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 by a drying step which was achieved within 1 h at room temperature. The screen-printed electrode (SPE) was coated by a polyanionic Nafion film in which electroactive cationic species could be accumulated. A detection limit of 10-9 M was thus obtained by cyclic voltammetric (CV) determination of [[(4-hydroxyphenyl)amino]carboxyl]cobaltocenium (P+) after 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 synthesized and the sensitive indirect CV determination of AP was performed 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 × 10-16 M was thus achieved in Tris buffer (pH 9) after hydrolysis of S- (10-4 M) to P+ (Michaelis constant Km = 48 μM) and simultaneous accumulation of P+ within Nafion for 1 h. The Nafion-SPE was stuck successfully to the bottom of a microwell, making it possible to work with solution volumes ranging from 50 to 250 μL, well adapted to enzyme immunoassays.