KR100453100B1 - High throughput screening assay system in microfluidic devices - Google Patents

Abstract

The present invention provides microfluidic devices and methods useful for performing high power screening assays. In particular, the devices and methods of the present invention are useful for screening many different compounds for effects on various chemical, preferably biochemical systems. The apparatus of the present invention includes a series of channels 110, 112, and any reagent channel 114 configured on the surface of the substrate. One or more such channels will generally have a very small cross-sectional dimension, for example from about 0.1 μm to about 500 μm. The apparatus of the present invention also includes reservoirs 104, 106 and 108 disposed at the ends of channels 110 and 114 and fluidly connected. As shown, sample channel 112 is used to introduce a number of different test compounds into the device. As such, the channels are generally fluidly connected to a plurality of sources of individual test compounds to introduce the test compounds individually into the same channel 112 and then into the channel 110.

Description

High Throughput Screening Assay System in Microscale Fluid Devices

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.

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 been interested in identifying compounds that can inhibit, reduce or even enhance the interaction 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 for 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 drugs. Significant interest in combinatorial chemistry and related technologies for generating and evaluating molecular diversity represents a significant milestone in the development of this model in 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 yields of solid phase coupling chemistry and synergy with biological methods for producing 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 realistic perception has recently led experimenters to extend the notion 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 RNAs) whose screening efficacy has also been screened by human genomic studies, such as the rapid increase in the number of test compounds that can be screened by modern combinatorial chemistry. Genes and gene products) 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 most 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, in fluid handling equipment and methods it has typically been impossible 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 associated equipment and apparatus that can perform repeat and accurate assay screening and can be operated in very low volumes.

The present invention fulfills these 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 in the first of the 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 comprise continuously flowing 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 one or more first surfaces with a plurality of reaction channels formed in the first surface. Each multiple reaction channel is also fluidly connected to two or more transverse channels formed within the surface. At least the first component of the biochemical system is introduced into a plurality of reaction channels, and a 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. The apparatus of the present invention also includes a source of different test compounds fluidly connected to a first channel of two or more cross channels and a source of one or more components of a biochemical system fluidly connected to a second channel of two or more cross channels. do. 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 into the second channel. In addition, the device of the present invention optionally includes a detection region in a 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 the minimum 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 apparatus 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 the first of the two or more transverse channels at a first point in each reaction channel, At a second point in each reaction channel is fluidly connected to the second transverse 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 of the transverse channels, 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 for detecting the effect of the test compound on the biochemical system.

1 schematically illustrates 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 depicts a “serial injection parallel reaction” microassay system in which 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 long term incubation.

6A schematically depicts a series of injection parallel reaction apparatus for use with a fluid based 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 chip arrangements used in a continuous flow assay screening system.

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

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

One. Application Field 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 microfluidic devices, and methods of using such devices to screen their effects on a variety of chemicals, preferably on 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, cell or membrane fractions) for receptor-ligand interactions, enzyme-substrate interactions, cell signaling pathways, bioavailability screenings. Transport reactions, and a variety of 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 materials listed above, for example polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, Polyimides 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, FM Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. And John Wiley & Sons, Inc., (through 1997 Supplement) (Ausubel); Watson et al. (1987) Molecular Biology of the Gene , Fourth Edition The Benjamin / Cummings Publishing Co., Menlo Park, 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, WH 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 also provides useful information for assaying biological systems, such as, for example, SIGMA chemical company (St. Louis, MO), R & D Systems (Minneapolis, Minnesota), Pharmacia LKB Pharmacia LKB Biotechnology (Pittscatware, NJ), CloneTech Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Aldrich Chemical Company (Milwaukee, WY), Glen Research, Inc., Glen Research, Inc., Gibco BRL Life Technologies, Inc. (GIBCO BRL Life Technologies, Inc., Gaiusburg, Maryland), Fluka Chemica-Biochemica Analytika (Buys, Switzerland), Invitrogen (San Diego, Calif.) And Applied Biosystems (Foster City, Calif.), 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. Subsequent tests can then be conducted on the compounds that show promising results in these screening assay methods to identify agents effective for the treatment of the disease or symptoms 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 a particular biochemical system in vitro, potential effectors for this system in vivo can be identified.

The biochemical system model used in the methods and devices of the present invention in its simplest form is intended for the effect of test compounds on the interaction between two components of the biochemical system, for example receptor-ligand interactions, enzyme-substrate interactions, and the like. Will be screened for. This type of biochemical system model will typically include two normal interacting components of the system to which the effector has been irradiated, such as 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, such as receptor-ligand binding or substrate turnover. do. 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 system function, for example the presence or absence of a labeling group (eg, in a binding reaction, transport screen) or a change in molecular weight, (in substrate turnover measurements) Change in the presence or absence of a reaction product or substrate or electrophoretic mobility (typically detected by a change in the 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 the effect of cellular responses, whole cells are optionally used. In addition, modified cell systems can be used in the screening systems included herein. For example, chimeric reporter systems are optionally used as indicators of test compound effects on certain biochemical systems. Chimeric reporter systems typically include heterogeneous reporter systems that are integrated into signaling pathways that signal binding of receptors to ligands. For example, the receptor is melted with a heterologous protein, for example an enzyme that can be easily assayed for activity. The heterologous protein subsequently detected is activated by activation of the receptor by ligand binding. As such, the alternative reporter system assays for receptor / ligand binding by generating a signal or result 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, FGF, PDGF, and their receptors, may, for example, promote cell proliferation and fractionation, activation of mediated 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, by regulating the interaction of its ligands with receptors, it is possible to modulate the biological response induced by this interaction.

