range of about 0.1 μm to about 100 μm. Although generally shown as straight channels, it will be appreciated that S-shaped, sawtooth or other channel shapes effectively employ longer channels within shorter distances in order to make full use of the space on the substrate.

Fabrication of these microscale elements on the surface of the substrate will generally be accomplished by any number of microfabrication well known in the art. Lithography is optionally used to produce, for example, glass, quartz or silicon substrates using methods well known in the semiconductor manufacturing industry, such as, for example, photolithographic etching, plasma etching, or wet chemical etching. Similarly, for polymer substrates, well known manufacturing techniques can also be used. These techniques include injection molding or stamp forming methods in which many substrates are optionally produced using rolling stamps that produce large sheet-like fine scale substrates, or polymer micromoulding techniques in which substrates are polymerized into micromechanical molders. do.

The device will typically include an additional planar element covering the channeled substrate that seals and fluidly seals the various channels to form a conduit. Attaching the flat cover element is accomplished by a variety of means, including thermal bonding, adhesives, or, in particular, natural bonding between the two components, for example glass or semi-rigid and non-rigid polymer substrates. . The flat cover element may further be provided with access ports and / or reservoirs for introducing the various fluid elements required for a particular screening.

The device shown in FIG. 1 includes reservoirs 104, 106 and 108 disposed at the distal ends of channels 110 and 114 and fluidically coupled. As shown, sample channel 112 is used to introduce a number of different test compounds into the device. As such, it will generally be fluidly coupled to a number of separate sources of test compound to be introduced separately into sample channel 112 and then into channel 110.

The introduction of a large number of individual, discrete volumes of test compound into the sample is carried out by a number of methods. For example, a pipette pipette may optionally be used to introduce test compounds into the device. In a preferred aspect, an electrical pipette fluidly connected to the sample channel 112 is used. Examples of such electric pipettes are described, for example, in US patent application Ser. No. 08 / 671,986 filed on June 28, 1996, which is incorporated herein by reference (Representative Document No. 017646-000500). In general, the electropipettor uses electroosmotic fluid induction as described herein to alternately sample multiple test compounds, or “test materials”, and spacer compounds. The pipette then delivers the individual or physically insulated sample or test compound volume in the desired material region in a series of ways into the sample channel for subsequent manipulation in the device. Individual samples are typically separated by spacer regions of low ionic strength spacer fluid. These low ionic strength spacer regions have higher voltage drops in the region of the desired material or test compound of high ionic strength, thereby inducing electrokinetic pumping. The test compound or desired material region, which is typically present in a solution of higher ionic strength, is the fluid region referred to as the first spacer region (also called the "protection band") in contact with the interface of the desired material region. These first spacer regions comprise a solution of high ionic strength to block the movement of the sample element into a low ionic strength fluid region, or a second spacer region that will cause electrophoretic bias. The use of the first and second spacer regions is described in detail in US patent application Ser. No. 08 / 671,986 filed on June 28, 1996, which is incorporated herein by reference (representative document number: 017646-000500).

Alternatively, sample channel 112 is optionally, individually fluidically connected to a plurality of separate reservoirs through separate channels. Each separate reservoir contains a separate test compound with additional reservoirs provided for suitable spacer compounds. Test compounds and / or spacer compounds are then transported into the sample channels from various reservoirs using suitable material induction designs. In either case, it is generally desirable to separate discrete sample volumes, or test compounds, having suitable spacer regions.

As shown, the device includes a detection window or region 116 in which signals from the biochemical system are optionally monitored. Such detection windows will typically include a transparent cover that allows for the detection of visible or optical observations and assay results, for example the observation of colorimetric or fluorescence reactions.

In a particularly preferred aspect, the monitoring of the signal in the detection window is accomplished using an optical detection system. For example, optical substrate signals are typically monitored using a laser activated fluorescence detection system using a laser light source at a suitable wavelength for activating a fluorescent indicator in the system, for example. The fluorescence is then detected using a suitable detector element, for example photomultiplier tube (PMT). Similarly for screens using colorimetric signals, a spectrophotometric detection system that guides the light source to the sample is optionally used to provide a measure of absorbance or transmission of the sample.

In an alternative aspect, the detection system may include a non-optical detector or sensor for detecting specific characteristics of the system disposed within the detection window 116. Such sensors include temperature, conductivity, potentiometer (pH, ions), ammeter (for compounds that are oxidized or reduced, for example O ₂ , H ₂ O ₂ , I ₂ , oxidative / reducible organic compounds, etc.).

In operation, a flowable first component of a biological system (eg, a fluid comprising a receptor or an enzyme) is located in the reservoir 104. This first component flows through the main channel 110, past the detection window 116 and into the waste reservoir 108. A second component of the biochemical system, such as a ligand or substrate, flows in a convection from side channel 114 into main channel 110, after which the first component and the second component can be mixed and interact. Deposition of these elements in the device is performed in a number of ways. For example, enzymes and substrates or receptor and ligand solutions can be introduced into the device through openings or sealable entry ports in a flat cover. Alternatively, these components are optionally added to their respective reservoir during manufacture of the device. For example, enzyme / substrate or receptor / ligand components are optionally provided in the device in lyophilized form. Prior to use, these components are easily configured by introducing a buffer solution into the reservoir. Alternatively, the component is lyophilized with a buffered salt, so that simple addition of water is all that is needed for reconstitution.

As noted above, the interaction of the first component with the second component is typically accomplished by a detectable signal. For example, in these embodiments where the first component is an enzyme and the second component is a substrate, the substrate is a pigment generating or fluorescence generating substrate that generates an optionally detectable signal when the enzyme acts on the substrate. When the first component is a receptor and the second component is a ligand, the ligand or receptor optionally comprises a detectable signal. In either case, the mixing and flow rate of the compound will typically remain constant such that the flow of the mixture of the first and second components through the detection window 116 will generate a steady state signal. "Static state signal" generally means a signal that has a regular, predictable signal intensity distribution. As such, the steady state signal may comprise a signal having a constant signal strength or, alternatively, a regular period of strength, with which a change in the normal signal distribution is measured. This latter signal is generated when the fluid flow is periodically interrupted, for example to load additional test compounds, as described in the description of the continuous flow system. Although the signal generated in the enzyme system described above will vary along the length of the channel, ie it increases with the exposure time the enzyme converts the fluorescence generating substrate to the fluorescent product, but at any particular point along the channel the signal will remain constant. And provide a constant flow rate.

From the sample channel 112, the test compound is periodically or continuously into the main channel 110 and into a stream of first and second components, such as a fluid region containing the test compound, also referred to as the "target material region". Is introduced. If these test compounds affect the interaction of the first element with the second element, the test compound will alter the detected signal in the detection window corresponding to the region of interest. As noted above, typically, various different test compounds to be injected through the channel 112 are separated by the first and, even, second spacer fluid regions, from one test compound to another test compound. Differentiation of effect, or lack of effect will be allowed. In embodiments in which an electroosmotic fluid induction system is used, the spacer fluid region may also act to reduce any electrophoretic bias that may occur in the test sample. The use of these spacer regions to induce electroosmotic flow of a fluid, as well as in the general removal of electrophoretic bias within a sample or test compound or region of interest, is filed on June 28, 1996, incorporated herein by reference. US Patent Application Serial No. 08 / 671,986 (Representative Document No. 017646-000500).

As one example, a constant continuous flow of enzyme and fluorescence generating substrate through main channel 110 will produce a constant fluorescence signal in detection window 116. If the test compound interferes with the enzyme, the introduction of the test compound (ie, within the desired material region) will result in a transient but detectable drop in the level of the signal in the detection window corresponding to the desired material region. The dropping timing in the signal will then be correlated with the particular test compound based on the injection known in the detection time-frame. In particular, the time required for the injected compound to produce the observed effect can be readily determined using a positive control.

