Comprehensive study of bioanalytical platforms:: Xanthine oxidase

A comprehensive study of a general bioanalytical platform for biosensor applications is presented using xanthine oxidase (XnOx) as a case study within the framework of developing approaches of broad applicability. In this context, emphasis is placed on amperometric biosensors based on XnOx, which has been immobilized by covalent binding to gold electrodes modified with dithiobis-N-succinimidyl propionate. The immobilized XnOx layers have been characterized using atomic force microscopy under liquid conditions and quartz crystal microbalance techniques. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy. Redox dyes of phenothiazine derivatives, specifically, thionine and methylene blue, have been found to work well as electron acceptors for reduced XnOx. The kinetic parameters and equilibrium constants of the mediated enzymatic oxidation of xanthine in the presence of the above-mentioned redox dyes have been calculated. The response of the enzymatic electrode to varying xanthine concentrations has been obtained in the presence of thionine or methylene blue as redox mediator in solution. Under these conditions, xanthine could be determined amperometrically at +0.2 V versus SSCE.