Acetylcholine enzyme sensor for determining methamidophos insecticide

Analytica Chimica Acta 434 (2001) 1–8

Acetylcholine enzyme sensor for determining methamidophos insecticide Evaluation of some genetically modified acetylcholinesterases from Drosophila melanogaster Gilvanda Silva Nunes a , Thierry Montesinos b , Paulo Brasil O. Marques a , Didier Fournier c , Jean Louis Marty b,∗ b

a Depto. de Tecnologia Qu´ımica, Centro Tecnológico/UFMA, Av. Portugueses s/n, CEP 65080-040 São Lu´ıs, MA, Brazil Centre de Phytopharmacie, Unversité de Perpignan, UMRA CNRS 5054, 52 Avenue de Villeneuve, 66860 Perpignan Cedex, France c Laboratoire de Synthèse et Physicochimie des Molécules d’Intérêt Biologique, Université Paul Sabatier, ESA 568, 118 Route de Narbonne, 31062 Toulouse, France

Received 29 June 2000; received in revised form 27 December 2000; accepted 8 January 2001

Abstract A sensitive screen-printed amperometric sensor suitable for rapid determination of the concentration of the insecticide methamidophos was developed. It was based on the principle of inhibition of acetylcholinesterase (AChE) activity. The first part of the study was focused on the screening of several genetically modified AChEs in order to select the most sensitive enzyme towards the methamidophos. Values for the bimolecular constant ki were also determined. In a second part of the study, we compared the lowest detectable methamidophos concentrations using different immobilised AChEs. The lowest detectable concentration in a standard solution was 1.4 ppb for the AChE(Dros)-B03 mutant compared to 4.8 ppb for the wild type AChE and 53 ppb for the electric eel source. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Screen-printed electrodes; Methamidophos; Recombinant acetylcholinesterases; Inhibition constant (ki )

1. Introduction The detection of anticholinesterases (anti-ChEs) in the environment is of major concern to the agricultural chemical industry, regulatory agencies and health care professionals. Acetylcholinesterase (AChE) is the target enzyme for two groups of widely used insecticides: organophosphates and carbamates. The inhibition of AChE activity disturbs normal neuronal function, resulting in a worst case scenario in ∗ Corresponding author. Tel.: +33-468662254; fax: +33-468662223.

the death of living organisms [1]. Insecticides that function by AChE inhibition are presently used extensively in the agricultural and forestry industries, and are preferred for their relatively low persistency compared to organochlorine pesticides [2,3]. On the other hand, organophosphates and carbamates usually show high acute toxicity, such that the recommended protection interval should be carefully followed in order to avoid possible health-related problems [3]. In developing countries, the widespread use of organophosphates has been accompanied by an appreciable increase in the incidence of poisoning with these agents. This is a result of their widespread

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 0 7 9 5 - 4

2

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

availability, indiscriminate handling and storage, and a lack of knowledge about the serious consequences of poisoning. Latest estimates from the World Health Organisation indicate that 1 million serious accidental poisonings and 2 million suicide attempts involving pesticides occur worldwide each year [4]. Studies on the application of organophosphates have demonstrated, for example, that during normal spraying, farmers are exposed to contamination by absorption through the skin of residues on clothing. In China alone, 27 provinces in 1995 reported a total of 48,377 poisoning cases, including 3204 fatalities. More than 7500 of these cases were mostly attributed to the normal agricultural use of parathion and methamidophos [5]. In Brazil, the insecticide methamidophos is widely used for the protection of soybean crops, mainly in the northeast region of the country, where basic information on suitable agricultural handling is still lacking. Because of this, environmental contamination and many cases of intoxication of farm workers have been reported [6]. For these reasons, fast, reliable and economically viable methods are required for the detection of such toxic compounds in the environment and in agro-food products, with the result being that growing interest in this field has seen the production of a variety of portable detection devices such as biosensors. Large numbers of samples for analysis necessarily requires the application of pre-screening methods, suitable for direct field use [1]. In this first step, the potentially hazardous samples could be identified and then further analysed in the laboratory using more expensive and time-consuming classical analytical methods, among them, the chromatographic methods involving HPLC [7,8] or GC [9,10]. Bioanalytical techniques could play an important role in this respect; a direct analysis is usually possible without any separation or cleanup steps, as the specific bioreagents could identify a single compound or a group of closely related analytes. In addition to immunochemical sensors using bioaffinity sensing principles and enzyme-linked immunoassay (ELISA) methods, which have been developed for the analysis of some ChE-inhibitor pesticides [11–14], inhibition of the biocatalytic activity of cholinesterase enzymes is widely employed for making electrochemical biosensors for environmental and food analysis. Such enzymes can be immobilised on electrode surfaces using different techniques, with

