Methods and compositions for characterizing a redox reagent system enzyme

• US 20060003400A1
• Filed: 06/30/2004
• Published: 01/05/2006
• Est. Priority Date: 06/30/2004
• Status: Abandoned Application

• Current Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Qian, Suyue, Hartz, Thomas P., Byrd, Patricia A.
• Application Number:
  US10/883,629
• Publication Number:
  US 20060003400A1
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 33/521 Single-layer analytical elements
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/558 using diffusion or migration of antigen or antibody immunochromatographic test strips G01N33/54387

Abstract

Methods and compositions for characterizing a redox reagent system enzyme are provided. In practicing the subject methods, a sample that includes a redox reagent system enzyme and a known amount of substrate is applied to an electrochemical cell that includes an enzyme-free reagent composition having a redox reagent system mediator. Also provided are electrochemical test strips that include the subject electrochemical cells, and systems and kits that include the same. The subject invention finds use in a variety of different applications, including redox reagent system characterization applications.

Claims

1. A method comprising:

(a) applying a sample comprising a first redox reagent system enzyme and a known amount of an enzyme substrate to an electrochemical cell comprising an enzyme-free reagent composition comprising a mediator of said redox reagent system; and

(b) detecting an electrical signal produced by said cell.

2. The method according to claim 1, wherein said enzyme is an oxidizing enzyme.

3. The method according to claim 2, wherein said oxidizing enzyme is chosen from an oxidase and a dehydrogenase.

4. The method according to claim 1, wherein said sample further comprises an enzyme cofactor.

5. The method according to claim 1, wherein said method further comprises using said detected electrical signal to determine analyte specificity of said first redox reagent system enzyme.

6. The method according to claim 5, wherein said method comprises detecting electrical signals produced from at least the following samples:

(i) a sample containing a first redox reagent system enzyme and a known first concentration of a first enzyme substrate;

(ii) a sample containing said first redox reagent system enzyme and a known first concentration of a second enzyme substrate;

(iii) a sample containing a second redox reagent system enzyme and said known first concentration of said first enzyme substrate; and

(iv) a sample containing said second redox reagent system enzyme and said known first concentration of said second enzyme substrate.

7. The method according to claim 6, wherein said method further comprises detecting electrical signals produced from the following samples:

(i) a sample containing said first redox reagent system enzyme and a known second concentration of said first enzyme substrate;

(ii) a sample containing said first redox reagent system enzyme and a known second concentration of said second enzyme substrate;

(iii) a sample containing said second redox reagent system enzyme and said known second concentration of said first enzyme substrate; and

(iv) a sample containing said second redox reagent system enzyme and said known second concentration of said second enzyme substrate.

8. The method according to claim 7, wherein said determined analyte specificity of said first redox reagent system enzyme is relative to said second redox reagent system enzyme.

9. The method according to claim 8, wherein said first redox reagent system enzyme is a non-naturally occurring redox reagent system enzyme.

10. The method according to claim 8, wherein said second redox reagent system enzyme is a naturally occurring redox reagent system enzyme.

11. The method according to claim 1, wherein said method further comprises using said detected electrical signal to determine activity of said enzyme in said sample.

12. The method according to claim 11, wherein said method comprises comparing said detected electrical signal to a reference.

13. The method according to claim 1, wherein said electrochemical cell is present on a test strip.

14. A method of determining substrate specificity of a first analyte dehydrogenase relative to a second analyte dehydrogenase, said method comprising:

(a) applying each of at least the following four samples;

(i) a sample containing a first analyte dehydrogenase and a known first concentration of a first enzyme substrate;

(ii) a sample containing said first analyte dehydrogenase and a known first concentration of a second enzyme substrate;

(iii) a sample containing a second analyte dehydrogenase and said known first concentration of said first enzyme substrate; and

(iv) a sample containing said second analyte dehydrogenase and said known first concentration of said second enzyme substrate;

to an electrochemical cell comprising an enzyme-free reagent composition comprising a mediator of a redox reagent system; and

(b) detecting an electrical signal produced for each of said samples to determine substrate specificity of said first analyte dehydrogenase relative to said second analyte dehydrogenase.

