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 whole blood containing 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 amperometry; 
 
       wherein the test target is glucose, the oxidation-reduction enzyme is CyGDH, and a Ru compound ruthenium(III) complex is used as the electron mediator, the method further comprising:
 applying constant voltage to the reagent layer; 
 dissolving the electron mediator directly in the whole blood containing the test target to form a solution; 
 detecting the solution by applying the voltage to the reagent layer for measurement; 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 electron mediator through a standard oxidation-reduction potential (versus a standard hydrogen electrode) between ferrocyanide ions and ferricyanide ions, 
       wherein the concentration of the test target is measured at any point between 3 and 15 seconds after the whole blood containing the test target is supplied to the reagent layer, and 
       wherein the system is configured to be without any interferent mitigating elements. 
     
     
       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:
 constructing a reaction system by supplying whole blood containing the test target directly to a reagent layer containing an oxidation-reduction enzyme and an electron mediator;   measuring the concentration of the test target by amperometry;   
       wherein the oxidation-reduction enzyme is selected from the group consisting of glucose oxidase, 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, NADH oxidase, uric acid oxidase (uricase), urease, and dihydrolipoamide dehydrogenase (diaphorase), and the electron mediator is a ruthenium(III) complex,
 the method further comprising: 
 applying voltage to the reagent layer; 
 dissolving the electron mediator directly in the whole blood containing the test target to form a solution; 
 detecting the solution by applying the voltage to the reagent layer for measurement; and 
 computing the concentration of the test target on the basis of a response current value from the reagent layer; 
 
       wherein the voltage is selected from a range of about 100 mV to 500 mV; 
       wherein the concentration of the test target is measured at any point between 3 and 15 seconds after the whole blood containing the test target is supplied to the reagent layer, and 
       wherein the system is configured to be without any interferent mitigating elements. 
     
     
       4. The method according to claim 3, wherein the voltage is selected from a range of about 100 mV to 350 mV. 
     
     
       5. The method according to claim 3, wherein the voltage is selected from a range of about 100 mV to 300 mV. 
     
     
       6. The method according to claim 3, wherein the test target is glucose and the oxidation-reduction enzyme is glucose oxidase. 
     
     
       7. A method for measuring a concentration of glucose, the method comprising:
 constructing a reaction system by supplying whole blood containing the glucose to a reagent layer containing an oxidation-reduction enzyme and an electron mediator;   measuring the concentration of the glucose by amperometry;   
       wherein the oxidation-reduction enzyme is glucose oxidase and the electron mediator is a ruthenium(III) complex
 the method further comprising: 
 applying voltage to the reagent layer; 
 dissolving the electron mediator directly in the glucose to form a solution; 
 detecting the solution by applying the voltage to the reagent layer for measurement; and 
 computing the concentration of the glucose on the basis of a response current value from the reagent layer; 
 
       wherein the voltage is selected from a range of about 100 mV to 350 mV; 
       wherein the concentration of the glucose is measured at any point between 3 and 15 seconds after the whole blood containing the glucose is supplied to the reagent layer, and 
       wherein the system is configured to be without any interferent mitigating elements. 
     
     
       8. A concentration measuring apparatus and a measurement test instrument, 
       wherein the measurement test instrument comprises
 a reagent layer; 
 a first electrode; and 
 a second electrode, 
 
       wherein the reagent layer contains an oxidation-reduction enzyme selected from the group consisting of glucose oxidase, 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, NADH oxidase, uric acid oxidase (uricase), urease, and dihydrolipoamide dehydrogenase (diaphorase); and a ruthenium(III) complex, and 
       wherein the concentration measuring apparatus comprises
 a voltage applier for applying voltage between the first and second electrodes; 
 a current value measurer for measuring the response current value when voltage selected from the range of about 100 mV to 500 mV is applied between the first and second electrodes; 
 a computer for computing the concentration of the test target on the basis of the response current value; and 
 a controller for controlling the current value measurement performed by the current value measurer, 
 
       wherein the controller is constituted such that the response current value necessary for computation by the computer is measured by the current value measurer at any point between 3 and 15 seconds after whole blood containing the test target is supplied directly to the reagent layer to form a solution with the ruthenium(III) complex, and 
       wherein the concentration measuring apparatus and the measurement test instrument are configured to be without any interferent mitigating elements. 
     
     
       9. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the test target is glucose and the oxidation-reduction enzyme is glucose oxidase. 
     
     
       10. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the voltage is selected from a range of about 100 mV to 350 mV. 
     
     
       11. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the voltage is selected from a range of about 100 mV to 300 mV. 
     
     
       12. The method according to claim 3, wherein the ruthenium(III) complex is provided as a micropowder in the reagent layer. 
     
     
       13. The method according to claim 3, wherein the ruthenium(III) complex is substantially uniformly dispersed in the reagent layer. 
     
     
       14. The method according to claim 7, wherein the ruthenium(III) complex is a micropowder in the reagent layer. 
     
     
       15. The method according to claim 7, wherein the ruthenium(III) complex is substantially uniformly dispersed in the reagent layer. 
     
     
       16. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the ruthenium(III) complex is provided as a micropowder in the reagent layer. 
     
     
       17. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the ruthenium(III) complex is substantially uniformly dispersed in the reagent layer. 
     
     
       18. The concentration measuring apparatus and a measurement test instrument according to claim 8, wherein the first and second electrodes are formed by screen printing with a carbon paste. 
     
     
       19. The method according to claim 1, wherein the concentration of the test target is measured at any point between 3 and 5 seconds. 
     
     
       20. The method according to any one of claims 3, 4, and 5, wherein the voltage that is applied to the reagent layer is constant voltage. 
     
     
       21. The method according to claim 4, wherein the voltage that is applied to the reagent layer is constant voltage. 
     
     
       22. The concentration measuring apparatus and a measurement test instrument according to any one of claims 8, 9, 10 and 11, wherein the voltage that is applied is constant voltage.