final sensitivity depends directly on the specific technique and on the choice of a suitable surface electrode mediator, in order to prevent matrix interferences. In previous studies, we have used cobalt(II) phthalocyanine (CoPC) as the mediator and acetylcellulose as a binder to obtain the desired sensitivity of ChE-graphite electrodes [15–17]. Thus, CoPC-modified-graphite sensors were successfully applied to the direct analysis of insecticide contamination of fruits and vegetables without any sample pre-treatment [18]. The main limitation of this ChE-electrode was its working potential, which was quite high (350 mV versus Ag/AgI, imposed by mediator) and could not effectively prevent oxidation of interference substances on the electrode surface. For this reason, and considering the need to improve the ChE-sensor sensitivity and stability, the main aims of the present work were (i) to evaluate the activity of several genetically modified AChEs obtained from Drosophila melanogaster (AChEs (Dros)) (GTP Technology, Toulouse, France), and their inhibition constant with respect to the insecticide methamidophos; (ii) to apply the screen-printing technology routinely used in Centre de Phytopharmacie (Perpignan, France) [19–21] and University of Stuttgart (Germany) [22], which involves the manufacture of disposable electrodes by incorporating a graphite paste containing tetracyanoquinodimethane (TCNQ) as electrochemical mediator to form the working electrode, and a printing Ag/AgCl paste to form the reference electrode.

2. Materials and methods 2.1. Reagents and biological substances Acetylcholinesterase (EC 3.1.1.7, specific activity of 500 U mg−1 ) from electric eel (EE) was purchased from Sigma Chemical Co. AChE from Drosophila melanogaster (Dros) was kindly provided by GTP (Toulouse, France); wild type and mutants were produced by in vitro expression in bacullovirus cells [23]. Acetylthiocholine chloride (ATCh) and 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB) were supplied by Sigma Co.; 7,7,8,8-tetracyanodimethane (TCNQ) from Aldrich, and medium viscosity hydroxy ethyl-cellulose (HEC) from Fluka. Photocrosslinkable poly(vinyl alcohol) bearing styrylpyridinium groups

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

(PVA-SbQ) in the normal form (polymerisation degree 1700; saponification degree 88; SbQ content 1.3 mol.%; solid content 13.5 wt.%; pH 6) was kindly provided by Toyo Gosei Kogyo Co. (Tokyo, Japan). Dowex (cation exchanger resin) was purchased from Sigma Co., NaCl (98.8% purity) was obtained from Merck (Augsburg, Germany). Standard of methamidophos (96.5% purity) was purchased from Dr. Ehrenstorfer (Augsburg, Germany). Stock and working solutions were made up in water, and these last were stored for a maximum 1 week, in the dark at −20◦ C. All other reagents were of analytical grade. 2.2. Preparation of the electrochemical sensors and measurement procedure A single-channel two-electrode sensing strip (approximate dimensions 7 mm × 44 mm × 0.5 mm) was screen-printed onto a PVC support by using a DEK 248 screen-printer (DEK printing machines Ltd., UK). The following layers were printed to form the working and reference electrodes (1) a conducting silver track (Electrodag PF410, Acheson), which forms the electrical connections between reference and working electrodes; (2) a graphite layer (Electrodag 423SS, Acheson) was printed over the silver tracking to prevent contact between the tracking and applied analyte solution; (3) for the reference electrode a silver/silver chloride ink (Electrodag 603SS, Acheson) was printed over the graphite layer on alternate silver tracks; (4) for the working electrode a paste prepared by mixing graphite T15 (Lonza) with TCNQ (mediator) and HEC (binder) was used and printed over the remaining graphite-coated silver tracks. The mixture was prepared as follows: 4 g of graphite was mixed with 100 mg TCNQ diluted in 300 ml acetone. The mixture was vacuum-evaporated for 30 min, and then 3.5 g of the resulting powder was mixed with 23 ml of a 3% (w/v) HEC solution. After homogenisation, the resulting paste was deposited by printing on the working electrode, and final graphite composites were ready for use after drying for about 1 day at room temperature; (5) the final layer (sensing layer; 2 ␮l), consisting of a mixture of PVA-SbQ and AChE solution (final concentrations: 30% (v/v) for PVA-SbQ; 1 U ml−1 for AChE (EE); 0.5 U ml−1 for AChE (Dros) and 1 U ml−1 for AChE (Dros-mutants)) was manually deposited over the TCNQ-modified graphite (working) electrode.

