Electrochemical immunoassay methods
DCFirst Claim
1. An electro-immunosensor comprising an electrode on which is deposited a colloidal gold adsorbed anti-analyte antibody bound to an enzyme/analyte conjugate.
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Abstract
An electro-immunosensor which can be used to determine the presence of a wide range of analytes in solution is described. Also described are methods of determining subnanogram levels of analytes in a one-step procedure. This novel immunosensor comprises an analyte binding agent bound to an electrode and a analyte/enzyme conjugate bound to the analyte binding agent as part of a catalytic electrical circuit. Displacement of the conjugate by analyte causes a proportional decrease in current. The immunosensor may also be adapted to detect two analytes in a single step by using different enzymes conjugated to the analyte binding agents.
189 Citations
47 Claims
- 1. An electro-immunosensor comprising an electrode on which is deposited a colloidal gold adsorbed anti-analyte antibody bound to an enzyme/analyte conjugate.
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6. An electrochemical enzyme-linked immunoassay comprising:
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(a) contacting a sample suspected of containing an analyte with an electrode on which is deposited a colloidal gold adsorbed anti-analyte antibody bound to a redox-enzyme/analyte conjugate; and (b) determining catalytic current decrease in the presence of a substrate of the redox enzyme when the electrode is coupled with a reference electrode wherein said decrease is related to the level of analyte present in the sample. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A dual site electro immunoassay for analyte determination, comprising:
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contacting a sample suspected of containing an analyte with an electro immunosensor comprising an electrode on which is deposited a colloidal gold adsorbed anti-analyte antibody having specificity for the analyte; incubating the sample with a redox enzyme/antibody conjugate wherein the antibody conjugated with the enzyme is specific for the analyte and lacks cross-reactivity with said surface bound antibody; and determining an electrochemical response generated from binding of the enzyme/antibody conjugate with the surface bound analyte when the electro immunobiosensor is coupled with a reference electrode wherein the response is a measure of the amount of analyte present in the sample. - View Dependent Claims (18, 19)
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20. An electro-immunosensor comprising:
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(a) a first anti-analyte antibody bound to protein-coated colloidal gold deposited on an electrode surface; (b) a second anti-analyte antibody bound to protein-coated colloidal gold deposited on the electrode surface;
wherein the first anti-analyte antibody is specific for an analyte which is different from a second analyte and non cross-reactive with the second anti-analyte antibody;(c) a first conjugate comprising a first redox enzyme bonded to a separate sample of said anti-analyte antibody; and (d) a second conjugate comprising a second redox enzyme bonded to a separate sample of said anti-analyte antibody. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28)
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29. A dual-analyte electro immunoassay, comprising:
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(a) contacting a sample suspected of containing a first analyte and a second analyte with an electro immunosensor comprising an electrode on which is deposited two colloidal gold adsorbed anti-analyte antibodies each having specificity for said first analyte or said second analyte wherein each analyte binds with its specific antibody to form an analyte/antibody complex; (b) incubating the bound analyte/antibody complexes with a first and a second redox enzyme/antibody conjugate wherein one conjugate is a first enzyme bound to an antibody specific for the first analyte that lacks cross reactivity with the first surface bound antibody and the other enzyme conjugate is a second antibody specific for the second analyte that lacks cross reactivity with the second surface bound antibody; and (c) measuring an electrochemical response generated from binding of the first and second enzyme-conjugated antibodies with the surface bound first or second analyte/antibody complex when the electro-immunosensor is coupled with a reference electrode to provide a measure of the amount of first or second analyte present in the sample. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36)
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- 37. A single-step electrochemical enzyme-linked immunoassay to detect subnanogram or picogram levels of an organic nitrogen compound in an aqueous sample, comprising contacting a sample suspected of containing said compound with the electrochemical immunosensor of claim I wherein the analyte/enzyme is said nitrogen compound bound to horseradish peroxidase and determining catalytic current decrease in the presence of H2 O2 when the immunosensor is coupled with a reference electrode.
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40. A method for preparing an electrochemical enzyme immunoassay sensor for determining subnanogram or picogram levels of an immunogenic analyte comprising:
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(a) immobilizing on colloidal gold an antibody that specifically reacts with an analyte; (b) depositing the immobilized antibody on an electrode surface; (c) separately immobilizing a peroxidase enzyme conjugate on colloidal gold wherein the peroxidase enzyme conjugate is formed between a peroxidase enzyme and the analyte; and (d) incubating the peroxidase enzyme/analyte conjugate with the electrode surface deposited antibody to form an antibody/peroxidase/conjugate complex.
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- 41. An electro immunosensor comprising an electrode on which is surface-deposited protein-coated colloidal gold bound to an analyte wherein the analyte is bound to an anti-analyte antibody/redox enzyme conjugate.
Specification