For receptor / ligand systems, similar changes in steady state signals can be observed. In particular, the receptor and its fluorescent ligands may have different flow rates depending on the channel. This can be accomplished by incorporating an intra-channel size exclusion matrix or, in the case of an electroosmotic method, altering the relative electrophoretic shift of the two compounds to allow the receptor to flow faster down the channel. Again, this is accomplished by the use of size exclusion matrices, or by the use of different surface charges in the channels that lead to the differential flow rate of the charge variable compound. If the test compound is bound to the receptor, it will generate a darker pulse of the fluorescence signal and then a brighter pulse. Without being bound to a particular theory of manipulation, it is believed that steady state signals cause both free fluorescent ligands and fluorescent ligands bound to the receptor. The bound signal travels at the same flow rate as the receptor, while the unbound ligand travels more slowly. If the test compound interferes with the receptor-ligand interaction, the receptor will not have a fluorescent ligand, thereby diluting the fluorescent ligand in the flow direction and leaving excess fluorescent ligand. This causes a temporary decrease in steady state signals followed by a temporary increase in fluorescence. Alternatively, when a scheme similar to the scheme used for the enzyme system is used, a signal exists that reflects the interaction of the receptor with the ligand. For example, a pH indicator that exhibits the pH effect of receptor-ligand binding can be incorporated into a device with a biological system, ie a biological system in the form of encapsulated cells, so that slight pH changes resulting from the binding can be detected [ References: Weaver, et al., Biol / Technology (1988) 6: 1084-1089. In addition, the activity of enzymes generated from receptor-ligand bonds, such as monitoring the activity of kinases, or upon activation by incorporation of fluorophores that are activated or quenched by structural changes to the enzyme upon activation Structural changes in the enzyme can be detected.

The flow and direction of fluid in the unscaled fluidic device is accomplished by a variety of methods. For example, the device includes an integrated microfluidic structure, such as a pipette pump and a pipette valve, or additional elements, such as a pump and a switching valve, for pumping and directing various fluids through the device. Examples of microfluidic structures are described in US Pat. Nos. 5,271,724, 5,277,556, 5,171,132 and 5,375,979. It is also described in published British patent application 2 248 891 and published European patent application 568 902.

Microfabricated fluid pumps and valve systems are readily used in the apparatus of the present invention, however, due to the cost and complexity associated with their manufacture and operation, the mass produced disposable devices planned by the present invention will not be available. For this reason, in one particularly preferred aspect, the device of the present invention will typically include an electroosmotic fluid induction system. Such a fluid induction system combines the precision of a fluid induction system lacking a moving part with the ease of manufacture, fluid control and low powder properties. Particularly preferred electroosmotic fluid induction systems are described, for example, in International Patent Application WO 96/04547, to Ramsey et al., The applicant of which is incorporated herein by reference.

In summary, these fluid control systems include electrodes disposed in a reservoir that is in fluid connection with a plurality of intersecting channels made into the surface of the substrate. The materials stored in the reservoirs are transported through a channel system that delivers a variety of materials in suitable volumes to one or more regions on the substrate to perform the desired screening assay.

Fluid and material transport and induction are accomplished through electroosmotic or electrokinetics. In summary, when a suitable material, typically a material comprising a fluid, is placed in a channel or other fluid conduit with functional groups present on the surface, these functional groups can be ionized. For example, if the surface of the channel contains hydroxyl functional groups on the surface, protons leave the surface of the channel and enter the fluid. Under these conditions, the surface will have a pure negative charge, while the fluid will, in particular, have an excess of protons or positive charges positioned close to the interface between the channel surface and the fluid. By applying an electric field along the channel section, the cations will flow toward the negative electrode. Movement of positively charged species in the fluid attracts the solvent with them. The steady state velocity of this fluid movement is generally given by the following equation:

Where

v is the solvent velocity,

ε is the dielectric constant of the fluid,

ζ is the zeta potential of the surface,

E is the electric field strength,

η is the solvent viscosity. As can be readily seen from the above equation, the solvent viscosity is directly proportional to the surface potential.

To provide a suitable electric field, the solvent generally includes a voltage regulator capable of simultaneously applying a selectable voltage level to each reservoir (including ground). Such voltage regulators can be performed using multiple voltage dividers and multiple relays to obtain selectable voltage levels. Alternatively, multiple, independent voltage sources can be used arbitrarily. The voltage regulator is electrically connected to each of the reservoirs via an electrode located or manufactured in each of the plurality of reservoirs.

Incorporating the electroosmotic fluid induction system into the device shown in FIG. 1 allows the incorporation of electrodes in respective reservoirs 104, 106, and 108 at the end of sample channel 112 or any fluid channel connected thereto. An integration, whereby the electrode is in electrical connection with a fluid disposed within each reservoir or channel. In the case of glass substrates, the etched channels will carry net negative charges generated from ionized hydroxyls that are naturally present on the surface. Alternatively, surface modification is optionally used to provide suitable surface charges, such as coating, induction, for example silanization, or saturation of the surface, to provide suitably charged groups on the surface. An example of such a treatment is described in Provisional Patent Application No. 60 / 015,498 (Agent No. 017646-002600), filed April 16, 1996, incorporated herein by reference.

In summary, suitable substrate materials are generally selected based on their compatibility with the conditions present in the particular operation to be performed by the device. Such conditions may include the extreme, temperature and salt concentration of pH. In addition, the base materials are also selected for their inertness to important components of the analysis and synthesis to be performed by the device. The polymeric base materials will be hard, semi-rigid or non-hard, opaque, semi-opaque or transparent depending on their intended use. For example, it will be common for devices comprising optical or visible detection elements to be at least partially fabricated from transparent polymeric materials to facilitate such detection. Alternatively, for example, a transparent window of glass or quartz can be incorporated into the device for this detection element. In addition, the polymeric material may have a straight or branched backbone and may cross or cross. Examples of polymeric materials are, for example, acrylics, in particular PMMA (polymethylmethacrylate); Examples of acrylics are Acryllite M-30 or Acrylite L-40, available from CYRO Industries, and Rockaway, available from Autohass North America. , NJ or PLEXIGLAS VS UVT; Macrocarbonate CD-2005 (Makrolon CD, available from Polycarbonate (e.g., Mobay Corporation (Pittsburgh, PA)) or The Plastics and Rubber, a subsidiary of Bayer Corporation) (2005), or Rezan Oak 1020L (LEXAN OQ 1020L) or Rezan Oak 1020) available from GE Plastics, polydimethylsiloxane (PDMS), polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone, Polycarbonates and the like. Optical, mechanical, thermal, electrical and chemical resistance properties for many plastics are well known (generally available from the manufacturer) or can be readily determined by standard analysis.

As described herein, electrokinetic fluid control systems used in the devices of the present invention generally employ materials with charged functional groups on these surfaces, such as hydroxyl groups present on the glass surface. As described, the apparatus of the present invention may also use plastic or other polymeric materials. In general, the materials of such materials have a hydrophobic surface. As a result, the use of electrokinetic fluid control systems in devices utilizing polymeric materials used in the present invention typically uses a modified material surface in contact with the fluid.

Surface modification of the polymeric material can be carried out in a variety of different forms. For example, the surface may be coated with a suitably charged material. For example, surfactants with charged groups and hydrophobic tails are preferred as coating materials. In short, the hydrophobic tail is located on the hydrophobic surface of the material, representing a charged head group in the fluid layer.

In one embodiment, the preparation of the surface charged on the material comprises exposing the surface to modify the channel and / or reaction chamber, for example, with a suitable solvent that partially dissolves or softens the surface of the polymeric material. The detergent is then contacted with the partially dissolved surface. The hydrophobic portion of the detergent molecule will be combined with the partially dissolved polymer. The solvent is then washed from the surface, for example with water, on which the polymer surface is cured with a detergent embedded in the surface, which appears at the fluid interface of the charged head group.

In another embodiment, the polymeric material, such as polydimethylsiloxane, may be modified by plasma spinning. In particular, plasma radiation of PDMS oxidizes methyl groups, liberates carbon, leaves hydroxyl groups at their positions, and creates their bonded hydroxyl groups, effectively creating a glassy surface on the polymeric material.

The polymeric material can be rigid, semi-rigid, non-rigid or a combination of rigid and non-rigid elements depending on where the device is used. In one embodiment, the material consists of one or more softeners, soluble material elements and one or more hardeners, harder rigid elements, channels and chambers made on the surface. As soon as the two materials are combined, the inclusion of soft elements allows the formation of effective fluid seals in the channels and chambers, eliminating the problems associated with rigid plastic components that are glued or melted.

Many additional elements are added to the polymeric material and provided to the electrokinetic fluid control system. Such elements may be added during the material formation process, ie during the forming or stamping step, or during the subsequent separation step. These elements typically include charged bolts, including electrodes for charged bolts for various fluid reservoirs and bolt detectors at various channel intersections.

The electrodes can be incorporated as part of the molding process. In particular, the electrodes are patterned in the mold so that when the polymeric material is introduced into the mold, the electrodes are properly positioned. Alternatively, the electrodes and other elements can be added after the material has been formed using chemical etching, after known microfabrication methods, such as sputtering or controlled vapor deposition methods.