3

The final AChE-sensors were kept under two 15 W neon lamps for 2 h at 4◦ C in order to facilitate polymerisation. Following these production steps, these sensors were ready to use after drying for a minimum of 18 h at 4◦ C. Amperometric measurements were carried out with a PRG-DEL potentiostat (Tacussel, France), and the working potential was poised at 100 mV (versus the Ag/AgCl reference electrode). Output current was recorded via a PC using PICO ADC software (PicoLog Software, release 3.07, Cambridge, UK). The biosensor strip was inserted vertically into a body of an analytical cell containing 5 ml of phosphate buffer (Na2 HPO4 /NaH2 PO4 ) at pH 7 under constant magnetic stirring. The 50 ␮l of substrate solution (0.1 M thiocholine (ATCh)) was then added (giving a final substrate concentration of 10−3 M) and the signal (steady-state current) initiated by the enzymatic reaction was recorded (dI0 ). After inhibition by immersing the sensing strip in an aqueous solution of methamidophos (different concentrations for different incubation times), another lower current intensity (dI1 ) was recorded in the same mode, after which relative inhibition (RI) could be calculated according the equation: RI (%) = [(dI0 – dI1 )/dI0 ] × 100. 2.3. Spectrophotometric determination of enzyme activities and inhibition constants For the determination of AChE activity and kinetic parameters of inhibition, spectrophotometric measurements were performed with the aid of a Hewlett-Packard diode array model 8451 A spectrophotometer. Determination of the enzymatic activity was carried out by applying the Ellman method [24], based on the reaction between enzymatic reaction product (thiocholine) and dithiobisnitrobenzoate (DTNB), resulting in the production of a coloured compound (thionitrobenzoate), according reactions as follows.

The assay was performed as follows. To 500 ␮l of a 2.5 × 10−3 M DTNB solution, 500 ␮l of a 2.0 ×

4

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

10−3 M ATCh solution was added. This was followed by the addition of a specified volume of enzyme suitably diluted with phosphate buffer at pH in order to obtain a final volume of 2 ml. Both DTNB and ATCh solutions were prepared in phosphate buffer at pH 7. An absorbance versus time curve was obtained, its slope recorded and then enzymatic activity, expressed in U ml−1 , calculated. A blank containing either no enzyme or no substrate was run in order to eliminate residual colour due to the DTNB. For the determination of inhibition constants, solutions of methamidophos were prepared by diluting a 10−2 M pesticide stock solution in distilled water. AChE stock solutions were prepared in 0.9% (w/v) NaCl solution, and then diluted in phosphate buffer at pH 7. Two wild type enzymes (AChE (EE) and AChE (Dros)) and six mutants from AChE (Dros) (B03; B03-23; B05; B06-23; B07, and B08-23.29.44) were assayed. Other mutants did not show activity. The procedure for determination of the enzyme inhibition constant (ki ) was adapted from the methodology described by Segel [25]. In this work, the procedure for the ki calculation in the presence of methamidophos was as follows. First, 200 ␮l of 2.5 × 10−3 M DTNB was added to 350 ␮l of phosphate buffer at pH 7, and then 50 ␮l of a mixture containing 2 ml of inhibitor

