Amperometric cholesterol biosensors based on the electropolymerization of pyrrole and the electrocatalytic effect of Prussian-Blue layers helped with self-assembled monolayers.

Three cholesterol biosensor configurations based on the formation of a layer of Prussian-Blue (PB) on a Pt electrode for the electrocatalytic detection of the H(2)O(2) generated during the enzymatic reaction of cholesterol with cholesterol oxidase (ChOx) were constructed. The enzyme was entrapped within a polypyrrole (PPy) layer electropolymerized onto the PB film. The influence of the formation of self-assembled monolayers (SAMs) on the Pt surface on the adherence and stability of the PB layer and the formation of an outer layer of nafion (Nf) as a means of improving selectivity were both studied. A comparative study was made of the analytical properties of the biosensors corresponding to the three configurations named: Pt/PB/PPy-ChOx, Pt/SAM/PB/PPy-ChOx and Pt/SAM/PB/PPy-ChOx/Nf. The sensitivity (from 600 to 8500nAmM(-1)cm(-2)) and selectivity of the developed biosensors permitted the determination of the cholesterol content in reference and synthetic serum samples. The detection limit for the Pt/SAM/PB/PPy-ChOx/Nf biosensor was 8muM. Formation of the SAM on the electrode surface and covering with a Nf film considerably improved the stability and lifetime of the biosensor based on the catalytic effect of the PB layer (as the PB layer was retained longer on the electrode), and the Nf layer protects the enzyme from the external flowing solutions. Lifetime is up to 25 days of use. The formation of the SAM also has an effect on the charge transfer and the formation of the PB layer.