Concentration measuring method, concentration test instrument, and concentration measuring apparatus

Abstract

The present invention relates to technology for constructing a reaction system including a test target, an oxidation-reduction enzyme, and an electron mediator, and measuring the concentration of the test target by an electrochemical process. A Ru compound is used as the electron mediator. The present invention provides a concentration test instrument including a substrate, first and second electrodes formed on the substrate, and a reagent layer formed as a solid. The reagent layer contains an oxidation-reduction enzyme and a Ru compound, and is constituted so as to dissolve and construct a liquid phase reaction system when a sample liquid is supplied.

The invention claimed is: 
     
       1. A method for measuring a concentration of a test target, the method comprising:
 constructing a reaction system by supplying the test target to a reagent layer containing an oxidation-reduction enzyme and an electron mediator; measuring the concentration of the test target by utilizing;   wherein the test target is glucose, the oxidation-reduction enzyme is CyGDH, and a Ru compound is used as the electron mediator,   the method further comprising:   applying constant voltage to the reagent layer; and   computing the concentration of the test target on the basis of a response current value from the reagent layer;   wherein the constant voltage is no greater than 300 mV and selected from a range from a standard oxidation-reduction potential (versus a standard hydrogen electrode) between an oxidant and a reductant of the Ru compound through a standard oxidation-reduction potential (versus a standard hydrogen electrode) between ferrocyanide ions and ferricyanide ions.   
     
     
       2. The method according to  claim 1 , wherein the Ru compound is oxidative Ru(III) complex expressed by the following chemical formula:
   [Ru(NH 3 ) 5 X] n+     (where X in the formula is NH 3  or a halogen ion, and n+ in the formula is the valence of the oxidative Ru(III) complex as determined by a type of X).   
     
     
       3. A method for measuring a concentration of a test target, the method comprising:
 contacting whole blood comprising the test target directly with an oxidation-reduction enzyme and an electron mediator disposed between electrodes disposed on a substrate such that the electron mediator is dissolved;   applying voltage to the electrodes; and   computing the concentration of the test target by amperometry, wherein   the oxidation-reduction enzyme is selected from the group consisting of GOD, GDH, αGDH, CyGDH, cholesterol dehydrogenase, cholesterol oxidase, lactic acid dehydrogenase, lactic acid oxidase, ascorbic acid dehydrogenase, ascorbic acid oxidase, alcohol dehydrogenase, alcohol oxidase, fructose dehydrogenase, 3-hydroxybutyric acid dehydrogenase, pyruvic acid oxidase, uric acid oxidase (uricase), urease, and dihydrolipoamide dehydrogenase (diaphorase), and   the electron mediator is an oxidative ruthenium(III) complex where the complex only contains monodentate ligands.   
     
     
       4. The method according to claim 3, wherein at least one of the oxidation-reduction enzyme and the electron mediator is directly allocated on the substrate. 
     
     
       5. The method according to claim 3, wherein the concentration of the test target is measured at any point between 3 and 15 seconds after detection of the supply of the whole blood.  
     
     
       6. The method according to claim 3, wherein the voltage is selected from a range of about 100 mV to 500 mV. 
     
     
       7. The method according to claim 3, wherein computing the concentration of the test target is configured to convert the response current value into a voltage value, and to check the voltage value against a predetermined calibration curve expressing the relationship between the voltage and the test target concentration. 
     
     
       8. The method according to claim 3, wherein the whole blood is applied to the oxidation-reduction enzyme and the mediator without passing a filter. 
     
     
       9. The method according to claim 3, wherein a voltage is applied to the electrodes disposed on the substrate before the whole blood directly contacts the oxidation-reduction enzyme and the electron mediator.  
     
     
       10. A concentration measuring apparatus and a measurement test instrument,
 wherein the measurement test instrument comprises
 an oxidation-reduction enzyme; 
 an electron mediator; 
 a first electrode; and 
 a second electrode, 
   wherein
 the oxidation-reduction enzyme is selected from the group consisting of GOD, GDH, αGDH, CyGDH, cholesterol dehydrogenase, cholesterol oxidase, lactic acid dehydrogenase, lactic acid oxidase, ascorbic acid dehydrogenase, ascorbic acid oxidase, alcohol dehydrogenase, alcohol oxidase, fructose dehydrogenase, 3-hydroxybutyric acid dehydrogenase, pyruvic acid oxidase, uric acid oxidase (uricase), urease, and dihydrolipoamide dehydrogenase (diaphorase); 
 the electron mediator is an oxidative ruthenium(III) complex where the complex only contains monodentate ligands, and 
 the concentration measuring apparatus comprises 
 a voltage applier configured to apply voltage between the first and second electrodes; 
 a current value measurer configured to measure the response current value when voltage is applied between the first and second electrodes; 
 a computer configured to compute the concentration of a test target on the basis of the response current value; and 
 a controller configured to control the current value measurement performed by the current value measurer, wherein 
 the controller is further configured such that the response current value resulting from direct contact of the whole blood with the oxidation-reduction enzyme and the electron mediator and necessary for computation by the computer is measured by the current value measurer. 
   
