Chemical sensor enhanced by direct coupling of redox enzyme to conductive surface

• US 7,364,886 B2
• Filed: 02/28/2006
• Issued: 04/29/2008
• Est. Priority Date: 02/28/2006
• Status: Expired due to Fees

• Current Assignee: Washington University In St Louis
• Original Assignee: University of Washington
• Inventors: Yager, Paul, Fu, Elain S., Hasenbank, Melissa S.
• Primary Examiner(s): Weber; Jon
• Assistant Examiner(s): MARTIN, PAUL C
• Application Number:
  US11/364,436
• Publication Number:
  US 20070202559A1
• Time in Patent Office: 791 Days
• Field of Search: None
• US Class Current: 435/174
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/28 involving peroxidase

Abstract

The invention provides a method, apparatus and system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme. In one embodiment, the method comprises immobilizing the redox enzyme on a first region of a conductive surface and contacting a substrate capable of producing a detectable signal upon oxidation or reduction with a second region of the conductive surface. The second region is electrically coupled with the first region and the redox enzyme is not present in the second region. The method further comprises exposing the immobilized redox enzyme to conditions that effect oxidation or reduction of the enzyme, and detecting oxidation or reduction of the substrate at the second region. The invention can be adapted for detecting a plurality of analytes.

Claims

1. A system for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme comprising:

(a) a continuous conductive surface having a first region and at least one additional region, wherein a redox enzyme is immobilized on the first region of the conductive surface, wherein the additional region is electrically coupled via the continuous conductive surface with the first region, and the redox enzyme is not present in the additional region; and

(b) means for detecting oxidation or reduction of a substrate capable of producing a detectable signal upon oxidation or reduction at the additional region, wherein the substrate is located at the additional region.

2. The system of claim 1, wherein the means for detecting oxidation or reduction comprises fluorescence or absorbance.

3. The system of claim 1, wherein the means for detecting oxidation or reduction comprises fluorescence, absorbance or surface plasmon resonance.

4. A method for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme comprising:

(a) exposing the immobilized redox enzyme on the conductive surface of the system of claim 1 to conditions that effect oxidation or reduction of the enzyme; and

(b) detecting oxidation or reduction of the substrate at the additional region on the conductive surface of the system.

5. The method of claim 4, wherein the substrate that produces a detectable signal upon oxidation or reduction comprises a colorimetric or fluorogenic enzyme substrate.

6. The method of claim 4, wherein the substrate produces the detectable signal via reaction with an agent and the detectable signal comprises a colorimetric, fluorogenic or precipitating product.

7. The method of claim 4, wherein the redox enzyme comprises horseradish peroxidase, glucose oxidase, alcohol oxidase, lactate oxidase, choline oxidase, cholesterol oxidase, glutamate oxidase or amino acid oxidase.

8. The method of claim 4, wherein the conditions that effect oxidation of the redox enzyme comprise contact with hydrogen peroxide.

9. The method of claim 4, wherein the substrate comprises tetramethylbenzidine (TMB), 4-chloro-1-naphthol (4-CN) and 3,3′

-diaminobenzidine (DAB) or dihydroxyphenoxazine (AMPLEX®

Red).

10. The method of claim 9, wherein the substrate is tetramethylbenzidine and the agent is dextran sulfate.

11. The method of claim 4, wherein the conductive surface comprises a metal film.

12. The method of claim 11, wherein the metal comprises gold, silver, copper, platinum or aluminum.

13. The method of claim 4, further comprising contacting a solution suspected of containing an analyte with the first region of the conductive surface.

14. The method of claim 4, wherein the electrochemical oxidoreduction activity is indicative of the presence of an analyte.

15. The method of claim 13, wherein the method further comprises introducing the substrate under flow or stopped flow conditions.