SUBFEMTOMOLAR DETERMINATION OF ALKALINE-PHOSPHATASE AT A DISPOSABLE SCREEN-PRINTED ELECTRODE MODIFIED WITH A PERFLUOROSULFONATED IONOMER FILM

A carbon-based ink composed of graphite particles and polystyrene was used in association with a manual screen-printer to prepare electrodes on a flexible polyester film. The screen-printing step was followed b y a drying step which was achieved within 1 h at room temperature. The screen-printed electrode (SPE) was coated by a polyanionic Nafion fil m in which electroactive cationic species could be accumulated A detec tion limit of 10(-9) M was thus obtained by cyclic voltammetric (CV) d etermination of [[(4-hydroxyphenyl)amino]carboxyl]cobaltocenium (P+) a fter accumulation for 60 min. Since this cationic phenol derivative P could be generated from the corresponding anionic ester phosphate (S- ) by alkaline phosphatase (AP) hydrolysis, the new S- substrate was sy nthesized and the sensitive indirect CV determination of AP was perfor med at a Nafion-coated SPE, The S- substrate did not interfere on the electrochemical response of P+ owing to the permselectivity of Nafion, An AP detection of 4 x 10(-16) M was thus achieved in Tris buffer (pH 9) after hydrolysis of S- (10(-4) M) to P+ (Michaelis constant K-m = 48 mu M) and simultaneous accumulation of P+ within Nafion for 1 h. Th e Nafion-SPE was stack successfully to the bottom of a microwell, maki ng it possible to work with solution volumes ranging from 50 to 250 mu L, web adapted to enzyme immunoassays.