solution plus 10 ␮l of AChE solution (in appropriate dilutions) was added to the cell. An absorbance versus time spectrophotometric curve was recorded from the 1 min time point, and the slope of the curve was taken each 3 min for 30 min (in this period, the AChE-inhibitor mixture was kept in a 25◦ C temperature-controlled bath for enzyme incubation with the pesticide). Parallel assays without inhibitor (control) and without enzyme-inhibitor mixture (blank) were carried out. 3. Results and discussion 3.1. Kinetic parameters for the AChE enzymes To estimate the inhibition constant ki , a spectrophotometric plot of residual activity versus incubation time was performed for each methamidophos concentration. As an example, six different pesticide concentrations were assayed for the mutant B03 (Fig. 1). Each plot was linear and values for the apparent reaction rate, Kobs , were obtained from the straight line slopes. In the absence of inhibitor, the activity response remained almost unchanged, but a discrete loss of activity could be observed with time even when the enzyme was mixed in water. Values for Kobs were

Fig. 1. Kinetic inhibition of genetically modified AChE (Dros) — B03 — against methamidophos insecticide in different concentrations.

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8 Table 1 Relative activities and inhibition constants of wild and mutant AChEs using methamidophos Relative activity (U ml−1 )

Inhibition constant (ki , mol−1 l min−1 )

Wild AChEs EEa Drosb

122.8 272.6

1.1 × 103 7.3 × 103

Mutant AChEsb B03 B03-23 B04-23 B05 B06-23 B07 B08-23.29.44

40.8 79.0 127.0 108.3 244.8 243.0 127.6

3.3 3.5 3.1 3.5 2.6 1.6 2.2

Enzyme (source)

× × × × × × ×

104 105 104 104 104 105 106

a

EE: from electric eel, obtained commercially. b Dros: from Drosophila melanogaster, obtained from Dr. D. Fournier (Paul Sabatier University, Toulouse, France).

directly proportional to the pesticide content, and inhibition constants could be obtained by plotting 1/Kobs versus 1/I (where I = inhibitor concentration, in M) for each AChE tested. Inhibition constants were calculated from the slope of this relation, where k i = 1/ slope. According to Table 1, two AChE (Dros)-mutants (B06-23 and B07) exhibited a relative activity similar to that of their parent enzyme. One hypothesis for this behaviour is that depending on the type of structural alteration for each AChE mutant, the affinity of the mutant for the substrate is affected differently. On the other hand, independent of the enzyme activity, ki values varied considerably with enzyme modification at low substrate concentrations. For example, in comparing the wild AChE (Dros) (relative activity = 272.6 U ml−1 ) to its generated mutant B08-23.29.44 (relative activity = 127.6 U ml−1 ), although the latter exhibited a decreased activity, this enzyme was found to be more strongly inhibited by methamidophos. The ki value of the B08-23.29.44 mutant was increased by a factor of about 103 compared to the wild AChE (Dros), allowing a suitable increase in enzyme sensitivity to be measured in response to the insecticide. Enzyme sensitivity provides an important parameter in consideration of the choice of the most suitable enzyme to be used in a biosensor. The appropriate enzyme should be the most sensitive to the investigated

