JP2002544523A - Target independent assay systems and methods - Google Patents

Abstract

(57) Abstract Prescreened libraries, such as prescreened chemical compositions useful for drug screening, are generated using a target-independent assay. The method typically includes prescreening the master library in a microfluidic device for effects associated with one or more target-independent parameters. Also includes multi-module workstations and integrated systems, such as microfluidic workstations, for performing target-independent assays.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] CROSS-REFERENCE present invention to RELATED APPLICATIONS Provisional Application USSN60 / 133,919, 5 May 13, 1999 filed; USSN60 / 164,218, 11 09, 1999 filed; and USSN6
0 / 195,159, filed April 6, 2000, and claims 35 UUSC 119 (e) and any other applicable statute or regulation.

Copyright notice 37C. F. R. In accordance with 1.71 (e), Applicants indicate that portions of the present disclosure include material that is subject to copyright protection. When revealing a patent application or record at the Patent and Trademark Office, the copyright holder did not object to facsimile reproduction of the patent content and disclosure by anyone, but all other copyrights were Anything remains.

[0003] In an effort to promote the process of background drug discovery of the present invention, the effort of a great deal of research and improvement has been spent. The main areas of investigation include improvements in conjugation chemistry techniques that facilitate the generation of a large number of different compounds, which are then evaluated to determine which generated compounds have pharmacologically significant activities. Clearly, as the number of compounds to be screened increases, there is a considerable need to increase the speed and throughput of the screening process.
Many methods have been improved to facilitate screening assays. It typically involves using complex and expensive robotic flow and plate handling systems to perform a large number of screening assays.

[0004] Once a compound has been screened and the potential activity has been demonstrated, the compound is screened again to determine the exact nature of its activity, and for example, toxicological effects, bioavailability Etc. are screened for a second effect. Various assays are typically performed, followed by an initial activity screening assay (or target-dependent assay) to assess (in pharmacological investigations) whether the active compound is useful in vivo. . Typically, many in vivo activities are
Potential effects of a drug on its absolute effectiveness, including toxicity of the compound, non-specific binding of the compound to other components, and passage of the compound through various biological barriers, etc. I can do it. These parameters are evaluated in a second or “target independent” assay. Generally, target-independent assays are performed in pharmacological studies following the identification of potential lead compounds. This is typically due to the high cost of reagents for performing these assays. It is desirable to be able to perform both the first screening assay and the second, or the target-independent assay, as quickly, cheaply and as quickly as possible, in order to maintain cost and facilitate drug improvement. The present invention addresses these and other needs.

SUMMARY OF THE INVENTION The present invention provides methods and assays for generating and characterizing large chemical composition libraries that are substantially devoid of compounds having undesirable effects in one or more target-independent assays. I do. Methods for evaluating large numbers of chemical compositions (including individual compounds, mixtures of compounds, etc.) in a cost-effective, high-throughput assay system suitable for screening libraries before performing target-dependent assays Is provided. The assays of the invention can be performed sequentially or simultaneously.
It is performed on a "parental" or "master" library, which usually exceeds 1,000, 10,000, 100,000 or 1,000,000 compositions, or on a continuously screened library from which to follow. Prescreened libraries can be physically separated from the master library,
Thereby, a pre-screened library can be generated or the designated components of the master library can be singly configured.

[0006] Prescreened libraries that are evaluated in one or more target-independent assays according to the methods of the invention are an aspect of the invention. The provided prescreened libraries are 100, 1000, 10,000, 10
Includes compounds that usually have more than 0000 or 1,000,000 compounds that exhibit the desired activity in one or more target-independent assays and / or that exhibit undesirable effects in target-independent assays. Lacks. In some embodiments, the library is a multi-well plate or other array.
Arranged in format. In some embodiments, a library is represented by one or more data sets associated with the results of one or more target-independent assays, with locations in the master library array. Typically, such a set of data is recorded on a computer-readable medium.

The present invention relates to target independent receptors that target serum half-life, cell uptake, oral efficacy, cell or organism viability, cell or organism toxicity, apoptosis, cell attachment, binding or activation, target Independent enzyme modulation activity, target-independent protein modulation activity, target-independent nucleic acid modulation activity, glucuronide binding modulation, sulfate binding modulation, amino acid binding modulation, acylation modulation, methylation modulation, transcription modulation, conversion modulation, protein folding ( modulation, modulation of cell growth, membrane permeability, membrane integrity, metabolic stability, temperature stability, solubility, solution viscosity, solution turbidity, acidity Provides assays to evaluate effects related to target-independent parameters such as degree, basicity, RedOx status, superoxide secretion, lipid peroxidation, octanol / water partitioning, precipitation in one or more buffers I do. A target-independent assay for assessing the binding of a chemical composition on a serum protein is provided by the method of the invention. One example of an important protein is human serum albumin (HSA). Due to the ubiquity of HSA in the human system (and similarly in other serum albumins in other mammalian systems), the interaction between HSA and important compositions has many effects, including in vivo May have efficacy of the composition at Typically, a composition that binds to HSA can be monitored, for example, by measuring the ability of the composition to inhibit the binding of a labeled substance to HSA in vitro. Therefore, in certain embodiments, the serum protein is HS
When A, the ability of the selected compound to bind to HSA is measured as a function of the inhibition of binding of a fluorescent dye such as dansyl-1-sulfonamide or dansyl sarcosine to HSA.

[0008] The present invention also provides a target-independent assay in which inhibition of an enzyme is assessed by components of a chemical composition library. The target-independent assays of the present invention include: glutathione-s-transferase, protease, peptidase, kinase, phosphatase, G protein, ATPase, cytochrome P450, ligase, carboxylesterase, epoxide hydrolase, aza- or nitroreductase, carbonyl reduction. Enzymes, disulfide reductase, sulfoxide reductase, quinone reductase, alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, xanthine oxidase, monoamine or diamine oxidase, prostaglandin synthase, flavin- Monooxidase,
And the effect of the composition on enzymes, including dehalogenases. In some embodiments, the effect in the enzyme is assessed using a fluorescent or fluorogenic material. In a preferred embodiment, the target-independent assay is performed in a microfluidic device. Some embodiments include a titrator element. In another embodiment, the target-independent assay involves flow cytometry in a microfluidic cell concentrator.

In certain embodiments, flow cytometry distinguishes living cells from dead cells. Flow cytometry, which distinguishes living cells from dead cells,
Different permeability, or calcein AM, BCECF AM, ethidium bromide, ethidium iodide, cationic dye, cationic membrane-permeable dye, neutral dye, neutral membrane-permeable dye, anionic dye, anionic membrane-permeable Based on binding to fluorescent dyes or dye bonds, such as dyes. In an alternative embodiment, the target-independent assay uses a fluorescent calcium indicator to assess the effect of the composition on calcium flux across the membrane of pre-loaded cells. In some embodiments, the master library is arranged in one or more multiwell plates, on a solid substrate such as a membrane or card, or in a bead matrix. Data sets associated with the results of one or more target-independent assays using one or more members of the master library are compiled in a small number of embodiments. In some cases, the dataset is correlated with the results of multiple target-independent assays using a master library component.

[0010] The present invention also provides a method and apparatus for performing a target-independent assay on a multi-module workstation. The multi-module workstation of the present invention comprises a screening module for performing a target-independent assay, a substrate corresponding to a composition library, a robot mechanism connected to the screening module, and a computer for controlling the movement of the substrate by, for example, a robot mechanism. Includes an auxiliary control system or alternatively a robotic mechanism between or within the substrates. In some embodiments, the screening module is arranged to perform a target-independent assay, such as a flow cytometry assay, wherein the assay evaluates the effect of the selected composition on the biochemical system;
Measure calcium flux across cell membranes, assess serum protein binding,
The binding to human serum albumin is evaluated, and the effect of the selected composition on the enzyme is evaluated. In a preferred embodiment, the screening module comprises a microfluidic device. Substrates include, for example, multi-well plates, solid substrates or bead arrays. Robotic mechanisms include, for example, conveyor belts, slide mechanisms, rollers, cable or pulley mechanisms, robotic armatures, or any combination thereof. The computer optionally includes a set of instructions for moving a substrate, moving a robotic mechanism, moving a screening module and / or selecting library members, etc., and relating to results of a target-independent assay using components of a library array. Data sets and user interfaces for program entry into the system. The invention further provides for the use of any pre-screened library or component composition in a target dependent assay. Kits for performing one or more target-independent and / or target-dependent assays for producing a prescreened library of the invention are also an aspect of the invention.

Introduction The high cost of drug improvement is largely due to the high rate of depletion for drugs that enter preclinical and clinical improvement. In addition to criteria such as toxicity and efficacy, the lead compound can exhibit desired properties with respect to properties such as absorption, bioavailability, metabolism, and the like. The ability to investigate and improve on the exclusion of chemical compounds that meet certain criteria in assays that signal biological events related to absorption, bioavailability, and metabolism is very important in the pharmaceutical industry. is there.

[0012] In the past, the cost of reagents (eg, key compounds) involved in performing a number of secondary screening assays (eg, absorption, bioavailability, metabolism, etc.) was very high. Therefore, only potentially active compounds were previously subjected to a second screening assay. Therefore, in standard compound screening methods,
A series of first screens (ie, screens for interesting activities) followed by a second screen on hits from the first screen (compounds identified as having significant activity). Therefore, typically, the separation round of the first screening is the separation round of the second screening. For example, many large pharmaceutical companies typically have a vast library of compounds that perform a first screen and identify hits, and it may be appropriate to rescreen each of these hits in a second assay .

[0013] In contrast, the present invention provides a method of producing a large library of chemical compositions prescreened for important secondary activities, providing a large number of libraries for selected clinical trials. These prescreened libraries are then screened for important activities. Thus, the present invention provides a method for pre-screening a library, a pre-screened library resulting from the performance of the method, an integrated system for performing a method and database of pre-screened compositions. For example, the chemical composition libraries of the present invention are prescreened in assays that predict biologically relevant properties prior to evaluation as potential drug leads. For the purposes of this disclosure, "pre-screened" means that a chemical composition library or collection of chemical compounds is more relevant to clinical behavior or other criteria of activity than, for example, characteristics characteristic of an important therapeutic target. Assess relevant properties 1
Indicates that it will be evaluated in a species or higher assay. Such an assay is a "target-independent assay", which screens for general properties besides the first important activity for a particular compound. In contrast, assays that assess the effects of a composition or library of compositions, for example, in a biological or other system for a therapeutic role, are distinguished from "target-dependent assays." The terms "target-independent" and "target-dependent" are considered to be related with respect to functional terms of a target or other assay component. For example, a kinase assay can be target dependent if the kinase of interest in the assay is the target of modulation by, for example, a compound or a component of a library. In contrast, kinase assays used, for example, to measure the effects of global phosphorylation state unrelated to the target (in some cases, the kinase itself) are target-independent assays.

The method of the present invention provides the significant benefit that a particular target-independent assay will be performed on a given library at one time, after each and every target-dependent assay. The basic methods and systems for performing these assays are described in US Pat. Nos. 5,942,443 and 199, issued Aug. 24, 1999.
U.S. patent application Ser. No. 08 / 883,638 filed Jun. 27, 7;
No. 09 / 173,469 filed on Mar. 14, and U.S. patent application Ser. No. 09 / 093,489, filed Jun. 8, 1998, which are incorporated herein by reference for all purposes. Introduced inside. A chemical composition library is composed of a collection of chemical compositions or compounds. Such libraries include chemical compounds, mixtures of chemical compounds such as polysaccharides, small organic or inorganic molecules, biological macromolecules such as peptides, proteins,
It can include a wide variety of different compounds, including nucleic acids, or extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, or naturally occurring or synthetic compositions. "Parent library" or "Master library"
By means a library whose components are evaluated for their ability to affect a particular biochemical system. Pre-screening these libraries can effectively prioritize further screening of those components of the library that have a positive or negative effect in these target-independent assays. Therefore, prescreened libraries can be selected for compounds that have a positive effect in these target-independent assays. Typically, the master library before the pre-screening assay is more than 1000, more typically more than 10,000, and often more than 10,000 or even just 1,000,000 or more, and the number of components For example, it is typically reduced by at least 10%, often 20%, in some cases 50% and sometimes 90% or more. In other words, target-dependent assays can be focused at only 90%, 80%, 50%, and potentially 10% or more of the starting or master library distribution after several target-independent assays. Is less than. A master or prescreened library can include an arrangement having one or more compound types for each position in the array, for example, the library can be arranged as a single compound set or as an array of a mixture of compounds Can be done. For ease of deconvolution (defined as a suitable component for the desired activity), the mixture is of the type of about 1,000 or less, typically about 100 or less, generally about 100 or less. 10 or less types, and often 5
Or less than the compound type of the compound.

In addition to removing less desirable members from the master library (physically or simply by noting the portion or presence of positive hits in the second assay), the second screening assay is a pre-screen It can also be used as a basis for adding additional members considering a given library. For example, where a compound of a given general structure is active in the second screen, additional structurally related library components can be generated, introduced into the tested and prescreened library. . Finally, in some embodiments, the complete master library is screened, despite the activity of the compounds in the second screening assay. Positive hits that are also active in the second assay are, of course, potential lead compounds. However, compounds that are negative in the second assay but positive in the target-dependent assay can be converted to potential lead compounds. For example, in certain embodiments, hits in a target-dependent assay can be improved for an improved second property (e.g., by chemical modification of the relevant compound) without eliminating activity in the target-dependent assay. Information from the target-independent library screen is used to modify such potential lead compounds, and, of course, the library can include such modified compounds for further secondary screening assays.

