Method for measuring concentrations of substrates in a sample liquid by using a biosensor
First Claim
1. A method for measuring concentrations of a first substrate and a second substrate in a sample liquid by using a biosensor having an electrically insulating substrate, an electrode system including a working electrode and a counter electrode formed on the insulating substrate, and a reaction layer including a first enzyme that can specifically react with the first substrate, a second enzyme that can specifically react with the second substrate and an electron acceptor;
- the method comprising the steps of;
(a) determining a relationship between a concentration of the first substrate and a response current value measured with an auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured for a known plurality of solutions having different known concentrations of the first substrate after set period of time T1, T1 beginning upon supplying a solution including the first substrate to the auxiliary testing biosensor;
(b) determining a relationship between the concentration of the first substrate and a response current value measured with the auxiliary testing biosensor after a set period of time T2, T2 beginning upon supplying the solution including the first substrate;
(c) determining a relationship between a concentration of the second substrate and a response current value measured with another auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured for a plurality of solutions having different known concentrations of the second substrate after the set period of time T2, T2 beginning upon supplying a solution including the second substrate to the auxiliary testing biosensor;
(d) supplying a sample liquid including the first and second substrates to the biosensor and applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T1, T1 beginning upon supplying the sample liquid, for measuring a response current value I1 ;
(e) applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T2, T2 beginning upon supplying the sample liquid, for measuring a response current value I2 ;
(f) obtaining a concentration C1 of the first substrate in the sample liquid corresponding to the current value I1 based on the relationship between the concentration of the first substrate and the response current value determined in step (a);
(g) obtaining a response current value I1 '"'"' corresponding to the concentration C1 of the first substrate in the sample liquid based on the relationship between the concentration of the first substrate and the response current value determined in step (b); and
(h) obtaining a concentration C2 of the second substrate in the sample liquid corresponding to a current value calculated by subtracting the current value I1 '"'"' from the current value I2 based on the relationship between the concentration of the second substrate and the response current value determined in step (c).
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Abstract
The present invention provides a method for measuring concentrations of the first and second substrates in a sample liquid by using a biosensor. The biosensor has an electrically insulating substrate, an electrode system, and a reaction layer including a first enzyme, a second enzyme and an electron acceptor. The method includes the steps of measuring a response current value I1 of a predetermined period of time T1 after supplying the sample liquid including the first and second substrates to the biosensor; measuring a response current value I2 of a predetermined period of time T2 after the supply of the sample liquid; obtaining the concentration C1 of the first substrate corresponding to the current value I1 based on the previously determined relationship between the concentration of the first substrate and the response current value of the predetermined period of time T1 after the supply; obtaining a response current value I1 '"'"' corresponding to the concentration C1 of the first substrate in the sample liquid based on the previously determined relationship between the concentration of the first substrate and the response current value of the predetermined period of time T2 after the supply; and obtaining the concentration C2 of the second substrate in the sample liquid corresponding to the current value calculated by subtracting the current value I1 '"'"' from the current value I2 based on the previously determined relationship between the concentration of the second substrate and the response current value of the predetermined period of time T2 after the supply.
174 Citations
20 Claims
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1. A method for measuring concentrations of a first substrate and a second substrate in a sample liquid by using a biosensor having an electrically insulating substrate, an electrode system including a working electrode and a counter electrode formed on the insulating substrate, and a reaction layer including a first enzyme that can specifically react with the first substrate, a second enzyme that can specifically react with the second substrate and an electron acceptor;
the method comprising the steps of; (a) determining a relationship between a concentration of the first substrate and a response current value measured with an auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured for a known plurality of solutions having different known concentrations of the first substrate after set period of time T1, T1 beginning upon supplying a solution including the first substrate to the auxiliary testing biosensor; (b) determining a relationship between the concentration of the first substrate and a response current value measured with the auxiliary testing biosensor after a set period of time T2, T2 beginning upon supplying the solution including the first substrate; (c) determining a relationship between a concentration of the second substrate and a response current value measured with another auxiliary testing biosensor having the same structure as the biosensor, the response current value being measured for a plurality of solutions having different known concentrations of the second substrate after the set period of time T2, T2 beginning upon supplying a solution including the second substrate to the auxiliary testing biosensor; (d) supplying a sample liquid including the first and second substrates to the biosensor and applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T1, T1 beginning upon supplying the sample liquid, for measuring a response current value I1 ; (e) applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T2, T2 beginning upon supplying the sample liquid, for measuring a response current value I2 ; (f) obtaining a concentration C1 of the first substrate in the sample liquid corresponding to the current value I1 based on the relationship between the concentration of the first substrate and the response current value determined in step (a); (g) obtaining a response current value I1 '"'"' corresponding to the concentration C1 of the first substrate in the sample liquid based on the relationship between the concentration of the first substrate and the response current value determined in step (b); and (h) obtaining a concentration C2 of the second substrate in the sample liquid corresponding to a current value calculated by subtracting the current value I1 '"'"' from the current value I2 based on the relationship between the concentration of the second substrate and the response current value determined in step (c). - View Dependent Claims (2, 3, 4, 5, 6, 7, 17, 18, 19, 20)
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8. A method for measuring concentrations of a first substrate and a second substrate in a sample liquid by using a biosensor having an electrical insulating substrate, an electrode system including a working electrode and a counter electrode, and a reaction layer including a first enzyme that can specifically react with the first substrate, a second enzyme that can specifically react with the second substrate and an electron acceptor;
the method comprising the steps of; (a) obtaining a response curve a indicating a relationship between a concentration of the first substrate and a response current value measured with an auxiliary testing biosensor for a plurality of solutions having different known concentrations of the first substrate after a set period of time T1, T1 beginning upon supplying a solution including the first substrate to the auxiliary testing biosensor, the set period of time T1 being a time required for at least part of the first substrate to react with the first enzyme; (b) obtaining a response curve b indicating a relationship between the concentration of the first substrate and a response current value measured with the auxiliary testing biosensor after a set period of time T2, T2 beginning upon supplying the solution including the first substrate to the auxiliary testing biosensor, the set period of time T1 being a time required for at least part of the second substrate to react with the second enzyme; (c) obtaining a response curve d indicating a relationship between a concentration of the second substrate and a response current value measured with the auxiliary testing biosensor for a plurality of solutions having different known concentrations of the second substrate after the set period of time T2, T2 beginning upon supplying the solution including the second enzyme; (d) applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T1, T1 beginning upon supplying the sample liquid including the first substrate and the second substrate to the biosensor, to measure a response current value I1 ; (e) applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T2, T2 beginning upon supplying the sample liquid including the first substrate and the second substrate to the biosensor, to measure a response current value I2 ; (f) obtaining a concentration of the first substrate in the sample liquid corresponding to the current value I1 based on the response curve a obtained in step (a); (g) obtaining a response current value I1 '"'"' corresponding to the concentration C1 of the first substrate in the sample liquid based on the response curve b obtained in step (b); and (h) obtaining a concentration C2 of the second substrate in the sample liquid corresponding to the current value calculated by subtracting the current value I1 '"'"' from the current value I2 based on the response curve d obtained in step (c). - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16)
Specification