EP0330517B2
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EP
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Method, system and devices for the assay and detection of biochemical molecules
Assignee: SolarCare Technologies Corp
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Filing: 1989-02-27
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Granted: 1997-02-05
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Expires: 2009-02-27
Abstract
Abstract Oxygen-independent methods, systems and devices for the enzymatic colorimetric assay and detection of biochemical analytes. Two systems are described, both of which produce less than one equivalent of dye per equivalent of substrate, maintaining dye concentrations in the range where Beer's law predicts a linear color-concentration relationship. One system produces an analog color signal from an analog analyte input, the other system produces a digital color signal from an analog analyte input.
Inventors
- 👤 John L. Palmer
- 👤 James B. Johnson
- 👤 Marsha W. Timmerman
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-07 09:00
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/52; C; C12; C12Q; C12Q1/00; C12Q1/26; Y; Y10; Y10S; Y10S435/00; Y10S435/805; Y10S435/81
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
176 words
A colorimetric assay system capable of measuring the presence or concentration of a biochemical analyte having electrons by a colour change which is indicative of the presence or concentration of the analyte, which system comprises, (1) an oxidase enzyme capable of electron transferase activity with the analyte and capable of oxidizing the analyte by removing, in the absence of oxygen, electrons from the analyte, thereby yielding a reduced enzyme, (2) an electron acceptor ("ea") having a half cell reduction potential greater than the reduced enzyme, which ea is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, and (3) a chromogen ("chr") capable of accepting electrons from the reduced enzyme and capable of changing colour in a visible range characterised in that the ea is not oxygen, is non-chromogenic and competes with the chr for electrons from the enzyme, and the colour change of the chromogen is less than one equivalent of colour per equivalent of analyte oxidized and is indicative of the presence or concentration of the analyte to be determined.
Claim 2
Dependent Claim
34 words
The colorimetric assay system of Claim 1 wherein the system also comprises an electron transfer agent ("eta") which promotes the transfer of electrons from the reduced enzyme to the ea, the chr, or both.
Claim 3
Dependent Claim
27 words
The colorimetric assay system of Claim 2 in which the eta is selected from the group consisting of flavins, phenazines, phenothiazines, phenoxazines, indophenols, substituted 1,4-benzoquinones and indamins.
Claim 4
Dependent Claim
107 words
The colorimetric assay system of Claim 3 in which the eta is a flavin selected from the group consisting of riboflavin, alloxazine and lumichrome, or a phenothiazine selected from the group consisting of PT+, TH, AA, AB, AC, MB, MG and TOL, or a phenazine selected from the group consisting of phenazine, phenazine methosulfate, phenazine ethosulfate, and safranine, or a phenoxazine selected from the group consisting of POA, BB3 and BCBA, or a substituted 1,4-benzoquinone selected from the group consisting of TFBQ, MMBQ, HMBQ, HMMBQ, DMBQ, DMMBQ and CCBQ, or an indamin selected from the group consisting of Bindschedler's green and phenylene blue, or the indophenol DCIP.
Claim 5
Dependent Claim
35 words
The colorimetric assay system of Claim 2 wherein the eta is phenothiazine, the chr is INT and ea is ferric chelate, or the eta is PMS, the chr is MTT and the ea is ferricyanide.
Claim 6
Dependent Claim
54 words
The colorimetric assay system of any one of Claims 2 to 5 in which the electron transferase enzyme is a copper-containing oxidoreductase or is an enzyme which contains a flavin-bound co-factor, a pteridine co-factor, a heme prosthetic group, a reversibly oxidisable dithiol group at the active site or non-heme iron in its reactive centre.
Claim 7
Dependent Claim
34 words
The colorimetric assay system of any one of Claims 2 to 6 which is an a/a assay system and wherein the colour change is directly proportional to the concentration of analyte to be measured.
Claim 8
Dependent Claim
32 words
The colorimetric assay system of Claim 2 or 7 wherein the ea is selected from the group consisting of TAR, TFBQ, 2,5-dihydroxy-1,4-benzoquinone and 2-cyclohexyl-5-hydroxy-1,4 -benzoquinone and the chr is a tetrazolium salt.
Claim 9
Dependent Claim
63 words
The colorimetric assay system of Claim 2 wherein the analyte is selected from the group consisting of ethanol, methanol, glucose, lactose, galactose, lactate, pyruvate, oxalate, ketones, albumin, triglycerides, cholesterol, bilirubin, hemoglobin, choline, oxalic acid, glycerol, sorbitol, lactic acid, pyruvic acid, ascorbic acid, uric acid, or a compound which reacts to generate said analyte in situ, and the enzyme is the corresponding electron transferase.
Claim 10
Dependent Claim
69 words
The colorimetric assay system of Claim 1 wherein the analyte is oxidized without a visible colour change until the ea is exhausted, at which point a visible change occurs as long as additional non-oxidized analyte is present, or no colour develops, which is indicative of the analyte being present at a concentration below the threshold concentration for colour change to be determined, which system is an a/d assay system.