Thus, in one aspect, the present invention will be useful for screening for compounds that affect the interaction between its ligand and receptor molecules. 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 considered to be an inhibitor of receptor / ligand binding. If binding is seen to be increased, the test compound is considered to be an enhancer or inducer of interaction.

In terms of efficiency, screening assays commonly performed in multi-well reaction plates, such as multi-well microplates, where concurrency is acceptable, are comparable to the screening of a large number of test compounds.

Similar, 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 enzyme activity to a substrate of an enzyme are screened by contacting the enzyme with a 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 with a control, ie, an enzyme contacted with the substrate in the absence of a 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", whereas compounds that make the activity prominent are called "derivatives".

In general, the 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. Test compounds are provided in accordance with certain embodiments to be practiced, for example injected in free form in solution, or optionally attached to a barrier, or solid support, such as beads. Many suitable solid supports are used to immobilize the test compound. 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 measure binding of labeled ligands to pathogens or pathogens or tumor components 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 antibody binding from a patient's blood to viral proteins such as HIV proteins is a common test to measure 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 the biological material is derived from animals other than humans, 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, e.g., 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 a device generally depends on the type of assay 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 electrical material delivery system" refers to the delivery and delivery of material between them through channels and / or chambers, by transporting and directing materials into structures containing channels and / or chambers connected to each other through electric field applications. While the cations are transported to the cathode, they include a system that causes material transport to transport the anions to the cathode.

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 specifically termed an 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 against the structure is applied. In brief, when the fluid is introduced into an etched glass channel or a channel having a surface with charged functional groups, for example hydroxyl groups, in the 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, creating a concentration of protons near the fluid / surface interface, or enclosing a large amount of fluid in the channel. To form a charged sheath. Application of the voltage gradient to the length of the channel will cause the proton sheath as well as the fluid it surrounds to be carried in the direction of the voltage drop, ie towards the cathode.

As used herein, “adjusted electrokinetic material transport and derivation” relates to an electrokinetic system that actively regulates the 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 comprise a body structure comprising two or more cross channels or fluid conduits, for example a closed chamber 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 the 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 inhibiting flow from other channels at the intersection. It works to induce 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 can be 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 a lower second voltage at the right end of the channel, or , By floating the right end (no voltage applied). However, this type of mass flow through these cross sections results in significant diffusion at the cross sections, which results from the convective effect at the cross sections as well as the natural diffusion properties of the materials carried in the medium used.

In controlled electrokinetic material delivery, the material carried across the intersection is suppressed by 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 intersection is "pinching", which prevents material from diffusing 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 side channel to reverse the material from the intersection.

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 arm to the right arm of the horizontal channel, is regulated from the vertical channel. It can be effectively controlled, interrupted and restarted by the flow, for example from the lower arm to the upper arm from the vertical channel. Specifically, in the "off" mode, the material is transported from the left arm to the upper arm through the intersection portion by applying a voltage gradient to the left and top ends. Inhibitory flow is directed from the lower arm to the upper arm by applying a similar voltage gradient along the path (lower end to upper end). A metered amount of material is then dispersed from the left arm of the horizontal channel to the right arm 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 for the four approaches for illustrative purposes, this controlled electrokinetic material transport system of cross-crossing can be easily adapted to each other for more complex interconnected channel networks, for example an array of interconnected parallel channels. .

B. Continuous Flow Assay System

In one preferred aspect, 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 to screen inhibitors or inducers of enzymatic activity, or agonists or antagonists of receptor-ligand binding. In brief, the continuous flow assay system comprises a continuous flow of a particular biochemical system along the microfabricated channels. 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 function of the stream is indicated by the occurrence 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 enzyme systems, the signal will be generated by, for example, the product of the catalytic action of the enzyme, generally on a pigment producing or fluorescence generating substrate. In binding systems, such as receptor ligand interactions, the signal typically involves binding 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 pigmentation and fluorescence generation signals, radioactive material decay, electron density, pH change, solvent viscosity, temperature and salt concentration are also appropriately 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 thickening reagents, enzymes (eg, those commonly used in ELISAs), biotin, dioxygenin, or hapten and antiserum or monoclonal antibodies. Include 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 matrices, carbohydrates or lipids. Detection is performed by one of several 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 as marker products or commonly used 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 generally characterized by one or more of the 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'-diaminostilbene, pyrene, quaternary phenanthridine, 9-aminoacridine , p, p'-diaminobenzophenone imine, anthracene, oxcarbocyanine, merocyanine, 3-aminoequilenine, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1,2 -Benzophenazine, retinol, bis-3-aminopyridinium salt, helebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene, 7 Hydroxycoumarins, phenoxazines, carboxylates, stropantidine, porphyrins, triarylmethanes and flavins. Individual fluorescent compounds which have a functional group for binding to an enzyme preferably detected in the device or assay of the present invention or which can be modified to incorporate the functional group include, for example, monosil 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-stilbene-2,2'-disulfonic acid; Pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate; N-phenyl-N-methyl-2-aminonaphthalene-6-sulfonate; Ethidium bromide; Stebrin; Auromine-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, Minneapolis), and Pharmacia Elkabi Biotechnology Pharmacia LKB Biotechnology: Fitzkataway, NJ), CloneTech Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company Company: Milwaukee, WY, Glen Research Inc., Gibco BIAL Technologies, Inc. (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD), Fluka Chemica Fluka Chemica-Biochemika Analytica (Bux, Switzerland), and Applied Biosyst ems (Foster City, Calif.) as well as other companies known to those skilled in the art.

