Electrochemical assay method with novel p-phenylenediamine compound

• US 5,609,749 A
• Filed: 12/29/1994
• Issued: 03/11/1997
• Est. Priority Date: 12/29/1993
• Status: Expired due to Fees

• Current Assignee: Mochida Pharmaceutical Company Limited
• Original Assignee: Mochida Pharmaceutical Company Limited
• Inventors: Yamauchi, Tadakazu, Terasawa, Hideyuki
• Primary Examiner(s): Tung, T.
• Application Number:
  US08/366,040
• Time in Patent Office: 803 Days
• Field of Search: 204/403, 204/418, 205/777.5, 435/817, 436/806
• US Class Current: 205/777.5
• CPC Class Codes: C07C 215/16 the nitrogen atom of the amino group being further bound to carbon atoms of six-membered aromatic rings
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: G01N 33/48707 by electrical means G01N33/49, G01N33/493 take precedence
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property

Abstract

An enzyme electrode, specific binding or the like electrochemical assay method capable of performing always stably high detection sensitivity (responsibility) and reappearance even in the case of blood, urine and the like samples that contain interfering substances and also of applying suitably to disposable use, and a novel p-phenylenediamine compound which is used in the assay method. Particularly, an electrochemical assay method in which a substance in a liquid sample is assayed using at least one oxidoreductase, wherein an oxidoreductase, an electron mediator and an electrode which performs electron transfer with the mediator are arranged in the assay system, and a compound of the following formula [I] or a salt thereof is used as the mediator which is highly soluble in water, dried (by freeze-, vacuum- or air-drying) easily and stable under dry condition, shows a high electron transfer rate with enzymes and is almost free from the influence of interfering substances in blood, urine and the like samples: ##STR1## wherein R¹ to R⁴ may be the same or different from one another and each means a hydrogen, a straight- or branched-chain alkyl group having 1 to 4 carbon atoms, optionally having a substituent, providing that at least one of R¹ to R⁴ has one or more groups selected from hydroxyl, mercapto, carboxyl, phosphonooxy and sulfo.

Claims

1. A method of measuring a substance in a liquid biological sample which comprises

contacting with a sample containing said substance at least one oxidation-reduction enzyme and an electron mediator wherein said electron mediator is a compound represented by the following formula (I) or a salt thereof:

##STR9## wherein R¹, R², R³ and R⁴ may be the same or different from one another and each represents a straight- or branched-chain alkyl group having 1 to 4 carbon atoms with the proviso that at least one of R¹, R², R³ and R⁴ has at least one group selected from the group consisting of hydroxyl, mercapto, carboxyl, phosphonooxy and sulfo; and

measuring said substance with an electrode capable of performing electron transfer with said electron mediator.

2. The electrochemical assay method according to claim 1 wherein said at least one group of R¹, R², R³ or R⁴ is selected from the group consisting of a hydroxyl group and a carboxyl group.

3. The electrochemical assay method according to claim 1 wherein said compound represented by the formula (I) is selected from the group consisting of;

##STR10##

4. The electrochemical assay method according to claim 1 wherein said substance is selected from the group consisting of a substrate, an inhibitor, a cofactor and an activator of said oxidation-reduction enzyme, and said measuring of said substance is through a modulation of an enzyme reaction generated in response to the amount of said substrate, inhibitor, cofactor or activator of said oxidation-reduction enzyme which is detected as a modulation of electrochemical signals at said electrode.

5. The electrochemical assay method according to claim 1 wherein said substance specifically reacts with at least one specific binding substance specific for said substance and said oxidation-reduction enzyme acts as a label.

6. The electrochemical assay method according to claim 5 further comprising,

introducing the sample containing said substance into a matrix through a sample-introducing means;

reacting said substance in said matrix with a first specific binding substance and a second specific binding substance which is labeled with an oxidation-reduction enzyme capable of forming an electron transfer species that generates a signal at the electrode, or

reacting said sample in an area outside said matrix with a first specific binding substance and a second specific binding substance which is labeled with an oxidation-reduction enzyme capable of forming an electron transfer species that generates a signal at the electrode and introducing said sample into said matrix; and

detecting changes in the distribution of at least one molecular species among a complex of said oxidation-reduction enzyme-labeled second specific binding substance and said substance, a complex of said oxidation-reduction enzyme-labeled second specific binding substance, said substance and the first specific binding substance and a free form of the oxidation-reduction enzyme-labeled second specific binding substance which occur in response to the amount of said substance by measuring a signal modulation at the electrode, wherein said signal modulation is limited by the mass transfer of the electron transfer species generated by the oxidation-reduction enzyme.

