Method and reagent system having a non-regenerative enzyme-coenzyme complex

• US 20050214891A1
• Filed: 05/16/2003
• Published: 09/29/2005
• Est. Priority Date: 05/16/2002
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Horn, Carina, Hoenes, Joachim, Knappe, Wolfgang-Reinhold
• Granted Patent: US 7,341,830 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

The invention relates to a method and reagent system for detecting an analyte in a sample by means of an enzymatic reaction, involving the use of an enzyme-coenzyme complex as a stoichiometric reaction partner for the analyte present in the sample.

Claims

1. A method for detecting an analyte in a sample by an enzymatic reaction, comprising the steps:

(a) contacting the sample with a detection reagent comprising an enzyme-coenzyme complex, under conditions with which no regeneration of the coenzyme takes place, and

(b) detecting a reaction of the analyte through a change in the enzyme-coenzyme complex.

2. The method as claimed in claim 1, characterized in that the enzymatic reaction comprises a redox reaction.

3. The method as claimed in claim 1 or 2, characterized in that an oxidoreductase is used as enzyme, and a change in the coenzyme due to oxidation or reduction is detected.

4. The method as claimed in claim 3, characterized in that the enzyme used is a dehydrogenase selected from a glucose dehydrogenase (E.C.1.1.1.47), lactate dehydrogenase (E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase (E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6), alcohol dehydrogenase (E.C.1.1.1.1) or amino-acid dehydrogenase, e.g.

L-amino-acid dehydrogenase (E.C.1.4.1.5).

5. The method as claimed in any of the preceding claims, characterized in that a coenzyme selected from flavin, nicotine and quinone derivatives is used.

6. The method as claimed in claim 5, characterized in that the coenzyme is selected from FAD, FADH₂, FMN, FMNH₂, coenzyme Q, PQQ, NAD⁺, NADH/H⁺, NADP⁺ and NADPH/H⁺.

7. The method as claimed in claim 1 to 6, characterized in that the change in the coenzyme is detected by optical methods.

8. The method as claimed in claim 7, characterized in that the change in the coenzyme is detected by measuring absorption, fluorescence, CD, ORD or refractometry.

9. The method as claimed in claim 8, characterized in that the change in the coenzyme is detected by measuring the fluorescence.

10. The method as claimed in any of the preceding claims, characterized in that a gel matrix with an enzyme-coenzyme complex embedded therein is used as detection reagent.

11. The method as claimed in claim 10, characterized in that the gel matrix has a layer thickness of ≦

50 μ

m, in particular of ≦

5 μ

m.

12. The method as claimed in claim 10 or 11, characterized in that a gel matrix based on photopolymerizable substances is used.

13. The method as claimed in any of the preceding claims, characterized in that an analyte in a body fluid is determined.

14. The method as claimed in claim 13, characterized in that a determination of glucose in blood is carried out.

15. The method as claimed in any of the preceding claims, characterized in that the duration of the reaction of the analyte is ≦

5 s.

16. The method as claimed in any of the preceding claims, characterized in that the reaction is carried out in the absence of mediators able to react with the coenzyme.

17. The method as claimed in any of the preceding claims, characterized in that the enzyme-coenzyme complex is employed as stoichiometric reactant in relation to the analyte.

23. The use of a reagent system as claimed in any of claims 18 to 22 in a method as claimed in any of claims 1 to 17.

18. A reagent system for detecting an analyte in a sample, comprising:

(a) a detection reagent comprising an enzyme-coenzyme complex in a form such that no regeneration of the coenzyme takes place, and

(b) a support to receive the detection reagent.

19. The reagent system as claimed in claim 18, characterized in that the enzyme-coenzyme complex is embedded in a gel matrix.

20. The reagent system as claimed in claim 18 or 19, characterized in that the support is at least partly optically transparent.

21. The reagent system as claimed in any of claims 18 to 20, characterized in that the support comprises an essentially planar structure.

22. The reagent system as claimed in any of claims 18 to 20, characterized in that the support comprises a plurality of different detection reagents.