Preferably, the fluorescent label absorbs light at wavelengths greater than about 300 nm, preferably greater than about 350 nm, more preferably greater than about 400 nm, and emits at a wavelength that is 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 shown but not a label that is characterized and not conjugated in any solvent.

Fluorescent labels are a preferred class of detectable labels because the inventors can identify 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 be luminescent 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 para-dimethylamino and para-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, microscopic, visual inspection, and digital cameras, charge coupling devices (CCDs) or photomultipliers and phototubes. It can detect with the photographic film using an electronic detector. 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 observing only the color associated with the label. For example, conjugated gold often shows pink, and various conjugated beads show the beads color itself.

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 generate a relatively constant level of signal in the window or detection region of the channel. Test compounds are introduced periodically and mixed with the system components. If these test compounds affect the system, they will deviate from a constant level of signal in the detection window. This deviation can then be correlated with the particular test compound being screened.

One embodiment of an apparatus for use in a series or continuous calibration geometry is shown in FIG. 1. As shown, the entire apparatus 100 is fabricated within the 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 selected according to their inertness to important components of the analysis or synthesis to be performed by the device.

Examples of useful substrate materials are polymeric substrates such as plastics, as well as glass, quartz and silicon. 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 rigid, semi-rigid, opaque, semi-opaque or transparent, depending on the use in which they are to be used. For example, a device comprising an optical or visible detection element will typically be made of a transparent material that, at least in part, permits 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 polymer substrate may have a straight or branched backbone, optionally crosslinked or uncrosslinked. Examples of particularly preferred polymer substrates are 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 within the surface of the substrate will generally be performed by any number of microfabrication techniques 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 wettable chemical etching. Similarly, for polymer substrates, well known manufacturing techniques can also be used. These techniques include injection molding or stamp molding, in which a large number of substrates are optionally produced, for example, using a rolling stamp that produces large sheet-like microscale substrates, or polymer micromoulding techniques in which the substrates are polymerized in a micromechanical molder. Included.

The device will typically include an additional planar element covering the channeled substrate that seals and fluidically 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 natural adhesion between the two components in the case of certain substrates, for example glass or semi-rigid and non-rigid polymer substrates. do. 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 apparatus shown in FIG. 1 includes reservoirs 104, 106 and 108 disposed at the distal ends of channels 110 and 114 and fluidly coupled. As shown, sample channel 112 is used to introduce a number of different test compounds into the device. As such, it will typically be fluidly coupled to a plurality of separate test compounds sources to be introduced separately into sample channel 112 and into channel 110.

Many individual and discrete volume test compounds are introduced into a sample by a number of methods. For example, a micropipetter is optionally 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. This low ionic strength spacer region has a higher voltage drop in the region of the desired material or test compound of high ionic strength, thereby inducing electrokinetic pumping. The test compound or region of interest that is typically present in a solution of higher ionic strength is the fluidic region referred to as the first spacer region (also referred to as the "guard band") in contact with the interface of the region of interest. 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 U.S. Patent Application Serial No. 08 / 671,986 filed on June 28, 1996, which is incorporated herein by reference (Representative Document No. 017646-000500). .

Alternatively, sample channel 112 is fluidly connected in a random and separate manner 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 that randomly monitors signals from the biochemical system. 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 signal in the detection window is monitored using an optical detection system. For example, optical substrate signals are typically monitored using a laser activated fluorescence detection system using, for example, a laser light source of a suitable wavelength for activating a fluorescent indicator in the system. 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 measure the absorbance or transmittance of the sample.

In an alternative aspect, the detection system may include a non-optical detector or sensor for detecting the 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 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 simultaneously from the side channel 114 into the 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 reservoirs during the 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 only water needs to be added during 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. "Normal 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 interrupted periodically, for example to load additional test compounds, as described in the description of the continuous flow system. The signal generated in the enzyme system will change along the length of the channel, ie it will increase 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 be constant. 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 to effect from one test compound to another test compound. Will allow change, or lack of effect. 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 generate 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 detectably lower signal level 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 ligand may have different flow rates depending on the channel. This can be accomplished by incorporating an intra-channel size exclusion matrix or by altering the relative electrophoretic shift of the two compounds in the case of electroosmotic methods to allow the receptor to flow faster down the channel. Again, this is accomplished by the use of size exclusion matrices, or by using different surface charges in the channels that lead to a flow rate difference 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 operation, it is believed that a steady state signal results in both a free fluorescent ligand and a fluorescent ligand 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 method similar to that used in 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. It is also possible to monitor the activity of enzymes generated from receptor-ligand bonds, for example the activity of kinases, or to incorporate fluorophores that are activated or quenched by structural changes to the enzymes upon activation of the enzymes upon activation. Structural changes can be detected.

The flow and direction of fluid in the microscale fluidic apparatus is accomplished by a variety of methods. For example, the device includes an integrated microfluidic structure, such as a pipette pump and 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 within 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 WO 96/04547, to Ramsey et al., The applicant of which is incorporated herein by reference.