5

inhibitor in order to obtain a detection threshold that is as low as possible. In this respect, enzyme modification could be a powerful tool to improve the sensitivity of enzymes to selected compounds (inhibitor). The inhibiting action of the organophosphorous pesticides is based on the formation of oxo-derivatives of the pesticide able to alkylate an active site of the enzyme (the serine aminoacid), which must remain free in order to bind the choline derivative [1–3]. By performing genetic alterations to the enzyme structure, sensitivity to insecticides can be increased but initial enzyme activity can be changed differently. 3.2. Acetylcholinesterase sensitive layers for methamidophos analysis Four different enzymes were immobilised on TCNQ-modified graphite electrodes for electrochemical studies: two wild enzymes (AChE from EE and Drosophila melanogaster (Dros)), and two AChE (Dros)-mutants (B03 and B08-23.29.44). For substrate biosensors, diffusion control of the response is preferred, because, in general, it provides a wider linear working range. Diffusional limitation is usually achieved for enzyme layers containing high loadings of activity; in our case, enzyme charge was fixed at 2 mU, but for AChE (Dros) it was changed to 1 mU in order to avoid supersaturation at the electrode surface in the initial seconds after the addition of substrate. Generally, the lower the enzyme loading, the greater is the observed sensitivity to inhibitors. This finding was confirmed in the present work and has been reported in other studies with different immobilisation procedures [18,26]. 3.2.1. Electrochemical response and reproducibility of the biosensor In this study, enzyme layers were prepared by photopolymerisation of AChEs with PVA-SbQ. This procedure is simple and provides good reproducibility of steady-state current generated from the reaction between enzyme and substrate. Fig. 2 shows the precision of 10 electrochemical measurements carried out on the same day with two TCQN-modified electrodes containing AChE (EE) and AChE (Dros). Coefficients of variation lower than 5% were observed for both cases.

6

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

Fig. 2. Reproducibility of steady-state current of TCNQ-modified graphite electrodes containing acetylcholinesterases during hydrolysis of the substrate (ATChCl: 10−3 M at pH 7).

3.2.2. Calibration curves for methamidophos Fig. 3 shows typical response curves obtained during hydrolysis of acetylthiocoline before (A) and after inhibition of the AChE (Dros) with methamidophos insecticide (B). A steady-state current is reached in phosphate buffer after about 3 min. After addition of substrate, a fall in current is observed, which then

Fig. 3. Methamidophos determination. Response of the enzyme sensor with AChE (Dros) immobilised in a TCNQ-modified graphite electrode by photopolimerisation with PVA-SbQ, in phosphate buffer pH 7. (A) Addition of substrate (initial signal) and (B) addition of substrate after inhibition of the enzyme with the pesticide.

begins to increase with enzymatic reaction until another plateau of the current is observed. The time required for incubation of the enzyme with inhibitor was first optimised in order to achieve good sensitivity. To this end, inhibitions achieved as a function of incubation time are shown in Fig. 4, with the pesticide concentration in solution maintained constant (7 × 10−6 M or 1 ppm), and all measurements performed after inhibition by immersing the enzyme electrode into an aqueous pesticide solution in order to simulate natural conditions (in river water, for example, samples are not buffered!). As the extent of inhibition is proportional to time, longer periods are preferred in order to achieve high sensitivity; thus, an incubation time of 10 min for wild and modified AChEs seemed to be reasonable for possible practical utilisation of these AChE-based electrodes in the field. As mentioned above, a further period of time after enzyme inhibition is necessary for the measurement of the remaining enzyme activity. It is clear that this approach is not very convenient for the rapid monitoring of samples in the field, but the time required for one analysis can be considerably reduced if the inhibition and measurement of enzyme activity are carried out simultaneously [26]. In the present work, this alternative approach was not tested. For subsequent experiments, an incubation time of 10 min for the enzyme inhibition was selected as the most appropriate. The inhibition curves obtained for the selected enzymes are non-linear curves, and strong inhibitions at lower concentrations of methamidophos were observed. This behaviour of inhibition curves has been previously reported in studies with ChE-sensors and carbamates [15,16,18], and appears to be similar for organophosphates [26]. The calibration linearity is possible for concentrations of methamidophos up to 1.4 × 10−7 M. Good sensitivity for the selected associated with the use of TCNQ electrode-modifier, which prevents oxidation of matrix interferents at the surface of the working electrode (by allowing the application of a lower working potential), is the major advantage of this sensor in routine-based and environmental monitoring analysis. For routine analysis of samples containing anti-ChEs, a more appropriate strategy would be to simply verify the initial relative inhibition, and, if the RI is higher than 50%, dilute the sample about 1000-fold in buffer assay in order to achieve a higher sensitivity.