As noted above, prior to the present invention, the scale and, therefore, the cost of prescreened chemical compounds in target-independent assays before assessing the effects in target-dependent assays has been increased. . Typically, libraries of chemical compounds are evaluated in target-dependent assays to determine their ability as drug leads. Prospective lead candidates are evaluated for parameters such as serum half-life, membrane permeability, toxicity, enzyme inhibition and the like. This approach maximizes the number of potential leads identified, but involves a relatively high level of wear and potential cost per potential lead. The use of reagents is also very expensive, as many leads do not meet the criteria of the second assay screen. As described above, the present invention provides libraries that have been prescreened in a target-independent assay, and therefore are substantially devoid of compounds having undesirable properties in a target-independent assay. Thus, a high percentage of identified compounds has the properties of a successful drug candidate, such as high bioavailability, low toxicity, stability, and the like. By utilizing the prescreened library of the present invention, downstream costs are minimized because fewer identified reads are eliminated during later stages of improvement, and are associated with higher improvement costs. Is done. Libraries of the invention are produced by evaluating large collections of chemical compositions in target-independent assays in a high-power format. Such high power formats include, for example, 96-well, 384-well, 1,53
It can be a solid substrate based on a 6-well plate based multi-well plate or as on a pin array. In a preferred embodiment, the high power format can include one or more assays in one or more microfluidic devices.

To facilitate the assessment of large collections of chemical compositions in one or more target-independent (and / or target-dependent) assays, the chemical compositions are preferably logically available sets, ie, Aligned in an "array".
The logical configuration of the array can be based on the location relationships of the array components (ie, where the array is in a grid-like configuration), or any configuration that can correspond to a look-up table that provides equivalent information. Can be Such arrays include master library arrays, as well as arrays of libraries prescreened in one or more target-independent assays. Library arrays also include prescreened libraries that have also been evaluated in one or more target-dependent assays. It is worth noting that, in addition to placing a single compound in the array, the array components can have a mixture of compounds instead. For example, where array components representing a mixture of compounds are tested, the components are further deconvoluted to identify which components are appropriate for activity. For example, if the components of the array components are known, they can be separately screened following the identification of the activity of the mixture. The library array of the present invention can include any material structure in which physical materials, including chemical compositions, can be maintained and utilized, such as multi-well plates, pin arrays, bead arrays, test tube arrays, and the like. For example, in a set of specific examples, for example, "MICROFLUIDIC SAMPLI"
NG SYSTEM AND METHODS "US Patent 6,042,709
The samples are arranged and used by a microfluidic device as described in the publication. A number of further useful library-microfluidic systems are described in "CLOSED-
LOOP BIOCHEMICAL ANALYZERS ”WO98 / 4548
No. 1. For more details on library systems and microfluidic systems, see ULTRA HIGH THROUGHPUT SAMPLING AN.
D ANALYSIS SYSTEMS AND METHODS, Walk et al., USSN 60 / 042,709, filed Jan. 6, 2000.

[0018] In other cases, it may be desirable to use locations in the library array to maintain a dataset related to the results of one or more target-independent assays. In the latter case, the data set is typically stored in a computer readable medium and optionally generated and / or accessed via an integrated system for performing the assay in question. In other embodiments, data storage and assay operations are maintained separately. The data set can be analyzed using a single target-independent assay, multiple target-independent assays, and / or one or more target-independent assays using the potential information identifying individual components of the chemical composition library array. It can be associated with the results of the assay. The array can be physically located on one multiwell plate, pin array, bead array, or the like, or a combination thereof. One or more library components are maintained at respective locations in the array. Further, a set corresponding to a data set or master library, and any number of prescreened libraries, can correspond to a single physical library array or any number of different and separate arrays. For example, a chemical composition library of more than 100,000 components that make up a master library may be a set of multi-well plates, for example, about 66 × 153.
It can be arranged in a 6-well plate, about a 261x384-well plate, or about a 1004x96-well plate, or various combinations thereof. Data sets matching the composition of each library component using locations in the array were typically established via the integrated system of the present invention. The library is then evaluated in the target-independent assays of the invention, and the results are correlated to locations within the array. Additional target-independent and target-dependent assays are performed sequentially or simultaneously, and the results are correlated to locations within the library array. Thus, one or more prescreened libraries of the present invention physically correspond to one or more master libraries of the present invention. Alternatively, the components of the selected library can be transferred and / or copied in a physically or spatially defined library array before performing any sequential assays.

Target-Independent Assays Target-independent assays involve a compound, regardless of the particular activity required.
It is usually an in vitro model system for measuring the effect of a chemical compound on a biological or biochemical system or molecule. In the pharmaceutical industry, whether the lead compounds are either chemical or biochemical compositions, or if they are naturally occurring,
Target-independent assays, whether isolated or synthetic, are used to evaluate potential lead compounds. The present invention
Methods for producing a library of chemical compositions that are evaluated for their effect in a species or higher target independent assay are provided. Therefore, the libraries produced here by the present method meet the specified criteria reflecting their effects in biochemical systems. Library components or components are associated with poor behavior in pre-clinical or clinical trials, and are therefore potentially known to lack substantial negative effects with inadequate therapeutics Provide a pool of lead candidates. Serum half-life, cell uptake, oral availability, cell or organism viability, cell or organism toxicity, apoptosis, cell adhesion, target-independent receptor binding or activity,
Target-independent enzyme modulation activity, target-independent protein modulation activity, target-independent nucleic acid modulation activity, glucuronide binding modulation, sulfate binding modulation, amino acid binding modulation, acylation modulation, methylation modulation, transcription modulation, conversion modulation, protein folding Modulation, modulation of cell growth, membrane permeability, membrane integrity, metabolic stability, heat stability, solubility, solution viscosity, solution turbidity, acidity, basicity, RedOx state, superoxidant secretions, lipid permeation Oxidation, octanol / water partitioning, and precipitation in one or more buffers,
Target independent assays that measure parameters such as can be performed in the context of the present invention. Indeed, any biochemical assay, if performed in a high power assay, whether known or later developed to describe a particular application, is suitable for the method of the invention. is there.

For example, cytotoxicity is assessed by assays such as different binding of live or fluorescent dyes and assessed by flow cytometry, which distinguishes between live and dead cells. Cultured cells, such as the human hepatocellular carcinoma cell line, Hep G2, or other suitable cells, are exposed to a composition of a chemical compound library and simultaneously or sequentially stained with a dye, eg, a live cell. Incubation is performed using propidium iodide, which shows different discoloration of dead cells (dead cells are bright PIs and live cells are dark PIs). This discoloration is then evaluated, and the percentage of dead cells is calculated, indicating an acceptable ratio of dead cells to those living cells, and determining cells exhibiting an acceptable level of toxicity. The bioavailability of a therapeutic composition may include other factors, solubility, stability in solution, water /
Affected by lipid splitting factor, brush border enzyme and protease-mediated intestinal degradation (eg, that occurs in the intestinal gut), and binding to serum proteins. These parameters are the subject of a target-independent assay. For example, the method of the present invention measures binding of a chemical composition library to serum proteins, such as human serum albumin (HSA). For example, the linkage can be one or more fluorescent dyes (eg, dansyl-1-sulfonamide,
Dansyl-sarcosine) as a function of the ability of the composition to affect or inhibit HSA binding.

[0021] The target-independent assays of the present invention also evaluate the binding and / or activity of cellular or reconstituted receptors. Examples of such assays include cellular or in vitro assays. Cellular assays measure binding, for example, as a function of cellular calcium flux. In vitro assays are based on competitive binding to a labeled ligand or other marker molecule, such as a dye. Examples of dye-based microfluidic cell assays are described in US Patent Application No. USSN 09 / 416,288, 19
No. USSN 60 / 158,323 filed Oct. 12, 1999, Jan. 1999
It can be found in the pending application on 08/8. Other target-independent assays measure the effect of a composition on the catalytic activity of an enzyme, eg, inhibition. Enzymes are typically either because they perform an essential or important cell housekeeping role, or because they represent a class of enzymes that play an important role in cell homeostasis, growth, differentiation, and division. Is selected. Such enzymes include: glutathione-s-transferase, protease, peptidase, kinase, phosphatase, G protein, ATPase, cytochrome P450, ligase, carboxylesterase, epoxide hydrolase, aza- or nitro reductase, carbonyl reductase, Sulfide reductase,
Including sulfoxide reductase, quinone reductase, alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, xanthine oxidase, monoamine or diamine oxidase, prostaglandin citase, flavin-monooxidase, and dehalogenase . Suppression of enzymatic activity by chemical compounds of these and other enzymes,
Or, conversely, enhancement can be assessed by a number of well-established assays known to those of skill in the art.

[0022] In contrast to the target-dependent assay target-independent assay, "target-dependent assay" chemical composition or special pharmacological screening target, for example, the desired result of the drug effect is required on the target Evaluate the effect of a library of compositions at a location. A target can be any biochemical molecule that plays a role in a biochemical pathway that mediates a process in an organism. As such, the target affects any or many biological processes involved within the health or disease of the organism. The therapeutic reagent is selected and has the desired effect on the target, or pathway or process involving the target. Target-dependent assays are performed to evaluate potential drug leads, eg, chemical compositions. Any in vitro used in measuring the effect of the chemical composition on potential therapeutic targets,
Cellular or in vivo assays are considered target-dependent assays for the purposes of the present invention. In vitro or cellular assays that measure aspects of the in vivo effect of the composition on the target are also considered target-dependent assays. Target-dependent assays are non-strictly distinguished from target-independent assays by the criteria of the type of assay or biochemical molecule (or pathway) that they evaluate, and the potential treatment of chemical compositions that are evaluated as biochemical molecules. Are distinguished in relation to the use of That is, target-dependent assays assess the biochemical effects of molecules on disease targets, for example, whereas target-independent assays assess efficiency,
The purpose is to evaluate the effect on the second problem affecting bioavailability and / or toxicity. Therefore, target-independent assays are not usually associated with any particular disease condition, but instead include systems or assays that work in normal healthy systems. Targets for target-dependent assays can be, for example, receptors, enzymes, signal molecules, membrane proteins, blood proteins, tissue proteins, ligands, ligands, substrates, cells, nucleic acids (DNA, RNA, etc.), membrane fractions, and the like. . Both target-independent and target-dependent assays can be performed in high power formats such as multiwell plates and microfluidic devices and combinations thereof.

In the present invention, a target-dependent assay can optionally be performed on a prescreened library. Obviously, it does not matter whether the target-dependent assay is performed before or after one or more target-independent assays are performed. However, in general, generating a prescreened library that is evaluated for several target-independent parameters, and then repeatedly using a valuable source as a basis for target-dependent assay screening design, is most likely Efficient and therefore least costly.

High Power Screening Format Prior to the present invention, it is very expensive to produce prescreened libraries that are evaluated in multiple target-independent assays, prior to identifying potential drug leads. Although basic assays have long been known to those skilled in the art, they have lacked the ability to reduce the size of the assay to make it economically desirable as a pre-screening tool. Depending on the particular embodiment being implemented, the chemical composition library can be provided, eg, freely injected in solution, or attached to a carrier or solid substrate, eg, beads. In a preferred embodiment, the chemical composition library (which can be a single compound or a mixture of compounds) can be arranged in an organized matrix or array. The array can be a composition in solution (eg, a microtiter plate), on a solid membrane, in a bead matrix, or other existing format. A number of solid substrates are suitable for immobilizing a chemical composition, including polymers, plastics, agarose, cellulose, dextran, carboxymethylcellulose, polystyrene,
Polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resin, plastic film, glass beads, polyamine methyl vinyl ether, maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer,
Including nylon, silk, etc.

In recent years, microtiter plates have become the standard format for the operation of high power screening assays. Standard microtiter plates are available with 96, 384 or 1536 wells, although the number of other commonly used wells includes 3456 and 9600. Well volumes range from 500 nanoliters to 200 microliters, depending on well depth and cross-sectional area.
Microtiter plates are typically selected for compatibility with current automated loading and robotic handling systems, and have the industry standard dimensions of 86 mm x 129 mm. Alternative formats have been proposed, for example, US Pat. No. 5,989,835, which is equally suitable for the method of the present invention. Other applicable systems are, for example, "MICROFLUI
DIC SAMPLING SYSTEM AND METHODS, US Patent No. 6,042,709, Parce et al. Many other useful library-microfluidic systems are described in "CLOSED-LOOP BIOCHEMI.
CAL ANALYZERS "WO 98/45481. Other library systems and microfluidic systems are available from ULTRA HIGH THROUG
HPUT SAMPLING AND ANALYSIS SYSTEMS A
ND METHODS ", Walk et al., USSN 60 / 042,709,200.
Filed Jan. 6, 2009.

Certain assays involve the detection of products by automated radiography, liquid scintillation counting, fluorescence measurements, etc., in which one or more assay components are used in a multiwell, eg, microwell It is preferably performed by moving to a filtration medium adapted to the titration format (see US Pat. No. 5,939,024). Assays of the invention, including target-independent and target-dependent assays, can be performed in the microtiter format described above. In some assays, the proper choice of well dimensions and reagents makes the required microliter scale in such a format feasible for generating prescreened libraries of the invention. But in many cases, the cost remains high.

