EP0125136A2
Expired / Lapsed
EP
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Assay systems utilising more than one enzyme
Assignee: Medisense Inc
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Filing: 1984-05-08
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Granted: —
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Expires: 2004-05-08
Abstract
Abstract This specification discloses assay systems which detect the presence of, or measure or monitor the extent of, an enzyme-catalysed reaction on a substrate. The systems of the present invention can be used to detect or measure the enzyme or the substrate, depending upon circumstances. The specification further discloses a method of assay of the type in which an electrode (1) poised at a suitable potential is contacted with a system comprising a first enzyme, (GO) a substrate (G) which undergoes a reaction catalysed by the said enzyme, and a mediator compound (F) which transfers charge to the electrode (1) from the first enzyme (GO) when it is catalytically active, whereby the current flowing in the electrode is a measure of the reaction taking place; in which at least one further enzyme (HK) and associated compound (ATP) is incorporated into the system, the further enzyme (HK) being productive of, or also being reactive with, the substrate (G) so as to affect its presence or level, but not being electrochemically linked by the mediator (F) to the electrode (1), whereby the consequent difference in electrode current flowing with, and in the absence of, the second enzyme (HK) and its associated compound is a measure of the extent of reaction of the further enzyme with its associated compound and thus permits the amount of one to be established if the amount of the other is known. There are two main subdivisions of the present disclosure (i) in which the electrode is provided at its surface with the first enzyme and the mediator compound to constitute a sensor electrode and (ii) in which the further enzyme exerts a catalytic change on a specific further substrate to yield substrateforthe first enzyme.
Inventors
- 👤 Graham Davis
- 👤 Hugh Allen Oliver Hill
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 00:31
CPC Classifications
A; A61; A61B; A61B5/00; A61B5/145; A61B5/1468; A61B5/1486; A61B5/68; A61B5/6846; A61B5/6847; A61B5/6848; C; C12; C12Q; C12Q1/00; C12Q1/001; C12Q1/004; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6825
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
172 words
A method of assay of the type in which an electrode poised at a suitable potential is contacted with a system comprising a first enzyme, a substrate which undergoes a reaction catalysed by the said enzyme, and a mediator compound which transfers charge to the electrode from the first enzyme when it is catalytically active, whereby the current flowing in the electrode is a measur, of the reaction taking place: in which at least one further enzyme and associated compound is incorporated into the system, the further enzyme being productive of, or also being reactive with, the substrate so as to affect its presence or level, but not being electrochemically linked by the mediator to the electrode, whereby the consequent difference in electrode current flowing with, and in the absence of, the second enzyme and its associated compound is a measure of the extent of reaction of the further enzyme with its associated compound and thus permits the amount of one to be established if the amount of the other is known.
Claim 2
Independent Claim
172 words
A method of assay of the type in which an electrode poised at a suitable potential is contacted with a system comprising a first enzyme, a substrate which undergoes a reaction catalysed by the said enzyme, and a mediator compound which transfers charge to the electrode from the first enzyme when it is catalytically active, whereby the current flowing in the electrode is a measur, of the reaction taking place: in which at least one further enzyme and associated compound is incorporated into the system, the further enzyme being productive of, or also being reactive with, the substrate so as to affect its presence or level, but not being electrochemically linked by the mediator to the electrode, whereby the consequent difference in electrode current flowing with, and in the absence of, the second enzyme and its associated compound is a measure of the extent of reaction of the further enzyme with its associated compound and thus permits the amount of one to be established if the amount of the other is known.
Claim 3
Dependent Claim
29 words
A method as claimed in claim 1 in which the electrode is provided at its surface with the first enzyme and the mediator compound to constitute a sensor electrode.
Claim 4
Dependent Claim
31 words
A method as claimed in claim 1 in which the further enzyme exerts a competitive reaction on the substrate and thus leads to a decrease in current flowing in the electrode.
Claim 5
Dependent Claim
99 words
A method as claimed in claim 3 in which (a) a sensor electrode provided at its surface with the first enzyme and with the mediator compound is contacted with a substrate to give a steady current reading (b) the second enzyme and associated compound one of which is in unknown quantity are added to set up a competitive reaction and hence decrease the electrode current and (c) the rate or extent of decrease, or the extent of substrate addition necessary to compensate for the decrease in electrode current, is noted as a measure of the mount of unknown component.
Claim 6
Dependent Claim
26 words
A method as claimed in claim 3 or 4 in which the further enzyme is a kinase and the associated compound is a high energy phosphate.
Claim 7
Dependent Claim
23 words
A method as claimed in claim 3 or 4, in which the further enzyme is a hexokinase and the associated compound is ATP.
Claim 8
Independent Claim
134 words
A method of assay for the unknown one of the pair of compounds hexokinase and ATP, used in a combination where the amount of one is known, which comprises: (a) contacting with a solution of glucose an electrode having at its surface a glucose oxidoreductase and a mediator compound to transfer charge from the said enzyme to the electrode when the enzyme is catalytically active, thereby to set up a steady electrode current based on the glucose level, (b) adding to the solution the hexokinase and ATP so as to set up with the glucose a competitive phosphorylation reaction to which the electrode is insensitive, whereby the steady current is correspondingly reduced; (c) deriving a value for the unknown level from the rate of, extent of, or glucose compensation for, the reduction in current.
Claim 9
Independent Claim
134 words
A method of assay for the unknown one of the pair of compounds hexokinase and ATP, used in a combination where the amount of one is known, which comprises: (a) contacting with a solution of glucose an electrode having at its surface a glucose oxidoreductase and a mediator compound to transfer charge from the said enzyme to the electrode when the enzyme is catalytically active, thereby to set up a steady electrode current based on the glucose level, (b) adding to the solution the hexokinase and ATP so as to set up with the glucose a competitive phosphorylation reaction to which the electrode is insensitive, whereby the steady current is correspondingly reduced; (c) deriving a value for the unknown level from the rate of, extent of, or glucose compensation for, the reduction in current.
