US5508171A — Assay method with enzyme electrode system

Claim 1

Independent Claim 254 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of working and counter electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials, for determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers substantially equal surface areas of working and counter electrodes in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid, wherein the reagent includes the oxidized form of a redox mediator and an enzyme, the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the oxidized form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the oxidized form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 2

Independent Claim 254 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of working and counter electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials, for determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers substantially equal surface areas of working and counter electrodes in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid, wherein the reagent includes the oxidized form of a redox mediator and an enzyme, the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the oxidized form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the oxidized form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 3

Dependent Claim 59 words

The method of claim 1, wherein the reagent further includes a buffer having a higher oxidation potential than the reduced form of the redox mediator, the buffer being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the oxidized form of the redox mediator.

Claim 4

Dependent Claim 20 words

The method of claim 2, wherein the reagent further includes at least one additional enzyme and an additional redox mediator.

Claim 5

Dependent Claim 28 words

The method of claim 2, wherein the reagent further includes a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.

Claim 6

Dependent Claim 43 words

The method of claim 4, wherein the reagent further includes: a surfactant of sufficient type and in sufficient amount to aid wetting of the reagent by the fluid, and a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

Claim 7

Dependent Claim 55 words

The method of claim 5, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide, the buffer is phosphate, the microcrystalline material includes microcrystalline cellulose and microcrystalline hydroxyethylcellulose, the enzyme is glucose oxidase, the surfactant is a nonionic surfactant, and the reagent stabilizer is glutamate, succinate, aspartate, blue dextran, or trehalose.

Claim 8

Dependent Claim 99 words

The method of claim 6, wherein the reagent includes: from about 1.1 to about 1.5 millimoles ferricyanide per gram of reagent, from about 1.3 to about 1.9 millimoles phosphate buffer per gram of reagent, from about 2,300 to about 10,400 units of glucose oxidase per gram of reagent, from about 50 to 71 milligrams microcrystalline cellulose per gram of reagent, from about 2 to about 3 milligrams microcrystalline hydroxyethylcellulose per gram of reagent, from about 2 to about 3 milligrams TRITON X-100 per gram of reagent, and from about 71 to about 102 milligrams glutamate per gram of reagent.

Claim 9

Dependent Claim 72 words

The method of claim 6, wherein the reagent includes, before drying, about 300 millimolar potassium ferricyanide, about 250 millimolar phosphate buffer, about 14 grams microcrystalline cellulose per liter of reagent, about 0-6 grams hydroxyethylcellulose per liter reagent, about 0.5 grams Triton X-100 surfactant per liter of reagent, about 37 millimolar succinate, and about 1.6 million tetramethyl benzidine units of glucose oxidase per liter of reagent, wherein the reagent pH is about 6.6.

Claim 10

Independent Claim 253 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of working and counter electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials, for determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers substantially equal surface areas of working and counter electrodes in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid, wherein the reagent includes the reduced form of redox mediator and an enzyme, the reduced form of the redox mediator being of sufficient type to donate at least one electron from a reaction involving enzyme, analyte, and reduced form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electroreduction is limited by the reduction of the oxidized form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the reduced form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the reduced form of a redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 11

Independent Claim 253 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of working and counter electrodes of substantially the same size, the electrodes being made of the same electrically conducting materials, for determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers substantially equal surface areas of working and counter electrodes in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid, wherein the reagent includes the reduced form of redox mediator and an enzyme, the reduced form of the redox mediator being of sufficient type to donate at least one electron from a reaction involving enzyme, analyte, and reduced form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electroreduction is limited by the reduction of the oxidized form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the reduced form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the reduced form of a redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 12

Dependent Claim 58 words

The method of claim 9, wherein the reagent further includes a buffer having lower reduction potential than the oxidized form of the redox mediator, the buffer being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the reduced form of the redox mediator.

