Reagent Composition and Methods of Using and Forming the Same

• US 20080199937A1
• Filed: 03/03/2006
• Published: 08/21/2008
• Est. Priority Date: 03/04/2005
• Status: Active Grant

• Current Assignee: Ascensia Diabetes Care Holdings AG (PHC Holdings Corporation), Syracuse University
• Original Assignee: Syracuse University
• Inventors: Chu, Amy H., Spradlin, Hope G.
• Granted Patent: US 8,921,065 B2
• Time in Patent Office: Days
• US Class Current: 435/200
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: G01N 27/3271 Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001

Abstract

A reagent composition containing GDH-PQQ as an enzyme-co-factor and screen-printed on working and counter electrodes of electrochemical biosensors, maintains activity of the enzyme reagents by proper selection of components. A preferred composition includes hydrophilic polymers, amorphous untreated silica, buffers, surfactants, and a mediator For example, the biosensor is useful in the amperometric determination of glucose.

Claims

1. A reagent composition for making electrochemical biosensors comprising:

(a) glucose dehydrogenase (GDH) and co-factor pyrrolo-quinoline quinone (PQQ) for oxidizing glucose in a biological sample;

(b) a hydrophilic polymer selected from the group consisting of cellulose derivatives, natural gums and gels, and water soluble synthetic polymers;

(c) a thickening agent selected from the group consisting of amorphous untreated silica powder, talc, mica, diatomaceous earth, and natural and modified clays;

(d) a buffer sufficient to maintain pH in the range of from about 4.5 to about 6.5 to 6.0;

(e) a surfactant; and

(f) a mediator.

2. The reagent composition of claim 1, wherein said buffer is an acetate, citrate, or succinate buffer.

3. The reagent composition of claim 1, wherein said surfactant is an alkyl aryl polyether alcohol.

4. The reagent composition of claim 1, wherein said mediator is potassium ferricyanide.

5. The reagent composition of claim 1, wherein the buffer is from 5.0 to 6.0.

6. The reagent composition of claim 5, wherein said composition has a viscosity of about 60,000 to about 180,000 cps (mPa·

s).

7. The reagent composition of claim 1, wherein said hydrophilic polymer is a cellulose derivative selected from the group consisting of sodium carboxymethyl cellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, and hydroxy propylmethyl cellulose.

9. The reagent composition of claim 1, wherein said thickening agent is amorphous untreated silica powder.

11. The reagent composition of claim 1, wherein 1-8 units of said GDH-PQQ is present for each milligram of the total weight of the composition.

12. The reagent composition of claim 2, wherein said buffer is present in about 30 to about 200 mM.

8. (canceled)

10. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. An electrochemical biosensor comprising:

(a) a non-porous substrate;

(b) working and counter electrodes disposed on said substrate;

(c) a reagent composition located on said electrodes, said composition comprising;

(1) glucose dehydrogenase (GDH) and co-factor pyrrolo-quinoline quinone (PQQ) for oxidizing glucose in a biological sample, for each milligram of the total weight of said composition;

(2) a hydrophilic polymer selected from the group consisting of cellulose derivatives, natural gums and gels, and water soluble synthetic polymers;

(3) a thickening agent selected from the group consisting of amorphous untreated silica powder, talc, mica, diatomaceous earth, and natural and modified clays;

(4) a buffer sufficient to maintain pH in the range of from about 4.5 to about 6.5,

(5) a surfactant, and

(6) a mediator; and

(d) a protective cover for said electrodes and said reagent composition.

19. The electrochemical biosensor of claim 18, wherein said buffer is about 30 to about 200 mM of an acetate, citrate, or succinate buffer.

20. The electrochemical biosensor of claim 18, wherein said surfactant is up to about 0.5 wt. % of an alkyl aryl polyether alcohol, based on the total weight of said composition.

21. The electrochemical biosensor of claim 18, wherein said mediator is about 10 to about 20 wt % of potassium ferricyanide, based on the total weight of said composition.

23. The electrochemical biosensor of claim 18, wherein said composition has a viscosity of about 60,000 to about 180,000 cps (mPa·

s).

24. The electrochemical biosensor of claim 18, wherein said hydrophilic polymer is about 2 to about 10 wt % of a cellulose derivative selected from the group consisting of sodium carboxy methyl cellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, and hydroxy propyl methyl cellulose, based on the total weight of said composition.

26. The electrochemical biosensor of claim 18, wherein said thickening agent is from about 1 to about 6 wt % of an amorphous untreated silica powder, based on the total weight of said composition.

27. The electrochemical biosensor of claim 26, wherein said amorphous untreated silica powder is hydrophilic.

28. The electrochemical biosensor of claim 18, wherein said reagent composition includes from about 5 to about 50 mM of calcium chloride.

22. (canceled)

25. (canceled)

29. A method of placing a reagent composition in an electrochemical biosensor, the method comprising screen printing a reagent composition, the reagent composition including:

(a) glucose dehydrogenase (GDH) and co-factor pyrrolo-quinoline quinone (PQQ) for oxidizing glucose in a biological sample;

(b) a hydrophilic polymer selected from the group consisting of cellulose derivatives, natural gums and gels, and water soluble synthetic polymers;

(c) a thickening agent selected from the group consisting of amorphous untreated silica powder, talc, mica, diatomaceous earth, and natural and modified clays;

(d) a buffer sufficient to maintain pH in the range of from about 4.5 to about 6.5;

(e) a surfactant; and

(f) a mediator.

31. The method of claim 29, wherein said thickening agent is amorphous untreated silica powder.

32. The method of claim 29, wherein the pH is maintained in the range of from 5.0 to 6.0.

30. (canceled)