15. The method according to claim 14, wherein said method further comprises detecting electrical signals produced from the following samples:

(i) a sample containing said first analyte dehydrogenase and a known second concentration of said first enzyme substrate;

(ii) a sample containing said first analyte dehydrogenase and a known second concentration of said second enzyme substrate;

(iii) a sample containing said second analyte dehydrogenase and said known second concentration of said first enzyme substrate; and

(iv) a sample containing said second redox analyte dehydrogenase and said known second concentration of said second enzyme substrate.

16. The method according to claim 14, wherein said first and second analyte dehydrogenases are glucose dehydrogenases.

17. The method according to claim 16, wherein said first glucose dehydrogenase is a naturally occurring glucose dehydrogenase and said second glucose dehydrogenase is a non-naturally occurring glucose dehydrogenase.

18. The method according to claim 17, wherein said first and second glucose dehydrogenases are soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenases.

19. The method according to claim 14, wherein said first and second enzyme substrates are reducing sugars.

20. The method according to claim 19, wherein at least one of said first and second enzyme substrates is glucose and said other of said enzyme substrates is a reducing sugar selected from the group consisting of galactose, maltose, xylose, or lactose.

21. The method of claim 14, wherein each of said samples further comprises a coenzyme.

22. The method according to claim 21, wherein said coenzyme is PQQ.

23. The method according to claim 14, wherein each of said samples further comprises a cofactor.

24. The method according to claim 23, wherein said cofactor is calcium.

25. The method according to claim 14, wherein said mediator of said enzyme free reagent composition is ferricyanide.

26. The method according to claim 14, wherein said enzyme free reagent composition further comprises a buffer.

27. The method according to claim 14, wherein said enzyme free reagent composition further comprises a mediator stabilizer.

28. A method of determining the activity of an analyte dehydrogenase in a sample, said method comprising:

(a) applying a sample comprising said analyte dehydrogenase and a known amount of a substrate therefore to an electrochemical cell comprising an enzyme free reagent composition comprising a mediator of a redox reagent system;

(b) detecting an electrical signal produced by said cell; and

(c) using said detected electrical signal to determine activity of said analyte dehydrogenase in said sample.

29. The method according to claim 28, wherein said method comprises comparing said detected electrical signal to a reference.

30. The method according to claim 29, wherein said method further comprises producing said reference.

31. The method according to claim 28, wherein said analyte dehydrogenase is a glucose dehydrogenase.

32. The method according to claim 31, wherein said glucose dehydrogenase is a soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase.

33. The method according to claim 28, wherein said sample further comprises a coenzyme.

34. The method according to claim 33, wherein said coenzyme is PQQ.

35. The method according to claim 28, wherein said sample further comprises a cofactor.

36. The method according to claim 35, wherein said cofactor is calcium.

37. The method according to claim 28, wherein said mediator of said enzyme free reagent composition is ferricyanide.

38. The method according to claim 28, wherein said enzyme free reagent composition further comprises a buffer.

39. The method according to claim 28, wherein said enzyme free reagent composition further comprises a mediator stabilizer.

40. An electrochemical cell comprising an enzyme-free reagent composition comprising a redox reagent system mediator.

41. The electrochemical cell according to claim 40, wherein said mediator is ferricyanide.

42. The electrochemical cell according to claim 40, wherein said composition further comprises a mediator-stabilizing buffer.

43. The electrochemical cell according to claim 40, wherein said reagent composition further comprises a stabilizer.

44. The electrochemical cell according to claim 43, wherein said stabilizer is a carbohydrate.

45. The electrochemical cell according to claim 44, wherein said carbohydrate is sucrose.

46. The electrochemical cell according to claim 40, wherein said cell is present in an electrochemical test strip.

47. A system comprising:

(a) an electrochemical cell comprising an enzyme-free reagent composition comprising a redox reagent system mediator; and

(b) a fluid medium comprising a redox reagent system enzyme and a known amount of an enzyme substrate.

48. The system according to claim 47, wherein said system comprises two or more of said electrochemical cells and two or more different fluid mediums.