Whether polymeric or other materials are used, the volt control is applied simultaneously to various reservoirs to provide the desired fluid flow characteristics, e. Affects. In particular, the deformation of the bolts charged to the various reservoirs can be moved and guides the fluid flow through the continuous channel structure of the device in a controlled manner, affecting the desired screening analysis and fluid flow to the device. .

2A shows a schematic of fluid induction during a conventional assay screen. In particular, it is injected into a continuous stream of enzyme-fluorescent mixture of test compounds (in the formulation region). As shown in FIG. 2A, with reference to FIG. 1, a continuous stream of enzyme flows from reservoir 104 along main channel 110. Test compound 120 separated by a suitable spacer region 121, eg, a low ionic strength spacer region, is introduced from sample channel 112 into main channel 110. Once introduced into the main channel, the test compound will interact with the flow enzyme stream. The mixed enzyme / test compound region then flows along main channel 110 past the intersection with channel 114. A continuous stream of fluorescent or colored material contained in reservoir 106 is introduced into sample channel 110, from which it is contacted with a continuous stream of enzyme comprising a test material region comprising test compound 122 and Are mixed. The action of the enzyme on the material will lead to an increase in fluorescence or coloring signal levels. This increased signal is perceived by increasing shading in the main channel to be close to the detection window. This signal direction also occurs in the test compound or test substance region, which region does not affect enzyme / substrate interaction, eg, test compound 126. Where the test compound does not affect the interaction of the enzyme with the substrate, the modification will appear as an induced signal. For example, a presumed fluorescent substance, ie a test compound that interferes with the interaction of an enzyme with a substrate, will induce less fluorescent product generated within the test substance region. This will lead to non-fluorescein in the flow stream through which the flow stream passes through the detection window 116, or to induce fewer fluorescent regions, the window corresponding to the test substance region. For example, as shown, the test substance region includes test compound 128, which is a presumed inhibitor of enzyme-substrate interaction and exhibits a lower detectable phosphor than the surrounding stream. This indicates a lack of shading of the test substance region 128.

A detector proximate to the detection window monitors the level of the fluorescence signal generated by enzymatic activity on the fluorescent or colored material. This signal is maintained at a relatively constant level for the test compound, and the compound does not affect enzyme-substrate interactions. However, when inhibitory compound compounds are screened, this will lead to a net drastic decrease in fluorescence signals indicating reduced or inhibited enzyme activity on the substrate. In contrast, upon screening, the derivative compounds induce an instant increase in fluorescence signal corresponding to increased enzyme activity on the substrate.

2B shows a schematic of the screen for an effector of receptor-ligand interaction. As shown in FIG. 2A, a continuous stream of receptors flows from reservoir 104 through main channel 110. A test compound or test substance region 150 separated by a suitable spacer fluid region 121 is introduced into the main channel 110 from the sample channel 112 and a continuous stream of fluorescent ligand from the reservoir 106 is connected to the side channel. Is introduced from 114. As shown in FIG. 2A, a continuous stream of fluorescent ligands and receptors across the detection window 116 will provide a constant signal intensity. The test substance region in the stream containing the test compound that does not affect receptor-ligand interactions provides the same or similar level of phosphor as the remaining ambient stream, eg, test compound or test substance region 152. something to do. However, the presence of a test compound that is opposite to the receptor-ligand interaction or has inhibitory activity may result in a lower level of the interaction in the portion of the stream in which the compound is located, eg, in the test compound or test substance region 154. Will induce. In addition, different flow rates for receptors bound to fluorescent ligands or free fluorescent ligands will result in detectable droplets at fluorescence levels corresponding to dilution of fluorescent materials derived from unbound faster moving receptors. Thereafter, there is an increase in the fluorescent material 156 corresponding to the accumulation of fluorescent ligands that move more slowly following a sudden decrease in the fluorescent material.

In some embodiments, it is desirable to provide additional channels for diverting or extracting the test substance region reaction mixture from the manipulation buffer and / or spacer regions. This is the case while retaining the reaction elements contained in the discontinuous fluid region during the reaction, while allowing these elements to separate during the data acquisition phase. As mentioned above, various elements of the reactants can be held together in the region of test substance moving through the reaction channel by incorporating a suitable spacer fluid region between the samples. In general, this spacer fluid region is selected to keep the sample within this original test substance region, in particular to prevent the sample from spreading into the spacer region even during extended reaction periods. However, this object can be achieved in the enzymatic separation of the assay, for example, where the assay takes place on the basis of the ligand-receptor assay mentioned above or where the reaction product is separated from the capillary. Thus, it may be desirable to eliminate these elements that prevent this separation during the initial portion of fluid induction.

A schematic illustration of one embodiment of an apparatus 500 for performing such sample or test substance separation or extraction is shown in FIG. 5. As shown, the test substance or test compound 504 is introduced into the device or chip through the sample channel 512. Again, they are typically introduced via a suitable injection device 506, for example a capillary pipette. The ionic strength and length of the first spacer region 508 and the second spacer region 502 are determined by the second spacer through the first spacer region 508 during the time that the sample with the highest electrophoretic movement is moved under the reaction channel. It is selected such that it cannot move to region 502.

Inferring the receptor ligand assay system, the test compound passes through the device 500 and the reaction channel 510 which are first mixed with the receptor. The first test compound / receptor in the form of a test substance region flows along the reaction channel in the incubation region 510a. Following this initial incubation, the test compound / receptor mixture is associated with a labeled ligand (eg, fluorescent ligand), where the mixture flows along the second incubation region 510b of the reaction channel 510. The flow rate and length of the incubation region of the system (measured by the potential applied at the ends of the respective reservoirs 514, 516, 518, 520, 522) and the sample channel 512 were incubated with the fluorescent ligand bound receptor and the test compound. Measure your time. The ionic strength of the solution containing the receptor and the fluorescent ligand and the flow rate of the material from the reservoir housing these elements into the sample channel are chosen so that the first and second spacer regions are unobstructed.

Separate test material regions containing receptors, fluorescent ligands, and test compounds flow along the reaction channel 510 by, for example, charging of potentials at the ends of reservoirs 514, 516, 518 and sample channel 512. Also at the opposite ends of reservoirs 520 and 522 and separation channel 524 the potential is charged to mix the potential at both ends of the mobile channel, so that the real flow across the mobile channel is zero. As the test material region passes through the intersection of the reaction channel 510 and the moving channel 526, the potential is suspended in the reservoirs 518 and 522, where the reservoirs 514, 516, 520 and the sample channel ( The charged potential at the end of 526 directs the separated test material region through the mobile channel to the separation channel 524. Once separated into the separation channel, the original potential is charged to all reservoirs, stopping the real flow flow through the moving channel 526. Thereafter, the conversion of the test substance can be repeated with each subsequent material region. Within the separation channel, the test substance region is exposed to a different state than that of the reaction channel. For example, different flow rates may be used and capillary treatment may be allowed for the separation of differently charged or different size pieces. In a preferred aspect, the test material is separated into a separation channel to place the test material into a capillary filled with high ionic strength buffer to remove low ionic strength spacer regions, thereby separating the various sample components outside the defined test substance region. Allow. For example, in the aforementioned receptor / ligand screen, the receptor / ligand mixture exhibits a different electrophoretic shift than when the ligand alone is present in the mobile channel, allowing the mixture to be separated from the ligand and detected. Can be.

Such modifications have a wide variety of uses and are particularly preferred for separating reaction products in the following reactions, for example cleavage reactions, cleavage reactions, PCR reactions and the like.

C. series of parallel analysis systems

More complex systems can also be derived within the scope of the present invention. For example, a schematic description of an alternative embodiment used in the “series of parallel injection reactions” is shown in FIG. 3. As shown, the apparatus 300 includes a planar material 302 as mentioned above. The surface fabricated material 302 is a series of parallel reaction channels 312-324. Also shown are three transverse channels fluidly connected to each parallel reaction channel. The three transverse channels comprise a sample injection channel 304, an optical seeding channel 306 and a collection channel 308. In addition, materials and channels are generally fabricated using materials and generally fabricated to the dimensions mentioned above. Although a series of parallel channels are shown and described, the reaction channels can also be fabricated in a variety of different directions. For example, rather than providing a series of parallel channels that are fluidly connected to a single transverse channel, the channels are optically selectively connected to a central reservoir, radially extending out therefrom, or optionally in some other non-parallel form. Are arranged. Also, while three transverse channels are shown, it will be appreciated that fewer transverse channels are used, for example, where biochemical system components are pre-deposited in the device. Similarly, as needed, more transverse channels are used to introduce additional elements into an optionally provided analysis screen. Accordingly, the series of parallel devices of the present invention is generally two or more, preferably three or four. It will include five or more transverse channels. Similarly, although reaction channels are shown in FIG. 7, it will be readily appreciated that the microdevice of the present invention may include more than seven channels depending on the needs of a particular screen. In a preferred aspect, the device will comprise 10 to about 500 reaction channels, more preferably 20 to about 200 reaction channels.