Claim 13

Dependent Claim 59 words

The method of claim 10, wherein the reagent further includes: a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent, a surfactant of sufficient type and in sufficient amount to wet a sample containing the analyte, and a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

Claim 14

Independent Claim 262 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of a working electrode and a counter electrode that is smaller than the work,ha electrode, the electrodes being made of the same electrically conducting materials, of determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers a smaller surface area of the counter electrode than the working electrode in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid wherein the reagent includes the oxidized form of a redox mediator and an enzyme, the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the oxidized form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the oxidized form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 15

Independent Claim 262 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of a working electrode and a counter electrode that is smaller than the work,ha electrode, the electrodes being made of the same electrically conducting materials, of determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers a smaller surface area of the counter electrode than the working electrode in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent to the fluid wherein the reagent includes the oxidized form of a redox mediator and an enzyme, the oxidized form of the redox mediator being of sufficient type to receive at least one electron from a reaction involving enzyme, analyte, and oxidized form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electrooxidation is limited by the oxidation of the reduced form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the oxidized form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the oxidized form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 16

Dependent Claim 59 words

The method of claim 12, wherein the reagent further includes a buffer having a higher oxidation potential than the reduced form of the redox mediator, the buffer being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the oxidized form of the redox mediator.

Claim 17

Dependent Claim 20 words

The method of claim 13, wherein the reagent further includes at least one additional enzyme and an additional redox mediator.

Claim 18

Dependent Claim 28 words

The method of claim 13, wherein the reagent further includes a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.

Claim 19

Dependent Claim 43 words

The method of claim 15, wherein the reagent further includes: a surfactant of sufficient type and in sufficient amount to aid wetting of the reagent by the fluid, and a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

Claim 20

Dependent Claim 55 words

The method of claim 16, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide, the buffer is phosphate, the microcrystalline material includes microcrystalline cellulose and microcrystalline hydroxyethylcellulose, the enzyme is glucose oxidase, the surfactant is a nonionic surfactant, and the reagent stabilizer is glutamate, succinate, aspartate, blue dextran, or trehalose.

Claim 21

Independent Claim 260 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of a working electrode and a counter electrode that is smaller than the working electrode, the electrodes being made of the same electrically conducting materials of determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers smaller surface area of the counter electrode than the working electrode in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent the fluid, wherein the reagent includes the reduced form of a redox mediator and an enzyme, the reduced form of the redox mediator being of sufficient type to donate at least one electron from a reaction involving enzyme, analyte, and reduced form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electroreduction is limited by the reduction of the oxidized form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the reduced form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the reduced form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 22

Independent Claim 260 words

A method, practiced with an electrochemical cell having a pair of electrodes consisting of a working electrode and a counter electrode that is smaller than the working electrode, the electrodes being made of the same electrically conducting materials of determining the concentration of an analyte in a fluid, comprising: a. contacting the fluid with a reagent that covers smaller surface area of the counter electrode than the working electrode in a sample receiving portion of the electrochemical cell, thereby exposing the entire reagent the fluid, wherein the reagent includes the reduced form of a redox mediator and an enzyme, the reduced form of the redox mediator being of sufficient type to donate at least one electron from a reaction involving enzyme, analyte, and reduced form of the redox mediator and being in sufficient amount to insure that current produced by diffusion limited electroreduction is limited by the reduction of the oxidized form of the redox mediator at the working electrode surface, and the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and the reduced form of the redox mediator; b. allowing the reaction involving the enzyme, analyte, and the reduced form of the redox mediator to go to completion; c. subsequently applying a potential difference between the electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; d. thereafter measuring the resulting diffusion limited current; and e. correlating the current measurement to the concentration of the analyte in the fluid.

Claim 23

Dependent Claim 58 words

The method of claim 18, wherein the reagent further includes a buffer having lower reduction potential than the oxidized form of the redox mediator, the buffer being of sufficient type and in sufficient amount to provide and maintain a pH at which the enzyme catalyzes the reaction involving enzyme, analyte, and the reduced form of the redox mediator.

Claim 24

Dependent Claim 61 words

The method of claim 19, wherein the reagent further includes: a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent, a surfactant of sufficient type and in sufficient amount to aid wetting of the reagent by the fluid, and a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1999-04-16	1999-10-16	2000-04-16	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2003-04-16	2003-10-16	2004-04-16	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2007-04-16	2007-10-16	2008-04-16	$7700.00	Window Closed / Lapsed

Assay method with enzyme electrode system

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