     
     
       11. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the measurement test instrument further comprises a substrate, wherein at least one of the oxidation-reduction enzyme and the electron mediator is directly allocated on the substrate.  
     
     
       12. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the response current value is measured by the current value measurer at any point after between 3 and 15 seconds after detection of the supply of the whole blood.  
     
     
       13. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the voltage is selected from a range of about 100 mV to 500 mV.  
     
     
       14. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the computer is configured to convert the response current value into a voltage value, and to check the voltage value against a predetermined calibration curve expressing the relationship between the voltage and the test target concentration.  
     
     
       15. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the measurement test instrument is configured without a filter. 
     
     
       16. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the voltage applier is configured to apply voltage to the first and second electrodes to detect the application of the whole blood before direct contact of the whole blood with the oxidation-reduction enzyme and the electron mediator.  
     
     
       17. The method according to claim 3, wherein the oxidative ruthenium(III) complex is expressed by the following chemical formula:
   [Ru(NH 3 ) 5 X] n+     
       where X is selected from the group consisting of NH 3 , a halogen ion, CN, pyridine, nicotinamide and H 2 O, and where X determines the valence n +  of the oxidative ruthenium(III) complex.  
     
     
       18. The method according to claim 17 wherein X is NH 3  and n + =3.  
     
     
       19. A device comprising a concentration measuring apparatus together with a concentration test instrument, 
       wherein:
 the concentration test instrument comprises a substrate; at least first and second electrodes formed on the substrate; a solid reagent layer comprising an oxidation-reduction enzyme selected from the group consisting of GOD, GDH, αGDH and CyGDH; and an oxidative ruthenium(III) complex where the complex only contains monodentate ligands, wherein the reagent layer is constituted so as to dissolve to a liquid phase reaction system when a sample liquid containing a test target is supplied; and 
 
       wherein:
 the concentration measuring apparatus comprises:
 a voltage applier for applying voltage between the first and second electrodes to oxidize a reductive ruthenium(II) complex to the oxidative ruthenium(III) complex; 
 a current value measurer for measuring the response current value correlated with a quantity of electrons released by the ruthenium(II) complex when voltage is applied between the first and second electrodes; and 
 a computer for computing the concentration of the test target on the basis of the response current value. 
 
 
     
     
       20. The device according to claim 19, wherein the GDH is a compound in which pyrroquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) serves as a coenzyme. 
     
     
       21. The device according to claim 19, wherein the concentration of the test target is measured at any point between 3 and 15 seconds after detection of the supply of the sample liquid containing the test target. 
     
     
       22. The device according to claim 19, wherein the voltage is selected from a range of about 100 mV to 500 mV.  
     
     
       23. The device according to claim 19, wherein the oxidative ruthenium(III) complex is expressed by the following chemical formula:
   [Ru(NH 3 ) 5 X] n+     
       where X is selected from the group consisting of NH 3 , a halogen ion, CN, pyridine, nicotinamide and H 2 O, and where X determines the valence n +  of the oxidative ruthenium(III) complex.  
     
     
       24. A method for measuring in blood a concentration of a test target, the method comprising:
 constructing a reaction system containing the test target, an electron mediator and an oxidation-reduction enzyme selected from the group consisting of GOD, GDH, αGDH and CyGDH;   measuring the concentration of the test target by amperometry, wherein an oxidative ruthenium(III) complex only containing monodentate ligands is used as the electron mediator, the method further comprising:   a first step of preparing a reductive ruthenium(II) complex in the reaction system;   a second step of applying voltage to the reaction system to oxidize the ruthenium(II) complex to the ruthenium(III) complex, and measuring the response current value corresponding to the quantity of electrons released by the ruthenium(II) complex; and   a third step of computing the concentration of the test target on the basis of the response current measured in the second step.   
     
     
       25. The method according to claim 24, wherein the GDH is a compound in which pyrroquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) serves as a coenzyme. 
     
     
       26. The method according to claim 24, wherein the concentration of the test target is measured at any point between 3 and 15 seconds after detection of the supply of the blood containing the test target. 
     
     
       27. The method according to claim 24, wherein the voltage is selected from a range of about 100 mV to 500 mV. 
     
     
       28. The method according to claim 24, wherein the oxidative ruthenium(III) complex is expressed by the following chemical formula:
   [Ru(NH 3 ) 5 X] n+     
       where X is selected from the group consisting of NH 3 , a halogen ion, CN, pyridine, nicotinamide and H 2 O, and where X determines the valence n +  of the oxidative ruthenium(III) complex. 
     
     
       29. The concentration measuring apparatus and measurement test instrument according to claim 10, wherein the oxidative ruthenium(III) complex is expressed by the following chemical formula:
   [Ru(NH 3 ) 5 X] n+     
       where X is selected from the group consisting of NH 3 , a halogen ion, CN, pyridine, nicotinamide and H 2 O, and where X determines the valence n + of the oxidative ruthenium(III) complex.