In particular, the device is useful for screening test compounds that are continuously injected into the device, and for use in parallel assay forms, where samples enter the device and allow for increased throughput.

In operation, the test compound in the separate test substance region is continuously introduced into the separated apparatus as mentioned above, wherein the separate injection substance region is the intersection of the reaction channels 310-324 and the parallel sample channel 304. Flow along the transverse sample injection channel 304 until it is close to. As shown in Figures 4A-4F, test compounds are optionally provided to be immobilized on individual beads. When the test compound is immobilized on the beads, the parallel channels are optionally fabricated to include bead stop wells 326-338 at the intersection of the sample injection channel 304 and the reaction channel. Arrow 340 represents the net fluid flow during this type of sample / bead injection. As individual bead positions into the stop wells, fluid flow through certain channels will generally be limited. Following the unrestricted fluid flow, the next series of beads flows to the next available stop well and is positioned in place.

At a point near the intersection of the parallel reaction channel and the sample injection channel, the test compound is directed to each of these reaction channels by redirecting the fluid flow down the channel. In addition, in the example of the test compound immobilized on the beads, the immobilization will generally be via a cleavable linker group, such as a photolysate, acid or base resolved linker group. Thus, test compounds are needed to escape from the beads by exposure to release agents such as light, acids, bases, etc. before the test compounds under the reaction channel flow.

In parallel channels, the test compound will be contacted in the biochemical system from which the effector compound is sought. As shown, the first component of the biochemical system is placed into the reaction channel using techniques similar to those described for test compounds. In particular, biochemical systems are typically introduced via one or more transverse channels 306. Arrow 342 indicates the direction of fluid flow in the seeding channel 306. The biochemical system is optionally a solution based on the enzyme / substrate or receptor-ligand mixtures mentioned above, for example, in a continuous flow, and in FIGS. It may be a bead with an immobilized enzyme / substrate system.

In embodiments in which the biochemical system is incorporated into particles, eg, cells or beads, the parallel channels may include particle retention regions 344. Typically, such retention zones will comprise porous gels or microstructures that do not pass through a particle sieve or filtration matrix, eg, particulate material but through which the flow of freed fluid passes.

Examples of microstructures of such filters include, for example, those described in US Pat. No. 5,304,487, which is incorporated herein by reference. In continuous systems, the fluid direction in more complex systems can generally be controlled using microfabricated fluid flow structures such as pumps and valves. However, the more complex the system, the more difficult this system is to control. Thus, the electroosmotic systems described herein are generally desirable for controlling such more complex systems. Typically, such systems couple electrodes in reservoirs located at the ends of the various transverse channels to regulate fluid flow through the device. In another aspect, it is desirable to include an electrode at the ends of all the various channels. This generally provides more direct control and also augments to more complex systems that are difficult to control. In order to reduce potential complexity, using fewer electrodes, parallel systems are preferred, for example two fluids are preferably moved at similar speeds in parallel channels proximate the various flow channels. In particular, as the channel length increases, the resistance will also increase with the channel. As such, the flow length between the electrodes must be designed identically, regardless of the parallel path chosen. This generally suppresses the generation of transverse electric fields, promoting the same flow in all parallel channels. To achieve substantially the same resistance between the electrodes, the shape of the channel structure can be modified to provide channel resistance independent of the traveled path. Alternatively, the resistance of the channel can be adjusted by selectively changing the cross section of the path, thereby keeping the resistance level independent of the path.

As with the test compounds shown through their respective parallel reaction channels, they will be in contact with the biochemical system in question. As mentioned above, in particular, biochemical systems will generally comprise a flowable labeling system, which system may, for example, be soluble labeling or additives such as colored or fluorescent substances, labeled ligands, or the like, or The relative action of particle based signals such as bead binding signaling groups. The flowable indicator then flows through each parallel channel to the collection channel 308, where a series of signals from each parallel channel flows past the detection window 116.

Referring to Figure 3, Figures 4A-4F show test compounds and biochemical system components injected into a "serial injection parallel reaction" device, exposing the system to the test compound, and flowing the generated signals from the parallel reaction channels. The process of passing through the detection window is shown. In particular, FIG. 4A illustrates the introduction of a test compound immobilized on beads 346 through sample injection channel 304. Similarly, biochemical system components 348 are introduced into reaction channels 312-324 through seeding channels 306. As described above, the components of the model system to be screened are optionally introduced into the reaction channel during manufacture, although they are introduced into the device together with the test compound. In addition, these components are optionally provided in liquid or lyophilized form to increase the shelf life of certain screening devices.

As shown, the biochemical system components are embodied in a cell or particle based system, but fluid components can also be used as described herein. As the particulate component flows into the reaction channel, they are optionally retained on the optical particles retained by the matrix 344, as mentioned above.

4B depicts leaving the test compound from the beads 346 by exposing the beads to the release agent. As shown, the beads are exposed to light from a suitable light source 352, e.g., a light source that generates light, at a wavelength suitable for photodegrading the linker group, and is coupled to their respective beads through a photodegradable linker group. Release the compound.

In FIG. 4C, the released test compounds flow along parallel reaction channels as shown by arrow 354 until these compounds come in contact with the biochemical system components. The biochemical system components 348 then perform their functions in the presence of the test compound, such as enzymatic reactions, receptor / ligand interactions, and the like. When several components of the biochemical system are immobilized on a solid support, the components can be released from these supports to provide the effect of initiating the system. Thereafter, a soluble signal 356 is generated corresponding to the functionalization of the biochemical system. As described above, the change in signal level that occurs is an indication that a particular test compound is an effector of a particular biochemical system. This is illustrated by the brightly painted signal 358.

4E and 4F, the soluble signal then flows from reaction channels 312-324 to detection channel 308 and passes through detection window 116 along the detection channel.

Again, a detection system as described above, located adjacent to the detection window, will monitor the signal level. In some embodiments, the beads containing the test compound are selectively regenerated to identify the test compound present thereon. This is generally accomplished by incorporation of the labeling group during the synthesis of the test compound on the beads. As shown, the spent beads 360, ie, the beads from which the test compound is released, are optionally transported from the channel structure through a port 362 to identify the test compound bound thereto. This identification is accomplished outside of the device by selectively inducing beads in the fragment collector, where test compounds present on the beads are selectively identified through identification of the labeling group or identification of residual compounds. The incorporation of labeled groups by a binding chemistry method has already been described as using chlorinated / fluorinated aromatic compounds or potential nucleotide sequences as labeling groups. Alternatively, the beads may optionally be transported to individual assay systems within the device itself when verification is performed.

FIG. 6A illustrates another embodiment of a “serial injection parallel reaction” device, which device can be used for fluid substrates as opposed to bead based systems. As shown, the apparatus 600 includes two or more transverse channels, as shown in FIGS. 3 and 4, that is, a sample injection channel 604 and a detection channel 606. These transverse channels are interconnected by a series of parallel channels 612-620 connecting the sample channel 604 to the detection channel 606.

The device shown also includes an additional channel set for directing the flow of fluid test compound into the reaction channel. In particular, the additional transverse pumping channel 634 is fluidly connected to the sample channel 604 by a series of parallel pumping channels 636-646. The pumping channel includes reservoirs 650 and 652 at its ends. The intersection of parallel channels 636-646 is staggered from the intersection of parallel channels 612-620 and sample channel 604, ie between them. Similarly, the transverse pumping channel 608 is connected to the detection channel 606 by parallel pumping channels 622-632. Again, the intersection of parallel pumping channels 622-632 and detection channel 606 is staggered at the intersection of reaction channels 612-620 and detection channel 606.