In sum, these fluid control systems include electrodes disposed in a reservoir placed in a fluid connection with a plurality of crossover channels fabricated within the surface of the substrate. The material stored in the reservoir is 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 electrokinesis. In sum, 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 may be ionized. For example, when hydroxyl functional groups are included on the channel 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 have an excess of protons or positive charges, especially at a location 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 represented 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 obtain selectable voltage levels by using multiple voltage dividers and multiple relays. 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.

Introducing the electroosmotic fluid induction system into the apparatus shown in FIG. 1 includes the introduction of an electrode into each reservoir 104, 106 and 108 at the end of the sample channel 112 or any fluid channel end connected thereto. Thus, the electrode is electrically connected with the fluid disposed in 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, the surface is optionally modified to provide a suitable surface charge such as coating, induction, eg silanization, or saturation of the surface to provide a suitably charged group on the surface. An example of such a treatment is described in split patent application No. 60 / 015,498 (agent document number 017646-002600), filed April 16, 1996, incorporated herein by reference.

In short, 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 material may be selected depending on their inactivation to important components of the analysis and synthesis to be performed by the device. The polymeric substrate materials will be hard, semi-rigid or non-hard, opaque, semi-opaque or transparent, depending on their use. For example, it will be common for devices comprising optical or visible detection elements to be at least partially fabricated from a transparent polymer substrate to facilitate such detection. Alternatively, for example, a transparent window of glass or quartz can be introduced 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 available from Polycarbonate (e.g., Mobay Corporation (Pittsburg, PA)) or The Plastics and Rubber, a subsidiary of Bayer Corporation. Makrolon CD-2005), or Rezan OQ 1020L or Rezan OQ 1020 available from GE Plastics; Polydimethylsiloxane (PDMS), polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone, polycarbonate 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 analytical methods.

As described herein, electrokinetic fluid control systems used in the devices of the present invention generally utilize materials having functional groups charged on these surfaces, such as hydroxyl groups present on the glass surface. As described, the device of the present invention may use plastic or other polymerizable substrates. In general, the materials of such materials have a hydrophobic surface. As a result, when using an electrokinetic fluid control system as a device using the polymerizable substrate used in the present invention, a modified substrate surface that is in contact with the fluid is typically used.

Surface modification of the polymerizable substrate can be performed 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 substrate, indicating a charged head group in the fluid layer.

In one embodiment, the preparation of a charged surface on a substrate includes exposing the surface to a suitable solvent that partially dissolves or softens the polymerizable substrate surface, such as to deform the channel and / or reaction chamber, for example. Included. Thereafter, the cleaner is 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 remaining on the surface, which appears at the fluid interface of the charged head group.

In another embodiment, the polymeric material, such as polydimethylsiloxane, can be modified by plasma spinning. In particular, plasma spinning of PDMS produces its bound hydroxyl functionalities, oxidizing methyl groups, freeing carbon, leaving hydroxyl groups at their positions, and effectively creating a glassy surface on the polymeric material.

The polymerizable substrate 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 substrate consists of one or more softeners, soluble material elements and one or more hardeners, more rigid rigid elements, channels and chambers fabricated on the surface. As soon as the two materials are combined, the inclusion of soft elements creates an effective fluid seal in the channels and chambers, and solves the problems associated with rigid plastic components that are glued or melted.

Numerous additional elements are added to the polymerizable substrate 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. Such elements typically include charged bolts, including electrodes for charged bolts for various fluid reservoirs and bolt detectors at various channel intersections.

The electrode may be included 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 to known microfabricated substrates, for example after sputtering or controlled vapor deposition methods, after the material is formed using chemical etching.

Whether polymeric substrates or other substrates are used, the bolt control is applied to various reservoirs simultaneously, such that continuous flow of receptors / enzymes to the waste reservoir, ligand / Influence temperament. In particular, the adjustment of the bolts applied to the various reservoirs can be moved and induces fluid flow through the intersecting 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. Specifically, injection of test compounds (in the formulation region) into a continuous stream of enzyme-fluorescent substrate mixture is shown. As shown in FIG. 2A, with reference to FIG. 1, a continuous stream of enzyme flows from the reservoir 104 along the 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 flowing enzyme stream. The mixed enzyme / test compound region then flows along main channel 110 via the intersection with channel 114. A continuous stream of fluorescent or colored substrates contained in the reservoir 106 is introduced into the sample channel 110, from which it is contacted and mixed with a continuous stream of enzyme comprising a test substance region comprising the test compound 122. do. The action of enzymes on the material will increase the level of fluorescence or coloring signals. 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. The test compound will appear as a modified signal where it does not affect the enzyme's interaction with the substrate. For example, a presumed fluorescent substrate, ie a test compound that interferes with the interaction of the enzyme with the substrate, will induce less fluorescent product generated in the test substance region. This will lead to a non-fluorescent region in the flow stream through which the flow stream passes the detection window 116, or to a less fluorescent region, the window corresponding to the test material region. For example, as shown, the test substance region includes test compound 128, which is a putative inhibitor of enzyme-substrate interaction and exhibits lower detectable fluorescence than the surrounding stream. This is indicated by the lack of shading of the test substance region 128.

A detector adjacent to the detection window monitors the level of the fluorescence signal generated by the enzyme activity on the fluorescent or coloring substrate. This signal is maintained at a relatively constant level for the test compound, and the compound does not affect enzyme-substrate interactions. However, if inhibitory compounds are screened, this will lead to a momentary drastic decrease in the fluorescence signal indicating reduced or inhibited enzyme activity on the substrate. In contrast, upon screening, the derivative compound instantaneously increases the fluorescence signal corresponding to increased enzyme activity against the substrate.

