Amperometric biosensor test strip

• US 6,541,216 B1
• Filed: 12/22/1999
• Issued: 04/01/2003
• Est. Priority Date: 12/22/1999
• Status: Expired due to Term

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Corporation (Roche Holding AG)
• Inventors: Burke, David W., Wilsey, Christopher Douglas
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/469,899
• Time in Patent Office: 1,196 Days
• Field of Search: 435/26, 435/19, 435/18, 435/15, 435/283.1, 435/287.1, 435/817
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

A reagent and method for determining the levels of 3-hydroxybutyric acid in a sample are provided. The reagent comprises a ferricyanide salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, and a catalytic amount of a second enzyme operative to catalyze the oxidization of the cofactor and the reduction of the ferricyanide. The reagent is incorporated into a test strip that generates an electrical output signal indicative of the level of 3-hydroxybutyric acid when the reagent is contacted with a sample.

Claims

1. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, and a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor.

2. The apparatus of claim 1, wherein the first enzyme is a dehydrogenase.

3. The apparatus of claim 2, wherein the first enzyme is hydroxybutyrate dehydrogenase.

4. The apparatus of claim 3, wherein the second enzyme is diaphorase.

5. The apparatus of claim 4, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

6. The apparatus of claim 3, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

7. The apparatus of claim 2 wherein the second enzyme is a diaphorase.

8. The apparatus of claim 7, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

9. The apparatus of claim 2, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

10. The apparatus of claim 1, wherein the second enzyme is diaphorase.

11. The apparatus of claim 10, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

12. The apparatus of claim 1, wherein the cofactor is selected from the group consisting of NAD⁺, NADP⁺, NADH₂, and NADPH₂.

13. The apparatus of claim 12, wherein the cofactor is NAD⁺.

14. The apparatus of claim 1, wherein the ferricyanide salt is potassium hexacyanoferrate (III).

15. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and being reactive with the sample in a manner effective to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, and a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor, wherein the reagent further comprises a filler.

16. The apparatus of claim 15, wherein the filler is a metal oxide.

17. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and being reactive with the sample in a manner effective to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor, and a polymer selected from the group consisting of polyethylene glycol/polyethylene oxide, polyvinyl-alcohol, polyvinyl-pyrrholidine, polystyrene sulfonate, polyvinyl-acetate and microemulsions of vinyl-acetate.

18. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and being reactive with the sample in a manner effective to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor, and a surfactant.

19. The apparatus of claim 18, wherein the surfactant is selected from the group consisting of sodium dioctyl sulfosuccinate and branched-nonyl phenoxypoly (ethylene-oxy)ethanol.

20. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and being reactive with the sample in a manner effective to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor, and an inorganic buffer.

21. The apparatus of claim 20, wherein the buffer is a pyrophosphate salt.

22. A single-use disposable test strip apparatus for detecting 3-hydroxybutyric acid in a liquid sample, the apparatus comprising:

a substrate,

at least one electrode positioned on the substrate, and

a single-use reagent in communication with the at least one electrode, the reagent being formed to dissolve in the liquid sample and being reactive with the sample in a manner effective to generate an electrical output signal indicative of the level of 3-hydroxybutyric acid in the sample, said reagent comprising a hexacyanoferrate (III) salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, a catalytic amount of a second enzyme operative to catalyze the electrochemical oxidation of a reduced form of the cofactor, and stabilizers selected from the group consisting of flavin mononucleotide, adenosine diphosphate, magnesium ions, lactose, and raffinose.

23. A method for determining information indicative of the level of 3-hydroxybutyric acid in a liquid sample, the method comprising the steps of:

reacting the sample with a single-use reagent formed to dissolve in the liquid sample, the reagent comprising a ferricyanide salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the sample, a cofactor corresponding to said first enzyme, and a catalytic amount of a second enzyme operative to catalyze the oxidization of the cofactor and the reduction of the ferricyanide,

generating an electrical output from the dissolved reagent indicative of the level of 3-hydroxybutyric acid in the sample,

measuring the electrical output; and

determining the level of 3-hydroxybutyric acid in the sample using information comprising the measured electrical output.

24. The method of claim 23, wherein the generating step includes applying an electrical potential to the reagent.

25. The method of claim 24, wherein the electrical potential is applied upon completion of the reaction of the sample and the reagent.

26. The method of claim 25, wherein the reaction is completed in about 60 seconds.

27. A method for determining information indicative off the level of 3-hydroxybutyric acid in a liquid sample, the method comprising the steps of:

providing a single-use disposable sensor including a working and counter electrodes and a reagent in communication with the electrodes, the reagent being formed to dissolve in the liquid sample and comprising a ferricyanide salt, a catalytic amount of a first enzyme operative to catalyze the oxidation of 3-hydroxybutyric acid in the fluid sample, a cofactor corresponding to said first enzyme, and a catalytic amount of a second enzyme operative to catalyze the oxidization of the cofactor and the reduction of the ferricyanide,

contacting the reagent with the liquid sample, and

applying a direct potential difference between the electrodes sufficient to generate an electrical output from the dissolved reagent indicative of the level of 3-hydroxybutyric acid in the fluid sample.

28. The method of claim 27, wherein the applying step occurs at least about 15 seconds after the contacting step.

29. The method of claim 28, wherein the applying step occurs about 50 seconds after the contacting step.

30. The method of claim 27, further comprising the step of measuring background impedance of the analyte and reagent.