Enzyme electrode system

• US 5,288,636 A
• Filed: 12/14/1990
• Issued: 02/22/1994
• Est. Priority Date: 12/15/1989
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Original Assignee: Boehringer Mannheim Corporation
• Inventors: Gerber, Martin T., Pollmann, Klaus H.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Beisner, William H.
• Application Number:
  US07/627,667
• Time in Patent Office: 1,166 Days
• Field of Search: 435/4.14, 435/25, 435/288, 435/291, 435/817, 204/153.11, 204/153.17, 204/403, 204/415
• US Class Current: 204/403.14
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

A biosensor that includes working and counter electrodes of substantially the same size and made of the same electrically conducting material. A reagent is contained in a reagent well in the biosensor. The reagent includes a redox mediator, an enzyme, and a buffer, and covers substantially equal surface areas of portions of the working and counter electrodes. When a sample, containing the analyte to be measured, is added to the reagent well, a reaction occurs that oxidizes the analyte and reduces the redox mediator. After this reaction is complete, an electrical potential difference is applied between the electrodes. The amount of oxidized form of the redox mediator at the counter electrode and the applied potential difference must be sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode. After a short time delay, current produced by the electrooxidation of the reduced form of the redox mediator is measured and the observed current is correlated to the amount of the analyte in the sample. In some cases, the analyte sought to be measured may be reduced and the redox mediator may be oxidized. In such cases, current produced by diffusion limited electroreduction of the oxidized form of the redox mediator is measured and correlated to the amount of analyte in the sample.

Claims

1. A device for analyzing an analyte, comprising:

a. a first electrical insulator;

b. 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 and being supported on the first electrical insulator;

c. a second electrical insulator, overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes substantially equal surface areas of the working and counter electrodes; and

d. a reagent, substantially covering the exposed electrode surfaces in the cutout portion and comprising the oxidized form of a redox mediator, an enzyme, and a buffer,

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,

the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and oxidized form of the redox mediator, and

the buffer having a higher oxidation potential than the reduced form of the redox mediator and 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 oxidized form of the redox mediator.

2. The device of claim 1, wherein the reagent further comprises a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.

3. The device of claim 2, wherein the reagent further comprises a surfactant of sufficient type and in sufficient amount to wet a sample containing the analyte.

4. The device of claim 3, wherein the reagent further comprises a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

5. The device of claim 4, 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 selected from a group consisting of glutamate, aspartate, blue dextran, and trehalose.

6. The device of claim 2, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide and the enzyme is glucose oxidase.

7. The device of claim 1, wherein the reagent further comprises at least one additional enzyme and an additional redox mediator.

8. The device of claim 1, wherein the electrically conducting material of the working and counter electrodes is selected from a group consisting of palladium, platinum, gold, silver, titanium, copper, and carbon.

9. The device of claim 1, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide, and the enzyme is glucose oxidase.

10. The device of claim 9, further comprising:

e. a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the oxidation of the reduced form of the redox mediator at the working electrode surface.

11. The device of claim 1, further comprising:

e. a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the oxidation of the reduced form of the redox mediator at the working electrode surface.

12. A device for analyzing glucose, comprising:

a. a first electrical insulator;

b. a pair of electrodes consisting of working and counter electrodes of substantially the same size, the electrodes being made of palladium and being supported on the first electrical insulator;

c. a second electrical insulator overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes substantially equal surface areas of the working and counter electrodes; and

d. a reagent, substantially covering the exposed electrode surfaces in the cutout portion and comprising

1) from about 1.1 to about 1.5 millimoles ferricyanide per gram of reagent,

2) from about 1.3 to about 1.9 millimoles phosphate buffer per gram of reagent,

3) from about 2,300 to about 10,400 units of glucose oxidase per gram of reagent,

4) from about 50 to about 71 milligrams microcrystalline cellulose per gram of reagent,

5) from about 2 to about 3 milligrams microcrystalline hydroxyethylcellulose per gram of reagent,

6) from about 2 to about 3 milligrams TRITON X-100 per gram of reagent, and

7) from about 71 to about 102 milligrams glutamate per gram of reagent.

13. The device of claim 12, wherein the second electrical insulator further includes an additional cutout portion exposing portions of the working and counter electrodes, and the device further comprises:

e. a power source in electrical connection with the working and counter electrodes at the additional cutout portion, the power source being capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the ferricyanide at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the oxidation of the reduced form of the ferricyanide at the working electrode surface.

14. A reagent incorporated into a sample receiving portion of an electrochemical device that measures an analyte and that has 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 and having substantially equal surface areas in the sample receiving portion, comprising:

the oxidized form of a redox mediator, an enzyme, and a buffer,

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,

the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and oxidized form of the redox mediator, and

the buffer having a higher oxidation potential than the reduced form of the redox mediator and 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 oxidized form of the redox mediator.

15. The reagent of claim 14, wherein the reagent further comprises at least one additional enzyme and an additional redox mediator.

16. The reagent of claim 14, further comprising:

a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.