[0027] In microfluidic devices the preferred embodiment, target-independent assays of the invention (and / or target-dependent assay) is performed in the microfluidic device or micro laboratory systems,
It allows for the integration of the elements required to perform the assay, automated equipment, and minimal environmental effects in the assay system, such as evaporation, contaminants, operator errors. These microfluidic devices have integration capabilities using, for example, microwell plates, solid substrates, bead arrays, and the like. For example, PCT / US9
Application Ser. No. 08/07723, filed Apr. 3, 1998, provides an example of a format for integrating microfluidic devices with microtiter plates, solid substrates, and the like. A number of devices that can be adapted for use in performing the assay methods of the invention are described by the inventor and coworkers. These devices are described in various PCT applications and U.S. Patents issued by the inventor and their co-workers, and are described in U.S. Pat. No. 5,699,157 issued Dec.
. Wallence Parce), published on 07/14/98, 5,779,8
No. 68 (J. Wallence Parce et al.), No. 5 published on 09/01/98
No., 800,690 (Calvin YH Chow et al.), 12/01/98.
No. 5,842,787 (Anne R. Kopf-Sill, etc.), 12
No. 5,852,495 issued on Feb. 22, 1998 (J. Wallance Park)
e), No. 5,869,004 issued on 02/09/99 (J. Wallence)
No. 5,876,675 issued on 03/02/99 (Coli).
n B. Kennedy), No. 5,880,071 issued on 03/09/99 (
J. Wallence Parce, etc.), No. 5,882 issued on 03/16/99
465 (Richard J. McReynolds), 03/23/99.
No. 5,885,470 (J. Wallence Parce et al.), 08
No. 5,942,443 issued on Jan. 24/99 (J. Wallance Park)
e, etc.), No. 5,948,227 issued on 09/07/99 (Robert S. et al.).
No. 5,955,028 (Calvi), issued on Feb. 21, 2009.
n Y. H. Chow), No. 5,957,579 issued on 09/28/99 (A
nn R.N. Kopf-Sill et al.), No. 5,958, issued on 09/28/99,
No. 203 (J. Wallence Parce et al.), No. 5,958,694 (Theo T. Nikiforov), published on 09/28/99, and 09/28.
No. 5,959,291 issued to J./199 (Morten J. Jensen);
As well as published PCT applications, such as WO 98/00231, WO 98/007
05, WO98 / 00707, WO98 / 02728, WO98 / 05424,
WO98 / 22811, WO98 / 45481, WO98 / 45929, WO9
8/46438, and WO98 / 49548, WO98 / 55852, WO98
/ 56505, WO98 / 56956, WO99 / 00649, WO99 / 10
735, WO 99/12016, WO 99/16162, WO 99/19056
, WO99 / 19516, WO99 / 29497, WO99 / 31495, WO
99/34205, WO99 / 43432, and WO99 / 44217.
Bead arrays in microfluidic systems are available, for example, from USSN by Mehta et al.
09 / 510,626 "Manilation of Micropart
mice in Microfluidic Systems ".

However, it will be appreciated that the particular arrangement of these devices will typically vary depending on the type of assay and / or the desired assay orientation. For example, in some embodiments, the screening methods of the invention can be performed using a microfluidic device having two intersecting channels or microchannels. For more complex assays and assay orientations, multi-channel / intersection devices can be used. The microscale, integrability and self-incorporation of these devices allows for virtually any assay orientation and is understood within the context of microlaboratory systems. For example, early technologies for providing cells based on microscale assays were
"High Throughput Screening Assay S
systems in Microscale Fluidic Devices "
Filed April 4, 1999 by WO 98/00231 and Mehta et al. Entitled "Manipulation of Microparticles In
Microfluidic Systems, 60 / 128,643. A complete integrated system with flow handling, signal detection, sample storage and sample access is available. For example, “High Thr
output Screening Assay Systems in Mi
Croscale Fluidic Devices "WO 98/00231 provides early technology for microfluidics and integration of sample selection and manipulation. Commercial products utilizing the microscale system are HP / Agilent Technologies HP2100 and Caliper HTS System (Caliper Technologies, Inc.).
Mountain View, California).

Normally, cells, enzymes, receptors, substrates, library components and other elements are transported in a microscale system by electrokinetic methods (including electroosmosis or electrophoresis) or by pressure-based flow mechanisms Alternatively, it can be flushed using a combination thereof. In particular, cells are desirably flowed using a pressure-based flow mechanism. Pressure is applied to the microscale element and fluid flow can be achieved using any of a variety of techniques. The flow of fluid (and the flow of suspended or dissolved matter in the fluid, including cells or other particles) is controlled by pressure based on mechanisms such as those based on fluid movement, eg, pistons, pressure bulkheads, vacuum pumps Adjust and replace liquids using probes, etc., and increase or decrease pressure at sites within the microfluidic system. The pressure is optionally by air, e.g., using a pressurized gas, or water pressure, to apply a force through a channel or conduit, e.g., to a pressurized liquid, or alternatively a positive displacement mechanism, i.e., a substance reservoir. Use an adaptive plunger or a combination of such forces. In another embodiment, a vacuum source is applied to a reservoir or well at one end of the channel to flow the suspension through the channel. The pressure or reduced pressure source is optionally provided to an external reduced pressure or pressure pump that is sealably fitted outside the device or system, e.g., at the inlet or outlet of the channel, or they are internal to the device, e.g., integrated within the device. And a micro-assembled pump operatively linked to the channel. Examples of microfabricated pumps are widely described in the art. See, for example, published International Application No. WO 97/02357.

Hydrostatic pressure, wicking and capillary forces are also used to provide pressure for a fluid flow of a substance such as a cell. For example, Alajo, filed February 5, 1999,
"METHOD AND APPARATUS FOR CONT"
INUOUS LIQUID FLOW IN MICROSCALE CHA
NNELS USING PRESSURE INJECTION, WIKI
NG AND ELECTROKINETIC INJECTION "USSN 09 / 245,627. In these methods, the adsorbent or branch capillary structure is placed in fluid contact with the part to which pressure is applied, thereby causing fluid to migrate to the adsorbent or branch capillary structure. A mechanism for reducing the adsorption of substances in a fluid-based stream is described in "PREVENTION OF SURFA" by Parce et al., Filed May 11, 1999.
CE ADSORPTION IN MICROCHANNELS BY AP
PLICATION OF ELECTRIC CURRENT DURING
PRESSURE-INDUCED FLOW "USSN 09 / 310,0
27. Briefly, the adsorption of cells, serum proteins, enzymes, substrates, test compounds or other substances to channel walls and other microscale elements is accomplished by applying an electric field that changes the current to the flowing substance. Can be reduced.

A mechanism for focusing cells and other components at the center of a microscale channel, which can be used to increase assay output by regulating flow rate, is disclosed in H. H., filed May 17, 1999. "FOCUSING O" by Garrett Wada and others
F MOCROPARTICLES IN MICROFLUIDIC SYS
TEMs "USSN 60 / 134,472. Briefly, cells are focused at the center of the channel by focusing the opposing flow stream into a main channel containing the cells or by other fluid manipulation. Diffusible substances, such as unbound or unreacted substances, can be washed from the cells by a buffer that continuously flows through the cell stream or channel, as described by Wada et al. Flow and buffers flow out of the channel and back. In an alternative embodiment, the microfluidic system can be introduced into a centrifuge rotor device, which spins in the centrifuge. Fluid and particle movement through the device depends on gravity and centripetal / centrifugal pressure.

One way to achieve the transport or transfer of substrates, ligands, library components and cells (particularly substrates and library components) through a microfluidic device is by the transport of electrokinetic substances. As used herein, an “electrokinetic mass transport system” refers to the transport and direction of a substance in a microchannel and / or chamber containing a structure through the application of an electric field to the substance, thereby causing the channel and the Cations move to the negative electrode while / or mass transfer occurs through and between the chambers, ie, while the anions move to the static electrode. For example, movement of fluid toward or away from the cathode or anode can cause movement of receptors, enzymes, serum proteins, cells, library components, etc., suspended in the fluid. Similarly, receptors, enzymes, serum proteins, cells, library components, etc. are charged and move in the opposite direction to the charged electrodes (in this case, the fluid flow reaches in one direction and the particle flow moves in the opposite direction). Is possible). In this embodiment, it can be fixed or flowing, and can include a matrix as in electrophoresis. Typically, electrokinetic mass transport and directing systems can use these systems depending on the electrophoretic mobility of charged species in an electric field applied to the structure. Such a system is specifically called an electrophoretic mass transport system. In electrophoretic applications, the walls of the internal channels of the electrokinetic transport system are optionally charged or uncharged. Typical electrokinetic transport systems include glass, charged polymers,
And made of uncharged polymer. The inner channel can optionally be coated using a material that changes the surface charge of the channel.

Various electrokinetic controllers and systems, as well as other references set forth herein, can be found in Ramsey, WO 96/0447, Parce et al., WO9.
8/46438 and Dubrow et al., WO 98/49548. The use of electrokinetic transport to control mass transfer within interconnected channel structures is described, for example, in Ramsey, in WO 96/04447 and US5.
, 858, 195. A typical controller is U.S.A. S. 5,800
690. The modulating voltage may be applied simultaneously to the various reservoirs to achieve the desired flow characteristics, such as a continuous or discontinuous (e.g., regular) oscillation of the labeled component towards the waste reservoir (e.g., causing the sample to oscillate in the direction of travel). A pulsed field) affects the flow. In particular, oscillations of the voltage applied in the various reservoirs can move and direct the fluid flow through the interconnected channel structure of the device.

Illustrative System FIG. 1A shows an example of a microfluidic device suitable for performing a target-independent assay used in the production of a prescreened library of the present invention. Among other things, target-independent and / or target-dependent assays that are fluorogenic assays, including protease, kinase, and phosphatase assays, are readily applicable to operation in the microfluidic device of FIG. 1A. In particular, a chemical composition comprising components of a master library, for example, from a master library array, such as a multiwell microtitration, through a pipettor element 101,
It flows into the main channel 102 where the reservoir 103 flows from the side channel 10
A continuous flow of enzyme flowing through 4 and into main channel 102 is contacted. Once introduced into the main channel, the library components interact with the enzyme stream. The mixed enzyme / library components flow along the main channel 102 without interaction with the channel 105. The fluorescing (or other labeled) substrate contained in the reservoir 106 flows along the continuous flow side channel 105 into the main channel 102 where it is contacted and mixed with the enzyme / library component continuous flow. The action of the enzyme on the substrate produces an increase in the level of the fluorescent signal. This increasing signal is indicated by an increase in fluorescence in the main channel when approaching the detection window 107. The increase in signal occurs in the absence of added library components or in the presence of library components that have no effect on enzyme / library component interactions. Where the library component has an effect on the interaction of the enzyme and the substrate, a change in the signal is seen. For example, assuming a fluorogenic substrate, library components that suppress the interaction of the enzyme with its substrate will produce less generated fluorescent product. This results in a non-fluorescent or less fluorescent source in the flow stream passing through the detection window 107, which corresponds to a source of interest. After passage through the detection window, the flow stream continues to the waste well 108.

A similar arrangement is the high / low voltage independent sources 110 and 11 shown in FIG. 1B.
2 is adapted by the addition of voltage channels 109 and 111 which are fluidly coupled to 2. An alternative device of the invention adapted to an assay, for example a GST assay, is illustrated in FIG. The microfluidic device includes a main flow channel 204 that operates vertically down the center of the body of the device. The main channel 204 is the buffer channel 20
6 and a fluid connection at the waste reservoir 208. Buffer channel 206
Fluidly couples with buffer reservoirs 210 and 212. The apparatus is shown to have multiple channels that intersect with main channel 204. For example, buffer channel 214 ends and fluidly couples near its starting point of main channel 204 and fluidly couples with buffer reservoir 216 at the other end. Sample introduction channel 218 also terminates and fluidly couples with main channel 204 and fluidly couples with sample reservoir 220 at the other end. Additional buffer / waste channels 222 and 224 and buffer / waste reservoirs 226 and 228 are also shown. Descriptors for the various wells and channels are primarily represented for the purpose of distinguishing the various channels and wells from each other. It is envisioned that different wells and channels can be used for a variety of different reagents depending on the analysis being performed.

Sources of assay components and integration with microfluidic formats Sources of assay components such as cells and isolated proteins, sources of substrates and ligands, potential components of library of chemical compositions of the present invention The source of the modulator can be in fluid communication with the microchannel shown here in any of a variety of ways. In particular, "Closed Loop Bio" by Knapp et al.
chemical Analyzers "(WO98 / 45481; PCT / U
S98 / 06723) and “High Throughput” by Parce et al.
Screening Assay Systems in Microscal
e Fluidic Devices "WO 98/00231 and, for example, Me
hta et al. “Manilation of Micropar
titles In Microfluidic Systems ”60/12
8,643, these systems including the sources of the substances mentioned in the application filed Apr. 4, 1999 apply. In these systems, a “titrator element” (ie, a channel through which components can move from a source to a microscale component such as a second channel or reservoir) is temporarily or permanently associated with a source of a substance. I do. The source is internal or external to the microfluidic device containing the titrator element, and can be, for example, a "siper chip" (essentially in a microfluidic device containing microscale channels such as capillaries). And external to the body of the microscale device). The device may be used, such as through a microwell plate, membrane or other solid substrate containing lyophilized components, bead matrices, wells or reservoirs, through the body of the microscale device itself and other titrator elements. Contact with external substance source. For example, the source of the cell type, assay component or library component is a microwell plate external to the body structure having at least one well with, for example, selected cells or reagents. Alternatively, a well located on the surface of the body structure containing the selected cell type, component or reagent, a reservoir located on the surface of the body structure containing the selected cell type, component or reagent; Body structure or liquid, partially liquid, encapsulated gel, dried, partially dried, lyophilized, containing at least one compartment containing the selected particle type or reagent, Or in an external container of solid phase structure containing selected cell types or reagents in other stabilized forms.

