Biosensor and method of quantitative analysis using the same

• US 5,496,453 A
• Filed: 10/12/1994
• Issued: 03/05/1996
• Est. Priority Date: 05/17/1991
• Status: Expired due to Term

• Current Assignee: Kyoto Daiichi Kagaku Company Limited
• Original Assignee: Kyoto Daiichi Kagaku Company Limited
• Inventors: Okuda, Hisashi, Uenoyama, Harumi
• Primary Examiner(s): Bell, Bruce F.
• Application Number:
  US08/321,604
• Time in Patent Office: 510 Days
• Field of Search: 204/403, 204/153.12, 204/415
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

A biosensor which electrochemically detects a material which relates to a reaction of a specific compound in a liquid sample with a biologically active substance or its related substance, in which the biologically active material or an optionally used mediator is placed at a part which is remote from a position of an electrode which acts as electrochemical detector means, and optionally the biological active material or the mediator is covered with a polymer layer.

Claims

1. A method of quantitative analysis of a specific compound in a liquid sample comprising supplying said liquid sample to a biosensor wherein said biosensor comprises an electrochemical means for detecting the product of a reaction of said specific compound with a biologically active material, and wherein said biologically active material is located at a position remote from the electrochemical detecting means, reading at least two electrochemical signals from the biosensor, which signals are at least a first electrochemical signal produced at the supply of the sample and generated by an electrochemically active substance present in the sample but not a product of a reaction of said specific substance with said biologically active material, and at least a second electrochemical signal generated later than said first signal by a product of a reaction of said specific compound with said biologically active material, and operating both signals, whereby the amount of said specific substance is quantitatively measured.

2. A method for the quantitative analysis of a material comprising:

(a) providing a biosensor comprising a measuring electrode, a mediator, and a biologically active material located at a distance from said measuring electrode;

(b) supplying a liquid sample of said material, comprising the object substance to be analyzed and an interfering substance, to said biosensor;

(c) reading a first electrochemical signal from said measuring electrode within a time period sufficient to allow said mediator to react with said interfering substance and the product of that reaction to reach said measuring electrode, but insufficient for a reaction product of said biologically active material and said object substance to reach said electrode;

(d) reading a second electrochemical signal from said measuring electrode after a time sufficient to allow said reaction product of said biologically active material and said object substance to reach said measuring electrode;

(e) determining a concentration of said interfering substance from said first electrochemical signal, and a combined concentration of said interfering substance and said reaction product from said second electrochemical signal;

(f) subtracting said concentration of said interfering substance from said combined concentration of said interfering substance and said reaction product; and

(g) obtaining thereby a concentration of said object substance.

3. The method of claim 2, wherein in step (c) the time for reading said first electrochemical signal from said measuring electrode is within a time period sufficient to allow said mediator to react with said interfering substance and the product of that reaction to reach said measuring electrode, but insufficient for a reaction product of said biologically active material and said object substance to react in a secondary reaction with said mediator, and for the product of the secondary reaction to reach said measuring electrode.

4. The method according to claim 3, wherein said biologically active material comprises a residue formed by the drying of a solution containing said biologically active material on a portion of said biosensor located at a distance from said measuring electrode.

5. The method according to claim 3, wherein said biologically active material is impregnated into an absorbing material, and dried, and wherein said absorbing material containing said dried biologically active material is placed in said biosensor at a distance from said measuring electrode.

6. The method according to claim 2, wherein said first electrochemical signal is measured approximately 0 to 6 seconds after said supplying of said liquid sample.

7. The method according to claim 2, wherein said second electrochemical signal is measured after approximately 50 seconds after said supplying of said liquid sample.

8. The method according to claim 2, wherein said biologically active material is selected from the group consisting of substrates of oxidoreductases, substances which perform an oxidation-reduction reaction using a reaction of a substrate or an enzyme, and substances measured by using an antibody-antigen reaction.

9. The method according to claim 8, wherein said biologically active material is selected from the group consisting of lactic acid oxidase, glucose oxidase, uricase, cholesterol oxidase, lactate dehydrogenase, isocitrate dehydrogenase, glutamic acid dehydrogenase, glucose 6-phosphate dehydrogenase, triglyceride, phospholipid, guanosine triphosphate, GOT, CPK, immunoglobulins and hormones.

10. The method according to claim 2, wherein said interfering substance is ascorbic acid.

11. The method according to claim 2, wherein said object substance is lactic acid or glucose.

12. A method for quantitative analysis of an analyte in a liquid sample which comprises:

i) contacting said sample with an electrode having coated thereupon a mediator substance which is electrochemically active upon reaction with a contaminant substance present in said sample;

ii) concurrently with step (i), contacting said sample with a composition comprising said mediator substance admixed with a biologically active substance wherein said biologically active substance specifically reacts with said analyte to produce an intermediate product moiety that is electrochemically reactive with said mediator;

iii) measuring a first electrical current at said electrode, at a first time, which results from the electrochemical reaction of said contaminant with said mediator;

iv) measuring a second electrical current at said electrode, at a second time later than said first time, which results from the electrochemical reaction of said intermediate product moiety with said mediator and also from the reaction of said contaminant with said mediator;

v) subtracting the current measured in step (iii) from the current measured in step (iv) to obtain a corrected current resulting only from the intermediate product moiety with said mediator; and

vi) comparing the corrected current obtained in step (v) to a calibration curve relating corrected current to analyte concentration to obtain a quantitative value of the analyte concentration.

13. The method of claim 12, wherein said biologically active substance is an oxidoreductase enzyme.

14. The method of claim 13, wherein said oxidoreductase enzyme is selected from the group consisting of lactate dehydrogenase, glucose oxidase, uricase, cholesterol oxidase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase and glutamic acid dehydrogenase.

15. The method of claim 12, wherein said mediator is ferricyanide.

16. The method of claim 12, wherein said electrode and said biologically active agent are each placed within a single vessel in separated locations.

17. The method of claim 12, wherein said biologically active substance is combined with a hydrophilic polymer.

18. The method of claim 12, said first time is between 0 and 6 seconds from the time of contact of said electrode with said sample.

19. The method claim 12, wherein said second time is 30 to 50 seconds from the time of contact of said electrode with said sample.

20. The method of claim 12, wherein said mediator is an electron mediator which can both accept and donate electrons.