A schematic diagram illustrating the operation of the present system is shown in FIGS. 6B-BC. As shown, a series of test compounds physically separated from separate test material regions are introduced into the sample channel 604 using the method described above. For an electroosmotic system, a voltage is applied to the reservoir 648 as well as the sample channel 604. Voltage is also applied to reservoirs 650: 652, 654: 656, and 658: 660. This allows zero net flow through a parallel channel array that flows fluid along the transverse channels 634, 604, 606 and 608 as shown by arrows and interconnects these transverse channels as shown in FIG. 6B. flow. As shown by the dark regions in FIG. 6B, a region of test substance containing the test compound is arranged in parallel reaction channels 612-620, sample channel parallel reaction channels 612-629, and the sample channel 604. Connection to the detection channel 606 stops the flow in all transverse directions by removing the applied voltage from the reservoir at the ends of these channels. As described above, the structure of the channel can be varied by maximizing the use of space on the substrate. For example, if the sample channel is straight, the distance between the reaction channels (and thus the number of parallel reactors that can be performed on the size limiting substrate) is defined by the distance between the test material regions. However, this limitation can be solved by changing the channel structure. For example, in some cases, the length of the first and second spacer regions can be accommodated by serpentine, square wave, sawtooth, or other reciprocating channel structures. This allows for the maximum number of reaction channel packings in a limited area of the substrate surface.

When the parallel reaction channels are aligned, the sample or test material then applies the first voltage to the reservoirs 650 and 652 and simultaneously applies the second voltage to the reservoirs 658 and 660 by parallel reaction channels 612-. 620, whereby the fluid flow through the parallel pumping channels 636-646 advances the test material into the parallel reaction channels 612-620 as shown in FIG. 6C. During this process, no voltage is applied to the ends of the reservoirs 648, 654, 656 or the chapel channel 604. Parallel channels 636-646 and 622-632 are generally length-adjusted so that the overall channel length, and thus the resistance levels from reservoirs 650 and 652 to channel 604, and reservoirs 658 and 660. The resistance level from to channel 606 will be the same in any path used. The resistance can generally be adjusted by adjusting the length or width of the channel. For example, the channel can be extended by including a fold or serpentine structure. Although this is not shown, to extend this channel length, channels 636 and 646 are the longest, and channels 640 and 642 are the shortest to generate a symmetrical flow, thereby advancing the sample to the channel. As can be seen, while the samples flow through channels 612-620, the resistances in these channels will be the same, as the individual channel lengths are the same.

After the reactant material is screened, the test substance region / signal member is moved to the detection channel 606 by applying a voltage from reservoirs 650 and 652 to reservoirs 658 and 660 and at the same time in the remaining reservoirs. The voltage is floating. Thereafter, the test substance region / signal is continuously moved past the detection window / detector 662 by applying a voltage to the reservoirs 654 and 656, while applying sufficient voltage to the ends of the remaining transverse channels in parallel. Suppress flow along the channel. Although shown as channels crossing at right angles, it will also be appreciated that other structures are suitable for continuous injection parallel reactions. For example, US patent application Ser. No. 08 / 835,101, filed Apr. 4, 1997, describes an advantage in channel and parabolic structures that vary in width to control fluid flow. In summary, the fluid flow in an electroosmotic system is controlled by the current flow between the electrodes and is thus related. The resistance in the fluid channel can vary as a function of the length and width of the path, so that channels of different lengths have an intermediate resistance. This resistance difference is not corrected, which can result in the generation of a lateral electric field, which can inhibit the device's ability to direct fluid flow to a particular area. The current, and thus fluid flow, follows the path of least resistance, for example the shortest path. Although this problem of transverse electric fields is solved by using a separate electrical system at the end of each parallel channel, i. E. A separate electrode, it is possible to control the device comprising all of these electrodes and the voltage applied to each of these electrodes. Producing a control system for this can be complicated, especially when dealing with hundreds to thousands of parallel channels in a single small scale device, for example 1-2 cm 2. Accordingly, the present invention provides a microfluidic device that affects continuous parallel switching by ensuring that current flow through each of the multiple parallel channels is at a suitable level to ensure the desired flow pattern through these channels or channel networks. will be. There are several suitable methods and substrate / channel types to achieve this goal.

In one example of a parabolic structure of a channel according to the device of the invention, the substrate comprises a main channel. The series of parallel channels terminates in the main channel. Opposite ends of these parallel channels connect parabolic channels. The current flow in each parallel channel is kept constant or even by adjusting the length of the parallel channel so that the channel structure connecting each parallel channel to their respective electrodes becomes parabolic. The voltage drop in the parabolic channel between the parallel channels is kept constant by adjusting the channel width to accommodate various channel currents due to the parallel current paths generated by these parallel channels. The parabolic shape of the channels is combined with their tapering structure to equalize the resistance along all parallel channels, thereby making the fluid flow the same regardless of the path selected. In general, the determination of the channel dimensions to favorably control the resistance between the channels can be performed by known methods and generally depends on factors such as the nature of the fluid being moved through the substrate.

While the description of the present invention generally describes a screening assay for identifying compounds that affect a particular interaction, the microexperimental systems described above must also affect the interaction between components and other members of the biochemical system. It can be used to screen compounds that specifically interact with components of the biochemical system that do not extend. Such compounds generally include binding compounds that can be used generally in the diagnostic and therapeutic art, for example, as therapeutic or marker groups, ie target groups for radionuclides, dyes, and the like. For example, such a system can optionally be used to screen test compounds that can bind a given component of a biochemical system.

II. Micro Experimental System

For ease of operation, although generally described for individual discrete devices, the described system is generally part of a larger system and can monitor and adjust the function of the device on each base or in parallel with several screen devices. have. An example of such a system is shown in FIG.

As shown in FIG. 7, the system may include a test compound processing system 700. The system shown includes a base 702 that can support a number of individual assay chips or devices 704. As shown, each chip includes a number of separate assay channels 706, each of which has a separate interface 708, such as a pipette, for introducing test compounds into the device. pipetter). The interface is used for sipping the test compound into the device, and first and second spacer fluids separated by the sipping are introduced into the device. In the illustrated system, the interface of the chip is inserted through the opening 710 from the bottom of the base 702, which base can be raised and lowered so that the interface is under the base, for example, on the conveyor system 712. Contact with a test compound or a wash / first spacer fluid / second spacer fluid contained in a multiwell microplate 711 located therein. In operation, a multiwell plate containing a large number of different test compounds is loaded 714 at one end of the conveyor system. The plate is placed on a separate conveyor by suitable buffer reservoirs 716 and 718 that can be filled into the buffer system 720. The plate is lowered from the conveyor and the test compound is sampled onto the chip and interspersed with a suitable spacer fluid region. After the test compound is loaded onto the chip, the multiwell plates are collected or loaded 722 at the end of the system. The overall control system includes a number of individual microexperimental systems or devices, as shown in FIG. Each device is connected to a computer system and properly programmed to control fluid flow and direction within the various chips and to monitor, record and analyze data obtained from screening assays performed by various devices. Such devices will typically be connected to the computer through an intermediate adapter module that provides an interface between the computer and each device to perform work instructions from the computer to the device and write data from the device to the computer. For example, adapters typically have suitable connections to the corresponding members of each device, e.g., electrical leads connected to electrodes in reservoirs used for electroosmotic fluid flow, power input and data output for detection systems, electrical Or optical relays, data relays for other sensor members incorporated into the device. The adapter device also provides periphery control for each device when control is needed, for example, to maintain each device at an optional temperature for performing a particular screening assay.

As shown, each device is also equipped with a suitable fluid interface, eg, a micropipette, for introducing test compounds into the individual devices. Such a device can be easily attached to a robotic system to allow test compounds to be sampled from multiple multiwell plates moving along a conveyor system. An intermediate spacer fluid region can also be introduced via the spacer solution reservoir.

III. Fluid electrode interface inhibits decomposition of chemical species in microchips

When pumping a fluid or other material by electroosmotic or electrophoresis through the device of the present invention, the species in the fluid may decompose when high voltage or current is applied, or when voltage is applied for a long time. Delaying the migration of the species from the electrode to the channel inlet or from the migration of the species to the electrode improves the performance of the chemical assay by reducing undesirable degradation of the species in the sample. This form is particularly desirable for assay systems in which voltage is applied for a long time, for example, from several hours to several days.

The shape of the electrode to reduce degradation of species in the assay of the present invention is shown in panel A-G of FIG. 12. This form delays the migration of the species from the electrode to the channel inlet, or the migration of the species to the electrode improves the performance of the chemical assay. 12 illustrates a representative electrode configuration, in which electrode 1211 is partially submerged in reservoir 1215 fluidly connected to fluid channel 1217.

In comparison, FIG. 12B utilizes salt bridges between frit 1219 and fluid reservoir 1221 fluidly connected to fluid channel 1223.

