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 constructed by a layer-by-layer nanothin film formation using cholesterol oxidase (COx) and poly(styrenesulfonate) on a monolayer of microperoxidase covalently-immobilized on Au–alkanethiolate electrodes. Cyclic voltammograms (CVs) of microperoxidase covalently-immobilized on mercaptopropionic acid- and aminoethanethiol-monolayer electrodes showed a redox wave with the formal potential of 0.40 V (versus Ag | AgCl | NaCl (sat.)). The formal potential and the apparent heterogeneous electron-transfer rate of immobilized microperoxidase were not specific with the inner Au–alkanethiolate layer. The biosensor shows a linear current response for cholesterol at the applied potential of 0 V in the concentration range of 0.2–3.0 mM with a correlation coefficient of 0.969. The current response of the biosensor for cholesterol was very rapid (response time <20 s) and was highly reproducible; sample standard deviation of the current response at the concentration of 1.5 mM in five individual determinations was 0.09. The magnitude of the amperometric response for cholesterol was 0.13 μA cm−2 at the concentration of 1.5 mM. The inertness and stability of the biosensor towards the potential electrical interferents, l-ascorbic acid, pyruvic acid and uric acid, was tested, and it was found that the biosensor rapidly responses to the addition of cholesterol even in the presence of these interferents.