2B shows a similar schematic of the screen for effectors of receptor-ligand interactions. As shown in FIG. 2A, a continuous stream of receptors flows from reservoir 104 through main channel 110. Test compound or test substance region 150 separated by suitable spacer fluid region 121 is introduced from sample channel 112 to main channel 110, and a continuous stream of fluorescent ligand from reservoir 106 is connected to the lateral channel ( 114). As shown in FIG. 2A, a continuous stream of fluorescent ligands and receptors through the detection window 116 will provide a constant signal intensity. The test substance region in the stream containing the test compound that does not have any effect on the receptor-ligand interaction may provide the same or similar level of fluorescence as the remaining surrounding stream, eg, the test compound or test substance region 152. will be. However, the presence of a test compound that is opposite to the receptor-ligand interaction or has inhibitory activity results in a lower level of the interaction in a portion of the stream in which the compound is located, eg, in the test compound or test substance region 154. Will induce. In addition, the flow rate difference for the receptor bound to the fluorescent ligand or free fluorescent ligand will detectably reduce the fluorescence level corresponding to the fluorescent dilution derived from the unbound faster moving receptor. Thereafter, not only the fluorescence decreases, but also the fluorescence 156 is increased corresponding to the accumulation of slower moving unbound fluorescent ligands.

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 keeps the reaction elements contained within the discontinuous fluid region during the reaction, while allowing these elements to separate during the data acquisition phase. As mentioned above, the various elements of the reactants can be held together in the region of test substance moving through the reaction channel by introducing an appropriate spacer fluid region between the samples. Generally, such spacer fluid regions are selected to keep the sample in this original test substance region, in particular, so that the sample does not spread to the spacer region even during extended reaction periods. However, this object can be achieved in analyte element separation, for example, where extra such assays are based on the ligand-receptor assays mentioned above or where the reaction product is separated in the capillary. Thus, it may be desirable to eliminate these elements that prevent this separation during the initial portion of fluid induction.

A schematic diagram 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 through the first spacer region 508 during the time that the highest electrically electrophoretic sample is moved below the reaction channel. It is selected such that it cannot move to the spacer region 502.

During estimating 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 test compound / receptor in the form of the 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 of the system (measured by the respective reservoirs 514, 516, 518, 520, 522 and the potential applied at the end of the sample channel 512) and the length of the incubation region are combined with the fluorescent ligand and the test compound. The incubation time of the receptor was measured. 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 application of a potential at the ends of reservoirs 514, 516, 518 and sample channel 512, for example. do. In addition, potentials are applied to opposite ends of reservoirs 520 and 522 and separation channel 524 so that the potentials match at both ends of the mobile channel, so that the net flow across the mobile channel is zero. As the test material region passes through the intersection of reaction channel 510 and mobile channel 526, the potential is suspended in reservoirs 518 and 522, where reservoirs 514, 516, 520 and sample channel 526. The potential applied at the end of leads the test material region through the moving channel 526 to the separation channel 524. Once separated into the separation channel, the original potential is reapplied in all reservoirs, stopping the net fluid flow through the moving channel 526. Thereafter, the conversion of the test substance can be repeated for 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 directed to a separate channel, placing the test material into a capillary filled with a buffer with high ionic strength to remove spacer regions of low ionic strength, thereby removing a variety of samples outside of the original test material region. Separate the components. For example, in the aforementioned receptor / ligand screen, the receptor / ligand complex exhibits a different electrophoretic shift than when the ligand alone is in the mobile channel, thus allowing better separation of the complex from the ligand and subsequent This can be detected.

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

In addition, more complex systems can be manufactured within the scope of the present invention. For example, a schematic description of an alternative embodiment used for the "serial injection parallel reaction" geometry is shown in FIG. 3. As shown, the apparatus 300 includes a planar substrate 302 as mentioned above. A series of parallel reaction channels 312-324 are fabricated within the surface of the substrate 302. 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, substrates 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 fluidly connected to a single transverse channel, the channels are optionally connected to a central reservoir and extend radially outward 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 where, for example, biochemical system components are predeposited in the device. Similarly, more transverse channels are used to introduce additional elements into optionally provided assay screens as needed. Accordingly, the series of parallel devices of the present invention will generally comprise two or more, preferably three, four, 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 not only useful for screening test compounds that are continuously injected into the device, but also for the use of parallel assay forms, and once the sample enters the device, throughput is increased.

In operation, the test compound in the separate test substance region is continuously introduced into the separated apparatus as mentioned above, with the separate injection substance region approaching the intersection of the reaction channels 310-324 and the parallel sample channel 304. Flow along the transverse sample injection channel 304 until 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, parallel channels are optionally fabricated to include the bead fixed 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 fixed 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 fixed well and positioned in place.

At a point close to the intersection of the parallel reaction channel and the sample injection channel, the test compound directs them to each reaction channel by redirecting the fluid flow down the channel. In addition, in the example of a test compound immobilized on the beads, it will generally be immobilized via a cleavable linker group such as a photolysate, acid or base cleaved linker. Thus, before the test compound flows down the reaction channel, the test compound is needed to escape from the beads, for example by exposure to release agents such as light, acids, bases, and the like.

