Electro chemical biosensor containing diazacyanine mediator for co-enzyme regeneration

Claim 2

An electrode suitable for the electrochemical regeneration of the co-enzymes dihydronicotinamide adenine dinucleotide (NADH), dihydronicotinamide adenine dinucleotide phosphate (NADPH) or analogs thereof, said electrode having on its surface a mediator of Formula I: ##STR25## in which: A represents the remaining members of an aromatic or quasiaromatic 5 or 6 membered heterocyclic ring which can be benzanellated; R 1 is alkyl, alkenyl, alkinyl, cycloalkyl or aralkyl; Z is the residue of a moiety of one of the formulae II, III or IV ##STR26## wherein: R 2 , R 3 , R 5 , R 6 and R 8 are independently hydrogen, alkyl, alkenyl, cycloalkyl, aralkyl, aryl or a saturated heterocyclic group; NR 2 R 3 is pyrrolidino, piperidino, morpholino, piperazino or N-alkylpiperazine; R 4 and R 7 are independently hydrogen, alkyl, alkoxy, halogen, hydroxy, nitro, cyano, alkanoylamino or alkylsulfonylamino; R 3 and R 4 together are a --CH 2 CH 2 -- or --CH 2 CH 2 CH 2 -- bridge which is substituted with alkyl; R 9 and R 10 are independently hydrogen, alkyl, alkoxy, halogen, hydroxy, nitro, cyano, alkanoyl or alkylsulfonyl; m, n and p are independently 0, 1 or 2; and X - is an anion.

Claim 3

The electrode of claim 1 in which ##STR27## is thiazole, benzothiazole, thiadiazole, pyrazole, indazole, imidazole, benzimidazole, triazole, pyridine, quinoline or pyrimidine.

Claim 4

The electrode of claim 1 wherein the mediator is a cationic dyestuff of Formula I in which the heterocycle: ##STR28## R 1 is C 1 to C 8 alkyl, C 3 to C 8 alkenyl, C 3 to C 8 alkinyl, C 4 to C 7 cycloalkyl or C 7 to C 9 aralkyl which are unsubstituted or substituted with fluorine, chlorine, bromine, hydroxy, C 1 to C 4 alkyl, C 1 to C 4 alkoxy, cyano or C 1 to C 4 alkoxycarbonyl; R 2 , R 3 , R 5 , R 6 , R 8 , R 14 and R 15 are independently hydrogen, C 1 to C 8 alkyl, C 3 to C 8 alkenyl, C 4 to C 7 cycloalkyl, C 7 to C 9 aralkyl or C 6 to C 10 aryl which are unsubstituted or substituted with halogen, hydroxy, C 1 to C 4 alkyl, C 1 to C 4 alkoxy, cyano, C 1 to C 4 alkoxycarbonyl, C 1 to C 4 alkanoylamino, C 1 to C 4 alkylsulfonyl or tetramethylsulfonyl; NR 2 R 3 and NR 14 R 15 are independently pyrrolidino, piperidino or morpholino; R 4 and R 7 are independently C 1 to C 8 alkyl; C 1 to C 8 alkoxy, hydroxy, fluoro, chloro, bromo, nitro, cyano, C 1 to C 8 alkanoylamino or C 1 to C 8 alkylsulfonylamino; R 3 and R 4 together are a --CH 2 CH 2 -- or --CH 2 CH 2 CH 2 -- bridge which is either unsubstituted or substituted with up to 3 methyl groups; R 9 and R 10 are independently hydrogen, C 1 to C 8 alkyl, C 1 to C 8 alkoxy, hydroxy, fluoro, chloro, bromo, nitro, cyano, C 1 to C 8 alkanoyl or C 1 to C 8 alkylsulfonyl; m, n and p are independently 0, 1 or 2; R 11 and R 12 are independently hydrogen, C 1 to C 8 alkyl, C 1 to C 8 alkoxy, C 4 to C 7 cycloalkyl, C 7 to C 9 aralkyl, C 6 to C 10 aryl, fluoro, chloro, bromo or cyano; R 13 is hydrogen, C 1 to C 8 alkyl, C 1 to C 8 alkoxy, fluoro, chloro, bromo or cyano; and X - is an anion wherein all of the alkyl, alkenyl, alkoxy and aralkyl groups are either straight chain or branched chain.

Claim 5

The electrode of claim 1 in which the mediator has the formula: ##STR29## in which R 1 means C 1 - to C 4 -alkyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is branched or unbranched; R 2 , R 3 , R 14 and R 15 independently of one another mean C 1 - to C 4 -alkyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is branched or unbranched, cyclopentyl, cyclohexyl, tetramethylensulfon-3-yl or phenyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methyl, ethyl, methoxy, ethoxy, cyano, nitro, acetyl, methylsulfonyl or acetylamino, and R 2 and R 14 independently of one another may mean in addition hydrogen or NR 2 R 3 and NR 14 R 15 independently of one another mean pyrrolidino, piperidino or morpholino, R 4a means hydrogen, fluoro, chloro, methyl or methoxy, R 4b means hydrogen, fluoro, chloro, methyl, methoxy, cyano, nitro, hydroxy, acetylamino, propionylamino or methylsulfonylamino and X - means an anion.

