Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides: Comparative study Original Research Article

A comparison between several acetylcholinesterase (AChE) immobilization procedures on the 7,7,8,8-tetracyanoquinodimethane (TCNQ)-modified graphite working electrodes is presented. The immobilization methods employed crosslinking with glutaraldehyde in presence of BSA protein and photopolymerization with poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ). The main variations were related to the enzyme charge in each electrode and the enzyme conditioning and storage conditions after immobilization. Initially, the enzyme–substrate reaction was carried out and the following parameters were chrono-amperometrically and -coulometrically monitored: current intensities, time to stabilize the current response, and the mass transfer represented by the Coulomb charge. The screen-printed biosensors that presented best characteristics were then used to perform the inhibition assays and to verify the sensitivity against the following NMC insecticides: aldicarb, carbaryl, carbofuran, and methomyl. In general, diffusion of electrons into the sensitive layer, mass transfer, and time to stabilize the current were adequate in all cases. The Cottrell law was followed before the 1 min of enzyme–substrate reaction. Adequate reproducibility within electrochemical measurements was also observed, with relative standard deviations varying from 6.5 to 18.6%. AChE immobilization with glutaraldehyde allow to obtain robust and reproducible biosensors, but they need a much higher enzyme content (80 mUA per electrode) to achieve current values comparable to that constructed by immobilizing the AChE through photopolymerization with PVA-SbQ (0.7 to 1 mUA per electrode). The limits of detection were determined with a minimum 10% inhibition, and varied from 10−9 to 8×10−9 M (0.2 to 1.5 ppb) by employing the enzyme immobilization through photopolymerization with PVA-SbQ. In practice, this kind of immobilization procedure is much simpler and produces good results: fast response, adequate reproducibility, large pesticides working ranges, and excellent sensitivities to N-methylcarbamates (NMCs) which in general do not present enzyme inhibition power as elevated as for the organophosphate pesticides.