12C provides an electrode 1225 that is immersed in a first fluid reservoir 1227 fluidly connected to a second fluid reservoir 1229 by a large channel 1231 that limits diffusion but has low electroosmotic flow. This reduces the decomposition of the species.

12D provides a similar dichotomy reservoir wherein electrode 1235 is fluidically connected to a second fluid reservoir 1241 by a small channel 1243 that is processed to reduce or eliminate electroosmotic flow. 1 submerged in fluid reservoir 1237.

FIG. 12E provides another similar bin reservoir wherein electrode 1245 is submerged 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 to reduce electroosmotic flow.

12F provides a modified dichotomy reservoir wherein electrode 1255 is submerged in a first fluid reservoir 1257 fluidly connected to a second fluid reservoir 1259 by a channel 1261. The fluid height of the second fluid reservoir 1259 is higher than the fluid height of the first fluid reservoir 1257 to direct fluid toward the electrode 1255.

FIG. 12G provides another modified bifurcation reservoir wherein electrode 1265 is immersed in first fluid reservoir 1267 fluidly connected to second fluid reservoir 1269 by channel 1271. . The diameter of the first fluid reservoir 1267 is small enough that the capillary force draws the fluid sufficiently into the first fluid reservoir 1267.

The methods and apparatus as described herein can be changed without departing from the spirit or scope of the invention as claimed, and the invention can be applied to many different uses, including the following uses:

To test the effect of each of a plurality of test compounds on a biochemical system, one or more first substrates having a first channel and a second channel intersecting the first channel, one or more of these channels being one or more Use with microfluidic systems having a cross-sectional dimension of 0.1 to 500 μm.

Use as a microfluidic system as described above, wherein the biochemical system can flow substantially continuously through one of the channels to test a plurality of test compounds continuously.

Use as a microfluidic system as described above, which may provide a plurality of reaction channels in a first substrate to concurrently expose a plurality of test compounds to one or more biochemical systems.

Use as a microfluidic system as described above, wherein each test compound is physically separated from adjacent test compounds.

Use as a substrate transport cross-channel for screening test substances that affect the biochemical system by flowing the test substance and biochemical system while using the channel.

Use as a substrate as described above wherein at least one channel has at least one cross-sectional dimension of 0.1 to 500 μm.

An assay using either a microfluidic system or a substrate as described above.

The present invention provides an apparatus for detecting the effect of a test compound on a biochemical system, in particular comprising a substrate having at least one surface with a plurality of reaction channels assembled to the substrate. The device as described above has two or more transverse channels assembled to a surface, each of the plurality of reaction channels being fluidly connected to the first channel of the two or more transverse channels at a first point of each reaction channel, Fluidly connected to the second transverse channel at the second point, the assay device comprising a device as described above.

Example

The following examples are for illustrative purposes and are not intended to be limiting. Those skilled in the art will recognize that there are a number of non-essential parameters that can yield substantially similar results.

Example 1 Enzyme Inhibitor Screen

Enzyme inhibition assay screen performance was demonstrated in planar chip form. A six ball planar chip with an arrangement as shown in FIG. 8 was used. The figure next to the channel represents the length in mm of each channel. Two voltage states were applied to the spheres of the chip. The first state (state 1) allowed the enzyme with buffer from the upper buffer wells to flow into the main channel. The second voltage state (state 2) disrupted the flow of buffer from the top well and introduced the inhibitor from the inhibitor well to flow with the enzyme in the main channel. In addition, a control test was performed in which the buffer was placed in the inhibitor wells.

The voltages applied to the spheres of the two applied voltage states are as follows.

State 1 State 2 Upper buffer well (I) 1831 1498 Inhibitor Well (II) 1498 1900 Enzyme Well (III) 1891 1891 Substrate well (IV) 1442 1442 Bottom buffer wells (V) 1442 1442 Detection / Wellwell (VI) 0 0

To demonstrate the efficiency of the system, the following enzymes / substrates / inhibitors were used to screen inhibitors of β-galactosidase of screen inhibitors:

Enzyme: β-galactosidase (180 U / mL in 50 mM Tris / 300 μg / mL BSA)

Substrate: Fluorescein-Dicalactoside (FDG) 400μM

Inhibitor: IPTG, 200 mM

Buffer: 20 mM Tris, pH 8.5

Enzyme and substrate were pumped through the main channel from each sphere under two voltage conditions. Inhibitors or buffers were alternately fed from each well to the subject by changing between voltage state 1 and voltage state 2. When no inhibitor was present at the detection end of the main channel, a baseline level of fluorescence product was produced. Upon introduction of the inhibitor, the fluorescence signal was greatly reduced, indicating inhibition of the enzyme / substrate interaction. Fluorescence data obtained from alternating supply of inhibitor and buffer to the main channel is shown in FIG. 9A. 9B, which overlays two data portions from FIG. 9A, compares inhibitor data directly with control (buffer) data. The control clearly shows that there is only a slight variation in the fluorescence signal by dilution of the enzyme substrate mixture, while the inhibitor screen shows a large decrease in the fluorescence signal indicating clear inhibition.

Example 2 Screening of Multiple Test Compounds

Assay screens were performed to identify inhibitors of the enzymatic reaction. A schematic of the chip to be used is shown in FIG. The chip has a 5 cm long reaction channel, 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 an enzyme solution and the side reservoir is filled with a fluorinating substrate. Each enzyme and substrate is diluted to provide a steady state signal at the detector with a linear signal range for the assay system. Voltage was applied to each reservoir (sample source, enzyme, substrate and waste) to achieve an applied electric field of 200 V / cm. This applied electric field resulted in a flow rate of 2 mm / sec. While the established sample passes through the chip, the sample will generally spread out and become wider. For example, for samples of low molecular weight, a benzoic acid diffusion range of about 0.38 mm and an electrophoretic shift of 0.4 mm, for example, were observed.

A test material region containing a test compound of 150 mM NaCl was introduced into the sample channel separated by a first spacer region of 150 mM NaCl and a second spacer region of 5 mM borate buffer. When introduced into the sample channel shown, the test material region required 12 seconds to shift the length of the sample channel to reach the incubation region of the reaction channel. This is the result of a flow rate of 2 mm / sec, which allows 1 second to move the sample pipettor from the sample to the spacer compound. To allow this interruption, the actual flow rate is 0.68 mm / sec. Another 12 seconds are required to move the mixture of enzyme / test compounds through the incubation zone to the junction of the substrate channel, where the substrate continues to flow into the reaction zone of the reaction channel. Each test substance region containing the test compound then requires 48 seconds to shift the length of the reaction region and pass the fluorescence detector. A schematic of the timing for the test material region / spacer region loading is shown in FIG. 11. The top panel shows the distribution of test material / first spacer area / second spacer area in the channel, and the bottom panel shows the timing required to load the channel. As shown, this schematic includes the loading (sipping) of the high salt (HS) first spacer fluid ("A"), the movement of the pipette ("B") to the sample or test substance, and the sipping of the sample or test substance (" C ″), pipette move to high salt first spacer fluid (“D”), pipette move to first spacer fluid (“E”), low salt (LS), or second spacer fluid (“F”), Two spacer fluid shiping ("G") and return of the first spacer fluid ("H"). This process is then repeated for each additional test compound.

A constant basic fluorescence signal is established at the detector in the absence of the test compound. Upon introduction of the test compound, it can be seen that the decrease in fluorescence is similar to that shown in FIGS. 9A and 9B, corresponding to the particular individual test compound over time. This test compound was uncertainly identified as an inhibitor of the enzyme, and further tests were conducted to confirm this and quantify the efficacy of this inhibitor.

While the invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art that various changes in form and detail may be made therein without departing from the true scope of the invention. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes, with equivalent qualifications indicating that each individual publication or patent document is very individual.

