Cholesterol oxidase modified gold electrodes as bioanalytical devices

Cholesterol oxidase (ChOx) has been immobilized by direct adsorption on gold electrodes. The resulting ChOx monolayers have been characterized using atomic force microscopy (AFM) under liquid conditions and quartz crystal microbalance (QCM) techniques. The immobilized enzyme retains its catalytic activity, thus spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). The replacement, in the enzymatic reaction, of the natural electron acceptor (O2) by an artificial mediator has been also evaluated, in particular, hydroxymethylferrocene (HMF), thionin, nile blue and azure A, have been studied as electron acceptors for reduced ChOx. In addition, the influence of the low cholesterol solubility on the experimental conditions using redox mediators was also discussed. Finally, the response of the enzymatic electrode to varying cholesterol concentrations has been obtained by measuring directly the H2O2 generated in the enzymatic reaction. Cholesterol can be determined amperometrically at +0.5 V (versus SSCE) with a detection limit of 60 μM and a sensitivity of 0.13 μA mM−1.