2. Background of the Prior Art U. S. Patent No. 5,534,132, issued July 9,1996 to Vreeke et al. disclosed an electrode for use in detecting an affinity reaction.

U. S. Patent No. 5,262,035, issued November 16,1993 to Gregg et al. disclosed a biosensor electrode using redox enzymes.

U. S. Patent No. 5,198,367, issued March 30,1993 to Aizawa et al. disclosed an amperometric immunoassay.

U. S. Patent No. 5,346,832, issued September 13,1994 to Aizawa et al. disclosed an amperometric immunoassay.

Vogtetal., 1965, Inorg. Chem. 4: 1157-1163describesruthenium-sulfurdioxide coordination compounds.

Ford et al., 1968, J. Amer. Chem. Soc. 90: 1187-1194 describes synthesis of pentaamine-ruthenium complexes of aromatic nitrogen heterocycles.

Yocum et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7052-7055 describes preparation of a pentaamineruthenium derivative of horse heart ferricytochrome c.

Nocera et al., 1984, J. Amer. C1lem. Soc. 106: 5145-5150 describes kinetics of intramolecular electron transfer from Ru"to Fe"'in ruthenium-modified cytochrome c.

Wright et al., 1986, Anal. Chem. 58: 2995-2998 describes a homogeneous enzyme-linked immunoassay with electrochemical detection of an electroactive product.

Embree et al., 1990, J. C'hromatogr. 526: 439-446 describes electrochemical detection of an electrochemically active derivative of digoxin.

Devlin et al., 1990, Science 249: 404-406 describes random peptide libraries.

Hirai & Varmus. 1990"llolec. Cell. Biol. 10: 1307-1318 describes site-directed mutagenesis of src homology domain 3.

Lam et al., 1991, Nature (London) 354: 82-84 describes random peptide libraries.

Burrows et al., 1991, Eur. J. Biochem. 202: 543-549 describes direct electrochemistry of proteins.

Wanatabe-Fukunaga et al., 1992, Nature (London) 356: 314-317 describesfas as an apoptotic factor.

Salamon et al., 1992, Arch. Biochem. Biophys. 299: 193-198 describes direct electrochemistry of thioredoxins and glutathione.

Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393-5397 describes random peptide libraries.

Scott et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5398-5402 describes random peptide libraries.

Hammer et al., 1992, J. Exp. Med. 176: 1007-1013 describes random peptide libraries.

Tsai & Weber, 1992, Anal. Chem. 64: 2897-2903 describes the influence of tyrosine on detection of copper-protein complexes.

O'Neil et al., 1992. Proteins : Structure, Function & Genetics 14: 509-515 describes GPIIb/IIIa antagonists obtained using phage display libraries.

Yu et al., 1992, Science 258: 1665-1668 describes src homology domain 3.

Dedman et al., 1993,. J. Biol. Cliem. 268: 23025-23030 describes random peptide libraries.

Chen et al., 1993, J. Amer. Chem. Soc. 115: 12591-12592 describes src homology domain 3-binding ligands using phage display libraries.

Kay et al., 1993, Gene 128: 59-65 describes random peptide libraries.

Hammer et al., 1993, Cell 74: 197-203 describes MHC-binding peptides.

Rozakis-Adcock et al., 1993, Nature (Lowldon) 363: 83-85 describes src SH3 domains.

Salamon et al., 1993. Proc. Natl. Acad. Sci. USA 90: 6420-6423 describes direct measurement of cyclic current-voltage responses using an electrode comprising an artificial lipid bilayer and integral membrane proteins.

Qureshi et al., 1993, Biomed. Chrornatog. 7: 251-255 describes methods for detecting HPLC fractions containing biologically active peptides.

Okada et al., 1993, J. Biol. Chem. 268: 18070-18075 describes SH3-deleted src mutants.

Koivunen et al., 1993, J. Biol. Chem. 268: 20205-20210 describes phage display libraries.

Koivunen et al., 1993, J. Cell Biol. 124: 373-380 describes phage display libraries.

Hiramatsu et al., 1994, J. Biochem. 115: 584-589 describes electrochemical detection of polyamines.

Suzawa et al., 1994, Anal. Chem. 66: 3889-3894 describes a homogeneous immunoassay with electrochemical detection.

Johnston et al., 1994, IiIorg. Chem. 33: 6388-6390 describes rhenium-mediated electrocatalytic oxidation of DNA at indium tin-oxide electrodes as a method for voltammetric detection of DNA cleavage in solution.

Picksley et al., 1994, Oncoges1e 9: 2523-2529 describes binding between p53 and MDM 2.

Fong et al., 1994, Drug. Develop. Res. 33: 64-70 describes scanning whole cells with phage display libraries.

Yu et al., 1994, Cell 76: 933-945 describes src homology domain 3 fragments.

Uchida & Kawakishi, 1994, J. Biol. Chem. 269: 2405-2410 describes the identification of oxidized histidine at the active site of Cu, Zn superoxide dismutase.

Biachini & Wild, 1994, Toxicol. Lett. 72: 175-184 describes electrochemical detection of 7-methyldeoxyguanosine.

Cummings et al. ? 1994, J. Chromatog. B 653: 192-203 describes electrochemical detection of neuropeptide growth factor antagonists.

Mitton & Trevithick, 1994, Methods Enzvmol. 233: 523-539 describes electrochemical detection of HPLC fractions for detecting antioxidant compounds in vertebrate lens.

Daniels & Lane, 1994, J. Molec. Biol. 243: 639-652 describes random peptide libraries.

Sparks et al., 1994, J. Biol. Cltem. 269: 23853-23856 describes si-c homology domain 3 fragments.

Cheadle et al., 1994, J. Biol. Chem. 269: 24034-24039 describes src homology domain 3 fragments.

Rickles et al., 1994, EMBO J. 13: 5598-5604 describes src homology domain 3-binding ligands using phage display libraries.

Iwabuchi et al., 1994, Proc. Natl. Acacl. Sci. USA 91: 6098-6102 describes p53 binding proteins.

Takahashi et al., 1994, Cell 76: 969-976 desc *bes I Goodson et al., 1994, Proc. Natl. Acad. Sci. USA 91: 7129-7133 describes urokinase receptor antagonists obtained using phage display libraries.

Scharf et al., 1995, Biochem. Biophys. Res. Commun. 209: 1018-1025 describes electrochemical studies on nitrate reductase using a biosensor.

Abrams & Zhao, 1995, J. Biol. Chem. 270: 333-339 describes src homology domain 3.

Ivanenkov et al., 1995, J. Biol. Chem. 270 14651-14658 14651-14658 describes random peptide libraries.

Takenaka et al., 1995, J. Biol. Chem. 270: 19839-19844 describes phage display libraries.

Martens et al., 1995, J. Biol. Chem. 270: 21129-21136 describes random peptide libraries.

Dyson & Murray, 1995, Proc. Natl. Acad. Sci. USA 92: 2194-2198 describes random peptide libraries.

Chen & Sudol, 1995. Proc. Natl. Acad. Sci. USA 92: 7819-7823 describes src homology domain 3.

Nagata & Golstein, 1995, Science 267: 1449-1456 describesfas as an apoptotic factor.

Saksela et al., 1995, EMBO J. 14: 484-491 describes src homology domain 3.

Sudol et al., 1995, FEBSLett. 369: 67-71 describes src homology domain 3.

Weng et al., 1995, Molec. Cell. Biol. 15: 5627-5634 describes src homology domain 3.

Rickles et al., 1995, Proc. Natl. Acad. Sci. USA 92: 10909-10913 describes src homology domain 3-binding ligands using phage display libraries.

Feng et al., 1995, Pi-oc. Natl. Acad. Sci. USA 92: 12408-2415 describes src homology domain 3.

Gold et al., 1995, Aiiii. Rev. Biochem. 64: 763-797 describes aptamers.

Phizicky & Fields, 1995, Microbiol. Rev. 59: 94-123 describe methods for detecting and analyzing protein-protein interactions.

Adey & Kay, 1996, Gene 169: 133-134 describes random peptide libraries.

Sparks et al., 1996, Proc. Natl. Acad. Sci. USA 93: 1540-1543 describes src homology domain 3 fragments.

Yanofsky et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7381-7386 describes high affinity interleukin type I antagonists obtained using recombinant peptide libraries.

Wrighton et al., 1996, Science 273: 458-463 describes small peptides isolated from random peptide libraries as mimetics for erythropoietin.

Holmes et al., 1996, Science 274: 2089-2091 describes src SH3 domains.

Fang et al., 1996, Biochem. Biophys. Res. Commlln. 220: 53-56 describes trypsin inhibitors obtained using phage display libraries.

Hahne et al., 1996, Science 274: 1363-1366 describesfas as an apoptotic factor.

Chan et al., 1996, EMBO J. 15: 1045-1054 describes formin binding proteins having domains that functionally resemble src SH3 domains.

SUMMARY OF THE INVENTION The present invention provides methods and apparatus for performing

electrochemical analysis for detecting binding between a biological binding pair. These methods and apparatus are useful for performing direct binding and competition binding experiments for detecting and analyzing compounds capable of inhibiting binding between the biological binding pair, thereby identifying compounds capable of interacting with biologically-active portions of the species comprising the biological binding pair. The methods of the invention are useful for performing rapid, high throughput screening of biologically active compounds for use as drugs that interact with one of the members of the biological binding pair and thereby interfere with or affect its biological function.

In a first aspect, the invention provides an apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair. The apparatus comprises a first electrode, the electrode comprising a conducting or semiconducting material. The apparatus also comprises a second, reference electrode, and a third, auxiliary electrode. Each of the electrodes is electrically connected to a potentiostat, and the electrodes are all in electrical contact with a reaction chamber containing a solution of an electrochemically reactive liquid. This electrochemically- reactive solution contains a first member of a biological binding pair. The solution further comprises a second member of the biological binding pair, wherein said second member is electrochemically labeled with a chemical species capable of participating in a reduction or oxidation reaction with the electrode under conditions whereby an electrical potential is applied to the electrodes. In the use of the apparatus. a current is produced in the apparatus when an electrical potential is applied to the electrodes; this current is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair. In preferred embodiments, either the first or the second member of the biological binding pair is present in the reaction chamber of the apparatus in concentration excess to the other member of the pair. In preferred embodiments, the first member of the biological binding pair is linked to a particle, most preferably a bead.