Claims (97)

A device for screening test compounds that affect a biochemical system, A substrate having one or more surfaces, At least two intersecting channels of at least one of the at least two intersecting channels, the at least one intersecting channel configured on the surface of the substrate having at least one cross-sectional dimension of from about 0.1 to about 500 microns A source of a plurality of different test compounds fluidly connected to the first cross channel of the at least two cross channels, A source of one or more components of the biochemical system fluidly connected to a second of the two or more cross channels, A fluid induction system for flowing one or more components in a second crossover channel of the two or more crossover channels and introducing different test compounds from the first crossover channel of the two or more crossover channels to the second crossover channel, Cover bonded with the surface, And a detection region in a second channel for detecting the effect of the test compound on the biochemical system. The method of claim 1, wherein the fluid induction system continuously flows at least a first component along a second of the two or more cross channels and periodically injects a test compound from the first channel to the second channel. Device. The method of claim 1, further comprising a third channel that fluidly couples a second component of the biochemical system, a third channel configured on the surface, and at least one of the two or more crossover channels with a source of the second component of the biochemical system. Containing device. The method of claim 3, wherein the fluid induction system continuously flows the mixture of the first component and the second component along a second cross channel of the two or more cross channels, periodically injecting a test compound from the first channel to the second channel. Device characterized in that. The device of claim 1, wherein the fluid induction system continuously flows a plurality of different compounds, each separated by a fluid spacer, from the first channel to the second channel of the at least two cross channels. The system of claim 1 wherein the fluid induction system is Three or more electrodes, each electrode in electrical contact with the two or more crossing channels at one different side of the intersection formed by the two or more crossing channels, and And a control system in which the movement of at least two test compounds or at least a first component in the cross-channel is controlled by simultaneously applying a variable voltage to each electrode. 2. The apparatus of claim 1, wherein the detection system includes a detection window in the second channel. 8. The apparatus of claim 7, wherein the detection system is a fluorescence detection system. The apparatus of claim 1 wherein the substrate is planar. 10. The apparatus of claim 1, wherein the substrate comprises etched glass. 2. The apparatus of claim 1, wherein the substrate comprises etched silicon. The apparatus of claim 1, further comprising a thermal insulation layer disposed on the etched silicon substrate. The apparatus of claim 1 wherein the substrate is a molded polymer. The device of claim 1, wherein at least one component of the biochemical system comprises an enzyme and a substrate that generates a detectable signal when reacted with the enzyme. 15. The device of claim 14, wherein the substrate is selected from the group consisting of chromogenic substrates and fluorogenic substrates. The device of claim 1, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, and at least one of the receptor and ligand has an associated detectable signal. The device of claim 1, wherein the first component of the biochemical system comprises a receptor / ligand binding pair, wherein the binding of the receptor to the ligand generates a detectable signal. 2. The system of claim 1, further comprising a plurality of electrodes in a plurality of reservoirs fluidly connected to one or more cross channels and a control system for simultaneously controlling the movement of the first component in the two or more cross channels by applying a voltage to each electrode. Containing device. 19. The device of claim 18, wherein the device minimizes degradation of species present in the reservoir or cross channel. The method of claim 19, Frits on one or more electrodes that reduce the electroosmotic flow to one or more electrodes A large channel located between two or more reservoirs, which limits the spread of the species and has a low electroosmotic flow rate, A small channel located between two or more reservoirs, which limits the spread of the species and is treated to reduce the electroosmotic flow rate, This allows a filled channel located between two or more reservoirs, having a low electroosmotic flow rate, by including a matrix to limit the transport of species, A high level reservoir having a fluid level higher than at least one low reservoir comprising an electrode, fluidly connected to the low reservoir, wherein the hydraulic pressure between the high reservoir and the low reservoir reduces the electroosmotic flow to the electrode side, and It is applied to receive one of the plurality of electrodes and is applied fluidly through the connecting channel to a second narrow diameter reservoir which is applied to draw the fluid by capillary electrophoresis to one electrode to count the electroosmotic flow rate in the connecting channel. And at least one component for reducing the electrical osmotic flow rate, selected from the group consisting of dual reservoir systems with connected first reservoirs. A device for detecting the effect of a test compound on a biochemical system, A substrate having one or more surfaces, Multiple reaction channels configured on the surface, Each of the plurality of reaction channels is in fluid communication with a first channel of two or more transverse channels at a first point of the reaction channel, and in fluid communication with a second channel of two or more transverse channels at a second point of the reaction channel; At least two transverse channels and at least two reaction channels each having at least one transverse channel having at least one cross-sectional dimension of from about 0.1 to about 500 microns, A source of one or more components of the biochemical system, fluidly connected to each of the plurality of reaction channels, A source of test compound fluidly connected to the first of the two or more transverse channels, A fluid induction system for controlling the movement of test compounds and components in two or more transverse channels and in multiple reaction channels, Cover bonded with the surface, An apparatus comprising a detection system for detecting the effect of a test compound on a biochemical system. The system of claim 21 wherein the fluid control system is A plurality of individual electrodes in electrical contact with each end of the at least two transverse channels, and And a control system for controlling the movement of the test compound and at least the first component in the two or more transverse channels and the plurality of reaction channels by simultaneously applying a variable voltage to each electrode. 22. The apparatus of claim 21, wherein each of the plurality of reaction channels comprises a bead stop well at a first point in the plurality of reaction channels. 22. The method of claim 21, wherein a source of one or more components of the biochemical system is fluidly connected to the plurality of reaction channels by a third transverse channel, the third transverse channel having one or more cross-sectional dimensions of 0.1 to 500 [mu] m. Device fluidly connected with each of the plurality of reaction channels at the third point of the channel. 22. The device of claim 21, wherein the third point of the reaction channel is halfway between the first point and the second point of the reaction channel. 27. The apparatus of claim 25, further comprising a particle retention region of each of the plurality of reaction channels between the third and second points of the plurality of reaction channels. 27. The apparatus of claim 26, wherein the particle retention region comprises a particle retention matrix. 27. The apparatus of claim 26, wherein the particle retention region comprises a microstructured filter. 22. The apparatus of claim 21, wherein the plurality of reaction channels comprise a plurality of parallel reaction channels configured on the surface of the substrate, wherein at least two transverse channels are connected to opposite ends of each parallel reaction channel. 22. The apparatus of claim 21, wherein at least two transverse channels are each configured on the substrate surface of the inner and outer concentric channels, and the plurality of reaction channels extend radially from the inner concentric channels to the outer concentric channels. 31. The apparatus of claim 30, wherein the detection system comprises a detection window in the second channel. 31. The apparatus of claim 30, wherein the detection system is a fluorescence detection system. 22. The apparatus of claim 21 wherein the substrate is planar. 22. The apparatus of claim 21, wherein the substrate comprises etched glass. 22. The apparatus of claim 21, wherein the substrate comprises etched silicon. 22. The apparatus of claim 21 further comprising a thermal insulation layer disposed on the etched silicon substrate. 22. The apparatus of claim 21 wherein the substrate is a molded polymer. 22. The device of claim 21, wherein at least one component of the biochemical system comprises an enzyme and a substrate that generates a detectable signal when reacted with the enzyme. 39. The device of claim 38, wherein the substrate is selected from the group consisting of chromogenic substrates and fluorinating substrates. The device of claim 21, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, and at least one of the receptor and ligand has an associated detectable signal. 22. The device of claim 21, wherein the first component of the biochemical system comprises a receptor / ligand binding pair, wherein the binding of the receptor to the ligand generates a detectable signal. In a method of determining whether a sample contains a compound that can affect a biochemical system, Providing a substrate having at least a first surface, and at least two cross channels configured on the first surface, at least one of the at least two cross channels having at least one cross-sectional dimension of 0.1 to 500 μm, Flowing a first component of a biochemical system of a first channel of at least two cross channels, Flowing the sample from the second channel to the first channel to contact the sample with the first component of the biochemical system; and Detecting the effect of one or more samples on the biochemical system. 43. The method of claim 42, wherein at least the first component of the biochemical system comprises at least one component of the antibody / antigen binding pair, and wherein the antibody specifically immunoreacts to the antigen. 43. The method of claim 42, wherein at least the first component of the biochemical system comprises an antibody and an antigen that specifically reacts with the antibody. 43. The method of claim 42, wherein one of the antibody and the antigen comprises a detectable labeling group. 43. The method of claim 42, wherein at least the first component of the biochemical system comprises one or more components of the receptor / ligand binding pair. 43. The method of claim 42, wherein at least the first component of the biochemical system comprises a receptor and a ligand capable of specifically binding to the ligand. 43. The method of claim 42, wherein the sample is derived from the patient. 