A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles

Abstract

A mediator-free phenol biosensor was developed. The low-isoelectric point tyrosinase was adsorbed on the surface of high-isoelectric point ZnO nanoparticles (nano-ZnO) facilitated by the electrostatic interactions and then immobilized on the glassy carbon electrode via the film forming by chitosan. It was found that the nano-ZnO matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retaining its activity to a large extent. Moreover, there is no need to use any other electron mediators. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at −200 mV (vs. saturated calomel electrode). The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within 10 s, and the sensitivity was as high as 182 μA mmol −1 L. The linear range for phenol determination was from 1.5 × 10 −7 to 6.5 × 10 −5 mol L −1 with a detection limit of 5.0 × 10 −8 mol L −1 obtained at a signal/noise ratio of 3. In addition, the apparent Michaelis–Menten constant ( K m app ) and the stability of the enzyme electrode were estimated. The performance of the developed biosensor was compared with that of biosensors based on other immobilization matrices.