Cholinesterase immobilisation on the surface of screen-printed electrodes based on concanavalin A affinity

This paper reports a new method for the immobilisation of acetylcholine esterase (AChE) on the surface of screen-printed electrodes (SPEs) based on the affinity between the glycoprotein enzyme and concavalin A (Con A). The surface of the working electrode has been modified with a Nafion layer that contains graphite, the mediator 7,7,8,8-tetracyanoquinodimethane (TCNQ) and heptylamine. The enzyme-free SPEs were characterised by cyclic voltammetry in buffer solutions and amperometry using cysteamine as analyte. The AChE immobilisation process leads to the sandwich structure: electrode-carbohydrate-Con A-enzyme. The first step of the immobilisation is the covalent activation of an amino group bound in a Nafion layer. The following steps are based on the affinity. The non-specific adsorption has been totally eliminated using BSA solutions at two different pHs. Various amounts of enzyme, from 0.1 to more than 2 mIU AChE, have been loaded on the electrode surface. The method offers the advantage of a free diffusion, which allows obtaining a response time of less than 2 min. An operational stability of more than 10 measurements was registered, while the active surface of the electrode was successfully reloaded for three consecutive times without any important change of the analytical performances. (C) 2004 Elsevier B.V. All rights reserved.