49. The method of claim 48, wherein the sample is blood derived. The method of claim 42, wherein the detecting step comprises measuring a parameter of the biochemical system in the presence and absence of the sample, and comparing the parameter measured in the presence of the sample with the parameter measured in the absence of the sample, Wherein the change in the parameter demonstrates the effect of the sample on the biochemical system. A device for screening test compounds that affect a biochemical system, A substrate having one or more surfaces, A substrate having at least one surface and comprising at least two cross channels configured on the surface of the substrate, wherein at least one cross channel of the at least two cross channels has at least one cross-sectional dimension of from about 0.1 to about 500 μm, A source of sample fluidly connected to the first of the two or more cross channels, A source of one or more components of the biochemical system fluidly connected to a second of the two or more cross channels, A fluid induction system for flowing one or more components in a second crossover channel of the two or more crossover channels and introducing different test compounds from the first crossover channel of the two or more crossover channels to the second crossover channel, A cover bonded to the surface, and And a detection region in the second channel for detecting the effect of the test compound on the biochemical system. In the method for screening a plurality of test compounds that affect the biochemical system, Providing a substrate comprising at least a first surface and at least two cross channels configured on the first surface, wherein at least one cross channel of the at least two cross channels has at least one cross-sectional dimension of about 0.1 to about 500 μm, Flowing a first component of a biochemical system of a first channel of at least two cross channels, Flowing at least the first test compound from the second channel to the first channel to contact the first test compound with the first component of the biochemical system, and Detecting the effect of at least the first test compound on the biochemical system. 53. The method of claim 52, wherein at least the first component of the biochemical system generates a detectable signal indicative of the function of the biochemical system. 53. The method of claim 52, wherein at least the first component of the biochemical system further comprises an indicator compound that interacts with the first component to generate a detectable signal indicative of the function of the biochemical system. 53. The method of claim 52, wherein at least the first component of the biochemical system comprises an enzyme and a substrate for the enzyme, and wherein the action of the enzyme on the substrate generates a detectable signal. 53. The method of claim 52, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, and at least one of the receptor and ligand has an associated detectable signal. 53. The method of claim 52, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, wherein the binding of the receptor to the ligand generates a detectable signal. 53. The method of claim 52, wherein at least the first component of the biochemical system is a biochemical barrier and the effect of at least the first component is the barrier penetrating ability of the test compound. 59. The method of claim 58, wherein the barrier is selected from the group consisting of epithelial and endothelial layers. 53. The method of claim 52, wherein at least the first component of the biochemical system comprises the cell and the detecting step comprises measuring the effect of the test compound on the cell. 61. The method of claim 60, wherein the cell is capable of generating a detectable signal corresponding to the cell function, and wherein the detecting step detects the effect of the test compound on the cell function by detecting the level of the detectable signal. 61. The method of claim 60, wherein the detecting step comprises detecting the effect of the test compound on the viability of the cell. In the method for screening a plurality of test compounds that affect the biochemical system, Providing a substrate comprising at least a first surface and at least two cross channels configured on the first surface, wherein at least one cross channel of the at least two cross channels has at least one cross-sectional dimension of about 0.1 to about 500 μm, Continuously flowing a first component of the biochemical system in a first channel of at least two cross channels, Periodically introducing different test compounds from the second channel of the at least two cross channels into the first channel, and Detecting the effect of at least the first test compound on the biochemical system. 64. The method of claim 63, wherein the step of periodically introducing comprises flowing a plurality of different test compounds physically separated from each of a plurality of different test compounds from a second channel of two or more cross channels from the second channel to the first channel. How to. 64. The method of claim 63, wherein at least a first component of the biochemical system generates a detectable signal indicative of the function of the biochemical system. 66. The method of claim 65, wherein the detecting step further comprises monitoring a detectable signal that is steady state intensity from the first component continuously flowing at a point in the first channel, wherein the interaction between the first component and the test compound And the effect on the deviation from the steady state strength of the detectable signal. 66. The method of claim 65, further comprising an indicator compound wherein at least the first component interacts with the first component to generate a detectable signal indicative of the function of the biochemical system. 68. The method of claim 67, wherein at least a first component of the biochemical system comprises an enzyme, the indicator compound comprises a substrate for the enzyme, and the action of the enzyme on the substrate generates a detectable signal. 66. The method of claim 65, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, and at least one of the receptor and ligand has an associated detectable signal. 70. The method of claim 69, wherein the receptor and the ligand flow along the first channel at different flow rates. 66. The method of claim 65, wherein the first component of the biochemical system comprises a receptor / ligand binding pair, wherein the binding of the receptor to the ligand generates a detectable signal. 64. The method of claim 63, wherein the first component of the biochemical system comprises the cell and the detecting step comprises measuring the effect of the test compound on the cell. 73. The method of claim 72, wherein the cell is capable of generating a detectable signal corresponding to the cell function, and wherein the detecting step detects the effect of the test compound on the cell function by detecting the level of the detectable signal. 73. The method of claim 72, wherein the detecting step comprises detecting the effect of the test compound on the viability of the cell. In the method for screening a plurality of different test compounds that affect the biochemical system, Providing a plurality of reaction channels configured at least on the first surface and the first surface, wherein each of the plurality of reaction channels is fluidly connected to at least two transverse channels configured on the surface, Introducing the first component of the biochemical system into the plurality of reaction channels, Flowing a plurality of different test compounds through at least one of the two or more transverse channels, each of which test compounds being introduced into one or more transverse channels of an individual test substance region, Inducing each of a plurality of different test compounds into a separate one of the plurality of reaction channels, and Detecting the effect of each test compound on one or more components of the biochemical system. 76. The method of claim 75, wherein at least a first component of the biochemical system generates a flow detectable signal indicative of the function of the biochemical system. 77. The detectable fluidity signal of claim 76, wherein the detectable fluidity signal generated in each of the plurality of reaction channels flows through and passes through the second transverse channel, wherein each detectable fluidity signal generated in each of the plurality of reaction channels is detectable fluidity. Physically separated from each other from the signal, wherein each detectable fluidity signal is detected separately. 77. The method of claim 76, wherein the flow signal comprises a soluble signal. 79. The method of claim 78, wherein the soluble signal is selected from fluorescent and colorimetric signals. 76. The method of claim 75, further comprising an indicator compound wherein at least the first component interacts with the first component to generate a detectable signal indicative of the function of the biochemical system. 81. The method of claim 80, wherein at least the first component of the biochemical system comprises an enzyme, the indicator compound comprises a substrate for the enzyme, and the action of the enzyme on the substrate generates a detectable signal. 76. The method of claim 75, wherein at least the first component of the biochemical system comprises a receptor / ligand binding pair, and at least one of the receptor and ligand has an associated detectable signal. 76. The method of claim 75, wherein the first component of the biochemical system comprises a receptor / ligand binding pair, wherein the binding of the receptor to the ligand generates a detectable signal. 76. The method of claim 75, wherein the first component of the biochemical system comprises a cell and the detecting step comprises measuring the effect of the test compound on the cell. 85. The method of claim 84, wherein the cell is capable of generating a detectable signal corresponding to the cell function, and wherein the detecting step detects the effect of the test compound on the cell function by detecting the level of the detectable signal. 86. The method of claim 85, wherein the detecting step comprises detecting the effect of the test compound on the viability of the cell. 76. The method of claim 75, wherein each of the plurality of different test compounds is immobilized on separate beads and inducing each of the plurality of different test compounds into a separate one of the plurality of reaction channels Receiving one of the individual beads at the intersection of the plurality of reaction channels and controllably releasing the test compound from each individual bead in each of the plurality of reaction channels. To test the effect of each of a plurality of test compounds on a biochemical system, one or more first substrates having a first channel and a second channel intersecting the first channel, one or more of these channels being one or more A method of using a microfluidic system having a cross-sectional dimension of 0.1 to 500 μm. 89. The method of claim 88, wherein the biochemical system can flow through one of the channels substantially continuously, thereby testing a plurality of test compounds continuously. 90. The method of claim 88 or 89, wherein a plurality of reaction channels can be provided in the first substrate to expose the plurality of test compounds in parallel to one or more biochemical systems. 91. The method of any one of claims 88, 89 and 90, wherein each test compound is physically separated from adjacent test compounds. A method of using a substrate carrying cross channel to screen a test substance that affects the biochemical system by flowing the test substance and the biochemical system while using the channel. 93. The method of claim 92, wherein at least one cross sectional dimension of the at least one channel is between 0.1 and 500 microns. 93. An assay method using the method of any of claims 88-93. An apparatus for detecting the effect of a test compound on a biochemical system, comprising a substrate having at least one surface, the substrate having a plurality of reaction channels. 97. The apparatus of claim 95, having two or more transverse channels assembled to a surface, each of the plurality of reaction channels being fluidly connected with a first of the two or more transverse channels at a first point of each reaction channel, And fluidly connect to the second transverse channel at a second point of the reaction channel. 96. An assay device comprising the device according to claim 1 or 95.