The chemical composition, including libraries, substrates, targets, reagents and other assay components, is typically provided in liquid form, eg, in solution, to a microfluidic device. Alternatively, the chemical composition, substrate, target, reagents, etc. are provided adhered or fixed to a particle matrix, such as a bead. The particle matrix can be essentially discrete and insoluble, and in some cases, can flow through a microscale system. Examples of particles can be beads or biological cells. For example, polymer beads (eg, polystyrene, polypropylene, latex, nylon, and many other materials), silica or silicon beads, ceramic beads, glass beads, magnetic beads, metal beads, and organic compound beads can be used. A great variety of particles are commercially available, such as those typically used in chromatography (eg, The 1999 Sigma “Bioch”
chemicals and Reagents for Life Science
es Research "Catalog from Sigma (Saint
Louis, MO) eg pp. 1921-2007; The 1999 S
upleco "Chromatography Products" Catalog
log and others), which are also commonly used in affinity purification (for example, Dynal, Dynabeads (registered trademark), and also, for example, Pro
mega, the Baxter Immunotherapy Group,
And many derivitized sources provided by many other sources.
beads), for example, various Derivatized Dynabeads® (eg, various magnetic Dynabeads®, which usually include binding reagents). Further details regarding the flow of particles can be found in the title "Manipulation of Microparticles" by Mehta et al.
In Microfluidic Systems "USSN 60 / 128,6
43, which can be found in the application filed April 4, 1999. As set forth herein, the definition for particles as intended herein includes biological and non-biological particulate matter. Therefore, cells are included within the definition of particle for the purposes of the present invention. The array particles are essentially of any shape, such as spheres, spirals, oblate, rods, cones, cubes, polygons, or combinations thereof (of course, as well as for certain cell-based particles). ) Irregular.

Cell-based microfluidic assays have been described in various publications by the inventors and their co-workers, and include, for example, “High Throughput” by Parce et al.
Screening Assay Systems in Microsca
"Le Flidic Devices" WO98 / 00231 and "Closed Loop Biochemical Analyzers" such as Knapp
(WO98 / 45481; PCT / US98 / 06723). It anticipates that one skilled in the art will culture cells and introduce them into a microfluidic device. In addition to Parce et al. And Knapp et al., Many references include bacteria, plants,
It can be used for the culture and production of many cells, including cells of animal (especially mammalian) and primitive bacterial origin, eg, Freshney (1994), Culture.
of Animal Cells, a Manual of Basic T
technique, 3rd edition Wiley-Liss, New York and also referred to herein as Sambrook, Ausubel and Ber.
ger (all above), Humason (1979) Animal Tissue
Techniques, 4th ed. H. Freeman and Comp
any; Ricciardelli et al., (1989) In Vitro Cell Dev. Biol. 25: 1016-1024; Payne et al. (1992).
Plant Cell and Tissue Culture in Liq
uid Systems John Wiley & Sons, Inc. New
York, NY (Paync); Gamborg and Phillips (
eds) (1995) Plant Cell, Tissue and Orange
n Culture; Fundamental Methods Spring
er Lab Manual, Springer-Verlag (Berlin
Heidelberg New York) (Gamborg); and At
las and Parks (eds) The Handbook of Mi
crobiological Media (1993) CRC Press, B
See oca Raton, FL (Atlas). Further information on culturing plant cells can be found in Sigma-Aldrich, Inc (St Louis
s, MO) (Sigma-LSRCCC) Life Science Re
search Cell Culture Catalog (1998),
And, for example, Sigma-Aldrich, Inc (St Louis, MO) (
Plant Culture Catalo, also made by Sigma-PCCS)
gue and found in commercially available commercial literature such as the Appendix (1997).
A particularly preferred use for cell-based microfluidic assays is a screen for binding and / or internalization of cell ligands, eg, cell receptor ligands, drugs, co-factors, and the like. This screening is further facilitated by arranging different sets of cells within an array of cells, the array of cells comprising a reagent containing any factor whose in vitro cell activity flowing through the set of cells is to be tested. Flow. Of course, cells can also be present in the flow of reagents and pass in contact with other array components.

The cells are optionally present as a set of particles in various formats of the invention. For example, cells can be immobilized on a solid substrate such as beads or other microparticles. Thus, the arrays of the present invention optionally include a heterogeneous particle comprising a solid support and cells or other critical components. In addition, for cells containing surface proteins and other molecules, cell binding molecules (cell receptor ligands, cell wall binding molecules, antibodies) to either the source of the channels in the microfluidic device or particles localized at locations within the microfluidic device Etc.) is convenient. A fill channel source is optionally fluidly associated with the titrator channel using an external port of the body structure. The filling channel uses the external port of the body structure,
Fluid coupling with the titrator channel and the well on the surface of the body structure using the pressure-based titrator channel and the internal port of the body structure using the electrotitrator channel and the external port of the body structure And an internal channel in the body structure that fluidly couples with a well in the body structure. As described more fully herein, the integrated microfluidic device of the present invention includes a very wide variety of storage elements for storing reagents to be evaluated. These include well plates, matrices, membranes and the like. The reagent is stored in liquid (eg, in a well in a microtitrator) or in a lyophilized state (eg, dried in a membrane or in a porous matrix such as a bead);
It can then be transported to the array components of a microinduction device using conventional robotics or electrotitrators or pressure titrator channels that fluidly couple with the reaction or reagent channels of a microfluidic system.

Typically, individual components of a library of chemical compositions are separately introduced into the assay systems described herein, and at least introduced into a relatively manageable pool of library components. . The relative effect of a library component (or pool of library components) on a particular biochemical system that is a target-independent or target-dependent assay is then measured relative to the control system and lacks the introduced library components. An increase or phenomenon associated with a cell, enzyme, binding, or other function indicates that the library component is an enhancer or inhibitor of a particular cell or molecular function, respectively.

Although the devices and systems specifically described herein generally relate to performing several or one special operation, the flexibility of the system makes it easier to operate further into these devices. It will be readily apparent from this disclosure that it is possible to integrate For example, the devices and systems described may, in fact, optionally include structures, reagents and systems for performing any number of operations both upstream and downstream from the actual systems described herein. . Such an upstream operation is
Sample handling and preparation operations include, for example, cell separation, extraction, purification, culture, expansion, cell activation, labeling, reaction, dilution, aliquoting, and the like. Similarly, downstream operations include similar operations, e.g., separation of sample components, labeling of components, assay and detection operations, components linked to cells or other assay components, or reactions separated from cells or of an assay of the invention. Or electrodynamic or pressure-based injection of components such as those produced by Further, the devices and systems optionally include structures for storage of one or more master and / or prescreened libraries, such as multiwell plates, pin arrays, bead arrays, and the like. For example, the system can include structures for cold storage, eg, refrigeration, freezing, storage of lyophilized material, liquid storage, and the like. Upstream and downstream assays and detection procedures include, without limitation, cell fluorescence assays, cell activity assays, molecular activity assays, receptor / ligand assays, immunoassays, and the like. Any of these elements can be fixed to an array component or, for example, a channel wall or the like. The target-independent assays of the present invention, as well as the desired upstream and downstream assay assays, are desirably performed in the context of an integrated system. The integrated system of the present invention includes, for example, one or more multi-well plates, robotic elements (eg, plate handling mechanisms, transport devices, etc.), controllers, microfluidic devices, detectors, and computers (eg, user input devices, Data output device etc.).

Instruments In the present invention, usually, cells, fluorescently labeled substrates and / or products are optionally monitored and / or detected so that functions such as enzyme activity can be measured. Based on the label signal, a series of decisions regarding the flow operation can be made, such as whether a particular modulator is assayed in detail and dynamic information is determined. The integrated system described herein comprises additional devices for controlling fluid movement, flow rate and direction within the device, detection instruments for detecting or detecting the results of operations performed by the system, a processor ( For example, to direct the controlling instrument according to programmed instructions, receive data from the detecting instrument, and analyze, store, and translate the data, and to generate the data and translate it into a readily accessible reporting format. And a microfluidic device as described above in connection with a computer. One example of an integrated system suitable for performing the target-independent assays (target-dependent assays) of the present invention is Caliper (
Caliper Technologies Corp. , Mountain
View, CA; www. callipertech. com. ), Labch
ip® Microfluidic High Power Screening System (HTS). The apparatus includes, for example, robots, flow engineering, plate handlers, detectors, and the like.

For a typical series of arrangements involving the transfer of components to or from a microtiter plate, a plate handling station is used. Several "off-the-shelf" plate handling stations capable of performing such transfers are commercially available, such as Zyma from Zymark Corporation.
rk system (Zymark Center, Hopkinton, MA; ht)
tp: // www. zymark. com /) and other stations utilizing an automatic titrator, eg, coupled with a robot for plate movement (eg, an ORCA® robot, eg, B, available in a variety of available laboratory systems).
Eckman Coulter, Inc. (Fullerton, CA). Further, CCS Packard (www.callcreative.com).
available micro-component systems, such as liquid handling (eg, dispensing, titration, etc.), transport and PlateTrak®, SideTrak (
®, and components for plate handling via the PlateStak ® element can be incorporated.

Controllers A variety of controlling instruments are connected to the microfluidic device to control the transport and orientation of fluids and / or substances in the device of the present invention, for example, by pressure based or electrokinetic controls. And can be used at will. For example, fluid transport and directing is often controlled, in whole or in part, using pressure based flow systems that introduce external or internal pressure sources to move the fluid. Internal sources include microfabricated pumps, such as diagram pumps, heat pumps, Lamb wave pumps, etc., which are described in the art. For example, U.S. Patent Nos. 5,271,724, 5,277,556 and
375, 979 and published PCT applications WO 94/05414 and WO 97/02357. As noted above, the systems described herein may also utilize electrokinetic material orientation and transport systems.

Preferably, an external pressure source is used and applied to the port at the end of the channel. These applied pressures or reduced pressures create a pressure differential across the length of the channel, moving fluid through them. In the interconnected channel network described herein, different flow rates in volume can be achieved by applying different pressures or reduced pressures in multiple ports, or preferably applying a single pressure in a common waste port, And optionally by arranging the various channels with appropriate resistances to produce the desired flow rates. Examples of systems are described, for example, in USSN 09 / 238,467, 1/28/99 application. Typically, the controller system will be appropriately positioned to accept or connect the microfluidic devices or system elements described herein. For example, the controller and / or detector may be equipped with the device of the present invention and the controller and / or detector.
Or optionally includes a stage to facilitate proper connection between the detector and the device. Typically, this stage includes appropriate mounting / array structural elements, such as nesting walls, alignment pins and / or holes, asymmetrical edges, etc. (to facilitate proper device alignment). Many such sequences are described in the references cited herein. The controlling device described above may also be used to provide an electrokinetic injection or withdrawal of material downstream of an important source for controlling upstream flow rates. The same equipment and techniques described above can also be used, and can be used to inject fluid into a downstream port and act as a flow control element.

Detector Here the device optionally comprises a single detector, for example, it detects atomic weight, fluorescence, phosphorescence, radioactivity, pH, charge, absorption, luminescence, temperature, magnetism, etc. Fluorescence detection is particularly preferred and is usually used for the detection of many compounds (but as shown
Upstream and downstream operations are performed on cells, assay components, library components, etc.
Other detection methods can be included). Besides fluorescence, evaporative light scattering and mass spectrometry
There are two additional detection mechanisms available in the context of the present invention. The detector optionally monitors one or more signals from downstream of the assay mixing point where the labeled substrate and cells or other assay components are mixed with the library components. For example, the detector can monitor multiple light signals that are consistent with "real-time" assay results.

Examples of detectors include photomultiplier tubes, spectrophotometers, mass spectrometers, CCD arrays, scanning detectors, microscopes, galvo-scans, and the like. Cells, labels or other components that emit a detectable signal flow past the detector, or alternatively, the detector moves relative to the array and can determine cell location (or CCD). As in the array, the detector can simultaneously monitor many spatial positions corresponding to the channel sources). More particularly, the detectors used in the devices and systems of the invention include, for example, one or more optical detectors, microscopes, CCD arrays, photomultipliers, photodiodes, emission photometers, fluorimeters , Phosphorescence meter, luminescence photometer, spectrophotometer, photometer, nuclear magnetic resonance photometer, electronic paramagnetic resonance photometer, electron spin resonance photometer, turbidimeter, Raman photometer, refractometer, interferometer, X-ray diffraction analyzer, electron diffraction analyzer, polarimeter, optical rotatory dispersion analyzer, circular dichroism photometer, potentiometer, chronopotentiometer, coulometer, ammeter, conductivity meter, hydrometer, mass analyzer, heat Includes hydrometer, titrator, differential scanning calorimeter, radioactive activation analyzer, radioactive isotope dilution analyzer, and the like. Detectors suitable for use in the methods and devices of the present invention include optical detectors and, for example, electrospray ionization mass spectrometers, which are, for example, adjacent to or associated with one or more microscale channels of the device. Specific examples of detectors that include mass analyzer elements are discussed further below.

