Electrochemical enzymic assay procedures
First Claim
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1. A method of assaying for activity of a first enzyme able to convert a non-mediator to a mediator, said mediator having a reduced form and an oxidized form, said method comprising:
- (a) providing a sample to be assayed for said activity of said first enzyme;
(b) contacting said sample with freely circulating non-mediator;
(c) providing a second enzyme, said second enzyme being catalytically active in the absence of said mediator;
(d) providing a substrate for said second enzyme;
(e) providing an electrode at an electrical potential which is intermediate between the redox potential of said mediator and said non-mediator, said mediator being capable of transferring charge between said second enzyme and said electrode, said electrical potential is such that said mediator is converted from said reduced form to said oxidized form at said electrode and said non-mediator is not oxidized or reduced at said electrode;
(f) measuring the current generated at said electrode, said current providing a measure of the conversion of said non-mediator to said mediator; and
(g) correlating the current generated at said electrode with the presence of said first enzyme.
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Abstract
Electrochemical detection of mediator level is employed in a method of assay using a redox enzyme and redox substrate to detect conversion to an effective mediator by an assay enzyme label of a compound which is non-mediating under the assay conditions.
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Citations
17 Claims
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1. A method of assaying for activity of a first enzyme able to convert a non-mediator to a mediator, said mediator having a reduced form and an oxidized form, said method comprising:
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(a) providing a sample to be assayed for said activity of said first enzyme; (b) contacting said sample with freely circulating non-mediator; (c) providing a second enzyme, said second enzyme being catalytically active in the absence of said mediator; (d) providing a substrate for said second enzyme; (e) providing an electrode at an electrical potential which is intermediate between the redox potential of said mediator and said non-mediator, said mediator being capable of transferring charge between said second enzyme and said electrode, said electrical potential is such that said mediator is converted from said reduced form to said oxidized form at said electrode and said non-mediator is not oxidized or reduced at said electrode; (f) measuring the current generated at said electrode, said current providing a measure of the conversion of said non-mediator to said mediator; and (g) correlating the current generated at said electrode with the presence of said first enzyme. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A system for detecting activity of a first enzyme in a sample, said first enzyme converting a non-mediator to a mediator, said mediator having a reduced form and an oxidized form, said system comprising:
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(a) said non-mediator which is capable of conversion by said first enzyme to said mediator; (b) a second enzyme which converts said mediator from said oxidized form to said reduced form; (c) a substrate for said second enzyme; and (d) an electrode and means for poising said electrode at an electrical potential which is intermediate between the redox potential of said mediator and said non-mediator, wherein said electrical potential is such that said mediator is converted from said reduced form to said oxidized form at said electrode and said non-mediator is not oxidized or reduced at said electrode. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17)
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Specification