Claim 11
Dependent Claim
234 words
A colorimetric assay system which is designed to measure the presence or concentration of enzymes having electron transferase activity by a colour change which is indicative of the presence or concentration of the electron transferase enzyme, which system comprises, 1) a substrate with which the electron transferase enzyme is capable of electron transferase activity and capable of oxidizing the substrate by removing, in the absence of oxygen, electrons from the substrate, thereby yielding a reduced enzyme, 2) a chromogen ("chr") which is capable of accepting electrons from the reduced enzyme and capable of changing colour in the visible range of the spectrum upon reduction, the colour change of the reduced chromogen being detectable and indicative of the presence or the concentration of the electron transferase enzyme to be determined, 3) an electron acceptor ("ea"), which is not oxygen, having a half-cell reduction potential greater than that of the reduced enzyme, which ea is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, and any colour change upon reduction of the ea not being indicative of the presence or the concentration of the electron transferase enzyme, and characterised in that the ea competes with the chr for electrons from the reduced enzyme and in that the enzyme is an oxidase, and in that the colour change of the chromogen is less than one equivalent of colour per equivalent of enzyme reduced.
Claim 12
Dependent Claim
33 words
The colorimetric assay system of Claim 11 wherein the system also comprises an electron transfer agent ("eta") which promotes the transfer of electrons from the reduced enzyme to the ea, chr, or both.
Claim 13
Dependent Claim
216 words
A diagnostic colorimetric device designed to measure the concentration of a biochemical analyte by developing a color signal when the analyte is at or above a threshold concentration and by the absence of a color signal when the concentration of the analyte is below the threshold, which device comprises, 1) an enzyme capable of electron transferase activity with the analyte and capable of oxidizing the analyte by removing, in the absence of oxygen, electrons from the analyte, thereby yielding a reduced enzyme, 2) a chromogen ("chr") which is capable of accepting electrons from the reduced enzyme and capable of changing colour in the visible range of the spectrum upon reduction, the colour change of the reduced chromogen being detectable and indicative of the presence or the concentration of the analyte to be determined, and 3) an electron acceptor ("ea"), which is not oxygen, having a half-cell reduction potential greater than the reduced enzyme, which ea is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner, with any colour change upon reduction not being indicative of the presence or the concentration of the analyte, characterized in that the enzyme is an oxidase and in that the colour change of the chromogen is less than one equivalent of colour per equivalent of analyte oxidized.
Claim 14
Independent Claim
248 words
A method for measuring the presence or the concentration of a biochemical analyte by the development of a colour change, which method comprises adding to the analyte the concentration or presence of which is to be measured a mixture which comprises, 1) an enzyme capable of electron transferase activity with the analyte and capable of oxidizing the analyte by removing, in the absence of oxygen, electrons from the analyte, thereby yielding a reduced enzyme, 2) a chromogen ("chr") which is capable of accepting electrons from the reduced enzyme and capable of changing colour in a visible range of the spectrum upon reduction, the colour change of the reduced chromogen being detectable and indicative of the presence or concentration of the analyte to be determined, and 3) an electron acceptor ("ea"), which is not oxygen, having a half-cell reduction potential greater than that of the reduced enzyme, which ea is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner with any colour change upon reduction not being indicative of the presence or the concentration of the analyte, noting the colour change which has or has not developed, the colour change being indicative of the presence, the absence or the concentration of the analyte to be determined, characterised in that the ea competes with the chr for electrons from the reduced enzymes, in that the enzyme is an oxidase, and in that the colour change of the chromogen is less than one equivalent of analyte oxidized.
Claim 15
Independent Claim
314 words
A method for the colorimetric assay of the presence or concentration of any enzyme having electron transferase activity by the development of a colour change which is indicative of the quantity of substrate consumed by the enzyme in a given time period, which method comprises adding to the enzyme the concentration or presence of which is to be determined a mixture which comprises, 1) a substrate with which the electron transferase enzyme is capable of electron transferase activity and capable of oxidizing the substrate by removing, in the absence of oxygen, electrons from the substrate, thereby yielding a reduced enzyme, 2) a chromogen ("chr") which is capable of accepting electrons from the reduced enzyme and capable of changing colour in the visible range of the spectrum upon reduction, the colour change of the reduced chromogen being detectable and indicative of the presence or the concentration of the electron transferase enzyme to be determined, 3) an electron acceptor ("ea"), which is not oxygen, having a half-cell reduction potential greater than the reduced enzyme, which ea is capable of accepting electrons from the reduced enzyme in a functionally irreversible manner with any colour change upon reduction not being indicative of the presence or the concentration of the electron transferase enzyme, noting immediately upon termination of a predetermined time period the colour change which has or has not occurred, determining by the colour change which has or has not occurred the quantity of substrate reacted during the pre-determined time period, and determining by the quantity of substrate reacted during the pre-determined time period, the presence or the concentration of the enzyme having electron transferase activity, characterized in that the ea competes with the chr for electrons from the reduced enzyme, and in that the enzyme is an oxidase, and in that the colour change of the chromogen is less than one equivalent per equivalent of enzyme reduced.
References Cited (Backward Citations)
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