The detector may include a computer or may be operatively connected, for example, having software to convert detector signal information into assay result information (eg, dynamic data of modulator activity) and the like. The signal is optionally calibrated, for example, by calibrating the microfluidic device by monitoring a signal from a known source. Microfluidic devices can also use multiple different detection systems to monitor the output of the system. The detection system of the present invention can be used to detect and monitor substances, particularly in the channel portion (or other reaction detection portion). Once detected, the flow rate in the channel and the cell velocity are also optionally measured and controlled, as described above. PCT / US98 / 11969 and 6
Calibration of dynamic information based on flow rates and other factors, as described in U.S. Pat. No. 0 / 142,984, is used to provide accurate dynamic information in flowing systems. As mentioned above, examples of detection systems include optical sensors, temperature sensors, mass sensors,
Includes pressure sensors, pH sensors, conduction sensors, etc. In these systems, the detector is connected to the device, channel or chamber on the sensor.
The microfluidic device or one or more channels, chambers, or conduits of the device, either in or adjacent to it, as ory communication with. As used herein, the phrase "connection on a sensor" of a particular part or element refers to a component in which the detector is a microfluidic device, part of a microfluidic device, or part of a microfluidic device for which the detector is intended. Means the position of the detector at a position where the characteristics of the detector can be detected. For example, a pH sensor placed on the sensor with the microscale channel can measure the pH of the fluid in the channel. Similarly, a temperature sensor placed on the sensor with the body of the microfluidic device can measure the temperature of the device itself.

Particularly preferred detection systems include optical detection systems for detecting the optical properties of substances in channels and / or chambers of a microfluidic device introduced into the microfluidic systems described herein. Such an optical detection system is typically located adjacent to the microscale channel of the microfluidic device and is connected to the channel and the sensor via an optical detection window located across the channel or chamber of the device. You. Optical detection systems include systems that can measure the light emitted from a substance in a channel, the transmission or absorption of the substance, as well as the spectral properties of the substance.
In a preferred embodiment, the detector measures the amount of light emitted from a substance, such as a fluorescent or chemiluminescent substance. Thus, the detection system typically includes focusing optics for collecting light based on the signal transmitted through the detection window and transmitting the light to a suitable photodetector. The power variation, field diameter and focal length microscopic object are immediately utilized as at least part of this light train. The photodetector is optionally a spectrophotometer, a photodiode, an avalanche photodiode, a photomultiplier, a diode array, or, in some cases, a charge-coupled device (CCD).
And the like. The detection system typically uses an analog-to-digital or digital-to-analog converter to transmit the detected optical data to a computer for analysis, storage and data translation,
Connect with computer.

In the case of fluorescent materials, such as labeled cells, labeled substrates and / or labeled products, the detector typically emits light at an appropriate wavelength to activate the fluorescent material. And a optic for directing the light source through the detection window to a substance contained within the channel or chamber. The light source can be any number of light sources that provide a suitable wavelength, including lasers, laser diodes, and LEDs. Other light sources are used in other detection systems. For example, broadband light sources are typically used in light scattering / transmission detection schemes and the like. Typically, light selection parameters are known in the art. As already indicated, mass spectrometry provides another preferred method for detecting the results of target-independent or target-dependent screening assays. Mass spectrometry is a widely used analytical technique, for example, information on the isotope ratio of atoms in a sample, various molecular structures, including biologically important molecules, and the qualitative and quantitative composition of complex mixtures. Can be used to provide A typical mass spectrometry system includes a system inlet, an ion source, a mass analyzer, and a detector, typically under reduced pressure. The detector is typically in operable connection with a signal processor and a computer. Desorption ion sources for use in the present invention include field desorption (FD), electrospray ionization (ESI), chemical ionization, matrix-assisted desorption / ionization,
(MALDI), plasma desorption (PD), fast atom bombardment (FAB), secondary ion mass spectrometry (SIMS), and thermal spray ionization (TS). ESI sources are particularly preferred.

[0051] Mass spectrometry is known in the art. Connecting a mass spectrometer using a microfluidic device
Are described, for example, in U.S. Pat. No. 5,571 to Karger et al.
, 398 “Precise Capillary Electrophore”
TIC INTERFACE FOR SAMPLE COLLECTION
OR ANALYSIS "and U.S. Patent No. 5,872,0 by Karger et al.
No.10 "MICROSCALE FLUID HANDRING SYSTEM"
"including. A common source of information about mass spectrometry is eg from Skoog et al. Principles of Instrumental Analysis (
5th edition) Hardcourt Brace & Company, Orlando
(1998). Usually, mass spectrometers are good for connecting to microfluidic devices.
Is suitable because the input into a normal microfluidic device is
Because In the present invention, this mass spectrometry capillary is a microscale.
Simply and fluidly with the channels in the system. To microfluidic systems
A method of adding an external capillary is described, for example, in US Pat.
No. 2,187 "ELECTROPIPETTOR AND COMPENSAT
ION MEANS FOR ELECTROPHORETIC BIAS "
Includes various sticking and / or drilling operations as described. Mass spectrometer
The advantage of connecting to a chromatograph is that the ionization chamber of the mass spectrometer is usually
That is. This vacuum is used as a negative pressure source for microfluidic devices
And provide an operating mechanism for the system. Another advantage is that for the detection of analytes,
Type is that no label is required.

A number of special microfluidic device configurations that specifically employed screening by mass spectrometry are described in US Pat. No. 60 / 176,093, January 14, 2000 by Parce et al.
Filed “ASSAYS AND MICROFLUIDIC DEVICE F”
OR TRANSPORTER, GRADIENT INDUCED, AND B
INDING ACTIVITIES ". In addition to the many microfluidic systems shown herein, target-independent assays, libraries and other automated tests using the methods of the invention can also be performed. For example, the FLIPR (Fluorescence Imaging Plate Reader) has been improved and quantitative optical screening for cell-based kinetic assays has been performed (Schroder and Neagle (1996) "FLI
PR: A New Instrument for Accurate, Hig
h Throughput Optical Screening " Journal of Biomolecular Screening 1 (2): 75
-80).

Computer As described above, either or both of the controller system and / or the detection system directs the operation of these instruments according to programmed or user input instructions, receives data and information from these instruments, It is coupled to a suitably programmed processor or computer that performs the translation, manipulation and reporting functions. Thus, the computer is typically suitably coupled to one or both of the devices (eg, an analog to digital or a digital to analog converter if necessary). The computer typically receives instructions from the user in the form of user input into configuration parameter fields, such as in a GUI, or programmed instructions, such as programmed for various different special operations. To
Including appropriate software. The software then translates those instructions into the appropriate language to direct the flow direction and operation of the transport controller and perform the desired operation. The computer then receives the data from one or more sensors / detectors included in the system, translates the data, provides it in a format understood by the user, and monitors and monitors flow rates, temperatures, applications, and programming. The data is used to initiate further controller instructions, such as in voltage and other controls. In the present invention, the computer typically includes software for monitoring the substance in the channel. In addition, the software is optionally used to control the injection or discharge of electrokinetically or pressure modulated substances. Injection or discharge is used to modulate the flow rate, mix the components, and the like.

Multi-Module Workstations In some embodiments, the target-independent and / or target-dependent assays of the present invention are performed in an integrated system that includes a multi-module workstation. The multi-module workstation of the present invention is for performing high power assays in microtiter plates, such as the ORCA® and PlateTrak® described above, in a plate reader, or directly from a pin or bead array. It also includes one or more microfluidic devices, as well as optionally including one or more devices. Microfluidizer or other microscale device, a multi-well plate reader (e.g. Bio-Tek Instruments, Inc: www.copali s.com; manufactured Elx800 and Bio-Rad: www.bio-rad.c om manufactured Ultramark and Benchmark Plate Reader Fluorometric, optical, colorimetric plate readers) and / or devices for automated instrumentation, such as robotic implementation of target-independent (and / or target-dependent) assays.
Or a screening module comprising any combination thereof is arranged to provide access to individual modules by a robotic mechanism. (Each module, which can be a single module or each module operated in conjunction with one or more additional modules in a multi-module workstation, is described in more detail above. As such, it includes the necessary methods and components for performing the identified targets or target-dependent assays, or sub-portions thereof (eg, sample preparation, incubation, etc.).

The robotic mechanism may be used to acquire and manipulate armatures, conveyor belts, or one or more assay components or library arrays, such as chemical composition libraries arranged in large numbers of multiwell plates. Transport mechanism. The armature or other transport mechanism can be movably mounted between the multi-module workstations (or the modules are movably mounted) and include, but are not limited to, library arrays, reagents, cells, substrates One or more assay components, including the like, can be transferred to individual modules (or alternatively the modules can be transferred to library members, or both components can be transferred to achieve an operable connection between the module and the library Can carry). The armature or other transport mechanism may include, for example, trucks, cables and pulleys, conveyors located adjacent to or between one or more modules, or between a screening module and one or more assay components. Mechanisms such as belts, rollers, robotic means, etc. can (and vice versa) transport between modules.

FIG. 9 illustrates an exemplary arrangement of such a workstation. For example, in one or more multi-well plates, the substrate containing the master library array is arranged in an organized array within the plate storage module 1012 (optionally including refrigeration elements, refrigeration elements, etc.). The user selects assay parameters via user input elements (eg, keyboard, touch screen, mouse, etc.) and sends a series of instructions through the controller element 1013 (PC computer, drive mechanism, etc.) to the track robot 10.
Carry to 10. The selected substrate is retrieved from the plate storage module 1012 by the robot armature 1011 and the truck robot 1010
Is transported through 1008 to one of the assay modules 1001. These assay modules typically have appropriate assay device elements (eg, microfluidic chips and detection elements) for performing well-defined assays. Upon completion of the first target-independent assay, the substrate is optionally transported to a second assay module for evaluation of a further target-independent or target-dependent assay. The results of one or more target-independent and / or target-dependent assays are described (although depicted as a single output device, multiple output elements can be used in the system, eg, one or Displayed on output device 1014 (coupled to further assay modules). The storage medium may be in a separate element such as a computer or a disk drive. Typically, a storage medium is operably linked to the assay module and stores the results of the assay. A database containing information on the location of the assayed component and the assayed component indicating the desired activity can be included in the controller element or other linked data storage system.

In this embodiment, and in certain alternative embodiments, a plurality of prescreening target-independent and / or target-dependent screening assays can be performed in each of the assay modules. Similarly, multiple target-independent assays and / or target-dependent screening assays can be used instead of multiple assay modules and achieve similar results.

Database One aspect of the present invention involves registering descriptors in a database for components of a library of prescreened compositions. Contains any relevant information describing the physical, logical or spatial properties or activities of the components of any pre-screened composition library (or any other library described herein). be able to. Typically, a database is embodied in a computer or computer-readable medium for access by a user and / or in an integrated system, for example, which operates on a library based on the information in the database (eg, One or more elements of an integrated system, such as plates, microfluidic components, etc. depending on information in a database).

A word processing software (for example, Microsoft Word (
(Registered trademark) or Corel WordPerfect (registered trademark)) database software (for example, Microsoft Excel, Corel Qua)
spreadsheet software such as troPro (R) or M
A standard desktop application (such as a database program such as Microsoft Access (R) or Paradox (R)) inputs a character string corresponding to a descriptor registered in the database. Thereby, the present invention can be adapted. For example, a computer system embodying a database may be, for example, a user interface (a GUI in a standard operating system such as a Windows, Macintosh, or LINUX system).
Software having the appropriate character string information to concatenate with and process strings of characters. The system for analysis of the present invention is a digital system with software for tracking library registrations, assay results, etc. and for using the information processing system (in pre-screening target-independent assay steps or target-dependent assay screening). It can include a computer. The computer may be, for example, a PC (any available operating system such as DOS®, OS2®, WINDOWS®, WI
NDWS NT (registered trademark), WINDOWS 95 (registered trademark), WINDO
WS98 (registered trademark), LINUX, Apple OS, Power PC, U
NIX (R), an Intel x86 or Pentium (R) chip-compatible computer running) or a commercially available conventional computer known to those skilled in the art. Software for processing information descriptor elements is available or available in Visualbasic, Fortran, Basi.
c, it can be easily created by those skilled in the art using Java (registered trademark) or the like.

Assay Kits The present invention also provides kits for producing the libraries of the present invention. In particular, these kits typically include microfluidic devices, systems, modules and workstations for practicing the present invention. The kit includes, but is not limited to, robotic elements (eg, truck robots, robotic armatures, etc.), plate handling devices, fluid handling devices, and computers (including input devices, monitors, cpu, etc.). Additional components for the assembly and / or operation of the multi-module workstation of the present invention may optionally be included. Typically, the microfluidic devices described herein are optionally packaged to include reagents for performing the appropriate functions of the device. For example, a kit is described with assay components (eg, buffers, reagents, enzymes, serum proteins, receptors, etc.) to perform a target-independent assay used to generate a prescreened library of the invention. An optional microfluidic device is optionally included. In the case of packaged reagents, pre-measured or pre-administered reagents (eg, pre-measured fluids that are easily reconstituted by the end user of the kit) are introduced immediately into the assay method without measurement. Part or pre-weighed or pre-measured solid reagent) can optionally be included.

