Method and sensor electrode system for the electrochemical determination of an analyte or an oxidoreductase as well as the use of suitable compounds therefor

Claim 2

A sensor electrode system for an electrochemical determination of the amount of an analyte in a liquid sample, comprising an admixture of an oxidoreductase and a first reducible substance, wherein the reducible substance has the property that the reducible substance is reduced by said oxidoreductase to produce a reduced substance in a non-reversible reaction, and that, in turn, said reduced substance, when oxidized, produces a second reducible substance which is different from the first reducible substance; means for storing the admixture of the oxidoreductase and the first reducible substance; means for contacting the admixture with the liquid sample which may contain the analyte; contacting means for electrically contacting the admixture in contact with the sample with two physically separated electrically conductive leads; said contacting means including an electrically conductive surface means for receiving electrons from the first reducible substance when the first reducible substance is reduced by said oxidoreductase to produce a reduced substance, and for oxidizing the reduced substance at the electrically conductive surface means to a second reducible substance which is different from the first reducible substance.

Claim 3

The sensor electrode system of claim 1, wherein said substance formed at said electrically conductive surface by oxidation is subsequently reduced by said oxidoreductase.

Claim 4

The sensor electrode system of claim 1 wherein said reducible substance is a compound of the formula I X--R (I) in which R is an electron-rich aromatic residue, and X is NO or NHOH.

Claim 5

The sensor electrode system of claim 1 wherein said reducible substance is a compound of the formula II HO--N=Y (II) in which Y is a quinoid system which is, after reduction, electron-rich in the aromatic state.

Claim 6

The sensor electrode system of claim 1 wherein said reducible substance is a nitrosoaniline derivative of the formula III ##STR27## in which R 1 is hydrogen, halogen, alkoxy, or alkylthio, R 2 is alkyl, and R 3 is hydroxyalkyl; or R 2 and R 3 are the same or different, and each may be dialkylaminoalkyl, hydroxyalkoxyalkyl, alkoxyalkyl or polyalkoxyalkyl wherein the alkyl residues may be unsubstituted or substituted with OH; or R 2 and R 3 form an alkylene interrupted by sulphur or nitrogen, in which the nitrogen is substituted with an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, dioxanylyl-alkyl, or polyalkoxyalkyl, and wherein the residue on the nitrogen is unsubstituted or substituted on the alkyl moiety by an OH; or if R 1 in the ortho position to NR 2 R 3 , R 2 and R 1 also may form an alkylene, and R 3 is an hydroxyalkyl, or, if the alkylene residue contains three carbon atoms, R 3 may be an alkyl residue; or R 1 is not hydrogen, R 2 and R 3 are the same or different and each is a hydroxyalkyl; or a salt of this derivative.

Claim 7

The sensor electrode system of claim 1 wherein said reducible substance is selected from the group consisting of N-(2-hydroxyethyl)-N'-p-nitrosophenyl-piperazine, N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline, o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, p-hydroxynitrosobenzene, N-methyl-N'-(4-nitrosophenyl)-piperazine, p-quinone dioxime, N,N-dimethyl-p-nitrosoaniline, N,N-diethyl-p-nitrosoaniline, N-(4-nitrosophenyl)-morpholine, N-benzyl-N-(5'-carboxypentyl)-p-nitrosoaniline, N,N-dimethyl-4-nitroso-1-naphthylamine, N,N,3-trimethyl-4-nitrosoaniline, N-(2-hydroxyethyl)-5-nitrosoindoline, N,N-bis-(2-hydroxyethyl)-3-chloro-4-nitrosoaniline,

Claim 8

4-dimethoxy-nitrosobenzene N,N-bis-(2-methoxyethyl)-4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline, N,N-dimethyl-3-chloro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline, and N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

Claim 9

4-dimethoxy-nitrosobenzene N,N-bis-(2-methoxyethyl)-4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline, N,N-dimethyl-3-chloro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline, and N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

Claim 10

The sensor electrode system of claim 1 wherein said reducible substance is N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline or N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)ethyl)-4-nitrosoaniline.

