Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures
First Claim
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1. An electrochemical reporter system comprising:
- (a) a recognition molecule capable of specifically binding an analyte in a structure restricted manner;
(b) an enzyme;
(c) a coupling element, for coupling with specificity the enzyme to the recognition molecule or the analyte;
(d) a substrate which in the presence of the enzyme is cleavable into a reporter molecule capable of exhibiting redox recycling; and
(e) a sensor for detecting the electrochemical reporter molecule, said sensor having a configuration such that the reporter molecule will exhibit redox recycling.
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Abstract
An immunochemical and molecular biologic endpoint reporter system in which reaction products, coupled to electrochemically active molecules susceptible to redox recycling or coupled to enzymes capable of proportional generation of said electrochemically active molecules, are detected and/or quantitated using amperiometry in conjunction with a silicon microchip possessing a closely spaced interdigitated array of nobel metal electrodes.
47 Citations
25 Claims
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1. An electrochemical reporter system comprising:
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(a) a recognition molecule capable of specifically binding an analyte in a structure restricted manner;
(b) an enzyme;
(c) a coupling element, for coupling with specificity the enzyme to the recognition molecule or the analyte;
(d) a substrate which in the presence of the enzyme is cleavable into a reporter molecule capable of exhibiting redox recycling; and
(e) a sensor for detecting the electrochemical reporter molecule, said sensor having a configuration such that the reporter molecule will exhibit redox recycling. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10)
(a) biotin conjugated to an antibody specific for the recognition molecule or the analyte and avidin conjugated to the enzyme;
(b) biotin conjugated to an antibody specific for the recognition molecule or the analyte and streptavidin conjugated to the enzyme;
(c) digoxigenin conjugated to an antibody specific for the recognition molecule or the analyte and digoxigenin-specific antibody conjugated enzyme (d) by cross-linking the enzyme to the recognition molecule; and
(e) by cross-linking the enzyme to an antibody specific for the recognition molecule.
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9. The electrochemical reporter system of claim 1 wherein the substrate is cleaved into at least one component comprising para-aminophenol.
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10. The electrochemical reporter system of claim 1 wherein the sensor is a microelectronic interdigitated array of electrodes having width between about 100 and about 800 nanometers and spaced between about 100 and about 800 nanometers from each other.
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11. An electrochemical immunoassay for detecting an analyte in a sample comprising the steps of:
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(a) having linked to a surface an antigen with an antibody specific for an analyte bound to the antigen, the antibody being coupled to an enzyme or having a coupling element for being specifically coupled to an enzyme;
(b) contacting the surface with a sample to be analyzed;
(c) collecting the antibodies displaced from the antigen by the analyte in the sample;
(d) adding a substrate to the collected antibodies, said substrate being cleavable by the enzyme into an electrochemical compound; and
(e) measuring the presence or amount of the electrochemical compound with an interdigitated array of electrodes capable of producing redox recycling of the electrochemical compound.
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12. An electrochemical immunoassay for detecting an analyte in a sample comprising the steps of:
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(a) having linked to a surface an antibody specific for an analyte and an antigen bound to the antibody, the antigen being coupled to an enzyme or having a coupling element for being specifically coupled to an enzyme;
(b) contacting the surface with a sample to be analyzed;
(c) collecting the antigen displaced from the antibody by the analyte in the sample;
(d) adding a substrate to the collected antigen, said substrate being cleavable by the enzyme into an electrochemical compound; and
(e) measuring the presence or amount of the electrochemical compound with an interdigitated array of electrodes capable of producing redox recycling of the electrochemical compound.
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13. An electrochemical immunoassay for detecting a specific analyte in a sample comprising the steps of:
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(a) having a recognition molecule linked to a non-magnetic surface, said recognition molecule capable of specifically binding the analyte in a structure restricted manner;
(b) contacting the non-magnetic surface with a sample to be analyzed;
(c) coupling with specificity an enzyme to the recognition molecule or the analyte;
(d) adding a substrate, which in the presence of the enzyme is cleaved into a reporter molecule capable of exhibiting redox recycling; and
(e) measuring the presence or amount of the reporter molecule with an interdigitated array of electrodes capable of producing redox recycling of the reporter molecule. - View Dependent Claims (14)
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15. An electrochemical reporter system comprising:
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a) an enzyme;
b) a coupling element, for coupling the enzyme to a nucleotide, oligonucleotide, branched oligonucleotide, nucleic acid, or analogs thereof;
c) a substrate, which in the presence of the enzyme is cleavable into a reporter molecule capable of exhibiting redox recycling; and
d) a sensor for detecting the reporter molecule, the sensor having a configuration such that the reporter molecule exhibits redox recycling. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23)
a) a covalent bond; b) biotinilated nucleotide, oligonucleotide, branched oligonucleotide, nucleic acid, or analogs thereof and avidin or streptavidin conjugated enzyme; and
c) digoxigenin labeled nucleotide, oligonucleotide, branched oligonucleotide, nucleic acid, or analogs thereof and digoxigenin-specific antibody conjugated enzyme.
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22. The electrochemical reporter system of claim 15, the substrate being cleaved into at least one component comprising pari-aminophenol.
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23. The electrochemical reporter system of claim 15, the sensor being a microelectronic interdigitated array of electrodes, wherein the electrodes have a width between about 100 and about 800 nanometers and wherein the electrodes are spaced between about 100 and about 800 nanometers from each other.
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24. An assay for detecting or quantitating a specific nucleic acid sequence in a sample comprising the steps of:
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a) having a first single stranded nucleic acid having a sequence complementary to a first segment of the specific nucleic acid sequence to be detected or quantified, the first single stranded nucleic acid being in a solution and bound to a surface;
b) contacting the first single stranded nucleic acid with a sample to be analyzed;
c) adding a labeled second single stranded nucleic acid to the sample to be analyzed, said second nucleic acid having a sequence complementary to a second segment of the nucleic acid sequence to be detected or quantified;
d) adding a substrate, said substrate in the presence of the second single stranded nucleic acid being cleaved into an electrochemical compound; and
e) detecting the presence or measuring the amount of electrochemical compound present in the solution with an interdigitated array of electrodes capable of producing redox recycling of the electrochemical compound.
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25. An electrochemical assay for detecting or quantitating a specific nucleic acid sequence in a sample comprising the steps of:
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a) amplifying the specific nucleic acid sequence in the presence of labeled nucleotides or labeled primer oligonucleotides such that the labeled nucleotides or the labeled primer oligonucleotides are incorporated in labeled newly synthesized nucleic acid;
b) adding a substrate, said substrate in the presence of the labeled newly synthesized nucleic acid being cleaved into an electrochemical compound; and
c) measuring the presence or amount of the electrochemical compound present with an interdigitated array of electrodes capable of producing redox recycling of the electrochemical compound.
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Specification