17. The reagent of claim 16, further comprising:

a surfactant of sufficient type and in sufficient amount to wet a sample containing the analyte.

18. The reagent of claim 17, further comprising:

a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

19. The device of claim 18, 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 selected from a group consisting of glutamate, aspartate, blue dextran, and trehalose.

20. A reagent incorporated into a sample receiving portion of an electrochemical device that measures glucose and that has 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 and having substantially equal surface areas in the sample receiving portion, comprising:

a. from about 1.1 to about 1.5 millimoles ferricyanide per gram of reagent;

b. from about 1.3 to about 1.9 millimoles phosphate buffer per gram of reagent;

c. from about 2,300 to about 10,400 units of glucose oxidase per gram of reagent;

d. from about 50 to about 71 milligrams microcrystalline cellulose per gram of reagent;

e. from about 2 to about 3 milligrams microcrystalline hydroxyethylcellulose per gram of reagent;

f. from about 2 to about 3 milligrams TRITON X-100 per gram of reagent; and

g. from about 71 to about 102 milligrams glutamate per gram of reagent.

21. A device for analyzing an analyte, comprising:

a. a first electrical insulator;

b. 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 and being supported on the first electrical insulator;

c. a second electrical insulator, overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes substantially equal surface areas of the working and counter electrodes; and

d. a reagent, substantially covering the exposed electrode surfaces in the cutout portion and comprising the reduced form of a redox mediator, an enzyme, and a buffer,

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,

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, and

the buffer having a lower reduction potential than the oxidized form of the redox mediator and 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.

22. The device of claim 21, wherein the reagent further comprises

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.

23. The device of claim 21, further comprising:

e. a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the reduction of the oxidized form of the redox mediator at the working electrode surface.

24. A reagent incorporated into a sample receiving portion of an electrochemical device that measures an analyte and that has 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 and having substantially equal surface areas in the sample receiving portion, comprising:

the reduced form of a redox mediator, an enzyme, and buffer,

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,

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, and

the buffer having a lower reduction potential than the oxidized form of the redox mediator and 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.

25. The reagent of claim 24, further comprising:

a microcrystalline material of sufficient type and in sufficient amount of 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.

26. A device for analyzing an analyte, comprising:

a. a first electrical insulator;

b. 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 and being supported on the first electrical insulator;

c. a second electrical insulator, overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes a smaller surface area of the counter electrode than the working electrode; and

d. a reagent, substantially covering the exposed electrode surfaces in the cutout portion and comprising the oxidized form of a redox mediator, an enzyme, and a buffer,

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,

the enzyme being of sufficient type and in sufficient amount to catalyze the reaction involving enzyme, analyte, and oxidized form of the redox mediator, and

the buffer having a higher oxidation potential than the reduced form of the redox mediator and 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 oxidized form of the redox mediator.

27. The device of claim 26, wherein the reagent further comprises a microcrystalline material of sufficient type and in sufficient amount to disperse the redox mediator in the reagent.

28. The device of claim 27, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide and the enzyme is glucose oxidase.

29. The device of claim 27, wherein the reagent further comprises a surfactant of sufficient type and in sufficient amount to wet a sample containing the analyte.

30. The reagent of claim 29, wherein the reagent further comprises a reagent stabilizer of sufficient type and in sufficient amount to stabilize the reagent.

31. The device of claim 30, 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 selected from a group consisting of glutamate, aspartate, blue dextran, and trehalose.

32. The device of claim 26, wherein the reagent further comprises at least one additional enzyme and an additional redox mediator.

33. The device of claim 26, wherein the electrically conducting material of the working and counter electrodes is selected from a group consisting of palladium, platinum, gold, silver, titanium, copper, and carbon.

34. The device of claim 26, wherein the analyte is glucose, the oxidized form of the redox mediator is ferricyanide, and the enzyme is glucose oxidase.

35. The device of claim 26, further comprising:

e. a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electrooxidation of the reduced form of the redox mediator at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the oxidation of the reduced form of the redox mediator at the working electrode surface.

36. A device for analyzing an analyte, comprising:

a. a first electrical insulator;

b. 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 and being supported on the first electrical insulator;

c. a second electrical insulator, overlaying the first electrical insulator and the electrodes and including a cutout portion that exposes a smaller surface area of the counter electrode than the working electrode; and

d. a reagent, substantially covering the exposed electrode surfaces in the cutout portion and comprising the reduced form of a redox mediator, an enzyme, and a buffer,

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,

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, and

the buffer having a lower reduction potential than the oxidized form of the redox mediator and 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.

37. The device of claim 36, wherein the reagent further comprises

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.

38. The device of claim 36, further comprising:

e. a power source in electrical connection with the working and counter electrodes and capable of supplying an electrical potential difference between the working and counter electrodes sufficient to cause diffusion limited electroreduction of the oxidized form of the redox mediator at the surface of the working electrode; and

f. a meter in electrical connection with the working and counter electrodes and capable of measuring the diffusion limited current produced by the reduction of the oxidized form of the redox mediator at the working electrode surface.