Such a device typically includes appropriate instructions for using the device and reagents, and, if it includes reagents for performing the desired functions of the device, within channels and / or chambers of the device. With appropriate instructions for introducing the reagents into the In the latter case, these kits include special auxiliary devices for introducing the substance into the microfluidic device, for example, suitably forming a syringe / pump or the like (in some preferred embodiments, the device itself is an internal device). Including a titrator element, such as an electrotitrator, for introducing a substance into the channels and chambers of the same). In the former case, such kits typically include a microfluidic device with the necessary reagents pre-administered in the channels / chambers of the device. Usually, such reagents are provided in a stabilized state. Such reagents are provided in a stabilized form to prevent prolonged storage degradation or other losses, for example, due to leakage. Many stabilization methods are used for stored reagents, which are chemical stabilizers (ie enzyme inhibitors, microsides / bacteriostats, anticoagulants), such as immobilization on a solid support, a matrix (ie, Physical stabilization of the substance through traps, freeze-drying, etc. in beads, gels, etc.). The elements of the kit of the invention are typically packaged with a single package or a set of related packages. The package can optionally include written instructions for performing one or more target-independent assays for the methods described herein. The kit can optionally also include a packaging material or container to maintain the microfluidic device, system or reagent element.

[0062]

【Example】

The following examples of target independent assays performed in a microfluidic device are provided by way of illustration and without limitation. Those skilled in the art will appreciate that while various parameters can be varied, substantially similar results are still achieved. Example 1: HSA assay Human serum albumin (HSA) is the most abundant protein in blood and is bound by a wide variety of biological materials. Many drugs bind reversibly to HSA and have high affinity. In drug discovery, measurement of drugs that bind to HSA is used to prioritize potential leads from target screening prior to subsequent animals and / or clinical trials. In the present invention, a large library of chemical compounds is first evaluated for binding to HSA. Then one
Provide a prescreened library to be evaluated in species or more target dependent or screening assays. The HSA assay protocol is more compatible with attempts to detect compounds that interact with human serum albumin in a published fluorogenic buffer (eg, Epps et al. (1995) Analytical Biochemistry 227: 342). Briefly, this assay is a competitive binding assay that measures the displacement of a dye that binds to HSA by a library component.

[0063] Two major sites on albumin have been measured and are important for binding both dyes and potential drugs. In this example, two different dyes are used to selectively bind to each of the major binding sites on albumin (Sites I and II) respectively: Dansyl-1-sulfonamide (DNSA) and Dansyl sarcosine (DS) . These dyes have properties of fluorescence with a greatly increased yield or binding to albumin. Compounds that bind albumin through one of these two sites displace the dye and result in a decrease in fluorescence that is optionally used to calculate the Kd and rank order of binding to albumin. The assay was performed using only one dye at a time, but has no effect on the spectrum and can be run simultaneously. The albumin-dye concentration is selected and the pre-measured K
a d-value can be produced, which can be about ４4 μM or more;
And the corresponding drug concentration is of the order of 20 μM. The HSA assay was performed in a microfluidic device of the design illustrated in FIG. 1A and has been previously described herein. The buffer system is p
H7.4 phosphate buffered saline (PBS), and typically the assay conditions are 10-60 seconds / compound time and 0.1 at one compound concentration.
Requires less than pmol / measurement of compound. De-Fatted
d) and essentially globulin-free human serum albumin were used in the assay.
The assay was as follows:

Embedded image

In this example, only Site I dye (DNSA) is used. The albumin concentration was 15 μM and the dye was a site I specific dye, 30 μM DNS
A. The experiments were performed at room temperature. Excitation of the fluorescence was centered at 330 nm and emission was 475-525 nm. The pressure applied to achieve the flow was 32 inches of water. The duration of the experiment was 10-60 minutes. The instrument used for data collection is shown in Figure IA. In this experiment, albumin and DNSA were premixed in the PBS buffer and added to wells 103 and 106 on the microfluidic device shown in FIG. IA (albumin and dye were transferred from well 103 to , It is also possible to supply albumin separately from well 106). The inhibitor in the HSA-drug binding plot (FIG. 3A) is phenylbutazone, which is known to bind to site I on HSA. Drug concentration is ~
7, 17, 35, 55, 75, 110, 150 and 180 μM. The assay cycle time for each sample is 25 seconds. The% inhibition values in the inhibition vs. phenylbutazone plot (FIG. 3B) are determined from the data shown in FIG. 3A (two replicates are shown). The assay was run for 〜45 minutes and the same titration (with eight different inhibitor concentrations) was run with 10 replicates.

In the HSA-binding plot for the six drugs (FIG. 3C), the different drugs are 175 μM in DMSO / PBS buffer. The cycle time was 30 seconds / sample, and the microfluidic device used (FIG. 1) was coated using a PEG (polyethylene glycol) coating. The six different drugs used are:
Phenylbutazone, sulfinpyrazone, acetylsalicylic acid, tolbudamide, warfarin (
warfarin) and ibuprofen. There are two different types of negative controls without drug, one with PBS buffer and one with 1-% DMSO / PBS buffer. Rankings for microfluidic devices and cuvettes were performed in rank order of drug plots binding to HSA and compared to literature values. The experimental concentrations and buffers for the cuvette experiments were essentially similar to those described for the microfluidic device above. The results are shown below: Rank order of drug binding to HSA (from strongest to weakest) Chip cuvette Literature Phenylbutazone Phenylbutazone Phenylbutazone Salphinepyrazone Salphinepyrazone Sarphinepyrazone Torbudamide Warfarin Torbudamide Acetylsalicylic acid = Tolvudamide warfarin warfarin = ibuprofen acetylsalicylic acid acetylsalicylic acid ibuprofen ibuprofen

In the HSA binding assay compilation embodiment, the consumption is within a given concentration range based on the above experimental conditions. The cycle time is the current, non-optimized time. The weight percentage of DMSO allowed in the assay is tested in a cuvette format. Rank order of binding agents is maintained up to 〜5% DMSO. The following is a compilation of embodiments of the HSA Binding Assay: Embodiments of the HSA Binding Assay Concentration Human Serum Albumin and Dye: 5-50 μM Compound: 10-500 μM Consumption Assay Reagent 50-500 femtomoles / data point; 10 μl / 8 hr Compound 10 -500 femtomoles / data point; 1 nl / sample time cycle time 25 seconds; 145 data points / hour DMSO weight percentage in assay 0.1-5%.

Example 2 GST Assay Glutathione-s-transferase (GST) plays an important role in antioxidant defense and is part of the animal's biochemical adaptation to environmental pressure. GST catalyzes reduced glutathione binding to a variety of endogenous and exogenous lipid-soluble xenobiotics, including dimethylaniline analogs, aminopyrine, imipramine, chlorpromazine, and related antidepressants and pesticides. As depicted, (1.) represents glutathione (γ-Glu-Cys-Gly).
2) shows a general schematic of the reaction on an electrophilic substrate catalyzed by GST. The GST assay consists of 100 mM bisTRIS pH 6.5, 1 M NDS
B (non-detergent sulfobetaine) can be performed using pentafluorobenzoyl phosphor (pFBF) at 10-20 μM (commercially available from Molecular Probes) in 5 mM NaCl. GST (rat liver)
Was purchased from Sigma Chemical (St Louis, MO). The assay was performed on a planar with the channel layout depicted in FIG.
r) Performed in a microfluidic device. FIG. 4, panel B shows the structure of an example of a substrate for performing a GST assay, and also shows the results of the assay (panel C). As depicted in FIG. 4B, (1.) is a colorimetric substrate (2,4-dinitro-chlorobenzene); (2.) is a fluorescent substrate (coumarin derivative) and (3.) is a fluorescein derivative. FIG. 4C shows 100 mM Bis in the planar chip experiment.
-Represents data for fluorescein derivatives in Tris, pH 6.5, 1 M NDSB, 5 mM NaCl.

Example 3 Protease Assay Prescreened libraries that have been evaluated for their effects as inhibitors or activators on cellular proteases, for example, are a feature of the invention. “Protease” is an enzyme that degrades proteins by hydrolyzing peptide bonds between amino acid residues. Kinds of protease include thiol protease, acid protease, serine protease and the like. Examples of proteases include, but are not limited to, carboxypeptidase A, subtilisin, papain, and pepsin. Protease assays are described in more detail, for example, in US patent application Ser. No. 09 / 093,542, 199, incorporated herein by reference.
It is described in the application filed on June 8, 2008. To perform a protease assay, a substrate, eg, a protein, is mixed or otherwise contacted with a protease. Various fluorogenic protease substrates are commercially available and include, for example, p-nitrophenol compounds. Proteases catalyze the reaction with the substrate, thus forming a product such as a protease cleavage product. In the present invention, protease assays can be used to evaluate chemical compositions for their ability to modulate, eg, inhibit or activate, proteases.

In the present invention, the protease inhibition assay is performed in a continuous flow format in a microscale channel of a microfluidic device. Typically,
The application of pressure or electrokinetic force as described above causes the reactants to move through the channel. If two reactants are introduced into the same channel, the two reactants mix and contact each other, react and form a product. For example, a protease optionally flows under pressure through a microfluidic device. The protease is contacted or mixed with a substrate, such as a protein, peptide, etc. The substrate typically labels, for example, a fluorogenic substrate. Proteases catalyze the hydrolysis of substrates to produce degraded protein products, for example, cleaved protein fragments. Various properties of the protease reaction, such as enzymatic mechanism, kinetics, inhibition, etc., are studied using protease assays. Protease suppression assays, for example, provide information on the types of compounds that inhibit proteases and the level of inhibition they provide. For example, pepstatin acts as a protease inhibitor by inhibiting the activity of carboxyl proteases.

The present invention provides a high power assay system suitable for testing many compounds in a short time protease inhibition assay. The chemical composition of the master library is optionally added before, during or after contact of the enzyme and substrate depending on the format of the assay. For example, to perform a protease suppression assay,
Library members are introduced into the microscale channel where the protease reaction is performed. The reaction is continued in the presence of the potential modulator. The detergent-like modulator compound is optionally removed from the microtiter plate containing the plurality of samples. The plurality of samples optionally include a potential modulator, inhibitor and / or activator. The results obtained in an exemplary protease inhibition assay test are shown in FIG.
Standard multiwell plate format and microfluidics (LabChi)
The assay parameters, enzyme consumption and substrate consumption statistics for the protease assays performed in the p® instrument) are compared in Table 1. A 125-fold reduction in enzyme consumption with a 6,700-fold reduction in substrate consumption is obtained in the microfluidic device as compared to a multiwell plate format. In addition, a significant increase in power was achieved by 4,000 assays performed per 12 hours.

[0071]

Table 1 Protease Assay II | Format | Parameters | Enzyme consumption │Substrate consumption │ ├──────┼──────┼─────────┼─────────┤ │Multiwell│60μl / well│0.075 pmole / ASSE│0.3 nmole / assay│ │microplate│1.25 nM enzyme│a││││││ │5 μM substrate│4.5 mg / 10 ⁶ enzyme│150 mg / 10 ⁶ assay│ ├──────┼── │ │Microfluidic device│10 seconds injection size│0.6 fmol / assay│45 fmol / analysis │ │Place │Cle │ │ │ │ │670 nM Enzyme│36.2μg / 10 ⁶ Ass│22.5μg / 10 ⁶ Analysis │ │ │50μM Substrate │A │ │ └──────┴──────┴───── ────┴─────────┘

Example 4 Kinase Assay Protein kinase catalyzes the phosphorylation of proteins by transferring a γ-phosphate group from ATP onto serine, threonine or tyrosine residues of the target peptide or protein. .
It is estimated that 2-3% of all genes in eukaryotic tissues encode protein kinases (Coc et al. (1991) Cell Regul 2: 965). Many of these have a wide variety of diseases, including cancer as a group
Although potential targets for drug improvement in therapy, they are important target-independent assay challenges. Kinase assays are described in US Pat.
No. 0 / 108,628. Kinase activity is traditionally radioactive AT
Assay using P and then isotopically labeled phosphate
Transfer from P to peptide or protein substrate. However, a typical kinase assay of the invention involves the use of a fluorescently linked peptide, such as "KEMPtide". Phosphorylation is detected as a shift in the movement of the labeled KEMPtide. For example: Tag-Leu-Arg-Arg -Ala-Ser-Leu-Gly ( "Kemptide;" Net charge: q) ↓ protein kinase A, ATP, cAMP Tag-Leu -Arg-Arg-Ala-Ser (OPO 3 2 ^- ) ^- Leu-G
ly (phosphorylated “Kemptide;” net charge: q ⁻² )

FIG. 6A schematically illustrates a kinase flux shift assay. As depicted, the bond creates a charge in the fluidity of the bond properties. The assay illustrates the assay results in the presence of varying levels of inhibitor in FIGS. 6B-E, which includes the calculation of rate constants (FIG. 6C). As depicted, FIG. 6B depicts ATP titration and detection for fluidity shift for the protein kinase A assay. Buffer conditions were 100 mM HE
PES, pH 7.5, 5 mM MgCl ₂ , 1 M NDSB-195, 10 mM DTT, 0.01% Triton. FIG. 6C shows the lineweaver-Burk for the protein kinase A assay.
) Represents plots and ATP titrations (■; ◆; represents two series of assays).
6D represents the results of a protein kinase A assay using a 10 μM H-9 inhibitor injection. Buffer conditions are 100 mM HEPES, pH 7.5, 5 m
M MgCl ₂ , 1 M NDSB-195, 10 mM DTT, 0.01% T
riton X-100, 0.05 mM ATP, 10 μM Fl-Kempt
ide substrate + 140 nM PKA catalytic subunit. 6E shows the protein kinase A assay results performed on a “siper chip” (a chip with an external capillary for sampling fluid from an external source of the chip, such as a titration dish). The results indicate titration of the inhibitor, with detection by mobility-shift. Buffer conditions were 100 mM HEPES, pH 7.5, 5 mM MgCl ₂ , 1 M NDSB.
-195, 10 mM DTT, 0.01% Triton X-100, 0.0
5 mM ATP, 100 nM PKA catalytic subunit 10 μM Fl-Kemp
Tide substrate.