In preferred embodiments, the electrochemical assay is a voltammetric, amperometric or coulometric assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In a preferred embodiment, the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof. In another preferred embodiment,

the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof. In yet another preferred embodiment, the first member of the biological binding pair is a first protein or fragment thereof that specifically binds to a second protein.

In preferred embodiments, the second member of the biological binging pair is a ligand, and antigen or a protein that binds to the first member of the biological binding pair. One of ordinary skill in the art will recognize the appropriate choice of first and second members of the biological binding pair (e. g., receptor/ligand, antigen/antibody, etc.).

In particularly preferred embodiments of the invention, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair, having a binding affinity of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (, uM), and most preferably from about 1 OnM to about 10 ut. Preferably surrogate surrogate ligand electrochemically labeled, more preferably with a ruthenium compound.

The apparatus of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the electrodes and reaction chambers of the invention, wherein each reaction chamber contains an electrolyte and is in electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus, and each of the electrodes in electrochemical contact with each reaction chamber is electrically connected to a potentiostat.

The apparatus of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the first and second members of the biological binding pair, wherein current is produced in the apparatus by participation in a reduction/oxidation reaction at the surface of the first electrode by each of the electrochemically labeled second members of the biological binding pairs at a particular electrical potential applied between the electrodes, wherein the current produced at each particular electrical potential is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair.

In preferred embodiments, the second member of the biological binding pair is electrochemically labeled with an organic or inorganic complex comprising a transition metal cation. In preferred embodiments, the electrochemical label is a ruthenium complex, preferably a pentaamineruthenium compound such as {Ru (NH,), CIICI,

Ru (NH3) 63-or Ru (NH3) 5 (H20) 2+. In other preferred embodiments, the electrochemical label is an iron complex, most preferably a ferrocene compound, or a cobalt or osmium complex.

The invention also provides a kit comprising at least one second member of the biological binding pair, preferably comprising a surrogate ligand having binding specificity for the first member of the biological binding pair characterized by a dissociation constant (Kd) of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (, uM), and most preferably from about 1 OnM to about 10 ßM, thus comprising an electrochemical probe. In certain embodiments of the kits of the invention, said second member of the biological binding pair is provided in an electrochemically labeled embodiment. In certain other embodiments of the kits of the invention, said second member of the biological binding pair is provided with reagents including an electrochemical label for preparing the electrochemically labeled embodiment by the user. The kit also preferably provides a first member of the biological binding pair specific for the second member of the biological binding pair provided therewith. Additional and optional components of the kits of the invention include buffers, reagents and electrodes as described herein.

Methods of using the apparatus of the invention are also provided. In a first embodiment, a method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to this aspect of the invention is provided. In this embodiment, the method comprises the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of an apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes is electrically connected to a potentiostat; wherein the first reaction chamber contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber contains an electrochemically-labeled second member of the biological binding pair and an unrelated species that does not specifically bind to the second member of the biological binding pair, or the first member of the biological

binding pair and an unrelated, electrochemically-labeled species that does not bind to the first member of the biological binding pair. The method further comprises the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber. wherein binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is detected by the production of a smaller current in the first reaction chamber than is produced in the second reaction chamber. Specific interaction between the members of the biological binding pair is detected by this comparison of the electrical current produced in each of the reaction chambers when an electrical potential is applied between the electrodes in each chamber. Specific binding of the first and second members of the biological binding pair in the first reaction chamber produces a lower current output in the first reaction chamber than is produced in the second reaction chamber, where there is no specific interaction between the second member of the biological binding pair and the unrelated species in that chamber, or between the first member of the biological binding pair in the second reaction chamber and the unrelated, electrochemically-labeled species contained in the second reaction chamber. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In an alternative embodiment of the methods of the invention provided for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair, the method comprises the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of an apparatus of the invention, wherein each of the electrodes is electrically connected to a potentiostat; and wherein the reaction chamber contains an electrochemically-labeled second member of the biological binding pair that specifically binds to a first member of the biological binding pair, and

b) performing a first electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus ; and c) adding to the reaction chamber a first member of the biological binding pair that is specifically bound by the electrochemically-labeled second member of the biological binding pair ; and d) performing a second electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus; and e) comparing the current produced in the first electrochemical assay to the current produced in the second electrochemical assay; wherein binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is detected by the production of a smaller current in the second electrochemical assay than is produced in the first electrochemical assay. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber. The level and amount of current produced in the first electrochemical assay in the presence of the second member of the biological binding pair and the absence of the first member of the biological binding pair in the reaction chamber is compared with the level and amount of current produced in the second electrochemical assay in the reaction chamber in the presence of both the first and second members of the biological binding pair. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair In preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In a second embodiment of the methods of the invention is provided a method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of this aspect of the invention, the method comprising the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the

apparatus of the invention, wherein each of the electrodes being electrically connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber further contains a compound to be tested to determine its ability to inhibit binding of the second member of the biological binding pair to the first member of the biological binding pair. The method further comprises the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber wherein an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the second reaction chamber than is produced in the first reaction chamber. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the chamber. The level and amount of current produced by specific binding of the first and second members of the biological binding pair in the reaction chamber is compared with the level and amount of current produced in the chamber in the presence of an inhibitor of specific binding. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In an alternative embodiment of the methods of the invention provided for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to claim 1, the method comprises the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus according to the invention, wherein each of the electrodes is electrically connected to a potentiostat and wherein the reaction chamber contains a first member of a biological binding pair and

an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, b) performing an electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus in the presence and absence of an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; and c) comparing the current produced in the electrochemical assay in the presence of the inhibitor to the current produced in the electrochemical assay in the absence of the inhibitor; wherein an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the electrochemical assay in the presence of the inhibitor than is produced in the electrochemical assay in the absence of the inhibitor.

In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber.

The level and amount of current produced in the electrochemical assay in the presence of an inhibitor of binding between the members of the biological binding pair is compared with the level and amount of current produced in the electrochemical assay in the reaction chamber in the absence of the inhibitor of binding of the first and second members of the biological binding pair.

In a preferred embodiment, the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber. In another preferred embodiment, the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber. In yet another preferred embodiment, the inhibitor is added to the reaction chamber together with the second member of the biological binding pair.

In additional preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In yet a third embodiment of the methods of the invention is provided a method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of this aspect of the invention, the method comprising the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes being electrically connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair; the method further comprising the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of claim 1 to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber wherein a complex mixture comprising an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the second reaction chamber than is produced in the first reaction chamber. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in each reaction chamber when an electrical potential is applied between the electrodes in the chamber. The level and amount of current produced by specific binding of the first and second members of the biological binding pair in the first reaction chamber is compared with the level and amount of current produced in the second reaction chamber in the presence of a complex chemical mixture comprising an inhibitor of specific binding. In a preferred embodiment, the second

member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In an alternative embodiment of the methods of the invention provided for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to claim 1, the method comprises the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, wherein each of the electrodes is electrically connected to a potentiostat, and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemically-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, b) performing an electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus in the presence and absence of a complex mixture comprising an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; and c) comparing the current produced in the electrochemical assay in the presence of the complex mixture to the current produced in the electrochemical assay in the absence of the complex mixture; wherein a complex mixture comprising an inhibitor of binding of the electrochemically labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the electrochemical assay in the presence of the complex mixture than is produced in the electrochemical assay in the absence of the complex mixture. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in each reaction chamber when an electrical potential is applied between the electrodes in the chamber. The level and amount of current produced by specific binding of the first and second members of the biological binding pair in the first reaction chamber is compared with the level and amount of current produced in the second reaction chamber in the presence of a complex chemical mixture

comprising an inhibitor of specific binding. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In a preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber. In another preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber. In yet another preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber together with the second member of the biological binding pair.

In additional preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In an additional aspect of this embodiment of the invention, the method is used to isolate and identify an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair. In this embodiment, the method comprises the additional steps of : -chemically fractionating the complex mixture having an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair, to produce fractionated submixtures; and performing the steps of the method on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair.

In this aspect, it will be recognized that all of the steps of the methods of the invention according to this aspect can be repeatedly performed on chemically fractionated submixtures to yield submixtures comprising increasingly purified preparations of the inhibitor. In preferred embodiments, the chemical fractionation includes chemical, biochemical, physical, and immunological methods for fractionation of chemical or biochemical species of inhibitor.

In preferred embodiments of each of the methods of the invention, the second member of a biological binding pair is an electrochemically labeled surrogate ligand characterized by a dissociation constant (Kd) for the first member of the biological

binding pair of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about I nanomolar (nM) to about 100 micromolar (, uM), and most preferably from about I OnM to about 10 ßM.

In a second aspect of the invention is provided another apparatus for performing an electrochemical assay for detecting binding between members of a biological binding pair. In this aspect of the invention, the apparatus comprises a first electrode, wherein the electrode comprises a conducting or semiconducting surface; a second, reference electrode comprising a conducting metal in contact with an aqueous electrolyte solution; and a third, auxiliary electrode comprising a conducting metal, wherein each of the electrodes is electrically connected to a potentiostat. The apparatus further comprises a reaction chamber containing a solution of an electrolyte, wherein each of the electrodes is in electrochemical contact therewith. The solution further contains a first member of a biological binding pair, and a second member of the biological binding pair, wherein said second member is bound to an electrochemical catalyst capable of participating in a reduction/oxidation reaction at the surface of the first electrode in the presence of a substrate for the electrochemical catalyst under conditions whereby an electrical potential is applied to the electrodes, and wherein the electrolyte in the reaction chamber further contains a substrate for the electrochemical catalyst. In the use of the apparatus of the invention, a current is produced in the apparatus when an electrical potential is applied to the electrodes and this current is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair.

In preferred embodiments, either the first or the second member of the biological binding pair is present in the reaction chamber of the apparatus in concentration excess to the other member of the pair. In preferred embodiments, the first member of the biological binding pair is linked to a particle, most preferably a bead.

