Method for measuring concentrations of substrates in a sample liquid by using a biosensor

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 T.sub.1, T.sub.1 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 T.sub.2, T.sub.2 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 T.sub.2, T.sub.2 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 T.sub.1, T.sub.1 beginning upon supplying the sample liquid, for measuring a response current value I.sub.1 ;
• (e) applying a voltage between the working electrode and the counter electrode in the electrode system after the set period of time T.sub.2, T.sub.2 beginning upon supplying the sample liquid, for measuring a response current value I.sub.2 ;
• (f) obtaining a concentration C.sub.1 of the first substrate in the sample liquid corresponding to the current value I.sub.1 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 I.sub.1 ' corresponding to the concentration C.sub.1 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 C.sub.2 of the second substrate in the sample liquid corresponding to a current value calculated by subtracting the current value I.sub.1 ' from the current value I.sub.2 based on the relationship between the concentration of the second substrate and the response current value determined in step (c).