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 K m = 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.