Surface acoustic wave method for in situ determination of the amounts of enzyme–substrate complex formed on immobilized glucose oxidase during catalytic reaction

Abstract

Surface acoustic waves (SAWs) have been applied to the in situ determination of the amounts of an enzyme–substrate complex formed on an immobilized enzyme surface during the catalytic reaction. The input and output interdigital transducer (IDT) electrodes were photolithographically fabricated on a LiNbO₃ crystal for the generation of Rayleigh SAWs and on LiTaO₃ crystals for shear horizontal SAWs. The characteristics of propagation of these SAWs in contact with liquid phases were examined. Glucose oxidase (GOX) was immobilized between two IDT electrodes by silane coupling, The functionalization of which was characterized by X-ray photoelectron spectroscopy. The simultaneous determination of the reaction rates and of the adsorbed amounts of glucose was performed by combining an electrochemical method with the SAW method. When the concentration of glucose in the solution was changed, the amount of the GOX–glucose complex formed varied in rough proportion to the reaction rate, which gives experimental evidence for the establishment of pseudo-equilibrium between the adsorbed glucose and glucose in the solution and hence for the validity of the Michaelis–Menten equation. The reaction rate per enzyme–substrate complex was nearly constant, independent of the amount of glucose adsorbed. The formation of the enzyme–substrate complex showed a bell-shaped change with increase in pH, having a maximum at pH 7, which is quite similar to that observed for the dependence of reaction rate on pH. The catalytic functions of the immobilized active GOX were concluded to be almost equivalent.