In preferred embodiments, the electrochemical assay is a voltammetric, amperometric or coulometric assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In a preferred embodiment, the first member of the biological binding pair is a receptor protein or ligand binding fragment thereof. In another preferred embodiment, the first member of the biological binding pair is an antibody protein or antigen binding fragment thereof. In yet another preferred embodiment, the first member of the biological binding pair is a first protein or fragment thereof that specifically binds to a

second protein.

In preferred embodiments, the second member of the biological binging pair is a ligand, and antigen or a protein that binds to the first member of the biological binding pair. One of ordinary skill in the art will recognize the appropriate choice of first and second members of the biological binding pair (e. g., receptor/ligand, antigen/antibody, etc.).

In particularly preferred embodiments of the invention, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair, having an affinity of binding of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (, uM), and most preferably from about 1 OnM to about 10 ßM. Preferably said surrogate ligand is electrochemically labeled, more preferably with a ruthenium compound.

In a preferred embodiment, the electrochemical catalyst bound to the second member of the biological binding pair is an enzyme, most preferably horse radish peroxidase. In additional preferred embodiments, the substrate of the electrochemical catalyst produces a detectable product upon undergoing an oxidation/reduction reaction with the electrochemical catalyst at the surface of the first electrode of the apparatus, most preferably a colored product.

The apparatus of this aspect of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the electrodes and reaction chambers of the invention, wherein each reaction chamber contains an electrolyte and is in electrochemical contact with one each of the three electrodes among the multiplicity of electrodes in the apparatus, and each of the electrodes in electrochemical contact with each reaction chamber is electrically connected to a potentiostat.

The apparatus of this aspect of the invention also includes embodiments wherein the apparatus further comprises a multiplicity of each of the first and second members of the biological binding pair, wherein current is produced in the apparatus by participation in a reductionioxidation reaction at the surface of the first electrode by each of the electrochemically labeled second members of the biological binding pairs at a particular electrical potential applied between the electrodes, wherein the current produced at each particular electrical potential is reduced upon binding of the second member of the biological binding pair to the first member of the biological binding pair.

As provided in this aspect of the invention, the second member of the biological

binding pair is labeled with an electrochemical catalyst. In preferred embodiments, the electrochemical catalyst is an enzyme, most preferably a redox enzyme capable of catalysis of its substrate to product by an oxidation/reduction mechanism wherein either functional groups on the enzyme or bound cofactors are involved in the oxidation/reduction cycle. In particularly preferred embodiments, the electrochemical catalyst is a peroxidase, for example horse radish peroxidase. In aspects of the apparatus requiring a oxidation/reduction of a bound cofactor, said cofactor is provided in the reaction chamber of the apparatus.

In the use of this embodiment of the invention, specific binding interactions between the members of the biological binding pair are detected by observation of an electrical current. The apparatus of the invention comprises a second member of the biological binding pair chemically linked with a species, preferably an enzyme, that is capable of being oxidized or reduced at the surface of the first electrode and to participate in cyclic oxidation/reduction reactions at the surface of the electrode in the presence of a third species present in the solution ; in embodiments wherein the electrochemical catalyst is an enzyme, the third species is a substrate for the enzyme.

As a consequence of these reactions, an observable current is produced in the apparatus of the invention. This third species, however, cannot be directly oxidized or reduced at the surface of the first electrode. In the use of this embodiment of the invention, specific binding interactions between the members of the biological binding pair are detected by observation of an electrical current. Said electrical current is produced at an electrode potential sufficient to activate (oxidize or reduce) the electrochemical catalyst attached to the second member of the biological binding pair. At said appropriate electrode potential, the electrochemical catalyst is reduced by (oxidized by) the first electrode, thereby activating the catalyst for its substrate. As substrate is consumed, the electrode potential permits cycles of oxidation/reduction of the electrochemical catalyst pair, thereby producing a current related to catalysis of the substrate by the electrochemical catalyst. In the practice of the invention, the amount of current produced by specific binding of the members of the biological binding pair is compared to the amount of current produced before addition of the first member of the biological binding pair, or to the amount of current produced upon addition of a known non-binding first member (thereby providing a negative control). Specificity of binding is determined by comparison of the current to that generated in the presence of a known inhibitor of

binding. Additional comparisons of the extent, capacity or rate of binding inhibition, activation or competition can be determined by analysis of the extent of produced current in the presence of putative inhibitors, competitors, activators or drug lead candidates, wherein specific details of the performance of such comparisons will be understood by those with skill in the art and are more fully disclosed below.

The invention also provides a kit comprising at least one second member of the biological binding pair, preferably comprising a surrogate ligand having binding specificity for the first member of the biological binding pair characterized by a dissociation constant (Kd) of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (M), and most preferably from about 1 OnM to about 10 uM, thus comprising an electrochemical probe. In certain embodiments of the kits of the invention, said second member of the biological binding pair is provided in an embodiment wherein it is linked to an electrochemical catalyst. In certain other embodiments of the kits of the invention, said second member of the biological binding pair is provided with reagents including an electrochemical catalyst label for preparing the electrochemical catalyst-linked embodiment by the user. The kit also provides a first member of the biological binding pair specific for the second member of the biological binding pair provided therewith.

Additional and optional components of the kits of the invention include buffers, reagents, substrates for the electrochemical catalyst and electrodes as described herein.

Methods of using this embodiment of the apparatus of the invention are also provided. In a first embodiment of these methods is provided a method for detecting binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to this aspect of the invention. In this embodiment, the method comprises the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes of the apparatus being electrically connected to a potentiostat; wherein the each of the reaction chambers contains a substrate for the electrochemical catalyst and wherein the first reaction chamber comprises a first member of the biological binding pair and a second member of the biological binding pair that

specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and wherein the second reaction chamber contains a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a chemical species that does not specifically bind to the second member of the biological binding pair, or wherein the second reaction chamber contains a first member of a biological binding pair and a chemical species that does not specifically bind to the second member of the biological binding pair, bound to an electrochemical catalyst; the method further comprising the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of the invention to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber wherein binding of the second member of the biological binding pair with the first member of the biological binding pair is detected by the production of a smaller current in the first reaction chamber than is produced in the second reaction chamber. Specific interaction between the members of the biological binding pair is detected by this comparison of the electrical current produced in each of the reaction chambers when an electrical potential is applied between the electrodes in each chamber. Specific binding of the first and second members of the biological binding pair in the first reaction chamber produces a lower current output in the first reaction chamber than is produced in the second reaction chamber, where there is no specific interaction between the second member of the biological binding pair and the unrelated species in that chamber, or between the first member of the biological binding pair and the unrelated, electrochemically-labeled species contained in the second reaction chamber.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a smaller amount of the detectable product in the first reaction chamber than is produced in the second reaction chamber.

In an alternative embodiment of this method for detecting binding of a second member of a biological binding pair that is labeled with an electrochemical catalyst with a first member of a biological binding pair using an apparatus according to this aspect of the invention, the method comprises the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of an apparatus according to the invention, and wherein each of the electrodes is electrically connected to a potentiostat; and wherein the reaction chamber contains an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to a first member of the biological binding pair and a substrate for the electrochemical catalyst, and b) performing a first electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus; and c) adding to the reaction chamber a first member of the biological binding pair that is specifically bound by the electrochemical catalyst-labeled second member of the biological binding pair; and d) performing a second electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus; and e) comparing the current produced in the first electrochemical assay to the current produced in the second electrochemical assay; wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is detected by the production of a smaller current in the second electrochemical assay than is produced in the first electrochemical assay. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber. The level and amount of current produced in the first electrochemical assay in the presence of the second member of the biological binding pair and the absence of the first member of the biological binding pair in the reaction chamber is compared with the level and amount of current produced in the second electrochemical assay in the reaction chamber in the presence of both the first and second members of the biological binding pair. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the

biological binding pair.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a smaller amount of the detectable product in the second electrochemical assay than is produced in the first electrochemical assay.

In preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In a second embodiment of the methods of this aspect of the invention is provided a method for identifying an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus according to the invention. In this embodiment, the method comprises the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes of the apparatus being electrically connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair, a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a substrate for the electrochemical catalyst, and wherein the second reaction chamber further contains an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; the method further comprising the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus according to this aspect of the invention to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber

wherein an inhibitor of binding of the second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the second reaction chamber than is produced in the first reaction chamber.

Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the chamber. The level and amount of current produced by specific binding of the first and second members of the biological binding pair in the reaction chamber is compared with the level and amount of current produced in the chamber in the presence of an inhibitor of specific binding.

In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the second reaction chamber than is produced in the first reaction chamber.

In an alternative embodiment of the methods of the invention for identifying an inhibitor of binding of a second member of a biological binding pair that is labeled with an electrochemical catalyst with a first member of a biological binding pair using an apparatus according to the invention, the method comprises the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus according to this aspect of the invention, and wherein each of the electrodes is electrically connected to a potentiostat and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to a first member of the biological binding pair and a substrate for the electrochemical catalyst, and b) performing an electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus in the presence and absence of an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; and

c) comparing the current produced in the electrochemical assay in the presence of the inhibitor to the current produced in the electrochemical assay in the absence of the inhibitor; wherein an inhibitor of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the electrochemical assay in the presence of the inhibitor than is produced in the electrochemical assay in the absence of the inhibitor.

Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber.

The level and amount of current produced in a first electrochemical assay in the presence of an inhibitor of binding between the members of the biological binding pair is compared with the level and amount of current produced in a second electrochemical assay in the reaction chamber in the absence of the inhibitor of binding of the first and second members of the biological binding pair.

In a preferred embodiment, the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber. In another preferred embodiment, the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber. In yet another preferred embodiment, the inhibitor is added to the reaction chamber together with the second member of the biological binding pair.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the electrochemical assay performed in the presence of the inhibitor than is produced in the electrochemical assay performed in the absence of the inhibitor.