In parallel channels, the test compound will be contacted in the biochemical system in 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. 4A-4E, and is incorporated into whole cell or bead based systems such as beads. 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 a porous structure or microstructure that does not pass through a particle sieving or filtration matrix, such as particulate material but through which a free flow of 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, fluid induction 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, electroosmotic systems as described above 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 the potential complexity using fewer electrodes, parallel systems are preferred, for example, it is desirable for two fluids to move at similar speeds in parallel channels, depending on the geometry of 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 geometry 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 arbitrarily changing the cross section of the path, thereby keeping the resistance level constant regardless of the path employed.

As with the test compounds derived through their respective parallel reaction channels, they will be in contact with the biochemical system in question. As mentioned above, special biochemical systems will generally comprise a flowable labeling system, which system may be a dissolvable labeling group such as a colorable or fluorescent substrate, labeled ligand, or the like, or a precipitating agent or The relative action of particle based signals such as bead bound 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 flow in series 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, the system is exposed to the test compounds, and the generated signals from the parallel reaction channels flow. 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 introduced into the reaction channel, optionally during manufacture, 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 included in the 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 any 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 a release agent. As shown, the beads are exposed to light from a suitable light source 352, e.g., a light source capable of generating light of a wavelength suitable for photodegrading the connector, thereby allowing each of these beads to be subjected to photodegradable connector. Release the compound to be coupled.

In FIG. 4C, the released test compounds will 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, eg, enzymatic reactions, receptor / ligand interactions, etc. in the presence of the test compound. 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 (FIG. 4D). As noted above, the change in signal level generated is an indication that a particular test compound is an effector of a particular biochemical system. This is indicated by the shading of the brighter portion of the 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 optionally 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 achieved outside of the device, optionally by inducing beads in the fragment collector, wherein test compounds present on the beads are optionally identified through identification of labeling groups or identification of residual compounds. The incorporation of labeled groups by a binding chemistry method has already been described using chlorinated / fluorinated aromatic compounds or potential nucleotide sequences as labeling groups (PCT Application WO 95/12608). In addition, 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 through a series of parallel pumping channels 636-646. At the end of the pumping channel includes reservoirs 650 and 652. 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 system is shown in FIGS. 6B-6C. 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 the arrows and interconnects these transverse channels as shown in FIG. 6B. flow. As shown by the shaded area of FIG. 6B, the region of test substance containing the test compound is aligned with the parallel reaction channels 612-620 connecting the sample channel 604 to the detection channel 606. By removing the voltage applied to the reservoir at the ends of these channels, the flow in all transverse directions is interrupted. 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 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 geometry of the channel. 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 the maximum number of reaction channels to be packed 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 at the same time a second voltage to the reservoirs 658 and 660 by parallel reaction channels 612-620. The flow of fluid through the parallel pumping channels 636-646 thus leads the test material to the parallel reaction channels 612-620 as shown in FIG. 6C. During this process, no voltage is applied at the ends of reservoirs 648, 654, 656 or sample channel 604. Parallel channels 636-646 and 622-632 are generally length adjusted so that the overall channel length, and thus the level of resistance from reservoirs 650 and 652 to channel 604, and the channels from reservoirs 658 and 660 The resistance level to 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 geometry. 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, while at the same time the voltage in the remaining reservoirs is floating. Let's do it. 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 to provide several parallel channels. Suppress the flow following. Although shown as channels crossing at right angles, it will also be appreciated that other geometries are suitable for continuous injection parallel reactions. For example, US patent application Ser. No. 08 / 835,101, filed Apr. 4, 1997, describes the advantages of varying widths and parabolic geometries to control fluid flow. In sum, in an electroosmotic system the fluid flow is controlled by the current flow between the electrodes and thus is relevant. The resistance in the fluid channel can vary as a function of the length and width of the path, so channels of different lengths have different resistances. 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 alleviated by using separate electrical systems, i.e., separate electrodes at the ends of each and all parallel channels, a device comprising all of these electrodes and the voltage applied to each of these electrodes Manufacturing a control system to control the control can be complicated, especially when dealing with hundreds to thousands of parallel channels in a single small scale device, for example 1 to 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 embodiment of the parabolic geometry of the 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 to sift the test compound into the device, wherein the 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 corresponding members of each device, e.g., electrical osmotic fluid flow for detection systems, electrical leads connected to electrodes contained in reservoirs used for power input and data output, 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. Intermediate spacer fluid regions 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 a salt bridge between frit 1219 and fluid reservoir 1221 fluidly connected to fluid channel 1223.

12C provides an electrode 1225 that is submerged 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. Reduces degradation of species.

FIG. 12D provides a similar dichotomy reservoir wherein electrode 1235 is first fluidically connected to second fluid reservoir 1241 by small channel 1243 processed to reduce or eliminate electrical osmotic flow. Locked in fluid reservoir 1237.

12E provides another similar dichotomy 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 bifurcation reservoir where electrode 1255 is submerged in first fluid reservoir 1257 fluidly connected to second fluid reservoir 1259 by 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.

12G provides another modified bifurcation reservoir wherein electrode 1265 is submerged 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 sufficiently attracts the fluid to 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 in fine fluid systems with cross-sectional dimensions of 0.1 to 500 μm.

Use as a microfluidic system as described above in which a 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.

Assays 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 processed in the surface. 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, each reaction Fluidly connected to the second transverse channel at a second point of the channel, the assay device comprising a device as described above.

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 figures next to the channels indicate 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.

