Pseudo-equilibrium approach to the design and use of enzyme-based amperometric biosensors evaluated using a sensor for hydrogen peroxide Original Research Article

This paper describes a pseudo-equilibrium approach to the measurement and data-processing steps in the use of enzyme-based amperometric biosensors for substrates. The goal of the method is to obtain the response corresponding to reaction of all substrate in a fixed volume of solution with the expectation that this will improve the linear range and ruggedness relative to the conventional steady-state option. The biosensor is used in a rotating disk mode such that data for current versus time can be monitored with and without rotation of the sensor. With the sensor rotating at moderately high speed, current is monitored until an initial steady-state response is obtained after which the rotation is stopped and current is monitored until a second steady-state response is obtained. Under controlled conditions, the transition form the first to the second steady-state condition corresponds to reaction of virtually all of the substrate in a small fixed volume of solution controlled by the dimensions of the diffusion layer. Integration of current as a function of time yields the charge corresponding to reaction of substrate in the fixed volume established by the diffusion process. Evaluation of this pseudo-equilibrium option using a peroxidase-based sensor for hydrogen peroxide yielded a linear range extending to at least four times the Michaelis constant and a pH dependence approximately 40-fold less than steady-state currents obtained from the same data sets.