EXAMPLE 5 As with phosphatase assays , enzymes that catalyze the dephosphorylation of peptides or proteins are also the subject of target-independent assays used to provide prescreened libraries of the invention. Briefly, the phosphatase assay fluorogenic substrate dFMU (
6,8-difluoro-4-methylumbelliferyl (methylumbeli)
feryl) phosphatase), which provides a fluorescent signal upon dephosphorylation (eg, incubation using phosphatase). The reaction is K
Opf-Sill et al. in U.S. patent application Ser. No. 09 / 093,542, filed Jun. 8, 1998, are described in detail. The phosphatase reaction is 50 mM
Implemented in HEPES, 1M NDSB-195, pH 7.9. Reagent concentrations are 125 nM LAR and 50 μM dFMUP. The system is programmed to perform a series of controls, followed by running enzymes and substrates, optionally including known inhibitors. The overall flowing flux remains constant in the main reaction channel with the ratio of the total flux from the reagents and buffer wells selected to provide the desired final reagent concentration in the main reaction channel. The fluorescence response was monitored at each stage. FIG. 7A represents typical data obtained from a phosphatase assay (substrate titration) performed in a microfluidic device. The raw data for K _m , V _max , k _cat and K _i are plotted in FIG. 7B. No inhibitor, 3
The least squares fit of the results for the 5 μM and 69 μM inhibitors was calculated (FIG. 7C) and compared to the results of the phosphatase reaction performed in the cuvette using a spectrophotometer (Table 2).

[0075]

[Table 2] Table 2. Phosphatase assay┌─────┬─────┬────┬──────┬─────────┐ │ │K _m │K _I │K _cat │Condition │ │ │ (μM) │ (μM) │ (μmol / min) │ │ ├─────┼─────┼────┼──────┼──────── LAR │ │ LAR / dFMUP │ Peptide │ nmol LAR │ │ ├─────┼─────┼────┼──────┼─────────┤ │ Cuvette │23.2 │167 │6 │1M NDSB-195 │ │ │ │ │ │50mM HEPES, pH7.5 │ │ │ │ │ │ │0.1 mg / ml BSA │ ├─────┼─────┼─ │ │Micro flow│18.7 │151 │4.74 │1M NDSB-195 │ │Equipment │ │ │ │ │50mM HEPES, pH7.5 │ └ ─────┴─────┴────┴──────┴─────────┘

Example 5 G-Protein Binding Receptor-Cell Based Assay A prescreened library of the invention is also provided by evaluating a large master library in a cell based assay. For example, the G-protein binding receptor assay is a target-independent assay of the present invention. G-
Protein binding receptor assays typically use a labeled calcium indicator to measure calcium flux across cell membranes, as schematically illustrated in FIG. 8A. In this example, the WT3-CHO cells are ATCC
From 10% FBS, 10 mM HEPES, Pen / Strep, 1
Cultured in RPMI 1640 containing mM sodium pyruvate, 2 mM L-glutamate, 100 μg / ml G418. Then cells (10 × 1
06 cells / 5 ml) was added to Fluo-3 (Molecular Probe Inc.).
. , Eugene, OR) with 1 mg / ml BSA, 20 mM HEP
ES, 0.04% pluronic acid, 2.5 m
Filling was performed using 4 μM Fluo-3-AM in Hank's balanced salt solution (HBSS) containing M probenecid by incubating at 37 ° C. for 40 minutes. Cells were incubated with Fluo-3 for 15 minutes at room temperature after loading with 1 μM Syto-62 (Molecular Probe I).
nc. ) Was used for counterstaining. Dye-loaded cells were washed twice by resuspending the pelleted cells in HBES containing HEPES and 1 mg / ml BSA. Cells are washed with the same washing medium and an additional density reagent,
e (Pharmacia Biotech) or Optiprep (Nycom
ed), resuspend in cell analysis buffer consisting of
8% (w / v) Ficoll-400 (Fl) to adjust to 5 centipoise
uka) to match the density.

The G-protein coupled receptor assay was etched using a 90 μm wide, 20 μm deep channel and in a microfluidic device (FIG. 8B) with a titrator element 801 in the arrangement shown in FIG. 8B. Will be implemented. The microfluidic device is prepared such that for a given buffer viscosity, samples in HBSS + HEPES are diluted 1/3 using buffer + cells, and agonists from agonist well 802 are diluted 1/5 using cell-sample mixtures. It is a design that looks like. Channels are filled with cell analysis buffer and wells are filled with 18 μl of assay medium. The waste well 804 connects to a vacuum source set at 0.8 lbs / in 2 and starts the buffer flow into this empty well. Cells are in cell well 8
Cells added to 06 and flowing through main channel 803 are sent to an incident fluorescence detector 805 downstream of the agonist addition point. The flow rate after agonist addition is 0.8 mm / sec, and the detector is set to allow a 20 second incubation time before detection and after agonist addition. Samples flow from 96-well microplates at 30 second intervals. The data flow is based on Caliper LabChip (R) Techno
2 in the Agilent 2100 Bioanalyzer using
Collected at 0 Hz. Fluo-3 emission at 520 nm using a green filter
And the Syte-62 emission was collected at 680 nm using a red filter: a blue LED excitation wavelength of 488 nm was used for both dyes. A software filter was used to separate the collected fluorescence emission records when cells were in the recording area of the detector. Fluo-3 / Syte-62 cell recording ratios were averaged during the 30 second sampling time after background subtraction. Fluorescein dye injection is used to mark the beginning and end of a group of 20 sample injections. The data is illustrated in FIGS. 8C-8F.

Example 6: Cytochrome P450 Assay The cytochrome P450 assay is an important tool for assessing metabolic and potential toxic effects of drug leads. The human cytochrome P450 preparation was obtained from Gente
It is commercially available from companies such as st Corporation. These enzymes perform metabolic oxidation of chemical compounds. There are many fluorescent substrates available for monitoring P450 activity, such as coumarin derivatives, resorufin derivatives, or fluorescein derivatives obtained from molecular probes. The utility of fluorogenic substrates to monitor the activity of these enzymes means that these assays can be performed in a continuous flow microfluidic device-based assay format. The following are examples of available fluorogenic substrates useful, for example, in p450 assays:
(1.) Coumarin derivatives, for example, 3-cyano-7-ethoxycoumarin → 3-cyano-7-hydroxycoumarin (Ex / Em maximum: 408/450 nm; extinction ratio: 43,000); (2.) resorufin derivatives, For example, phenoxazoline → resorufin (Ex / Em maximum: 571/585 nm; extinction ratio: 54,000)
(3.) fluorescein derivatives such as fluorescein diethyl ether → fluorescein (Ex / Em maximum: 490/520 nm; extinction ratio: 70,000
).

The kinetic parameters for the four different human P450 isoenzyme and substrate combinations were obtained from Gentest Website (Table 3). Using these enzyme reaction parameters and the known flow parameters for the microfluidic device design of FIG. 1A, calculate the product formation rate and the total amount of product forming for a given starting concentration of enzyme and substrate. (Table 4). The ratio of the amount of product formed to the concentration detection limit estimated for these molecules provides an indication of the feasibility of running a continuous flow assay. In this calculation, the enzyme concentration is Gent
Using the known dilution factor for the est P450 preparation and the microfluidic device of FIG. 1A, it was assumed to be the maximum obtained using a 30 second sampling time. The final substrate concentration was set close to their K _m values were assumed detection limit of 50μM for fluorogenic reaction product. Calculations show that all four of these enzymatic reactions were observed in the microfluidic design, despite the very slow reversal rates exhibited by these enzymes. The percent inhibition expected to be observed for the hypothetical inhibitor is also calculated using the same set of assay parameters, ensuring that the assay is sensitive to the inhibitor under these conditions.

[0080]

[Table 3]

[0081]

[Table 4]

The net consumption of the reagents in the microfluidic device format was calculated from the assumed reaction conditions and compared to that calculated for the microplate assay protocol listed on the Gentest Website (Table 5). In this example, the performance of the assay in the microfluidic device format uses an estimated output of about 2000 assays per 12 hour day, reducing enzyme consumption by 2%.
An 80-fold reduction results in a 16,500-fold reduction in substrate consumption.

[0083]

[Table 5]

All publications, patents, patent applications, etc., so that each publication, patent, patent application, Internet web page, and other article are specifically and individually incorporated by reference for all purposes. , Internet web pages, and other articles are incorporated herein by reference for all purposes. While the invention has been described in detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[Brief description of the drawings]

FIG. 1A depicts a microfluidic channel array useful for performing a target-independent assay.

FIG. 1B depicts a microfluidic channel array useful for performing a target-independent assay.

FIG. 2 depicts a microfluidic channel array for performing a target-independent assay.

FIG. 3A shows an HSA-drug binding plot.

FIG. 3B shows a plot of inhibition versus phenylbutazone concentration.

FIG. 3C depicts data for HSA binding to six drugs.

FIG. 4A shows an overview of the glutathione-s-transferase (GST) reaction.

FIG. 4B depicts useful substrates for GST assays.

FIG. 4C depicts the results of an assay performed in a microfluidic device.

FIG. 5 shows protease assay results.

FIG. 6A shows migration shifts in a protein kinase assay.

FIG. 6B shows migration shift data in a protein kinase assay.

FIG. 6C. Line for measurement of rate data in PKA assay.
3 represents an eaver-Burk plot.

FIG. 6D depicts PKA inhibitor data.

FIG. 6E depicts data obtained in a microfluidic sipper device.

FIG. 7A depicts phosphatase assay data from a microfluidic assay.

FIG. 7B represents raw data.

FIG. 7C depicts the measurement of K _i from raw data.

FIG. 8A depicts G-protein binding to receptor cells based on an assay optionally performed in the microfluidic device depicted in FIG. 8B.

FIG. 8B depicts a microfluidic device.

FIG. 8C depicts data from G-protein binding receptor cells based on assays performed in a microfluidic device.

FIG. 8D depicts data from G-protein binding to receptor cells based on assays performed in a microfluidic device.

FIG. 8E depicts data from G-protein binding receptor cells based on assays performed in a microfluidic device.

FIG. 8F depicts data from G-protein binding to receptor cells based on assays performed in a microfluidic device.

FIG. 9 depicts a multi-module workstation useful for performing multiple target-independent assays, for example, to generate a prescreened library.