In additional preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In yet a third embodiment of the methods of this aspect of the invention is

provided a method for screening a complex chemical mixture for an inhibitor of binding of an electrochemically labeled second member of a biological binding pair with a first member of a biological binding pair using an apparatus of the invention. These methods comprise the steps of : a) providing a first reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, and a second reaction chamber in electrochemical contact with each of the electrodes of the apparatus of the invention, each of the electrodes being electrically connected to a potentiostat; wherein each of the reaction chambers contains a first member of a biological binding pair, a second member of the biological binding pair that specifically binds to the first member of the biological binding pair, bound to an electrochemical catalyst, and a substrate for the electrochemical catalyst, and wherein the second reaction chamber further contains a portion of the complex mixture comprising an inhibitor of binding of the second member of the biological binding pair; the method further comprising the steps of : b) performing an electrochemical assay in each of the first and second reaction chambers of the apparatus of this aspect of the invention to produce a current in the electrodes of the apparatus; and c) comparing the current produced in the electrochemical assay in the first reaction chamber to the current produced in the electrochemical assay in the second reaction chamber wherein a complex mixture comprising an inhibitor of binding of the second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the second reaction chamber than is produced in the first reaction chamber. Specific interaction between the members of the biological binding pair is detected by a comparison of the electrical current produced in each reaction chamber when an electrical potential is applied between the electrodes in the chamber. The level and amount of current produced by specific binding of the first and second members of the biological binding pair in the first reaction chamber is compared with the level and amount of current produced in the second reaction chamber in the presence of a complex chemical mixture comprising an inhibitor of specific binding. In a preferred embodiment, the second member of the biological binding pair

is a surrogate ligand for the first member of the biological binding pair.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the second reaction chamber than is produced in the first reaction chamber.

In an additional aspect of this embodiment of the invention is provided a method for screening a complex chemical mixture for an inhibitor of binding of a second member of a biological binding pair that is labeled with an electrochemical catalyst with a first member of a biological binding pair using an apparatus according to this aspect of the invention, the method comprising the steps of : a) providing a reaction chamber in electrochemical contact with each of the electrodes of the apparatus wherein each of the electrodes is electrically connected to a potentiostat, and wherein the reaction chamber contains a first member of a biological binding pair and an electrochemical catalyst-labeled second member of the biological binding pair that specifically binds to the first member of the biological binding pair, and a substrate for the electrochemical catalyst, b) performing an electrochemical assay in the reaction chamber to produce a current in the electrodes of the apparatus in the presence and absence of a complex mixture comprising an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair; and c) comparing the current produced in the electrochemical assay in the presence of the complex mixture to the current produced in the electrochemical assay in the absence of the complex mixture; wherein a complex mixture comprising an inhibitor of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is identified by the production of a larger current in the electrochemical assay in the presence of the complex mixture than is produced in the electrochemical assay in the absence of the complex mixture.

Specific interaction between the members of the biological binding pair is

detected by a comparison of the electrical current produced in the reaction chamber when an electrical potential is applied between the electrodes in the reaction chamber.

The level and amount of current produced in a first electrochemical assay in the presence of a complex chemical mixture comprising an inhibitor of specific binding between the members of the biological binding pair is compared with the level and amount of current produced in a second electrochemical assay in the reaction chamber in the absence of a complex chemical mixture comprising an inhibitor of specific binding of the first and second members of the biological binding pair. In a preferred embodiment, the second member of the biological binding pair is a surrogate ligand for the first member of the biological binding pair.

In preferred embodiments, the substrate for the electrochemical catalyst produces a detectable product, most preferably a colored product, and wherein inhibition of binding of the electrochemical catalyst-labeled second member of the biological binding pair with the first member of the biological binding pair is further detected by the production of a larger amount of the detectable product in the electrochemical assay in the presence of a complex mixture comprising an inhibitor of specific binding than is produced in the electrochemical assay in the absence of the complex mixture comprising an inhibitor of specific binding between the members of the biological binding pair.

In a preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber after the second member of the biological binding pair is added to the reaction chamber. In another preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber before the second member of the biological binding pair is added to the reaction chamber. In yet another preferred embodiment, the complex mixture comprising the inhibitor is added to the reaction chamber together with the second member of the biological binding pair.

In additional preferred embodiments, the electrochemical assays performed using the methods of the invention are voltammetric, amperometric or coulometric assays, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In an additional aspect of this embodiment of the invention, the method is used to isolate and identify an inhibitor of binding of the second member of the biological binding pair to the first member of the biological binding pair. In this embodiment, the method comprises the additional steps of : d) chemically fractionating the complex mixture having an inhibitor of

binding of the second member of the biological binding pair to the first member of the biological binding pair, to produce fractionated submixtures; and e) performing the steps of the method on each of the fractionated submixtures to identify the submixtures that have an inhibitor of binding of the biological binding pair.

In this aspect, it will be recognized that all of the steps of the methods of the invention according to this aspect can be repeatedly performed on chemically fractionated submixtures to yield submixtures comprising increasingly purified preparations of the inhibitor. In preferred embodiments, the chemical fractionation includes chemical, biochemical, physical, and immunological methods for fractionation of chemical or biochemical species of inhibitor.

In preferred embodiments of each of the methods of the invention, the second member of a biological binding pair is an electrochemically labeled surrogate ligand characterized by a dissociation constant (Kd) for the first member of the biological binding pair of from about from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (uM), and most preferably from about 1 OnM to about 10, uM.

In additional preferred embodiments of the apparatus and methods of this invention are provided an electron donor species contained in the reaction chambers of the apparatus of the invention, wherein the electron donor species is oxidized with transfer of an electron to the electrochemical label at the surface of the first electrode.

In these embodiments of the apparatus and methods of the invention, oxidation/reduction of the electrochemical label at the surface of the first electrode is accompanied by the production of a detectable amount of chemiluminescence that is diminished upon binding of the second member of the biological binding pair with the first member of the biological binding pair.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram illustrating the principle of diffusion limits on the rate of interfacial electrochemistry between an electrode surface and an electrochemically active species in solution.

Figure 2 is a graph showing cyclic voltammetric analysis of an electrochemically labeled peptide under the conditions described in Example 2.

Figure 3 is a graph showing the results of the expriment described in Example 3 and illustrating the decrease in collectable, Faradaic current upon binding of a small, electrochemically labeled, biotinylated peptide to neutravidin.

Figure 4 is a graph showing the determination of the effective diffusion coefficent of a small, electochemically labeled, biotinylated peptide free in solution, in the presence of biotin-saturated neutravidin, and bound to a stoichiometric excess of neutravidin as described in Example 3.

Figure 5 is a graph illustrating the increase in collectable, Faradaic current observed when a small electrochemically labeled species is displaced from a larger species by competitive binding of an unlabeled species as described in Example 4.

Figure 6 is a graph depicting the changes in peak anodic current as a function of added competitor under the conditions described in Example 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides apparatus and methods for detecting specific interactions, particularly including binding interactions, between members of a biological binding pair. For the purposes of this invention, the term"biological binding pair"is intended to encompass any two biologically-derived or isolated molecules, or any chemical species that specifically interact therewith, that specifically bind with a chemical affinity measured by a dissociation constant of at least 50mM. Specifically included in this definition of a biological binding pair are proteins that interact with other proteins, including fragments thereof ; proteins and peptides; proteins and ligands; proteins and co-factors; proteins and allosteric or cooperative regulators; proteins and nucleic acids; proteins and carbohydrates; antigens and antibodies; lipids, including fatty acids, triglycerides and polar lipids that interact with proteins or peptides; receptors and ligands, particularly cytokines; virus-receptor pairs; enzymes and substrates; and

enzymes and inhibitors. Also encompassed with this definition are any chemical compound or mixture that interacts with at least one member of a biological binding pair. The members of the biological binding pairs of the invention are intended to encompass molecules that are naturally-occurring, synthetic, or prepared by recombinant genetic means or biochemical isolation and extraction means. Synthetic embodiments of a member of a biological binding pair will be understood to typically share structural similarity with at least a portion of any naturally-occurring analogue which they resemble or are constructed to resemble or mimic. These definitions are non-exclusive and non-limiting, and are intended to encompass any two biological or chemical species capable of specifically interacting with the defined chemical affinity.

The apparatus of the invention comprises a first, conductive or semiconductive electrode. Examples of materials useful forpreparing the conductive or semiconductive electrodes of the invention include metallically-impregnated glass, such as tin-doped indium oxide or fluorine-doped tin oxide glass, gold, carbon orplatinum. The apparatus also includes an auxiliary electrode, comprised of any conducting metal, including for example, platinum (Pt)-wire auxiliary electrodes, and a reference electrode, including silver wire electrodes, silver/silver chloride (Ag/AgCl) reference electrodes, saturated calomel, and others know to those with skill in the art. The apparatus comprises one or a multiplicity of reaction chambers, each having one of each of these electrodes, and each of the electrodes being electrically connected to a potentiostat or other device that applies and maintains a constant potential under varying current conditions. For the purposes of this invention, the term"electrically connected"is intended to mean, inter alia, that the components are connected such that current can flow through the electrodes.

The apparatus of the invention also provides a second member of a biological binding pair, wherein said second member is electrochemically labeled. Electrochemical labels are defined as chemical species, either complexes of transition metals or organic species that are capable of participating in a reduction/oxidation (redox) reaction with the first electrode of the apparatus when an electrical potential is applied between the electrodes in the reaction chamber of the apparatus. Electrochemical labels include cationic species such as transition metal cations including ruthenium, iron, osmium and cobalt. Electrochemical labeling strategies include coordination of one or more nucleophilic atoms of a second member species to form a transition-metal complex. For

second members of a biological binding pair comprising a peptide, electrochemical labels are attached to the peptide via reactive functional groups including but not limited to histidine, lysine or cysteine side chains or the amino terminus. Inorganic complexes such as Ru2-3+-amine complexes, ferrocenes, and osmium-or cobalt-polypyridyl complexes are attached to the peptide via histidine or cysteine residues or at the amino terminus. Redox-active organic molecules, such as paraquat derivatives and quinones, are attached to peptides by conjugating the redox-active organic moiety via lysine or cysteine residues or at the amino terminus. Alternatively, organic labels or the organic ligands of transition-metal complexes can include reactive substituents that allow direct incorporation of these complexes in the context of automated biopolymer synthesis. The choice of the electrochemical label is optimized for detection of the label to the exclusion of other redox couples present in the solution and for possible interference of the label with the specific binding of the second member of the biological binding pair with a first member of a biological binding pair.

