# US 5,346,832 A · Homogeneous amperometric immunoassay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,346,832 A - Document ID: US5346832A - Status: Granted - Kind: A - Application number: 07/993,224 - Filed: 1992-12-18 - Granted: 1994-09-13 - Earliest claimed: 1989-06-09 - Assignees: Ciba Corning Diagnostics Corp. - Inventors: Masuo Aizawa; Brenda D. Manning; Miki Hidaka; Laura S. Uretsky - Web page: https://patentgrep.com/patent/US5346832A ## CPC - G01N 33/5438: {Electrodes} - G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching - Y10S 435/817: Enzyme or microbe electrode - Y10S 436/806: Electrical property or magnetic property - Y10S 436/817: Steroids or hormones - Y10S 436/818: Human chorionic gonadotropin - Y10S 530/807: Hapten conjugated with peptide or protein - Y10S 530/812: Peptides or proteins is immobilized on, or in, an organic carrier - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract A method is described for measuring the amount of analyte present in a sample containing the analyte using a homogeneous amperometric immunoassay. The analyte is chemically bonded to a suitable carrier molecule, which is also chemically bonded to an electroactive molecule. The electroactive molecule, such as ferrocene carboxylic acid, contains a redox center which is capable of transferring a charge to an electrode. A preferred carrier molecule is bovine serum albumin (BSA), while suitable analytes include digoxin, theophylline and HCG. The immunoassay is conveniently performed by applying a voltage to a set of electrodes. ## Claims ### Claim 1 A method for determining the concentration of an analyte present in a sample of biological fluid contained in an electrochemical cell equipped with a set of electrodes, said method comprising the steps of: a) forming an electroactive complex containing an antigenic analyte chemically bonded to a carrier molecule, said carrier molecule also being chemically bonded to an electroactive molecule capable of transferring a charge to an electrode; b) adding the biological fluid, predetermined amounts of the electroactive complex, and an antibody to the analyte to the cell; c) applying a voltage across the electrodes and measuring the current in the fluid at different voltage levels to form a voltage/current relationship for the sample fluid; and d) comparing the voltage/current relationship for the sample fluid with standard voltage/current relationships generated using known amounts of analytes to determine the concentration of analyte present in the sample. ### Claim 2 (depends on 1) The method of claim 1 wherein the analyte is selected from the group consisting of theophylline, thyroxine, digoxin and HCG. ### Claim 3 (depends on 1) The method of claim 1 wherein the carrier molecule is bovine serum albumin. ### Claim 4 (depends on 1) The method of claim 1 wherein the electroactive molecule is a ferrocene derivative. ### Claim 5 (depends on 4) The method of claim 4 wherein the ferrocene derivative is selected from the group consisting of ferrocene carboxylic acid and ferrocenylacetic acid. ### Claim 6 (depends on 5) The method of claim 5 wherein the ferrocene derivative is ferrocene carboxylic acid. ### Claim 7 (depends on 1) The method of claim 1 wherein the analyte is digoxin, the antibody is a anti-digoxin antibody, the carrier molecule is bovine serum albumin, and the electroactive molecule is ferrocene carboxylic acid. ### Claim 8 (depends on 1) The method of claim 1 wherein the working and counter electrodes are fabricated from platinum or graphite. ### Claim 9 (depends on 1) The method of claim 1 wherein the biological fluid is whole blood, plasma, serum or urine. ### Claim 10 (depends on 1) The method of claim 1 wherein a buffer is added to the fluid sample. ### Claim 11 An electroactive complex comprising at least one antigenic analyte chemically bonded to a carrier molecule, said carrier molecule also being chemically bonded to at least one electroactive molecule. ### Claim 12 (depends on 11) The electroactive complex of claim 11 wherein the analyte is selected from the group consisting of theophylline, thyroxine, digoxin, and HCG. ### Claim 13 (depends on 11) The electroactive complex of claim 11 wherein the carrier molecule is bovine serum albumin. ### Claim 14 (depends on 11) The electroactive complex of claim 11 wherein the electroactive molecule is ferrocene carboxylic acid. ### Claim 15 (depends on 11) The electroactive complex of claim 11 wherein the analyte is digoxin, the carrier molecule is bovine serum albumin, and the electroactive molecule is ferrocene carboxylic acid. ### Claim 16 (depends on 15) The electroactive complex of claim 15 which is prepared by first covalently binding ferrocene carboxylic acid to bovine serum albumin, and subsequently covalently binding digoxin to the bovine serum albumin. ### Claim 17 (depends on 11) The electroactive complex of claim 11 wherein the chemical bonds are covalent bonds. ### Claim 18 (depends on 11) The electroactive complex of claim 11, wherein the carrier molecule is selected from the group consisting of bovine serum albumin, bovine gamma globulin, ribonuclease, cytochrome C, and insulin. ### Claim 19 (depends on 11) The electroactive complex of claim 11, wherein the carrier molecule is a molecule having a molecular weight less than 150,000. ### Claim 20 (depends on 11) The electroactive complex of claim 11, wherein the carrier molecule is selected from a molecule having one or more amino and/or one or more carboxyl groups.