Surface-modified electochemical biosensor

• US 5,286,364 A
• Filed: 03/29/1991
• Issued: 02/15/1994
• Est. Priority Date: 06/08/1987
• Status: Expired due to Term

• Current Assignee: Alexander M. Yacynych
• Original Assignee: Rutgers University
• Inventors: Reynolds, Eugene R., Piznik, Sylvia S., Geise, Robert J., Yacynych, Alexander M.
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): BELL, BRUCE F
• Application Number:
  US07/677,384
• Time in Patent Office: 1,054 Days
• Field of Search: 204/78, 204/418, 204/59 R, 204/403, 205/317, 205/198, 435/817
• US Class Current: 205/83
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

An electrode for a biosensor (e.g., a glucose biosensor) has a layer of an electrically insulating polymer formed in situ on its operating surface by electropolymerization. For example, a diaminobenzene and a dihydroxybenzene (e.g., 1,3-diaminobenzene and resorcinol) are copolymerized on the electrode'"'"'s surface by immersing the electrode in a circulating dilute solution of the monomers in deaerated phosphate buffer, and applying a small, continuously cycling voltage between that electrode and another electrode (e.g., from 0.00 V to 0.80 V) until current flow between the electrodes decreases to a minimum. Because the polymer is electrically insulating, polymerization ceases while the polymer layer is still very thin (e.g., 10 nm). An analyte sensing agent, e.g., an enzyme such as immobilized glucose oxidase, is imbedded in the polymer, but with a number of its analyte recognition sites unblocked. The polymer layer shields the electrode surface from interferrents and fouling agents such as uric acid and proteins, but it is sufficiently porous to permit smaller electroactive molecules (e.g., hydrogen peroxide) generated through contact of the enzyme with the analyte molecules to diffuse through to the electrode surface. Preferably a ferrocene compound (e.g., alpha-hydroxy-ethylferrocene or 1,1'"'"'-dimethylferrocene), which functions as an electron mediator, is applied to the polymer film, and held there by adsorption.

Claims

1. A method of making a sensing element for use in a biosensor that amperometrically measures the concentration of an analyte in a biological liquid, said method comprising the following steps:

a) obtaining a first electrode having a sensing surface;

b) immersing said sensing surface of said first electrode in a solution of monomer wherein said monomer is electropolymerizable into an electrically non-conducting polymer;

c) flowing an electric current from a cathode, through said solution, to said first electrode, at a voltage and amperage sufficient to cause the monomer to be electropolymerized on the sensing surface of the first electrode;

d) ceasing the current flow after the amperage of the current as measured at the first electrode declines to a relatively steady minimum, thereby yielding a sensing surface coated with an adherent layer of electrically non-conducting polymer;

e) disposing with the polymeric coating on the sensing surface of the first electrode a chemical sensing agent that, when contacted by a specific analyte in a biological liquid, generates an electroactive molecule that can be detected amperometrically.

3. A method according to claim 1 comprising ceasing the current flow when a stable polymer film is formed.

4. A method according to claim 1 comprising embedding said chemical sensing agent in said electrically non-conducting polymer during electropolymerization.

5. A method according to claim 1 comprising entrapping said chemical sensing agent in said electrically non-conducting polymer during electropolymerization.

6. A method according to claim 1 comprising disposing said chemical sensing agent on said layer of electrically non-conducting polymer during electropolymerization.

2. A method of making a sensing element for use in a biosensor that amperometrically measures the concentration of an analyte in a biological liquid, said method comprising the following steps:

a) obtaining a first electrode having a sensing surface;

b) applying to said sensing surface of said first electrode a chemical sensing agent that, when contacted by a specific analyte in a biological liquid, generates an electroactive molecule that can be detected amperometrically.

c) immersing said sensing surface of said first electrode in a solution of monomer wherein said monomer is electropolymerizable into an electrically non-conducting polymer;

d) flowing an electric current from a cathode, through said solution, to said first electrode, at a voltage and amperage sufficient to cause the monomer to be electropolymerized on the sensing surface of the first electrode; and

e) ceasing the current flow after the amperage of the current as measured at the first electrode declines to a relatively steady minimum, thereby yielding a sensing surface coated with an adherent layer of electrically non-conducting polymer having said chemical sensing agent disposed therewith.

7. A method according to claim 2 comprising ceasing the current flow when a stable polymer film is formed.

8. A method according to claim 2 comprising embedding said chemical sensing agent in said electrically non-conducting polymer during electropolymerization.

9. A method according to claim 2 comprising entrapping said chemical sensing agent in said electrically non-conducting polymer during electropolymerization.

10. A method according to claim 2 comprising disposing said chemical sensing agent on said layer of electrically non-conducting polymer during electropolymerization.

11. A method of making a sensing element for use in a biosensor for amperometrically measuring the concentration of an analyte in a biological liquid, said method comprising:

a) obtaining an electrode having a sensing surface;

b) applying a layer of electrically non-conducting polymer attached to and covering the sensing surface of said electrode by forming said polymer by electropolymerizing a monomer directly onto said sensing surface by using said electrode as a working electrode to conduct the electric current driving the polymerization, said polymer layer being sufficiently solid to shield the electrode surface from contact with interferents in a biological liquid when the sensing element is immersed therein;

c) disposing a chemical sensing agent partially embedded in said layer of polymer, said sensing agent having analyte recognition sites that are not blocked by said polymer, said sites being operable, when contacted by a specific analyte in a biological liquid sample, to generate an electroactive molecule that can be detected amperometrically if it reaches the sensing surface of the electrode;

d) permeating said polymer layer by diffusing said electroactive molecule through said polymer layer to contact the sensing surface of the electrode.