Method for quantitative measurement of a substrate

• US 6,225,078 B1
• Filed: 07/27/1998
• Issued: 05/01/2001
• Est. Priority Date: 07/29/1997
• Status: Expired due to Term

• Current Assignee: PHC Holdings Corporation
• Original Assignee: Matsushita Electric Industrial Company Limited (Panasonic Holdings Corporation)
• Inventors: Ikeda, Shin, Yoshioka, Toshihiko, Nankai, Shiro
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US09/122,591
• Time in Patent Office: 1,009 Days
• Field of Search: 435/14, 435/25, 435/28, 204/403, 204/412, 204/415
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/006 for glucose

Abstract

The invention provides a method for quantitative measurement of a substrate with high accuracy by electrochemically oxidizing an electron mediator which has been reduced by enzyme reaction thereby determining the substrate concentration based on a current flowing during electrochemical oxidation from which adverse effects of an easy-to-oxidize substance on the oxidation process have been minimized. The quantitating method in accordance with the present invention comprises a first step for causing a substrate contained in a sample to react with a specific oxidoreductase to the substrate in the presence of an electron mediator in oxidized state, and a second step for electrochemically reducing the electron mediator in oxidized state which remains non-reduced by the enzyme reaction in the first step, thereby obtaining a current flowing during electrochemical reduction.

Claims

1. A method for quantitative measurement of a substrate in a sample solution without interference from the presence of an easily oxidizable substance with a biosensor comprising an electrically insulating base plate, an electrode system having at least a working electrode and a counter electrode formed on said base plate, and a reaction layer including an oxidoreductase and an electron mediator in oxidized state disposed on said electrode system, said method comprising the steps of:

contacting the sample solution with said reaction layer to react the substrate with said oxidoreductase to produce reduced oxidoreductase, wherein said reduced oxidoreductase reacts with said electron mediator to cause a reduction of at least some of said electron mediator, wherein reduced and

unreduced electron mediator are obtained,

applying a voltage between the working electrode and the counter electrode to electrochemically reduce said unreduced electron mediator at the working electrode, and measuring a reduction current flowing between the working electrode and the counter electrode, thereby quantifying the substrate.

2. The method for quantitative measurement of a substrate in a sample solution in accordance with claim 1, wherein said electrode system further comprises a reference electrode.

3. The method for quantitative measurement of a substrate in a sample solution in accordance with claim 2, wherein said reaction layer further includes a hydrophilic polymer.

4. The method for quantitative measurement of a substrate in a sample solution in accordance with claim 2, wherein said reaction layer further includes additional electron mediator in reduced state to measure low concentrations of the substrate.

5. The method for quantitative measurement of a substrate in a sample solution in accordance with claim 1, wherein said reaction layer further includes a hydrophilic polymer.

6. The method for quantitative measurement of a substrate in a sample solution in accordance with claim 1, wherein said reaction layer further includes additional electron mediator in reduced state to measure low concentrations of the substrate.