Method and reagent system having a non-regenerative enzyme-coenzyme complex
View Patent ↗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.
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 coenzyme takes place, whereby the enzyme-coenzyme complex is employed in an at least stoichiometric amount relative to the analyte present in the sample, 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 , 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).
5. The method as claimed in claim 1 , characterized in that a coenzyme selected from nicotine derivatives is used.
6. The method as claimed in claim 5 , characterized in that the coenzyme is NAD + .
7. The method as claimed in claim 1 , 2 , 3 , 4 , 5 or 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, circular dichroism, optical rotary dispersion 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 claim 1 , 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 μm.
12. The method as claimed in claim 10 or 11 , characterized in that a gel matrix based on photo-polymerizable substances is used.
13. The method as claimed in claim 1 , 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 claim 1 , characterized in that the duration of the reaction of the analyte is ≦5 s.
16. The method as claimed in claim 1 , characterized in that the reaction is carried out in the absence of mediators able to react with the coenzyme.
17. A method for detecting an analyte in a sample by an enzymatic reaction, comprising the steps:
contacting the sample with a detection reagent comprising a detection reagent comprising an enzyme-coenzyme complex in a form such that no regeneration of the coenzyme takes place, whereby the enzyme-coenzyme complex is employed in an at least stoichiometric amount relative to the analyte present in the sample, and a support to receive the detection reagent, under conditions with which no regeneration of coenzyme takes place, and
detecting a reaction of the analyte through a change in the enzyme-coenzyme complex.