Layer-by-layer construction of an active multilayer enzyme electrode applicable for direct amperometric determination of cholesterol

A biosensor for direct amperometric determination of cholesterol was constr ucted by a layer-by-layer nanothin film formation using cholesterol oxidase (COx) and poly(styrenesulfonate) on a monolayer of microperoxidase covalen tly-immobilized on Au-alkanethiolate electrodes. Cyclic voltammograms (CVs) of microperoxidase covalently-immobilized on mercaptopropionic acid- and a minoethanethiol-monolayer electrodes showed a redox wave with the formal po tential of 0.40 V (versus Ag \ AgCl \ NaCl (sat.)). The formal potential an d the apparent heterogeneous electron-transfer rate of immobilized microper oxidase were not specific with the inner Au-alkanethiolate layer. The biose nsor shows a linear current response for cholesterol at the applied potenti al of 0 V in the concentration range of 0.2-3.0 mM with a correlation coeff icient of 0.969. The current response of the biosensor for cholesterol was very rapid (response time < 20 s) and was highly reproducible; sample stand ard deviation of the current response at the concentration of 1.5 mM in fiv e individual determinations was 0.09. The magnitude of the amperometric res ponse for cholesterol was 0.13 muA cm(-2) at the concentration of 1.5 mM. T he inertness and stability of the biosensor towards the potential electrica l interferents, L-ascorbic acid, pyruvic acid and uric acid. was tested, an d it was found that the biosensor rapidly responses to the addition of chol esterol even in the presence of these interferents. (C) 2001 Elsevier Scien ce B.V. All rights reserved.