Analytica Chimica Acta 523 (2004) 107–115 Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides Comparative study Gilvanda Silva Nunes a,∗ , Gérard Jeanty b , Jean-Louis Marty b a Núcleo de Análises de Res´ ıduos de Pesticidas—NARP, Depto. Tecnologia Qu´ ımica, CCET-Universidade Federal do Maranhão. Av. Portugueses, s/n. CEP 65080-040, São Lu´ ıs, MA, Brazil b Centre de Phytopharmacie, Laboratoire BIOMEM, Unversité de Perpignan. 52, Avenue Paul Alduy. 66860, Perpignan, Cedex, France Received 30 January 2004; received in revised form 22 March 2004; accepted 22 March 2004 Available online 18 August 2004 Abstract 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 (80mUA 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 1.5ppb) 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. © 2004 Elsevier B.V. All rights reserved. Keywords: AChE-screen-printed-based biosensors; TCNQ mediator; N-Methylcarbamate insecticides 1. Introduction Organophosphorus and carbamate pesticides, which present low environmental persistence but a high acute toxicity, have come into widespread use [1]. Among these, N-methylcarbamate (NMC) insecticides have been exten- sively used for pest control in many countries, due to ∗ Corresponding author. E-mail address: jlmarty@univ-perp.fr (G. Silva Nunes). their efficacy, low bioaccumulation, and moderately rapid degradation in the environment. Nevertheless, a significant amount of these compounds is transferred to surface runoff and subsurface drainage from agriculture land and, can cause a spectrum of toxic effects on aquatic organisms and human beings [2]. These pesticides are toxic because they act as inhibitors of acetylcholinesterase (AChE) that hydrolyses the neuro- transmitter acetylcholine (ACh). This enzyme is present in vertebrates and insects; its inhibition can disrupt the trans- mission of nerve impulses [1,3]. So, the presence of residues 0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.03.100