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

• US 20070202559A1
• Filed: 02/28/2006
• Published: 08/30/2007
• Est. Priority Date: 02/28/2006
• Status: Active Grant

• Current Assignee: Washington University In St Louis
• Original Assignee: University of Washington
• Inventors: Yager, Paul, Fu, Elain, Hasenbank, Melissa
• Granted Patent: US 7,364,886 B2
• Time in Patent Office: Days
• US Class Current: 435/11
• 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 method for detecting electrochemical oxidoreduction activity mediated by a redox enzyme at a site remote from the enzyme comprising:

(a) contacting a substrate that produces a detectable signal upon oxidation or reduction with the at least one additional region of the conductive surface of the system of claim

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

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

2. The method of claim 1, wherein the substrate that produces a detectable signal upon oxidation or reduction comprises a colorimetric or fluorogenic enzyme substrate.

3. The method of claim 1, wherein the substrate produces the detectable signal via reaction with an agent and the detectable signal comprises a colorimetric, fluorogenic or precipitating product.

4. The method of claim 1, wherein the redox enzyme comprises horseradish peroxidase, glucose oxidase, alcohol oxidase, lactate oxidase, choline oxidase, cholesterol oxidase, glutamate oxidase or amino acid oxidase.

5. The method of claim 1, wherein the conditions that effect oxidation of the redox enzyme comprise contact with hydrogen peroxide.

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

-diaminobenzidine (DAB) or dihydroxyphenoxazine (Amplex®

Red).

7. The method of claim 6, wherein the substrate is tetramethylbenzidine and the agent is dextran sulfate.

8. The method of claim 1, wherein the conductive surface comprises a metal film.

9. The method of claim 8, wherein the metal comprises gold, silver, copper, platinum or aluminum.

10. The method of claim 1, further comprising contacting a solution suspected of containing an analyte with the first region of the conductive surface.

11. The method of claim 1, wherein the electrochemical oxidoreduction activity is indicative of the presence of an analyte.

12. The method of claim 1, wherein the contacting of step (a) comprises introducing the substrate under flow or stopped flow conditions.

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

(a) a 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 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.

14. A method for detecting a plurality of analytes comprising:

(a) contacting a solution suspected of containing a plurality of analytes with a plurality of sets of electrically coupled regions, wherein each set of regions is electrically isolated from the other sets of regions and a first region of each set of regions comprises an immobilized redox enzyme that is oxidized or reduced upon contact with an analyte,

(b) contacting at least one additional region of each set of regions with a substrate that produces a detectable signal upon oxidation or reduction, wherein the redox enzyme is not present in the additional region;

(c) exposing the immobilized redox enzymes to conditions that effect oxidation or reduction of the enzymes; and

(d) detecting oxidation or reduction of the substrates at the additional region of each set of regions.

15. The method of claim 14, wherein the contacting of steps (a) and (b) comprises introducing the solution under flow or stopped flow conditions.

16. The method of claim 14, wherein the contacting of steps (a) and (b) is concurrent.

17. A sensor for detecting a plurality of analytes comprising a plurality of sets of electrically coupled regions, wherein each set of regions is electrically isolated from the other sets of regions and a first region of each set of regions comprises an immobilized redox enzyme that is oxidized or reduced upon contact with an analyte, and at least one additional region of each set of regions is electrically coupled with the first region, and the redox enzyme is not present in the additional region, whereby, upon contact with a substrate that produces a detectable signal upon oxidation or reduction, oxidation or reduction of the substrate is detected.