Claim 11

The sensor electrode system of claim 1 wherein said reducible substance is selected from the group consisting of 2,2'-[(3-fluoro-4-nitrosophenyl)imino]bis-ethanol, 2,2'-[(3-chloro-4-nitrosophenyl)imino]bis-ethanol, 2,2'[(3-methoxy-4-nitrosophenyl)imino]bis-ethanol, 2,2'[(3-methylmercapto-4-nitrosophenyl)imino]bis-ethanol, 2-[(2-hydroxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol, 2-[(2-methoxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol, 1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-methoxy-2-propanol, 1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-(2-hydroxyethoxy-2-propanol, 1-methyl-4-(4-nitrosophenyl)-piperazine, 4-(4-nitrosophenyl)-1-piperazine-ethanol, 5-nitroso-1-indoline ethanol, 1-methyl-6-nitroso-1,2,3,4-tetrahydroquinoline, 6-nitroso-3,4-dihydro-1(2H)quinoline ethanol and their salts.

Claim 12

A sensor electrode system for an electrochemical determination of the amount of an oxidoreductase in a liquid sample, comprising an admixture of a corresponding enzyme substrate and a first reducible substance, wherein the reducible substance has the property that the reducible substance is reduced by said oxidoreductase to produce a reduced substance in a non-reversible reaction, and that, in turn, said reduced substance, when oxidized, produces a second reducible substance which is different from the first reducible substance; means for storing the admixture of the enzyme substrate and the first reducible substance; means for contacting the admixture with the liquid sample which may contain the oxidoreductase; contacting means for electrically contacting the admixture in contact with the sample with two physically separated electrically conductive leads; said contacting means including an electrically conductive surface means for receiving electrons from the first reducible substance when the first reducible substance is reduced by said oxidoreductase to produce a reduced substance, and for oxidizing the reduced substance at the electrically conductive surface means to a second reducible substance which is different from the first reducible substance.

Claim 13

A sensor electrode system for an electrochemical determination of the amount of an oxidoreductase in a liquid sample, comprising an admixture of a corresponding enzyme substrate and a first reducible substance, wherein the reducible substance has the property that the reducible substance is reduced by said oxidoreductase to produce a reduced substance in a non-reversible reaction, and that, in turn, said reduced substance, when oxidized, produces a second reducible substance which is different from the first reducible substance; means for storing the admixture of the enzyme substrate and the first reducible substance; means for contacting the admixture with the liquid sample which may contain the oxidoreductase; contacting means for electrically contacting the admixture in contact with the sample with two physically separated electrically conductive leads; said contacting means including an electrically conductive surface means for receiving electrons from the first reducible substance when the first reducible substance is reduced by said oxidoreductase to produce a reduced substance, and for oxidizing the reduced substance at the electrically conductive surface means to a second reducible substance which is different from the first reducible substance.

Claim 14

The sensor electrode system of claim 9 wherein said reducible substance is a compound of the formula I X--R (I) in which R is an electron-rich aromatic residue, and X is NO or NHOH.

Claim 15

The sensor electrode system of claim 9 wherein said reducible substance is a compound of the formula II HO--N═Y (II) in which Y is a quinoid system which can, for reduction, be denoted electron-rich in the aromatic state.

Claim 16

The sensor electrode system of claim 9 wherein said reducible substance is a nitrosoaniline derivative of the formula III ##STR28## in which R 1 hydrogen, halogen, alkoxy, or alkylthio, R 2 is alkyl, and R 3 is hydroxyalkyl; or R 2 and R 3 are the same or different, and each may be dialkylaminoalkyl, hydroxyalkoxyalkyl, alkoxyalkyl or polyalkoxyalkyl wherein the alkyl residues may be unsubstituted or substituted with OH; or R 2 and R 3 form an alkylene interrupted by sulphur or nitrogen, in which the nitrogen is substituted with an alkyl, hydroxyalkyl, hydroxyalkoxyalkyl, alkoxyhydroxyalkyl, dioxanylyl-alkyl, or polyalkoxyalkyl, and wherein the residue on the nitrogen is unsubstituted or substituted on the alkyl moiety by an OH; or if R 1 is in the ortho position to NR 2 R 3 , R 2 and R 1 form an alkylene, and R 3 is an hydroxyalkyl; or, if the alkylene residue contains three carbon atoms, R 3 may be an alkyl residue; or if R 1 is not hydrogen, R 2 and R 3 are the same or different and each is an hydroxyalkyl; or a salt of this derivative.