In the practice of the invention, preferred compounds comprising the second member of the biological binding pair are"surrogate"ligands to the first member of the specific binding pair. For the purposes of this invention, the term"surrogate ligand"is intended to define a set of biologically-active compounds that specifically bind to any defined target comprising a first member of a biological binding pair. Although this definition is intended to encompass a variety of ligands of a target, particularly a target protein, comprising the first member of a biological binding pair, including a natural ligand. the surrogate ligands of the invention preferably comprise those ligands that specifically bind to the target protein with a chemical affinity measured by a dissociation constant (Kd) of from about 50 picomolar (pM) to about 0.5 mM, more preferably from about 1 nanomolar (nM) to about 100 micromolar (, uM), and most preferably from about lOnM to about 10 ßM. Such surrogate ligands are preferred because they bind with sufficient affinity that the concentration of the electrochemical label at the surface of the first electrode of the apparatus of the invention is sufficient to produce an experimentally-detectable current, while at the same time the binding affinity is weak enough to be displaced by competitors and inhibitors at concentrations of these compounds that are economical and can be experimentally achieved. Surrogate ligands therefore provide both the required degree of specificity and the required degree of easy dissociability to enable the methods and apparatus of the invention to detect binding

inhibition by competitor species. Some of the targets for which binding peptides have been identified are listed in Table I.

In the practice of the methods of the invention, second members of the biological binding pair that are electrochemically-labeled surrogate ligands include, but are not limited to, peptides, nucleic acids, carbohydrates, and small molecules. In embodiments of the methods of the invention using peptides as second members of the biological binding pair, the peptides are preferably obtained from phage-displayed combinatorial peptide libraries (see co-owned and co-pending U. S. patent application, Serial No.

08/740,671, filed October 21,1996, incorporated by reference herein) as well as other means. such as synthetic peptides prepared on pins or beads. Such peptides that contain only naturally-occurring amino acids must be electrochemically-labeled, because they lack sufficient redox potential under the voltage conditions tolerated by the first member of the biological binding pair. Peptides and proteins comprising the electrochemical probes and targets of the apparatus and methods of this invention can be prepared by synthetic methods, including solid phase peptide synthesis, biochemical isolation and modification techniques including partial proteolysis, and by recombinant genetic <BR> <BR> <BR> <BR> methods understood by those with skill in the art (see Sambrook et al., 1990, Molecular Clonino, 2d ed, Cold Spring Harbor Laboratory Press, N. Y.).

An example of a useful electrochemical labeling method is addition of a ruthenium group (Ru (NH3) 5 (OH,), 2-) to histidine residues within a peptide sequence.

Another example of a useful electrochemical labeling method is addition of a ferrocene carboxylic acid to side chains of lysine residues via the use of selective deprotection stratégies during automated peptide synthesis. Ferrocenes are also attractive labels because addition of substituents to the organic cyclopentadienyl-ligands can alter the redox potential by several hundred millivolts, thereby allowing observation of binding of multiple labels in one assay. Alternatively, electrochemical labels can be added to the amino-or carboxyl termini of peptides or protein fragments post-synthetically, or to the reactive side chain thiol groups of a cysteine residue, the hydroxyl group of a serine or threonine residue (or on a carbohydrate moiety), or the amino group of a lysine residue of the peptide. In addition, Fmoc derivatives of"unnatural"amino acids (such as D- amino acids or amino acid analogues such as £-aminOCaproiC acid) that can be incorporated during peptide synthesis can be used. For example, cobalt or osmium complexes with polypyridyl ligands possessing carboxylic acid substituents can be coupled to the s-amine of lysine using standard peptide chemistry.

Table I. Targets for which binding peptides have been identified from combinatorial libraries Targets References Streptavidin 1,2,3 HLA-DR 4,5 concanavalin A 6,7 calmodulin 8,9 S100 10 p53 11 SH3 domains 12-18 Urokinase receptor 19 bFGF-R integrin IIb/IIIa/avB1 20-23 Hsc70 24 tissue factor VIIa atrial naturiuretic peptide A receptor fibronectin 25 E-selectin 26 CD I-B2M complex 27 tissue-type plasminogen activator 28 core antigen of Hepatitis B virus 29 HIV-I nucleocapsid protein NCp7 30 erythropoietin receptor 31 trypsin 32 chymotrypsin 33 interleukin-1 receptor 34 References: 1. Devlin el al., 1990, Science 249: 404; 2. Lam et al., 1991, Nature 354: 82; 3. Fowlkes, 1993, Gene 128: 59; 4. Hammer et al., 1992, J. Exp. Med. 176: 1007; 5. Hammer et a/., 1993, Cell 74: 197; 6. Scott et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5398; 7. Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393; 8. Dedman et al., 1993, J. Biol. Chef. 268: 23025; 9. Adey et al., 1996, Gene 169: 133; 10.

Ivanenkov et al., 1995, J. Biol. Chenil. 270: 14651; 11. Daniels et al., 1994, J. Mol. Biol.

243: 639; 12. Sparks et al., 1996, Proc. Natl. Acad. Sci. USA 93: 1540; 13. Sparks et al., 1994, J. Biol. Chem. 269: 23853; 14. Cheadle et al., 1994, J. Biol. C'hem. 269: 24034; 15. Rickles et al., 1994, EMBO J. 13: 5598; 16. Rickles et al., 1995, Proc. Natl. Acad. <BR> <BR> <P>Sci. USA 92: 10909; 17. Chen et al., 1993, J. Amer. Chem. Soc. 115: 12591; 18. Yu et al., 1994, Cell 76: 933; 19. Goodson et al., 1994, Proc. Natl. Acad. Sci. USA 91: 7129; 20. O'Neill et al., 1992, Proteins 14: 509; 21. Fong et al., 1994, Drug Dev. Res. 33: 64; 22. Kolvunen et al., 1993, J. Biol. Chem. 268: 20205 ; 23. Kolvunen et al., 1993, J. Cell Biol. 124: 373; 24. Takenaka et al., 1995, J. Biol. Chez. 19839; 25. J. Cell Biol. 130: 1189; 26. Martens et al., 1995, J. Biol. Chem. 270: 21129; 27. Science 269 : 223; 28.

Proc. Natl. Acad. Sci. USA 92: 7627; 29. Dyson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 2194; 30. FEBSLett. 361: 85; 31. Wrighton et al., 1996, Science 273: 458; 32. Fang et al., 1996, Biochem. Biophys. Res. C'ommun. 220: 53; 33. J. Chromatography 711: 119; 34. Yanofsky et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7381.

In addition, a variety of non-peptide surrogate ligands can be adapted to this electrode system. For example, nucleic acids (e. g., RNA and DNA species, including poly-and oligonucleotides) that specifically bind to a target molecule can be obtained from combinatorial nucleic acid libraries; these molecules have been termed"aptamers" (as disclosed in Gold et al., 1995, Aszn. Rev. Biochem. 64: 763). Such aptamers can be electrochemically-labeled with a labeling group at either the 3'or 5'termini, or modified nucleotide triphosphate that binds an electrochemical labeling group can be incorporated into oligonucleotides by non-discriminating RNA or DNA polymerases during the in vitro generation of the aptamer. Finally, certain small molecules can be electrochemically-labeled in a way that does not destroy their binding activity. For example, cyclic AMP (cAMP) can be electrochemically-labeled without diminishing binding to protein kinase A, thereby providing a biological binding pair for electrochemical analysis of compounds that affect cAMP binding to protein kinase A.

Solutions of electrochemically-reactive liquids (also termed electrolyte solutions herein) useful in the apparatus of the invention include any electrolyte solution at physiologically-relevant ionic strength (equivalent to about 0.15M NaCI) and neutral pH. Nonlimiting examples of electrolyte solutions useful with the apparatus of the invention include phosphate buffered saline (PBS), HEPES buffered solutions, and sodium bicarbonate buffered ion solutions.

In the practice of the methods of the invention, when an appropriate potential is applied to the electrode. electrons are transferred to or from the electrochemical label from or to the electrode. The efficiency of electron transfer is proportional to the square root of the species'diffusion coefficient which, in turn, is inversely proportional to the species'radius. Therefore, codiffusion of the surrogate ligands with the larger target protein slows the frequency with which the electrochemical labels encounter the electrode with the result that less current is produced. This schema is illustrated in Figure 1.

In preferred embodiments of the apparatus and methods of this invention are provided an electron donor species contained in the reaction chambers of the apparatus of the invention, wherein the electron donor species is oxidized with transfer of an electron to the electrochemical label at the surface of the first electrode. In these embodiments of the apparatus and methods of the invention, oxidation/reduction of the electrochemical label at the surface of the first electrode is accompanied by the

production of detectable amount of chemiluminescence that is diminished upon binding of the second member of the biological binding pair with the first member of the biological binding pair. For the purpose of this invention, the term"electron donor species"is intended to encompass any chemical species having a readily-oxidized electron pair, including but not limited to amines (such as triethylamine, tripropylamine, triethanolamine), phosphines, arsines and the like. Preferably, the chemiluminescence produced by oxidation/reduction of the electrochemical label in the presence of the electron donor species has a wavelength in the visible portion of the electromagnetic spectrum, although the invention encompasses detectable chemiluminescence at any wavelength.

The invention provides methods of using the apparatus described herein to perform electrochemical assays. For the purposes of this invention, the term "electrochemical assay"is intended to encompass any assay performed in an apparatus of the invention, wherein the oxidation/reduction of an electrochemical label attached to a second member of a biological binding pair is achieved at a first electrode on the invention upon application of an electric potential between the electrodes. In preferred embodiments, the electrochemical assays of the invention include but are not limited a voltammetric, amperometric or coulometric assay, most preferably cyclic voltammetry, chronoamperometry, or chronocoulometry.

