Electrochemical detection of nadh or naph

• US 20050067303A1
• Filed: 02/21/2003
• Published: 03/31/2005
• Est. Priority Date: 02/22/2002
• Status: Abandoned Application

• Current Assignee: Isis Innovation Limited
• Original Assignee: Isis Innovation Limited
• Inventors: Bell, Stephen, Wong, Luet Lok
• Application Number:
  US10/505,341
• Publication Number:
  US 20050067303A1
• Time in Patent Office: Days
• US Class Current: 205/777.500
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase

Abstract

A method for detecting the presence or absence of, or for determining the concentration of, NADH or NADPH in a sample is provided, wherein the method comprises contacting a reductase and a redox active agent to said sample; and measuring the quantity of reduced redox active agent produced by the reductase, by electrochemical means. The method may be used to quantify the amount or activity of a redox enzyme or its substrate, wherein the redox enzyme uses NAD⁺, NADP⁺, NADPH or NADP as a cofactor.

Claims

1. An electrochemical method for detecting the presence or absence of, or for determining the concentration of, NADH or NADPH in a sample comprising;

(a) contacting a reductase and a redox active agent with said sample; and

(b) measuring the quantity of reduced redox active agent produced by the reductase, by electrochemical means.

2. A method for monitoring the amount or activity of a redox enzyme or substrate, wherein the redox enzyme uses NADP, NADPH or NAD⁺ or NADP⁺ as a co-factor comprising carrying out the method of claim 1, wherein electron transfer between the redox active agent and an electrode is correlated to the amount or activity of the redox enzyme or substrate.

3. A method according to claim 1, wherein the NADH or NADPH are produced by the reduction of NAD⁺ or NADP⁺ by a redox enzyme which concomitantly oxidises a substrate.

4. A method according to claim 3, wherein the amount of NADH or NADPH formed is proportional to the amount of the redox enzyme present or the amount of its substrate and hence allows the detection, or quantification, of the enzyme or substrate in the sample.

5. A method according to claim 2, wherein the redox enzyme is a dehydrogenase.

6. A method according to claim 1 wherein the reductase is capable of accepting two electrons from NADH or NADPH.

7. A method according to claim 1 wherein the reductase is selected from putidaredoxin reductase of the cytochrome P450_cam enzyme system from Pseudonomas putida, the flavin (FAD/FMN) domain of the P450_BM-3 enzyme from Bacillus megaterium, spinach ferrodoxin reductase, rubredoxin reductase, adrenodoxin reductase, nitrate reductase, cytochrome b₅ reductase, corn nitrate reductase, terpredoxin reductase and yeast, rat, rabbit and human NADPH cytochrome P450 reductase or a functional derivative of any thereof.

8. A method according to claim 1 wherein the redox active agent is Fe(CN)₆³⁻

, RU(NH₃)₆³⁺, or ferrocenium monocarboxylic acid (FMCA).

9. A method according to claim 1 wherein the reductase is specific for NADH.

10. A method according to claim 9 wherein the reductase is a putidaredoxin reductase.

11. A method according to claim 1 wherein the reductase is specific for NADPH.

12. A method according to claim 11 wherein the reductase is the flavin domain of P450_BM-3 or is spinach ferrodoxin reductase.

13. A method according to claim 1 which allows a monitoring of the amount of the substrate, enzyme, NADH or NADPH over time.

14. A method according to claim 1 wherein the redox active agent is not diaphorase or an organic dye.

16. An electrochemical cell, which can be used to carry out a method as defined in claim 1, wherein the electrochemical cell comprises:

(a) sample holding means;

(b) a source of reductase;

(c) a redox active agent; and

(d) means for detecting and/or quantifying any current generated.

15. An electrochemical cell comprising:

(a) sample holding means;

(b) a source of reductase;

(c) a redox active agent; and

(d) means for detecting and/or quantifying any current generated.