Claim 6

The electrode of claim 4 wherein: R 1 means methyl, ethyl, hydroxyethyl, methoxyethyl, cyanoethyl or benzyl, R 2 , R 3 , R 14 and R 15 independently of one another mean methyl, ethyl, propyl, hydroxyethyl, hydroxypropyl, cyanoethyl, benzyl, cyclohexyl, phenyl, methylphenyl, methoxyphenyl, cyanophenyl or tetramethylensulfon-3-yl and R 14 additionally means hydrogen or NR 2 R 3 and NR 14 R 15 independently of one another mean pyrrolidino, piperidino or morpholino, R 4a means hydrogen, methyl or methoxy, R 4b means hydrogen, chloro, methyl, methoxy, cyano, acetylamino or methylsulfonylamino and X - means an anion.

Claim 7

The electrode of claim 1 wherein the mediator has the formula: ##STR30## wherein: R 1 means C 1 - to C 4 -alkyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is optionally branched or unbranched, R 2 and R 3 independently of one another mean C 1 - to C 4 -alkyl, which is optionally unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is branched or unbranched, cyclopentyl, cyclohexyl, tetramethylensulfon-3-yl or phenyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methyl, ethyl, methoxy, ethoxy, cyano, nitro, acetyl, methylsulfonyl or acetylamino, and R 2 may mean in addition hydrogen or NR 2 R 3 means pyrrolidino, piperidino or morpholino, R 4a means hydrogen, fluorine, chlorine, methyl or methoxy, R 4b means hydrogen, fluorine, chlorine, methyl, methoxy, cyano, nitro, hydroxy, acetylamino, propionylamino or methylsulfonylamino, R 13 means hydrogen, fluorine, chlorine, methyl, methoxy or ethoxy and X - means an anion.

Claim 8

The electrode of claim 6 wherein: R 1 means methyl, ethyl, hydroxyethyl, methoxyethyl, cyanoethyl or benzyl, R 2 and R 3 independently of one another mean methyl, ethyl, propyl, hydroxyethyl, hydroxypropyl, cyanoethyl, benzyl, cyclohexyl, phenyl, methylphenyl, methoxyphenyl, cyanophenyl or tetramethylensulfon-3-yl or NR 2 R 3 means pyrrolidino, piperidino or morpholino, R 4a means hydrogen, methyl or methoxy, R 4b means hydrogen, chlorine, methyl, methoxy, cyano, acetylamino or methylsulfonylamino, R 13 means hydrogen or methoxy and X - means an anion.

Claim 9

The electrode of claim 1 wherein the mediator has the formula: ##STR31## in which R 1 means C 1 -C 4 -alkyl, which is unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is optionally branched or unbranched, R 8 means C 1 -C 4 -alkyl, which is unsubstituted or substituted by fluorine, chlorine, hyddroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is optionally branched or unbranched, cyclopentyl, cyclohexyl or phenyl, R 9 and R 10 independently mean hydrogen, fluorine, chlorine, methyl, methoxy, cyano, nitro, acetyl or methylsulfonyl, R 14 and R 15 independently mean C 1 -C 4 -alkyl, which is optionally unsubstituted or substituted by fluorine, chlorine, hydroxy, methoxy, ethoxy, cyano, methoxycarbonyl or phenyl and which is optionally branched or unbranched, cyclopentyl, cyclohexyl, tetramethylensulfon-3-yl or phenyl, which is optionally unsubstituted or substituted by fluorine, chlorine, hydroxy, methyl, ethyl, methoxy, ethoxy, cyano, nitro, acetyl, methylsulfonyl or acetylamino, R 14 may mean in addition hydrogen, NR 14 R 15 means pyrrolidino, piperidino or morpholino and X - means an anion.

Claim 10

The electrode of claim 8 wherein: R 1 means methyl, ethyl, hydroxyethyl, methoxyethyl, cyanoethyl or benzyl, R 8 means methyl, ethyl, hydroxyethyl, cyanoethyl or benzyl, R 9 means cyano, nitro, acetyl or methylsulfonyl, R 10 means hydrogen, chlorine, methyl or cyano, R 14 and R 15 independently of one another mean methyl, ethyl, propyl, hydroxyethyl, hydroxypropyl, cyanoethyl, benzyl, cyclohexyl, phenyl, methylphenyl, methoxyphenyl, cyanophenyl or tetramethylensulfon-3-yl and R 14 additionally means hydrogen or NR 14 R 15 means pyrrolidino, piperidino or morpholino and X - means an anion.

Claim 11

A method of modifying the performance of an electrochemical biosensor having a working electrode and a reference electrode which operates with a dehydrogenase catalyst and a co-enzyme as an energy transferring redox couple, which method comprises using the electrode described in claim 1 as the working electrode of the biosensor.