In one application of the methods of the invention are provided high-throughput screens of natural product and combinatorial chemical libraries for antagonists of protein-protein interactions. Such low molecular weight chemical antagonists of specific protein-protein interactions are of value to the pharmaceutical industry as potential drug leads for developing therapeutic agents. In the practice of these methods of the invention, a target protein comprising a first member of a biological binding pair is dissolved in an appropriate electrolyte. The first electrode is placed in a reaction chamber of the apparatus of the invention, preferably a microtiter plate well, said reaction chamber containing the target protein, an electrochemically-labeled surrogate ligand. and a compound or mixture of compounds to be tested for the ability to inhibit binding of the surrogate ligand to the target protein. (It will be understood that fragments of biologically-active proteins retaining the specific binding properties thereof are also encompassed within the scope of the target proteins of the electrodes of the invention.) For example, each of the reaction chambers or microtitre sample wells in a

representative experiment contain discrete combinatorial compounds or purified natural products (such as polyketides or fermentation broth components). After incubating the compounds in the presence of the electrode, voltammetric analysis of the current produced in the reaction chamber is performed. The results of these analyses are compared for wells containing the electrochemically-labeled surrogate ligand in the presence and absence of the compound or mixture of compounds to be tested.

Compounds that inhibit the binding of the electrochemically-labeled surrogate ligand to the target protein on the electrode surface yield an increased amount of current compared with compounds that do not bind to the target, which show no effect on surrogate ligand binding. In the practice of this invention, appropriate controls for detecting increase in observed current due to target protein denaturation are included in each determination. These controls include testing the target protein in a reaction chamber freshly prepared with a solution comprising an electrolyte and electrochemically-labeled surrogate ligand in the absence of the compounds to be tested, and by testing these compounds with unrelated target proteins. Optimally, the methods of the invention are practiced on a minimal volume of solution, either on a 96-well microtitre plate whereby 96 electrodes are configured to be utilized simultaneously, or in the context of emerging microfluidic technology.

Alternatively, the competition binding assays are performed to detect compounds that affect specific binding between the target protein and the electrochemically-labeled surrogate ligand by causing a conformational change in the target protein. In these embodiments of the methods of the invention, the target protein is first incubated with the electrochemically-labeled surrogate ligand, and then placed in a reaction chamber containing the compound or compounds to be tested. Compounds that bind to an available site on the target and induce a conformational or allosteric change in the target cause release of the electrochemically-labeled surrogate ligand, and are detected by the production of an increase in the observed current in the reaction chamber as detected, for example, by cyclic voltammetric analysis. As above, appropriate control reactions are performed to detect loss of surrogate ligand binding due to target protein denaturation.

The invention also provides methods for measuring the binding affinity of interaction between members of a biological binding pair, such as protein-peptide and protein-protein interactions. These measurements are useful for determining the

dissociation constant (Kd) of the interaction between the components of the biological binding pair. These methods provide an alternative to existing methods for measuring binding affinities and dissociation constants, such as surface plasmon resonance instruments (e. g., BIAcore"", Pharmacia). The methods of the present invention are advantageous compared with such previously-disclosed technologies because the present methods are more rapid, less costly and require less biological material. In addition the methods of the invention constitute a homogeneous assay whereas the methods known in the prior art require immobilization of one member of the biological binding pair. In addition, the methods of the present invention can be practiced using electrochemical probes and electrochemical ligands having molecular weights of 300 daltons or more.

In contrast, the methods known in the prior art require ligands that are at least about 5 kilodaltons (kD) in size, since the signal strength using prior art methods is proportional to the size of the binding ligand. This limitation prevents analysis of binding interaction properties of molecules having molecular weights less than the cutoff threshold, 5kD.

This limitation is important, since small molecular weight compounds form a large percentage of potential drug lead compounds. In addition, assay conditions using the methods and apparatus of this invention are more permissible than the assay conditions required using the methods of the prior art, including but not limited to conditions of probe concentration, salt concentration and assay performance in the presence of organic solvents.

The invention also provides methods and apparatus for determining the binding affinity and chemical"strength"of the interactions between members of a biological binding pair. Knowing the strength of the interaction between two members of a biological binding pair is important for determining whether the interaction has potential as a good target for drug discovery. The ability to detect these interactions with a rapid, inexpensive and convenient assay can greatly accelerate both target validation and screening. The methods of the present invention provide the ability to screen any two members of a biological binding pair for the capacity to specifically bind or otherwise specifically interact. The invention also provides methods for mapping region (s) of interaction between the members of the pair, using various truncated or altered forms of either or both members of the binding pair. For protein-protein interactions, there are several currently of interest in drug discovery, that are listed in Table II.

In yet another embodiment ofthe methods ofthe invention are provided methods

for detecting specific binding and other interactions between members of biological binding pairs in complex mixtures of chemical and biochemical molecules. In one embodiment, protein: protein interaction methods are provided. Such interactions are difficult to detect or characterize using existing technology. Using the methods of the present invention, a particular target protein is incubated with an electrochemically- active solution containing an electrochemically-labeled surrogate ligand and a cell extract comprising a protein (s) that specifically interacts with the target protein. As described with other embodiments of competition binding experiments using the methods of the invention, binding of the interacting protein instead of the electrochemically-labeled surrogate ligand results in an increased amount of current produced during electrochemical analysis, e. g., cyclic voltammetry.

This inventive method for detecting protein-protein interactions is advantageous compared with currently-available methods, as illustrated by a comparison with current methods for assaying column fractions during protein purification. The currently- available techniques include enzymatic assays of chromatographic column fractions that generate a radioactive product and that are only applicable to proteins having known enzymatic activity. For proteins with unknown enzymatic activity, ELISA assays, band shift assays using a radiolabeled target, or co-immunoprecipitations (that require antibodies to a radiolabeled target) are used. Each of these methods is time-consuming and tedious, and frequently require the use of radiochemical detection methods that are disadvantageous in terms of safety and regulatory concerns.

In contrast, the methods of the current invention are rapid, specific, and inexpensive. An additional advantage of the electrochemical screening methods of the present invention is that such screening methods are able to detect weak protein-protein interactions that cannot be detected by existing techniques. The methods of the present invention are also applicable to a variety of alternative embodiments of protein purification techniques, including analysis of chromatographic fractions, tissue distribution surveys for the presence of the target binding protein in tissue samples from tumors, for example, and for cell-cycle specific interactions, for example, by using extracts from synchronized cells.

Table II Interactions of Interest Molecule 1 Molecule 2 fibronectin integrin antigen antibody calmodulin-20 effector molecules tubulin microtubule associated proteins actin actin binding proteins, Dnase I p53 MDM2 cdk cyclin, p21 ras raf fos jun TBP RNA polymerases Sos Grb2 p53 p53BP2 K-channel src various proteins WW domain containing proteins ptyr proteins SH2 domains, PTB domains, phosphatases UGI UDG regulatory subunit PKA catalytic subunit PKA enhancer elements enhancer binding proteins DNA transcription factors RNA RNA binding proteins concanavalin A lectins lipids lipoproteins fatty acids (FA) FA binding proteins steroids steroid hormone receptors cytomegalovirus DNA polymerase polymerase accessory factor BPTI trypsin Rb E2F, E 1 A, SV40 T antigen References: Iwabuchi et al., 1994, Proc. Natl. Acad. Sci. USA 91: 6098; Holmes et al., 1996, Science 274: 2089; Rozakis-Adcock et al., 1993, Nature 363: 83; Phizicky et al., 1995, Microbiol. Rev. 59: 94; Chan et al., 1996, EMBO J. 15: 1045; Chen et al., 1995, Proc. Natl. Acad. Sci. USA 92: 7819; Sudol et al., 1995, FEBS Lett. 369: 67.

The methods and apparatus of the invention are advantageous over the analytical techniques and equipment known in the art for the following reasons. First, the sensitivity of the methods of the invention permit detection of specific binding interactions between the members of a biological binding pair over 4-5 orders of magnitude of concentration (i. e., 10,000- to 100,000-fold). This invention provides detection methods having the sensitivity of radiochemical detection methods without the health, safety and regulatory concerns that accompany radiochemically-based methods.

The invention also affords detection of biological binding interactions with high sensitivity over a wide range of binding affinities. Second, the assays are rapid, inexpensive and are performed in vitro. Third, the reagents used in the practice of the invention (i. e., the electrodes and electrochemically-labeled surrogate ligands) are stable and have a relatively long shelf-life compared with,/bfea/Mp/e, radiochemical reagents.

Fourth, structure-activity relationships can be determined quantitatively, based on the determination of changes in binding kinetics observed using cyclic voltammetry, for example. Fifth, the analyses can be multiplexed, that is, each reaction can be performed in a reaction chamber comprising more than one biological binding pair, wherein the electrochemical label of each of the second members of the binding pair present in the reaction chamber has a different redox potential, so that one or a mixture of potential drug lead compounds can be analyzed for binding to a variety of potential targets. Sixth, the methods and apparatuses of the invention are amenable to automation, including but not limited to the use of multiwell (such as 96-well microtitre) assay plates and robotic control of electrodes and electrochemical components of the reaction chambers thereof.

Seventh, the sensitivity of the electrochemical assays of the invention permit detection of small amounts of either surrogate ligand, inhibitory compounds, or both, thereby increasing the efficiency of performing assays such as drug screenings. Eighth, these increases in efficiency result in higher throughput screening, addressing a maj or obstacle to drug development. Ninth, the invention provides methods for determining dissociation constants for biological binding pair interactions that are more rapid, less expensive and require less sample than known methods (including, for example, equilibrium dialysis, analytical ultracentrifugation, analytical microcalorimetry and BIAcore-analysis). Tenth, the assays provided by the present invention can be performed in the absence of any information on the identity of the binding partner for any target protein or surrogate ligand. This advantage eliminates the requirement that

the biological activity of a target protein be known before the protein can be characterized. Eleventh, the assays of the invention are flexible, and allow analysis of binding or competition binding for any biological binding pair. Moreover, either of the binding pairs can be electrochemically-labeled, and under appropriate assay conditions, the diffusion behavior of both members of the biological binding pair in the electrochemically active solution can be observed and characterized.