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 supplied from each well to the main channel 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 overlaps 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 in the linear range of signals for the assay system at the detector. 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 to 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. An additional 12 seconds is required to move the mixture of enzyme / test compounds through the incubation zone to the intersection of the substrate channels, 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 Figures 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 (68)

An apparatus for screening a plurality of test compounds for effects on one or more first biochemical components, the apparatus comprising: (i) a body structure comprising two or more microscale channels disposed therein; (ii) a plurality of test compound sources fluidly connected to at least one of the at least two microscale channels; And (iii) a source of a first biochemical component fluidly connected to at least one of the at least two microscale channels. The apparatus of claim 1, wherein at least one of the two microscale channels comprises at least one cross sectional dimension of from about 0.1 to about 500 μm. 2. The apparatus of claim 1, wherein the at least two microscale channels comprise cross channels or cross channels. The apparatus of claim 1 further comprising a plurality of reaction channels fluidly connected to one or both of the two or more microscale channels. The apparatus of claim 4, wherein at least one of the plurality of reaction channels comprises at least one cross sectional dimension of between about 0.1 μm and about 500 μm. 5. The device of claim 4, wherein the plurality of reaction channels comprise a plurality of parallel reaction channels disposed within the body structure, wherein at least two microscale channels are fluidly connected to opposite ends of each parallel reaction channel. 5. The apparatus of claim 4, wherein at least two microscale channels are disposed within the body 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. 5. The device of claim 4, wherein each reaction channel comprises a bead resting well, a particle resting well, or a particle retention zone. 9. The apparatus of claim 8, wherein the particle retention region comprises a particle retention matrix or microstructured filter. The device of claim 4, wherein a plurality of sources of test compound are fluidly connected to each channel of the plurality of microscale channels. The device of claim 1, wherein the source of the plurality of test compounds comprises a plurality of reservoirs disposed within the body structure. The method of claim 1, wherein the source of the plurality of test compounds comprises a pipettor element, wherein the pipette element is fluidly connected to one or more of the two or more microscale channels during operation of the device. Characterized in that the device. The device of claim 1, comprising a capillary channel fluidly connected to the first end for at least one of the two or more microscale channels. The apparatus of claim 13, wherein the capillary source is fluidly connected to a second end for the plurality of test compound sources. The device of claim 1, wherein the plurality of test compound sources comprise a plurality of particles disposed in one or more of the two or more microscale channels. The device of claim 1, wherein the plurality of test compound sources comprises a plurality of test compounds. The device of claim 1, wherein the source of the first biochemical component is disposed within the surface of the body structure. The method of claim 1, wherein the body structure is thermally thermally bonded to a second substrate overlying a first substrate having two or more microscale channels etched therein. By laminating with a device. 19. The apparatus of claim 18, wherein the first substrate and the second substrate comprise a glass substrate. 2. The apparatus of claim 1, wherein the body structure comprises etched glass, etched silicon or molded polymer. 21. The apparatus of claim 20, wherein the body structure comprises etched silicon and an insulating layer disposed on the etched silicon. 2. An apparatus according to claim 1, wherein the body structure is made by stamp-molding or injection-molding. The device of claim 1, wherein the first biochemical component comprises an enzyme and an enzyme substrate, which generates a detectable signal when the enzyme substrate reacts with the enzyme. The device of claim 23, wherein the enzyme substrate is a colored or fluorescent enzyme substrate. The device of claim 1, wherein the first biochemical component comprises a receptor / ligand binding pair, the receptor or ligand comprising a detectable labeling group. The device of claim 25, wherein a detectable signal is generated by binding of the receptor to the ligand. The device of claim 1, wherein the first biochemical component comprises one or both of the antibody / antigen binding pairs, wherein the antibody specifically immune response to the antigen. The device of claim 27, wherein the antibody or antigen comprises a detectable label. 29. The device of claim 25 or 28, wherein the detectable labeling group comprises a fluorescent group. The device of claim 1, wherein the first biochemical component or the plurality of test compounds are derived from blood or from a patient. The device of claim 1, further comprising a source of a second biochemical component, said source being fluidly connected to at least one of the at least two microscale channels. The method of claim 1, further comprising a fluid induction system, wherein the fluid induction system induces a first biochemical component through at least one of the at least two microscale channels, and wherein the plurality of test compounds is supplied to a plurality of test compound sources. From to at least one of the two or more microscale channels. 33. The method of claim 32, wherein the fluid induction system forms a continuous flow of the first biochemical component along at least one of the at least two microscale channels, wherein at least one component of the plurality of test compounds is at least two microscale. Device periodically injected into one or more of the channels. 34. The device of claim 33, wherein the spacer is introduced into one or more of the two or more microscale channels by a fluid induction system, wherein the spacer separates different components of the plurality of test compounds. 33. The system of claim 32, wherein the fluid induction system is At least three electrodes, each electrode being in electrical contact with at least two microscale channels on different sides of the intersection formed by the at least two channels, and And a control system in which the movement of a test compound or a first biochemical component in two or more channels is controlled by simultaneously applying a variable voltage to each electrode. 33. The apparatus of claim 32, wherein the fluid induction system comprises a pressure source. 37. The apparatus of claim 36, wherein the pressure source comprises a pump. The system of claim 1, further comprising a source of a second biochemical component fluidly connected to at least one of the fluid guide system and the at least two microscale channels, wherein the fluid guide system comprises one of the at least two microscale channels. In accordance with the above, a continuous flow of the mixture of the first biochemical component and the second biochemical component is formed, wherein at least one component of the plurality of test compounds is periodically injected into at least one of the at least two microscale channels. Device. The device of claim 1, further comprising a detection region in one or more of the two or more microscale channels to detect effects on one or more biochemical components. 