Claim 10
Independent Claim
157 words
A method of assay for creatine kinase, which comprises: (a) contacting with a mixed solution of hexokinase, adenosine diphosphate, creative phosphate and glucose an electrode having at its surface a glucose oxidoreductase enzyme and a mediator compound to transfer charge from the enzyme to the electrode when the enzyme is catalytically active, thereby to set up a steady electrode current based on the glucose level, (b) adding to the solution creatine kinase to be assayed, under conditions in which adenosine diphosphate (ADP) is converted to adenosine triphosphate (A T P) by reaction of the creatine phosphate and so that consequentially the glucose is reacted, in competition with the oxidoreductase enzyme reaction, to glucose-6-phosphate by the hexokinase and ATP phsophorylation to which the electrode is insensitive, whereby the steady current is correspondingly reduced, and (c) deriving a value for the unknown creatine kinase level from the rate of, extent of, or glucose compensation for, the reduction in current.
Claim 11
Independent Claim
157 words
A method of assay for creatine kinase, which comprises: (a) contacting with a mixed solution of hexokinase, adenosine diphosphate, creative phosphate and glucose an electrode having at its surface a glucose oxidoreductase enzyme and a mediator compound to transfer charge from the enzyme to the electrode when the enzyme is catalytically active, thereby to set up a steady electrode current based on the glucose level, (b) adding to the solution creatine kinase to be assayed, under conditions in which adenosine diphosphate (ADP) is converted to adenosine triphosphate (A T P) by reaction of the creatine phosphate and so that consequentially the glucose is reacted, in competition with the oxidoreductase enzyme reaction, to glucose-6-phosphate by the hexokinase and ATP phsophorylation to which the electrode is insensitive, whereby the steady current is correspondingly reduced, and (c) deriving a value for the unknown creatine kinase level from the rate of, extent of, or glucose compensation for, the reduction in current.
Claim 12
Independent Claim
151 words
A method of assay for creatine which comprises : (a) contacting with a mixed solution of hexokinase, adenosine triphosphate (ATP), creatine kinase and glucose an electrode having at its surface a glucose oxidoreductase enzyme and a mediator compound to transfer charge from the enzyme to be electrode when the enzyme is catalytically active, thereby to set up a steady electrode current depending on the available glucose level remaining after the competitive ATP/hexokinase phosphorylation reaction to glucose-6-phosphate to which the electrode is insensitive, whereby the steady current is correspondingly reduced; (b) adding to the solution creatine to be assayed under conditions in which ATP is converted to ADP, with the formation of creatine phosphate by the creatine kinase, and thereby decreases the amount of ATP available for the competitive glucose phosphorylation reaction. (c) deriving a value for the unknown creatine level from the rate of or extent of alteration in electrode current.
Claim 13
Independent Claim
151 words
A method of assay for creatine which comprises : (a) contacting with a mixed solution of hexokinase, adenosine triphosphate (ATP), creatine kinase and glucose an electrode having at its surface a glucose oxidoreductase enzyme and a mediator compound to transfer charge from the enzyme to be electrode when the enzyme is catalytically active, thereby to set up a steady electrode current depending on the available glucose level remaining after the competitive ATP/hexokinase phosphorylation reaction to glucose-6-phosphate to which the electrode is insensitive, whereby the steady current is correspondingly reduced; (b) adding to the solution creatine to be assayed under conditions in which ATP is converted to ADP, with the formation of creatine phosphate by the creatine kinase, and thereby decreases the amount of ATP available for the competitive glucose phosphorylation reaction. (c) deriving a value for the unknown creatine level from the rate of or extent of alteration in electrode current.
Claim 14
Dependent Claim
28 words
A method as claimed in claim 1 in which the further enzyme exerts a catalytic change on a specific further substrate to yield substrate for the first enzyme.
Claim 15
Dependent Claim
35 words
A method as claimed in claim 10 in which the electrode is provided with the first enzyme, the mediator compound therefor, and the further enzyme to constitute a sensor electrode for the said further substrate.
Claim 16
Dependent Claim
47 words
A method as claimed in claim 11 in which the electrode is coated with sarcosine oxidase or dehydrogenase, creatinase, and a mediator to transfer a charge from the sarcosine to the electrode, and is contacted with a solution containing creatine for assay thereof by conversion to sarcosine.
Claim 17
Dependent Claim
47 words
A method as claimed in claim 11 in which the electrode is coated with sarcosine oxidase or dehydrogenase, creatinase, creatininase, and a mediator to transfer charge from the sarcosine to the electrode, and is contacted with creatinine for assay thereof by serial conversion to creatine and sarcosine.
Claim 18
Dependent Claim
23 words
A method as claimed in claim 1, 2, 3 ,4, 7, 8 or 9 in which the first enzyme is a glucose oxidase.
Claim 19
Dependent Claim
23 words
A method as claimed in claim 1, 2, 3, 4, 7, 8 or 9 in which the first enzyme is a glucose dehydrogenase.
Claim 20
Dependent Claim
25 words
A method as claimed in claim 1, 2, 3, 4, 7 8, 9, 10, 11, 12 or 13 in which the mediator is a ferrocene.
Claim 21
Dependent Claim
25 words
A method as claimed in claim 1, 2, 3, 4, 7, 8, 9, 199, 11, 12 or 13 in which the mediator is 1,1 -dimethylferrocene.
References Cited (Backward Citations)
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