Electrochemical system for rapid detection of biochemical agents that catalyze a redox potential change

• US 5,567,302 A
• Filed: 06/07/1995
• Issued: 10/22/1996
• Est. Priority Date: 06/07/1995
• Status: Expired due to Fees

• Current Assignee: Molecular Devices
• Original Assignee: Molecular Devices
• Inventors: Song, Herking, Hafeman, Dean G.
• Primary Examiner(s): Bell, Bruce F.
• Application Number:
  US08/483,249
• Time in Patent Office: 503 Days
• Field of Search: 205/777.5, 204/418, 204/403, 204/406, 204/412, 422/82.01, 422/82.03, 422/68.1, 435/4, 435/817, 435/29, 435/288, 435/291
• US Class Current: 205/777.5
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The present invention relates to a system for detecting, in a reliable, precise and highly sensitive manner, biochemical agents such as enzymes that catalyze a redox potential change. One electrode is used to measure redox potential changes in an aqueous electrolyte containing the biochemical agents. Another electrode is used to deliver a feedback current to the electrolyte in response to measured Changes in electrolyte redox potential. The amount of feedback current or charge delivered through the electrode to the electrolyte is sufficient in magnitude to maintain a constant redox potential. Quantitation of the amount of feedback current or charge necessary to maintain the constant redox potential may then be used to determine the amount of biochemical agents present. Alternatively, the redox potential need not be kept constant, but instead may be allowed to reach a new steady-state. Thus, the current, or charge, conducted by a feedback electrode to maintain a new steady-state potential in the presence of an enzymatic reaction may be used to quantitate the amount of enzymatic activity present. The present invention provides precision in the quantitation results, high sensitivity in the enzyme detection, and a wider dynamic range for quantitation of the biochemical agent.

Claims

1. A method for detecting biochemical agents that catalyze a redox potential change comprising the steps of:

(a) electrochemically contacting an electrolyte containing the biochemical agents with a first electrode and a second electrode;

(b) measuring a change in the redox potential of the electrolyte through the first electrode;

(c) delivering coulometric feedback to the electrolyte through the second electrode, in response to a measured change in the redox potential of the electrolyte, in an amount sufficient to maintain the electrolyte at a substantially constant redox potential; and

(d) quantitizing the amount of coulometric feedback necessary to maintain the electrolyte at the substantially constant redox potential.

2. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 1, including the step of electrochemically contacting the electrolyte with a first electrode comprising a metalized sensing region on an insulated surface of a semiconductor.

3. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 2, including the steps of applying a bias potential to the semiconductor, illuminating an area on the semiconductor under the metalized sensing region to produce a photocurrent in an external circuit connected to the semiconductor, and measuring the photocurrent to determine the change in redox potential of the electrolyte.

4. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 3, including the step of delivering a beam of intensity-modulated light to illuminate the area on the semiconductor.

5. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 1, including the step of electrochemically contacting the electrolyte with a carbon sheet electrode as the first electrode.

6. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 1, including the step of delivering coulometric feedback to the electrolyte through a current source.

7. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 6, including the step of delivering coulometric feedback to the electrolyte through a variable and direct current source.

8. A method for detecting biochemical agents that catalyze a redox potential change comprising the steps of:

(a) electrochemically contacting an electrolyte containing the biochemical agents with an insulated semiconductor having a metalized sensing region;

(b) applying a bias potential to the semiconductor;

(c) illuminating an area on the semiconductor under the metalized sensing region to produce a photocurrent in an external circuit connected to the semiconductor;

(d) measuring the photocurrent;

(e) delivering coulometric feedback to the electrolyte through a controlling electrode, in response to a measured change in the redox potential of the electrolyte, in an amount sufficient to maintain the electrolyte at a substantially constant redox potential; and

(f) quantitizing the amount of coulometric feedback necessary to maintain the electrolyte at the substantially constant redox potential.

9. A circuit for detecting biochemical agents that catalyze a redox potential change comprising a first electrode and a second electrode for electrochemical communication with an electrolyte containing the biochemical agents, means operatively associated with the first electrode for measuring a change in redox potential of the electrolyte, and means operatively associated with both the means for measuring a change in redox potential of the electrolyte and the second electrode for delivering coulometric feedback to the electrolyte in response to a measured change in redox potential of the electrolyte.

10. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 9, wherein the first electrode comprises a metalized sensing region on an insulated surface of a semiconductor.

11. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 10, wherein the means for measuring a change in redox potential of the electrolyte includes means for applying a bias potential to the semiconductor, means for illuminating an area on the semiconductor under the metalized sensing region to produce a photocurrent in an external circuit connected to the semiconductor, and means for measuring the photocurrent.

12. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 11, wherein the means for illuminating an area on the semiconductor is a light source that delivers a beam of intensity-modulated light.

13. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 9, wherein the first electrode is a carbon sheet electrode.

14. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 9, wherein the means for delivering coulometric feedback to the electrolyte in response to a measured change in redox potential of the electrolyte includes a current source.

15. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 14, wherein the current source is a variable and direct current source.

16. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 9, the circuit further comprising means operatively associated with the means for delivering coulometric feedback for quantitating the amount of coulometric feedback delivered to the electrolyte.

17. A circuit for detecting biochemical agents that catalyze a redox potential change comprising a semiconductor, an insulator on at least one surface of the semiconductor to insulate the semiconductor from an aqueous electrolyte containing the biochemical agents, a metalized sensing region on a surface of the insulator for electrochemical communication with the aqueous electrolyte, a controlling electrode for electrochemical communication with the aqueous electrolyte near the metalized sensing region, means for applying a bias potential to the semiconductor, means for illuminating an area on the semiconductor under the metalized sensing region to produce a photocurrent in an external circuit connected to the semiconductor, means for measuring the photocurrent, and means operatively associated with both the means for measuring the photocurrent and the controlling electrode for delivering coulometric feedback to the aqueous electrolyte in response to the measured photocurrent.

18. A circuit for detecting biochemical agents that catalyze a redox potential change as set forth in claim 17, the circuit further comprising means operatively associated with the means for delivering coulometric feedback for quantitating the amount of coulometric feedback delivered to the aqueous electrolyte.

19. A method for detecting biochemical agents that catalyze a redox potential change comprising the steps of:

(a) electrochemically contacting an electrolyte containing the biochemical agents with an electrode; and

(b) measuring a steady-state current conducted on the electrode as a result of the redox potential change.

20. A method for detecting biochemical agents that catalyze a redox potential change as set forth in claim 19, including the step of electrochemically contacting the electrolyte with a carbon sheet electrode as the first electrode.