[Explanation of symbols]

101 Titrator Element 102 Main Channel 103 Reservoir 104, 105 Side Channel 106 Reservoir 107 Detection Window 108 Waste Well 109 Voltage Channel 110 High / Low Voltage Source 111 Voltage Channel 112 High / Low Voltage Source 204 Main Channel 206 Buffer Channel 208 Waste Reservoirs 210, 212 Buffer reservoir 214 Buffer channel 216 Buffer reservoir 218 Sample introduction channel 220 Sample reservoir 222, 224 Buffer / waste channel 226, 228 Buffer / waste reservoir 1001, 1002, 1003, 1004, 1005, 1006, 1007,
1008 Assay module 1010 Truck robot 1011 Robot armature 1012 Plate storage module 1013 Controller element 1014 Output device 1015 Input device

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/26 C12Q 1/26 1/34 1/34 1/48 1/48 1/527 1/527 G01N 33/15 G01N 33/15 Z 33/566 33/566 37/00 101 37/00 101 (31) Priority claim number 60 / 195,159 (32) Priority date April 6, 2000 (2000.04) .6) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW State 94028 Portra Valley Minoca Road 30 (72) Theo T. Nikiforov, United States 95112 San Jose, South Sixtyth Street, California 37 (72) Inventor Jay Worrace Perth United States of America California 94303 Palo Alto Los Robles Avenue 754 (72) Inventor, Calvin YH Chow United States California 94028 Port Lavalry Minoca Road 455 F term (reference) 2G045 AA40 BB25 CB01 DA20 DA36 DA77 FB01 FB02 FB07 FB11 FB12 GC12 GC15 JA01 JA07 4B029 AA07 AA27 BB01 BB11 BB16 CC01 FA04 FA10 FA11 Q12 QA13Q QQ89 QR01 QR02 QR06 QR10 QR18 QR20 QR74 QR77 QS31 QS39 QX02 QX10

Claims (71)

[Claims] 1. A method of generating a prescreened chemical composition library: prescreening a large master library in at least one target-independent assay, thereby providing a library of prescreened composition. Said method. 2. The pre-screening step physically separates the components of the master library to produce a library of pre-screened compositions.
the method of. 3. The method of claim 2, wherein the components of the master library are physically separated based on the positive or negative activity of the pre-screening step. 4. The method of claim 1, further comprising registering descriptors in a database for components of a library of prescreened compositions, wherein the database is embodied in a computer or computer readable medium. The method. 5. A database embodied in a computer or computer readable medium created by the method of claim 4. 6. The method of claim 7, further comprising screening a master library, a library of prescreened compositions, or a second library of prescreened compositions in at least one additional target-independent assay. The method described in. 7. The master library, or library of prescreened compositions, is further screened in at least one additional target-independent assay, thereby generating a second library of prescreened compositions. The method of claim 1, comprising: 8. The method of claim 7, further comprising registering descriptors in a database for components of the second library of prescreened compositions, wherein the database is embodied in a computer or computer readable medium. . 9. A database embodied in a computer or computer readable medium created by the method of claim 8. 10. The method of claim 1, wherein the large master library is created by integrating one or more components of the master library. 11. The method of claim 1, wherein the library of prescreened compositions comprises at least about 1000 or more compositions. 12. The method of claim 11, wherein the library of prescreened compositions comprises at least about 10,000 or more compositions. 13. The method of claim 12, wherein the library of prescreened compositions comprises at least about 100,000 or more compositions. 14. The method of claim 13, wherein the library of prescreened compositions comprises at least about 1,000,000 or more compositions. 15. Target-independent assays include: serum half-life, cell uptake, oral efficacy, cell or organism viability, cell or organism toxicity, apoptosis, cell adhesion modulation, target-independent receptor modulation, target-independent Enzyme activity modulation, target-independent protein activity modulation, target-independent nucleic acid activity modulation, glucuronide binding modulation, sulfate binding modulation, amino acid binding modulation, acylation modulation, methylation modulation, transcription modulation,
Conversion modulation, protein folding modulation, cell growth modulation, membrane permeability, membrane integrity, metabolic stability, temperature stability, solubility, solution viscosity, solution turbidity, acidity, basicity, RedOx state, superoxidation 2. The method of claim 1, wherein the effect is associated with one or more target-independent parameters selected from secretion, lipid peroxidation, octanol / water partitioning, precipitation in one or more buffers, and the like. Method. 16. The method of claim 15, wherein the target independent assay is performed by flow cytometry in a cell focusing device. 17. The method of claim 16, wherein flow cytometry distinguishes between live and dead cells. 18. The method of claim 17, wherein flow cytometry detects the fluorescence of the dye or dye-bound, wherein the dye or dye-bound is calcein A.
M, BCECF AM, ethidium bromide, ethidium iodide, cationic dye,
The method selected from the group consisting of cationic membrane-permeable dyes, neutral dyes, neutral membrane-permeable dyes, anionic dyes, and anionic membrane-permeable dyes. 19. The method of claim 15, wherein the target independent assay evaluates a selected composition on calcium flux across the cell membrane, wherein the cell membrane is a component of the cell and the cell uses a fluorescent calcium indicator. The method is pre-filled. 20. The method of claim 15, wherein the target-independent assay assesses binding of the selected composition on serum proteins. 21. The method according to claim 20, wherein the serum protein is human serum albumin (HSA). 22. The target-independent assay assesses the ability of the selected composition to inhibit HSA binding by at least one fluorescent dye.
Wherein the fluorescent dye is selected from dansyl-1-sulfonamide (DNSA) or dansyl sarcosine (DS). 23. The method of claim 22, wherein the HSA is present at a concentration of about 15 μM; the concentration of HSA and the concentration of the dye are selected to provide a Kd of about 4 μM. 24. The method of claim 15, wherein the target-independent assay evaluates inhibition of the enzyme, wherein the enzyme is: glutathione-s-transferase, protease, peptidase, kinase, phosphatase, G protein, ATPase, cytochrome P450. , Ligase, carboxylesterase, epoxide hydrolase,
Aza or nitro reductase, carbonyl reductase, disulfide reductase, sulfoxide reductase, quinone reductase, alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, xanthine oxidase, monoamine or diamine oxidase, prostaglandin Prostaglandin system
ase), flavin-monooxidase, and dehalogenase. 25. The enzyme is glutathione-s-transferase, and a target-independent assay assesses the ability of the selected composition to inhibit the conversion of substrate to product by glutathione-s-transferase. The method of claim 24. 26. The method of claim 25, wherein the substrate is a fluorogenic or chromogenic substrate for glutathione-s-transferase, and wherein the fluorogenic or chromogenic assay is 2,4-dinitro-chlorobenzene, a coumarin derivative or a fluorescein derivative. Method. 27. When the substrate is pentafluorobenzoylfluorescein (pFB
28. The method of claim 26, wherein F) is present and pFBF is present at a concentration of about 10-20 [mu] M. 28. The method of claim 24, wherein the enzyme is a protease and the target independent assay evaluates the effect of the selected composition on the fluorescence signal resulting from cleavage of the fluorogenic substrate by the protease. 29. The method of claim 28, wherein said fluorogenic substrate is AMC. 30. The method of claim 24, wherein the enzyme is a kinase and a target-independent assay assesses the effect of the selected composition on the shift of migration caused by the kinase on the substrate. 31. The method of claim 30, wherein the substrate is a peptide that binds fluorescently. 32. Fluorescently binding peptide is continuous: Tag-Leu-A
32. The method of claim 31, comprising rg-Arg-Ala-Ser-Leu-Gly. 33. The method of claim 24, wherein the enzyme is a phosphatase and the target-independent assay assesses the effect of the selected composition on the fluorescent signal resulting from dephosphorylation of the fluorogenic substrate by the phosphatase. 34. The method of claim 33, wherein the fluorogenic substrate is dFMU. 35. The method of claim 24, wherein the enzyme is cytochrome P450 and the target-independent assay evaluates the effect of the selected composition on the fluorescence signal generated by conversion of the fluorogenic substrate by cytochrome P450. 36. The substrate is a coumarin derivative, resofugin.
36) The method of claim 35 which is a derivative or a fluorescein derivative. 37. The method of claim 24, wherein the enzyme is a G-protein and the target-independent assay evaluates the effect of the selected composition on the fluorescent signal produced by the action of the G-protein. 38. The method of claim 37, wherein the fluorescent signal is generated by a calcium flux binding to the G-protein. 39. Calcium flux crosses the cell membrane and the membrane is a receptor.
39. The method of claim 38, comprising a G-protein complex. 40. The method of claim 39, wherein the cell membrane is a component of the cell and the cell is pre-filled using a fluorescent calcium indicator. 41. The method of claim 1, comprising performing a target-independent assay in a microfluidic device. 42. The method of claim 41, wherein the microfluidic device includes one or more titrator channels. 43. The master library is arranged in one or more multi-well plates, on one or more solid substrates, or in one or more bead matrices, thereby generating a master library array. 42. The method of claim 41, wherein 44. The multi-well plate comprises one or more: a 96-well plate, a 384-well plate and a 1536-well plate; or wherein the solid substrate is immobilized on one or more: a membrane. 44. The method of claim 43, comprising a membrane having a plurality of components of the array; or the bead matrix comprises one or more microfluidic bead arrays. 45. The method further comprising a compiling data set, wherein the data set comprises at least one component using one or more master library components.
44. The method of claim 43, wherein the results of the species target independent assay are correlated. 46. The method of claim 45, wherein the dataset correlates results of the plurality of target independent assays to a plurality of components of the master library. 47. The method of claim 43, further comprising performing at least one additional target independent assay to generate one or more additional prescreened libraries. 48. The method according to claim 48, wherein the first and one or more further prescreened libraries are in the same one or more multi-well plates, on the same one or more solid substrates or 47. The method of claim 46, wherein the methods are arranged in the same one or more bead matrices. 49. The method of claim 46, comprising performing the target-independent assay on a multi-module workstation that includes one or more modules that perform different target-independent assays. 50. The method of claim 1, further comprising assaying at least one composition identified in the pre-screening step in a target-independent assay. 51. The prescreened library of claim 1. 52. The prescreened library of claim 51, wherein the prescreened library comprises more than about 100 compositions. 53. The prescreened library of claim 51, wherein the prescreened library comprises more than about 1,000 compositions. 54. The prescreened library of claim 51, wherein the prescreened library comprises more than about 10,000 compositions. 55. The prescreened library of claim 51, wherein the prescreened library comprises more than about 100,000 compositions. 56. The pre-screened library comprises about 1,000,000
52. The prescreened library of claim 51, comprising more than zero compositions. 57. The prescreened library of claim 51, wherein said library comprises one or more components having a desired activity in a target-independent assay. 58. The prescreened library of claim 57, wherein the prescreened library is substantially devoid of components having a negative effect in a plurality of target independent assays. 59. The pre-screened library is represented by a set of data, wherein the data set is one using the well portion of the master library array.
58. Associated with the result of a species or more target independent assay.
A prescreened library of. 60. The prescreened library of claim 59, wherein the data set is stored in a computer readable medium. 61. A multi-module workstation for performing a plurality of target-independent assays: a plurality of screening modules structurally configured to perform a target-independent assay; at least one type comprising a plurality of chemical compositions Substrate: a robotic mechanism adjacent and operatively connected to the plurality of screening modules for at least one substrate, wherein at least one substrate is at least one of the plurality of screening modules during operation of the multi-module workstation. A mechanism for moving to a contiguous position or into one of a plurality of screening modules; and a computer assisted robot control system operatively coupled to the robotic mechanism, wherein the control system is controlled by the robotic mechanism. Without even multi-module workstation comprising the system for controlling the movement of one of the substrates. 62. The multi-unit according to claim 61, wherein during operation of the apparatus, the robotic mechanism and the second robotic mechanism position one or more screening modules or portions thereof adjacent to one or more substrates. Module workstation. 63. A multi-module workstation for performing a plurality of target-independent assays: a plurality of screening modules structurally configured to perform a target-independent assay; at least one comprising a plurality of chemical compositions Substrate; a robotic mechanism adjacent to at least one of the plurality of screening modules, wherein the mechanism arranges at least one of the plurality of screening modules adjacent to or in one or more substrates; and a robot. A computer-assisted robot control system operatively connected to the mechanism, wherein the control system controls at least one movement of the plurality of screening modules by the robot mechanism. Down. 64. During operation of the device, the robot mechanism and the second robot mechanism
64. The multi-module workstation of claim 63, wherein one or more at least one substrate or portion thereof is located adjacent to a plurality of species or more screening modules. 65. The workstation of claim 61 or 63, wherein the screening module comprises at least one microfluidic device. 66. The workstation of claim 65, wherein the at least one microfluidic device includes at least one titrator channel. 67. At least one of a plurality of target-independent screening modules is formed: serum half-life, cell uptake, oral availability, cell or organism viability, cell or organism toxicity, apoptosis, cell adhesion , Target-independent receptor binding or modulation, target-independent enzyme activity modulation, target-independent protein activity modulation, target-independent nucleic acid activity modulation, glucuronide binding modulation, sulfate binding modulation, amino acid binding modulation, acylation modulation, methylation modulation, Transcriptional modulation, transduction modulation, protein folding modulation, cell growth modulation, membrane permeability, membrane integrity, metabolic stability,
Thermal stability, solubility, solution viscosity, solution turbidity, acidity, basicity, RedOx state,
Perform assays to evaluate effects associated with one or more target-independent parameters selected from: superoxidant secretions, lipid peroxidation, octanol / water partitioning, and precipitation in one or more buffers. 64. The workstation of claim 61 or 63. 68. At least one of a plurality of target-independent screening modules is formed: a flow cytometry assay, an assay to assess the effect of a selected composition in a biochemical system, measuring calcium flux across cell membranes 64. Perform an assay selected from the group consisting of an assay, an assay to assess serum protein binding, an assay to assess binding to human serum albumin, and an assay to assess the effect of a selected composition on an enzyme. Workstation. 69. The at least one substrate is one or more: a solid substrate;
64. The workstation of claim 61 or 63, wherein the workstation is selected from a bead array and a multiwell plate. 70. A robot mechanism comprising: one or more of: a conveyor belt positioned between at least two of the plurality of target-independent assay modules; a slide positioned between at least two of the plurality of target-independent assay modules. A mechanism, a roller located between at least two of the plurality of target independent assay modules, a cable and a pulley located between at least two of the plurality of target independent assay modules, one or more target independent screening modules. 64. The workstation of claim 61 or 63, comprising: a robot armature located adjacent to the one, a robot armature located adjacent to one or more substrates. 71. A computer assisted robot control system comprising: (A) moving one or more substrates by a robotic mechanism between at least two of the plurality of screening modules; Positioning of the substrate by a robotic mechanism adjacent thereto, movement of at least one of a plurality of screening modules connected to at least one of the one or more substrates, or selection of library components: (B) one or more sets of data corresponding to the results of one or more target-independent assays using one or more components of the master library array; and (C) Programming input into the robot control system 64. The workstation of claim 61 or 63, wherein said workstation comprises one or more components selected from: a user interface for selectively inputting control to a robotic mechanism.