7. The electrochemical assay method according to claim 5 further comprising

introducing the sample containing said substance into a matrix through a sample-introducing means;

competitively reacting said substance with a specific binding substance or a substance which competes with said substance to be assayed for said specific binding substance, or

introducing said sample into said matrix after competitively reacting said substance with a specific binding substance or a substance which competes with said substance to be assayed for the specific binding substance; and

detecting changes in the distribution of at least one oxidation-reduction enzyme-labeled molecular species among a complex of said specific binding substance and said substance to be assayed, a complex of said specific binding substance and the substance which competes with said substance for said specific binding substance, a free form of said specific binding substance and a free form of said substance which competes with said substance for said specific binding substance, which occur in response to the amount of said substance by measuring a signal modulation at the electrode, wherein said signal modulation is limited by the mass transfer of the electron transfer species generated by the oxidation-reduction enzyme.

8. A method of measuring a substance in a liquid sample comprising contacting with a sample containing said substance, at least one oxidation-reduction enzyme, and an electron mediator wherein said electron mediator is a compound represented by the following formula (II) or a salt thereof:

##STR11## wherein R⁵, R⁶, R⁷ and R⁸ may be the same or different from one another and each represents a hydrogen, a straight- or branched-chain alkyl group having 1 to 4 carbon atoms with the proviso that at least one of R⁵, R⁶, R⁷ and R⁸ is an alkyl group which has at least one group selected from the group consisting of hydroxyl, mercapto, carboxyl, phosphonooxy and sulfo, and at least one of R⁵, R⁶, R⁷ and R⁸ is hydrogen; and

measuring said substance with an electrode capable of performing electron transfer with an electron mediator.

9. The electrochemical assay method according to claim 8 wherein said at least one group of R⁵, R⁶, R⁷ and R⁸ has a hydroxyl group.

10. The electrochemical assay method according to claim 8 wherein said substance is selected from the group consisting of a substrate, an inhibitor, a cofactor and an activator of said oxidation-reduction enzyme, and said measuring of said substance is through a modulation of an enzyme reaction generated in response to the amount of said substrate, inhibitor, cofactor or activator of said oxidation-reduction enzyme which is detected as a modulation of electrochemical signals at said electrode.

11. The electrochemical assay method according to claim 8 wherein said substance to be assayed specifically reacted with at least one specific binding substance specific for the substance to be assayed and said oxidation-reduction enzyme acts as a label.

12. The electrochemical assay method according to claim 11 further comprising

introducing the sample containing said substance into a matrix through a sample-introducing means;

reacting said substance in said matrix with a first specific binding substance and a second specific binding substance which is labeled with an oxidation-reduction enzyme capable of forming an electron transfer species that generates a signal at the electrode, or

reacting said sample in an area outside said matrix with a first specific binding substance and a second specific binding substance which is labeled with an oxidation-reduction enzyme capable of forming an electron transfer species that generates a signal at the electrode and introducing said sample into said matrix; and

detecting changes in the distribution of at least one molecular species among a complex of said oxidation-reduction enzyme-labeled second specific binding substance and said substance, a complex of said oxidation-reduction enzyme-labeled second specific binding substance, said substance and the first specific binding substance and a free form of the oxidation-reduction enzyme-labeled second specific binding substance which occur in response to the amount of said substance by measuring a signal modulation at the electrode, wherein said signal modulation is limited by the mass transfer of the electron transfer species generated by the oxidation-reduction enzyme.

13. The electrochemical assay method according to claim 11 further comprising

introducing the sample containing said substance into a matrix through a sample-introducing means;

competitively reacting said substance with a specific binding substance or a substance which competes with said substance to be assayed for said specific binding substance, or

introducing said sample into said matrix after competitively reacting said substance with a specific binding substance or a substance which competes with said substance to be assayed for the specific binding substance; and

detecting changes in the distribution of at least one oxidation-reduction enzyme-labeled molecular species among a complex of said specific binding substance and said substance to be assayed, a complex of said specific binding substance and the substance which competes with said substance for said specific binding substance, a free form of said specific binding substance and a free form of said substance which competes with said substance for said specific binding substance which occur in response to the amount of said substance by measuring a signal modulation at the electrode, wherein said signal modulation is rate-limited by the mass transfer of the electron transfer species generated by the oxidation-reduction enzyme.