In these embodiments of the apparatus of the invention are also comprised a second member of a biological binding pair, preferably a peptide or nucleic-acid surrogate ligand as defined herein, coupled to an electrochemical catalyst comprising an electrochemically activated catalytic species. Examples of such electrochemical catalysts are enzymes such as glucose oxidase and horseradish peroxidase, which effect the oxidation or reduction of their substrates and are electrochemically reactivated at potentials that are insufficient to effect direct electrochemistry of the substrate. Such enzymes are understood in the art to achieve catalysis by lowering an electrochemical barrier in the redox chemistry of the substrate, so that judicious choice of electrode potential allows selective electrochemical detection of the enzyme-catalyzed reaction in the vicinity of the electrode. Also, several synthetic transition-metal complexes such as those of oxoruthenium (IV), oxoosmium (IV), oxomolybdenum (IV), dioxomolybdenum (VI) and dioxorhenium (VI) are capable of oxidizing or reducing a variety of organic functional groups in a substrate at potentials at which direct electrochemistry is impossible. (For examples, see Stultz et al., 1995, J. Am Chem. Soc. <BR> <BR> <BR> <BR> <P>117: 2520; Cheng et al., 1995, JAm. Chem. Soc. 117: 2970, Neyhart et al., 1993, J. Am Chem. Soc. 115: 4423; Schultz et al., 1993, J. Am Chem. Soc. 115: 4244; Thorp et al., 1989, Inorg. Chem. 28: 889).

In the use of this embodiment of the invention, binding of the second member of the biological binding pair to the first member of the biological binding pair results in codiffusion of the electrochemical catalyst and the target protein. Redox reactions between the electrochemical label, the substrate and the electrode results in current flow at the electrode, due to transfer of redox equivalents to the substrate. For example, using a surrogate ligand linked chemically to horseradish peroxidase, binding of the surrogate with the first member of a biological binding pair causes the horse radish peroxidase enzyme to be reduced at a rate limited by codiffusion with the target. This reduced form of the enzyme is the active form, which can therefore act catalytically to transfer

electrons to hydrogen peroxide in the solution, producing oxygen and water. The enzyme is constitutively reduced by the electrode after each catalytic cycle and, as the entire process is repeated, the binding of the surrogate ligand-enzyme conjugate is detected and quantitated as decrease in current flowing through the electrode to the solution. Thus, under the appropriate conditions of substrate concentration and applied potential at the electrode, the amount of current produced is related to the amount and extent of binding between members of the biological binding pair.

In particularly preferred embodiments of this aspect of the invention, the substrate of the electrochemical catalyst produces a detectable product upon undergoing an oxidation/reduction reaction with the electrochemical catalyst at the surface of the first electrode of the apparatus. For the purposes of this invention, the term"detectable product"is intended to encompass a product that can be differentially detected in a solution containing the biological binding pairs, the electrochemical catalyst, and the substrate therefore. Exemplary of a detectable label, but not limited thereto is a colored product, that is, a product that absorbs electromagnetic radiation, preferably within that portion of the spectrum comprising visible light, so that the extent of the reaction can be monitored colorimetrically. Also within the meaning of the term"detectable product"is any product that can be detected spectrophotometrically, including for example differences in the oxidation state of the NADT/NADH or NADP+/NADPH redox pair, as well as other cofactors including for example riboflavin. In this aspect, the detection of the detectable product can be used as a control for stability of the electrochemical catalyst, the substrate therefor or both, or for depletion of the substrate by the catalyst during the reaction (i. e., saturation), and can be used as a surrogate for current production or as an internal validating control for the observed current produced in the reaction chamber of the apparatus.

In another application of the invention, binding or lack of binding of the conjugate is used to determine the occupancy of the available binding sites by an electrochemically inactive species. Typically, this species will be a single drug candidate from a large library of either natural products or combinatorially synthesized molecules. Binding of the drug candidate can be ascertained by at least three related techniques. In a first embodiment, the first member of the biological binding pair comprising a target can be preincubated with the drug candidate to allow all possible binding interactions between the candidate drug and the first member of the biological

binding pair to occur prior to adding the electrochemically-labeled surrogate ligand. The increase in current upon addition ofthe electrochemically-labeled surrogate ligand, when compared with current produced in the absence of the drug candidate, is a measure of the extent of occupancy of the available binding sites of the electrode-immobilized first member of the biological binding pair by the drug. In a second embodiment, a drug candidate is added concurrently with an electrochemically-labeled surrogate ligand conjugate at different concentrations, and the effect of the presence of the drug candidate on the produced current used to determine the inhibition constant of the drug for surrogate ligand binding. In a third embodiment, the target can be saturated with electrochemically-labeled surrogate ligand prior to the addition of the drug, whereby loss of observable current indicates the capacity of the drug candidate to displace surrogate ligand binding. Those of ordinary skill will recognize the utility of these methods for characterizing the binding and inhibitory properties of drug candidates for any biological binding pair of interest.

Additional features of the invention are more fully illustrated in the following Examples. These Examples illustrate certain aspects of the above-described method and advantageous results. These Examples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Preparation of Electrochemicallv Labeled Peptides Electrochemically labeled peptides were prepared using art-recognized techniques (see Yocom et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7052-7055; Nocera et al., 1984, J. Amer. Chem. Soc. 106: 5145-5150). In one example, the derivatized peptide EC-1, having the amino acid sequence: Lys (s-biotin)-Ser-Lys (£-ferroceneacetic acid)-Ser-NH, (SEQ ID No.: 1) was synthesized as follows. The first (underlined) lysine incorporated was protected as an a-Fmoc-, s-Boc-derivative. After removal of the Boc-group, ferroceneacetic acid was coupled to the £-amine via standard benzotriazolyl-N-oxy- tris (dimethylamino) phosphonium hexafluorophosphate/hydroxy-benzotriazole (BOP/HOBt) chemistry. The remainder of the peptide was synthesized via standard procedures.

EXAMPLE 2 Cvclic Voltammetry Cyclic voltammograms were collected using a BAS 100B potentiostat/galvanostat (BAS, West Lafayette, IN) with a single compartment voltammetric cell equipped with a glassy carbon (GC) working electrode (area = 0.032 cl23, platinum (Pt)-wire auxiliary electrode and silver/silver chloride (Ag/AgCl) reference electrode (see Johnston etal, 1994, Inorg. Chem. 33: 6388-6390). An example of such a voltammogram is shown in Figure 2 and was acquired under the following conditions. The reaction chamber of the apparatus of the invention contained 100 pM EC1 peptide as disclosed in Example 1 dissolved in a buffered aqueous solution containing 10 mM Tris, 150 mM NaCI, and 0.05% Tween 20. Cyclic voltammetry was performed where the applied voltage was scanned at 50 mV s-'. The cyclic voltammogram shown in Figure 2 illustrates the electrochemical behavior of the EC1 peptide in the absence of a first member of a biological binding pair that specifically binds to the peptide.

EXAMPLE 3 Electrochemical Assay for Detecting Specific Binding The electrochemical analysis apparatus and methods of the invention were used to detect and analyze the specific binding interaction between neutravidin and the biotinylated EC1 peptide as follows. Cyclic voltammetry was performed using a solution in the reaction chamber as disclosed in Example 2 at applied voltage scan rates of 25,50,100,200, and 400 mV s-'. The voltammogram acquired at 50 mV s-'is shown in Figure 3 (the curve designated"A"). Voltammograms were then acquired under identical peptide and scan-rate conditions in the presence of stoichiometric excesses of neutravidin (shown in Figure 3, the curve designated as"B") and biotin-saturated neutravidin. These results showed that the presence of neutravidin resulted in binding between the neutravidin and the biotinylated EC 1 peptide, and that this binding reduced the currents observed in the cyclic voltammograms.

This reduction in the observed currents was used to determine the effective diffusion coefficients of the peptide-neutravidin complexes formed in the reaction chamber. The effective diffusion coefficient of the peptide under these conditions was calculated via a plot of peak anodic current versus the square root of scan rate according

to the Randles-Sevcik equation: ip = 269000n"VCAD where ip is the peak current, n is the number of electrons transferred, v is the square root of scan rate in V s', C is concentration in moles/cm', A is electrode area in cm2, and D is the diffusion coefficient in cm2 s'. These plots are shown in Figure 4. The effective diffusion coefficients of EC-1 alone was determined to be approximately 3.8 x 10-'cm2 s-', and in the presence of biotin-saturated neutravidin was determined to be approximately 3.4 x 10-7 cm2 s~', respectively. These results were consistent with experimental expectations, since the neutravidin-biotin binding affinity is sufficiently high that dissociation of neutravidin-biotin and complex formation between the freed neutravidin and biotinylated EC-1 peptide was considered highly unlikely. In the presence of neutravidin, the diffusion coefficient calculated from the observed current was reduced to approximately 6.8 x 1 O~8 cm2 s~', consistent with a loss of approximately 50 percent of the Faradaic current. These results demonstrated that the peptide codiffused with the 60 kD neutravidin protein only when specific interaction of the biotin label of the EC-1 peptide with the biotin-binding site of neutravidin occurred.

These results thus demonstrated that specific binding between the first member of a biological binding pair (neutravidin) with an electrochemically-labeled second member of a biological binding pair (the EC-1 peptide) could be detected using the methods and apparatus of the invention, and that the observed current reduction could be used to characterize the biological binding pair.

EXAMPLE 4 Electrochemical Assav for Screening Combinatorial Libraries An electrochemical assay of the invention is used to screen combinatorial chemical libraries to detect samples that perturb the electrochemical signal in cyclic voltammetry resulting from the interaction between a target protein and electrochemically-labeled binding peptide. For the electrochemical assay to be useful for screening libraries of chemical compounds for compounds that disrupt the target: peptide interaction, the conditions of the screen must not be easily perturbed, or the voltammetry output diminished thereby. Optimally, such a screening assay will function over a wide range of pH and salt concentrations, and is not sensitive to common contaminants (such as coupling reagents) that are frequently encountered in

combinatorial chemical libraries.