40. The apparatus of claim 39, wherein the detection region comprises a detection window in one or more of the two or more microscale channels. The device of claim 1, further comprising a detection system for detecting effects on one or more biochemical components. 42. The apparatus of claim 41, wherein the detection system comprises a fluorescence detection system. The method of claim 1, further comprising: a plurality of electrodes in a plurality of reservoirs fluidly connected to at least one of the at least two microscale channels, And applying a voltage to each electrode simultaneously to control the movement of the first biochemical component or the plurality of test compounds in the two or more microscale channels. 44. The device of claim 43, wherein the device minimizes degradation of one or more species present in the plurality of reservoirs. 45. The frit of claim 44, wherein the frit on one or more of the plurality of electrodes reduces the electroosmotic flow rate toward one or more of the plurality of electrodes, A large channel located between two or more of the multiple reservoirs, which limits the diffusion of one or more species and has a low electroosmotic flow rate, Narrow channels located between two or more of the multiple reservoirs, which limit the diffusion of one or more species and have low electroosmotic flow rates, A filled channel located between two or more of the reservoirs, the channel having a low electroosmotic flow rate, comprising a matrix to restrict the transport of one or more species, A high reservoir having a fluid level higher than at least one low reservoir comprising an electrode and fluidly connected to the low reservoir, wherein the high reservoir has reduced electroosmotic flow to the electrode side by hydraulic pressure between the high reservoir and the low reservoir, and It is adapted to receive one of the plurality of electrodes and is adapted to aspirate fluid by capillary electrophoresis to one of the plurality of electrodes fluidly through the connection channel to a second narrow diameter reservoir that counts the electroosmotic flow rate in the connection channel. And at least one component for reducing the electrical osmotic flow rate, selected from a dual reservoir system having a first reservoir connected to the outlet. A method for screening a plurality of test compounds for effects on one or more biochemical components, Flowing at least one biochemical component through the first microscale channel, contacting the at least one biochemical component with a first component of the plurality of test compounds, and subjecting the at least one biochemical component to the plurality of test compounds for the at least one biochemical component. Detecting the effect of one component. 47. The method of claim 46, comprising continuously flowing one or more biochemical components through the first microscale channel. 48. The method of claim 47, wherein the detecting step comprises continuously monitoring a detectable signal comprising a steady state intensity from one or more biochemical components flowing in series, wherein the plurality of test compounds for the one or more biochemical components Wherein the effect of the first component of deviates from the steady state intensity of the detectable signal. 47. The method of claim 46, comprising periodically contacting one or more biochemical components with different components in the plurality of test compounds. 50. The method of claim 49, comprising physically separating different components of the plurality of test compounds. 51. The method of claim 50, comprising physically separating different components of the plurality of test compounds with one or more spacers. 47. The method of claim 46, further comprising: (i) flowing one or more biochemical components into the plurality of microscale reaction channels, and (ii) different components of the plurality of test compounds into each component in the plurality of microscale reaction channels. Flowing the method. 47. The method of claim 46, wherein the one or more biochemical components comprise one or both of the antibodies / antigens, wherein the antibody specifically immunoreacts to the antigen. The method of claim 53, wherein the antibody or antigen comprises a detectable label. The method of claim 46, wherein the one or more biochemical components comprise one or both of the receptor / ligand binding pairs, wherein the receptor specifically binds to the ligand. 56. The method of claim 55, wherein the receptor or ligand comprises a detectable label group. 59. The method of claim 54 or 56, wherein the detectable labeling group comprises a fluorescent group. 47. The method of claim 46, wherein at least one biochemical component comprises an enzyme and an enzyme substrate, wherein a detectable signal is generated by the action of the enzyme on the enzyme substrate. 47. The method of claim 46, wherein the at least one biochemical component comprises a biological barrier and the effect of the first component in the plurality of test compounds comprises the ability of the first component in the plurality of test compounds to cross the barrier. Characterized by the above. 60. The method of claim 59, wherein the biological barrier comprises an epithelial layer or an endothelial layer. 47. The method of claim 46, wherein the at least one biochemical component comprises at least one cell and the detecting step comprises measuring the effect of the first component in the plurality of test compounds on the at least one cell. 62. The method of claim 61, wherein a detectable signal corresponding to cell function is generated by one or more cells, and the detecting step detects the effect of the first component in the plurality of test compounds on the cell function by detecting the detectable signal level. Method comprising the step of: a. 62. The method of claim 61, wherein measuring the effect of the first component in the plurality of test compounds on the one or more cells comprises detecting the effect on the viability of the one or more cells. . The method of claim 46, wherein the one or more biochemical components or a plurality of test compounds are derived from blood or from a patient. 47. The method of claim 46, wherein the detecting comprises measuring a parameter of the biochemical system comprising at least one biochemical component in the presence and absence of the first component in the plurality of test compounds, wherein the change in the parameter is biochemical. Characterized by exhibiting an effect on the biological system or biochemical component. 47. The method of claim 46, wherein the at least one biochemical component generates a detectable signal representative of the function of the biochemical component. 67. The method of claim 66, wherein the detectable signal comprises a fluorescent or chromatic signal. 47. The method of claim 46, wherein the at least one biochemical component comprises an indicator compound that interacts with the first biochemical component to generate a detectable signal representative of the function of the biochemical component.