Claim 17

The sensor electrode system of claim 9 wherein said reducible substance is selected from the group consisting of N-(2-hydroxyethyl)-N'-p-nitrosophenyl-piperazine, N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline, o-methoxy-[N,N-bis-(2-hydroxyethyl)]-p-nitrosoaniline, p-hydroxynitrosobenzene, N-methyl-N'-(4-nitrosophenyl)-piperazine, p-quinone dioxime, N,N-dimethyl-p-nitrosoaniline, N,N-diethyl-p-nitrosoaniline, N-(4-nitrosophenyl)-morpholine, N-benzyl-N-(5'-carboxypentyl)-p-nitrosoaniline, N,N-dimethyl-4-nitroso-1-naphthylamine, N,N,3-trimethyl-4-nitrosoaniline, N-(2-hydroxyethyl)-5-nitrosoindoline, N,N-bis-(2-hydroxyethyl)-3-chloro-4-nitrosoaniline,

Claim 18

4-dimethoxy-nitrosobenzene N,N-bis-(2-methoxyethyl)-4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline, N,N-dimethyl-3-chloro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline, N-(2-hydroxyethyl-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline, and N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

Claim 19

4-dimethoxy-nitrosobenzene N,N-bis-(2-methoxyethyl)-4-nitrosoaniline, 3-methoxy-4-nitrosophenol, N-(2-hydroxyethyl)-6-nitroso-1,2,3,4-tetrahydroquinoline, N,N-dimethyl-3-chloro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-fluoro-4-nitrosoaniline, N,N-bis-(2-hydroxyethyl)-3-methylthio-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(2-(2-methoxyethoxy)-ethyl)-4-nitrosoaniline, N-(2-hydroxyethyl)-N-(3-methoxy-2-hydroxy-1-propyl)-4-nitrosoaniline, N-(2-hydroxyethyl-N-(3-(2-hydroxyethoxy)-2-hydroxy-1-propyl)-4-nitrosoaniline, and N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)-ethyl)-4-nitrosoaniline.

Claim 20

The sensor electrode system of claim 9 wherein said reducible substance is N,N-bis-(2-hydroxyethyl)-p-nitrosoaniline or N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)ethyl)-4-nitrosoaniline.

Claim 21

The sensor electrode system of claim 9 wherein said reducible substance is selected from the group consisting of 2,2'-[(3-fluoro-4-nitrosophenyl)imino]bis-ethanol, 2,2'-[(3-chloro-4-nitrosophenyl)imino]bis-ethanol, 2,2'[(3-methoxy-4-nitrosophenyl)imino]-bis-ethanol, 2,2'[(3-methylmercapto-4-nitrosophenyl)imino]bis-ethanol, 2-[(2-hydroxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol, 2-[(2-methoxyethoxy)ethyl-(4-nitrosophenyl)amino]ethanol, 1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-methoxy-2-propanol, 1-[N-(2-hydroxyethyl)-(4-nitrosoanilino)]-3-(2-hydroxyethoxy-2-propanol, 1-methyl-4-(4-nitrosophenyl)-piperazine, 4-(4-nitrosophenyl)-1-piperazine-ethanol, 5-nitroso-1-indoline ethanol, 1-methyl-6-nitroso-1,2,3,4-tetrahydroquinoline, 6-nitroso-3,4-dihydro-1(2H)quinoline ethanol and their salts.