To evaluate the validity of the electrochemical analysis assay for screening combinatorial libraries, theoretical cyclic voltammograms were calculated using the BAS DigiSim cyclic voltammetry computer simulation software. The mathematical model assumes a target protein with a diffusion coefficient of 2.0 x 10-'cm s'bound to an electrochemically-labeled surrogate ligand which, unbound, has a diffusion coefficient of 4.0 x 10~6 cm2 s~'. Binding ofthe surrogate ligand and protein is governed by a dissociation constant of 1.0 x 10-'M and the binding reaction rate constant is fixed at a diffusion-controlled value of l. Ox 109 M-'s'. Cyclic voltammograms were calculated assuming protein and surrogate ligand concentrations of 100 uM under conditions of increasing concentrations of a competitor with a target-binding affinity of 50 nM, using a theoretical microfluidic cell with electrode area of 8.5 x 10-6 cnr and using a potential scan rate of 50 mV s-'.

The cyclic voltammograms are shown in Figure 5; a plot of peak anodic current versus concentration of added competitor (Figure 6) demonstrates that the surrogate ligand is displaced from the target at inhibitor concentrations of-10 to 100 aM, consistent with stoichiometric displacement of the surrogate ligand by a higher-affinity species.

EXAMPLE 5 Electrochemical Analvsis for Determining the K, for a Known Protein: Peptide Interaction The electrochemical analysis methods of the invention are also useful for determining the Kd of the interaction between a protein and specific binding peptides therefor, for example, the Src-SH3 domain and a number of short, proline-rich specific binding peptides. The interaction of the Src SH3 domain with short, proline-rich peptides such as Arg-Pro-Leu-Pro-Pro-Leu-Pro (SEQ ID No.: 2) and Ala-Pro-Pro-Val- Pro-Pro-Arg (SEQ ID No.: 3) have been intensively studied, and Kd values have been determined by validated means such as BIAcore' (Pharmacia, Upsalla). On average, these peptides have been shown to bind to the Src SH3 domain with a Kd Of 5 llM. These data provide a solid basis for comparison of the ability of the electrochemical assay of the invention to provide an accurate Kd value for the same interaction.

The Kd value for the interaction of GST-Src SH3 and SH3 binding peptides is

determined using the electrochemical analysis methods of the invention to provide a comparison with apharmacologically-validated method. Solutions of the GST-Src SH3 fusion protein are mixed with electrochemically-labeled species of the proline-rich SH3- domain specific binding peptides shown above. Electrochemical signal is generated using cyclic voltammetry as described in Examples 2-4 above, and the signal is monitored over time as described in Example 4 above. Electrochemical signal data are collected at various concentrations of the peptide, and the electrochemical signal is then used to calculate a Kd value for the peptide. Kd values are also determined using the BIAcoret method as a validated control, and a comparison of the results between the two analytical methods used to validate the values determined using the electrochemical analysis assays of the invention.

EXAMPLE 6 Electrochemical Analvsis for Detecting Protein: Peptide Interactions in Complex Mixtures The electrochemical analytic methods and apparatus of the invention are used to detect protein: peptide interactions in a complex mixture. A variety of different target molecules and sources of specific binding peptides are used in these experiments.

Although it is usually possible to find a surrogate ligand for a protein by using phage display or by screening combinatorial libraries (see co-owned and co-pending U. S. patent application, Serial No. 08/740,671, filed on October 31,1996, incorporated herein by reference), the natural ligand for a protein is more difficult to identify. The electrochemical screening assays of the invention provide a relatively simple means for identifying natural ligands. To demonstrate this aspect of the electrochemical analytic methods of the invention, the natural ligand for the Src SH3 domain in cell extracts is detected. GST-Src SH3 domain fusion protein solutions are incubated with electrochemically-labeled Src-SH3 binding peptide to specifically"load"the SH3 domain with the electrochemically-labeled peptide. The solutions are then incubated with whole cell extracts from about 107 108 HeLa cells and cyclic voltammetry performed. Data analysis is performed to determine the extent of increase in the electrochemical signal resulting from displacement of the electrochemically-labeled peptide by compound (s) present in the HeLa cell extract. The cell extract is then

fractionated using conventional biochemical fractionation techniques, including a variety of chromatographic methods (such as anion exchange chromatography using DEAE Sepharose, cation exchange chromatography using carboxymethyl Sepharose, and size exclusion chromatography using Sephadex and Sepharose). After each fractionation, fractions are analyzed for the presence of a compound (s) that can displace binding of the electrochemically-labeled specific binding peptide as detected by cyclic voltammetry.

Only those fractions containing such activity are carried through subsequent steps of the biochemical fractionation. After several such biochemical purification steps, active fractions are analyzed by SDS-polyacrylamide gel electrophoresis to determine the relative purity of the fractions. Microsequencing of homogeneous protein-containing "bands"is then used to isolate and identify the active protein (s) comprising the fraction having specific peptide displacement activity. The methods of the invention thereby provide a sensitive method for detecting protein-protein interactions from heterogeneous mixtures of biological compounds.

The electrochemical analysis assays of the invention are also used to determine functions for orphan receptors isolated and identified by recombinant genetic methods.

Frequently, DNA sequences are discovered encoding regions resembling receptor coding domains. When these sequences are discovered, it is presently quite difficult to determine the biological function or activity of the encoded receptor or the natural ligand of these receptors. For an unknown receptor sequence, the extracellular domain of the receptor is expressed and used as the target for screening phage displayed peptide libraries to identify a surrogate ligand. The surrogate ligand is then used in a number of ways. Electrochemical screens of combinatorial libraries are conducted to identify antagonists of the assay. These compounds are then used in model biosystems (for example, mammalian cells transformed with a recombinant expression construct encoding the receptor that express the receptor at the cell surface) to decipher the biological role of the receptor (for example, to determine if the receptor is linked to adenylate cyclase).

The surrogate ligands are also used in an electrochemical screening assay to identify the natural ligand. Cell lysats, supernatants or tissue extracts are fractionated and assayed by the electrochemical screening assays of the invention to identify fractions containing a molecule that displaces the labeled surrogate ligand from the electrode-bound target. Protein or peptide ligands isolated thereby can be then identified

by sequencing. Small molecule ligands may be identified by mass spectral analysis and other analytical systems. Such assays systems can also be applied to molecules found in biological fluids (such as mammalian brain extracts) to indicate the existence of the natural ligand in such biological extracts.

An example of such use of the electrochemical analysis assays of the invention is the identification of ligands for thefas receptor (see Hahne et al., 1996, Science 274: 1363; Nagata et al., 1995, Science 267: 1449; Takahashi et al., 1994, Cell 76: 969; Wanatabe-Fukunaga et al., 1992, Nature 356: 314). The fas receptor, which is expressed in almost all cell types, triggers the apoptotic (programmed cell death) pathway when it is bound by its ligand. The expression of the ligand for thefas receptor is much more restricted. Apoptosis is triggered when the ligand on one cell interacts with the receptor on another cell. This is a therapeutically useful target since it has recently been demonstrated that the expression of the fas ligand on the surface of some melanoma cells triggers apoptosis in body's immune cells, thereby allowing cancer cells to evade the host immune response.

The extracellular domain of the fas receptor is expressed for use as a target in phage display to identify a surrogate ligand (using, for example, the methods disclosed in co-owned and co-pending U. S. patent application, Serial No. 08/740.671, filed on October 31,1996, incorporated herein by reference). Surrogate ligands so identified are <BR> <BR> <BR> then electrochemically-labeled and incubated with the fas receptor extracellular domain.

Plasma cell supernatants are fractionated, and the fractions assayed by cyclic voltammetry and electrochemical screening as described herein to detect those fractions that contain activity capable of displacing the labeled surrogate ligand from the electrode. After a series of fractionations by conventional chromatographic techniques, thefas receptor ligand is detected. In addition, the function of thefas receptor is identified using the electrochemical analytic methods of the invention. In these assays, the extracellular domain of thefas receptor is used to obtain a surrogate ligand via phage display library as described above. The labeled surrogate ligand is then used in an electrochemical screen to identify compounds from a combinatorial chemical library that displace or compete the labeled ligand from the. fas receptor protein. The identified compound may either be an agonist or an antagonist of fas receptor activity. The compound (s) identified in this screen are tested in a model biological system to study receptor function. For example, the compound is added to cells in culture that express

thefas receptor and the biological response of the cells observed. A receptor antagonist blocks the apoptotic pathway in the presence of thefas ligand while a receptor agonist mimics the fas ligand and results in stimulation of the apoptotic pathway. Thus, detection of apoptosis provides a sensitive assay that can be used in conjunction with the electrochemical analysis assays of the invention to analyze fas receptor function.

EXAMPLE 7 Electochemiluminescence Detection of Codiffusion of Members of a Biological Binding Pair The electrochemical assay of the invention is used to screen combinatorial chemical libraries to detect samples that perturb a chemiluminescence signal resulting from the interaction between a target protein and an electrochemically labeled binding peptide. A variety of different target molecules and sources of specific binding surrogate ligands are used in these experiments.

Although it is accurate to describe an electrochemical current as a sum ofohmic, capacitive, and Faradaic components, it can be in practice difficult to account accurately and precisely for the relative contributions from each source. An advantageous feature of some electrochemical reactions is the production of a photon from a luminescent product. If the rate of the electrochemical reaction is governed by diffusion, then it becomes possible to measure this diffusion rate as a function of the quantity of light emitted. For example. oxidation of both a trisbipyridine ruthenium (II) complex and triethanolamine occurs at a potential of +1.0 V versus a Ag/AgCI reference electrode.

The two oxidized products then react to yield a luminescent ruthenium (II) species.

Because the bimolecular rate constant is a function of the sum of the diffusion coefficients of the two species, the yield of photons is a direct measure of these diffusion coefficients. Thus, if the ruthenium complex is attached to the surrogate ligand, codiffusion with the larger member of the biological binding pair will result in a decrease in the signal from electrochemiluminescence. In this embodiment of electrochemical drug screening assays as provided by the invention, compounds that displace the surrogate ligand from the target will effect faster diffusion of the preluminescent species resulting in a larger amount of photons emitted.

It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.