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

7

Fig. 4. Optimisation of incubation times for the inhibition of the enzyme with the methamidophos (concentration fixed at 7 × 10−6 M). The relative inhibitions were measured as a decrease in stead-state current for the substrate (1 mmol l−1 ) after the addition of pesticide.

In general, enzymatic-amperometric sensors have a clear advantage over spectrometric or fluorimetric methods in that they can also be used when the solutions to be analysed (containing the inhibitor) are turbid or coloured. On the other hand, the most significant problem in the analytical application of all of these enzyme-inhibition sensors comes from their sensitivity to many commercial organophosphorous pesticides. In this way, a rigorous analytical determination is possible in the presence of only one pesticide in the sample to be analysed, unless a preliminary separation procedure is carried out, or enzymes of different origin — known to be more specific in the presence of a selected pesticide — are used. The sensor can be used as a toxicity indicator of the sample. The lowest detected concentrations of methamidophos determined by incubation are compared in Table 2 for the various selected AChEs and mutants. For the incubation method, a minimum 10% relative inhibition of the enzyme was considered appropriate for the calculation of the detection limit of the sensors. Depending on the enzymatic source, the enzymes exhibited different sensitivities toward methamidophos insecticide, although the

Table 2 Comparison of the lowest detected methamidophos concentrations (in M and ppb) using different immobilised cholinesterases

In M In ppb

AChE (EE)

AChE (Dros)

AChE (Dros)-B03

3.71 × 10−7 53

3.40 × 10−8 4.8

9.93 × 10−9 1.4

results for pesticide detection were around the ppb level.

4. Conclusions The present study focused on an examination of the response to the insecticide methamidophos of an AChE-based biosensor containing AChEs from different sources. For this purpose, disposable sensors were used, and the enzymes showing higher sensitivities were selected for further studies. It was demonstrated that TCNQ-modified graphite electrodes could be conveniently used for amperometric measurements of the activity of AChEs. These

8

G.S. Nunes et al. / Analytica Chimica Acta 434 (2001) 1–8

electrodes are more stable and cheaper when compared with probes based on the measure of AChE inhibition through the activity of the choline oxidase (bi-enzyme sensors). Some AChE mutants obtained by modification of the structure of AChE from Drosophila melanogaster exhibited increased sensitivities. These were found to be suitable for the preparation of cholinesterase biosensors for detection of the methamidophos insecticide in water and agro-food samples, and can be used for field analysis since an amperometric portable biosensor is convenient under such conditions.

Note: The modification of amino-acids in the different mutants are not indicated because these enzymes are patented. Acknowledgements The authors would like to thank the “Programme Environnment et Santé” no. 98123 of the Ministère de l’Aménagement du Territoire et de l’Environnement and the program INCO-Copernicus IC.15-CT96-0804 for their financial support. References [1] C. Fest, K.J. Schmidt, The Chemistry of Organophosphorus Pesticides: Reactivity, Syntesis, Mode of Action, Toxicology, Bayer AG Lab, New York, 1973, pp. 164–201. [2] R. Cremlyn, Pesticides — Preparation and Mode of Action, Wiley, Chichester, 1978. [3] A.K. Hassal, The Biochemistry and Uses of Pesticides: Structure, Metabolism, Mode of Action and Uses in Crop Protection, 2nd Edition, VCH, Weinheim, New York, Basel, Cambridge, 1990.

[4] J. Jeyaratnam, Acute pesticide poisoning: a major global health problem, World Health Stat. Q. 43 (1990) 139–144. [5] UNEP — United Nations Environmental Program, Convention on Trade in Dangerous Chemicals and Pesticides: Final Round of Talks Started, home-page: http://www.unep.ch/iuc/submenu/press/press/pic/pr3-98.htm. [6] G.S. Nunes, Roubar Futuro, Jornal O Imparcial, São Lu´ıs, MA, Brazil, Agosto, 1999. [7] C. Schlett, Fresenius J. Anal. Chem. 339 (1991) 344–347. [8] S. Lacorte, D. Barceló, Anal. Chem. 68 (1996) 2464–2470. [9] S.B. Lartiges, P. Garrigues, Analusis 23 (1995) 418–421. [10] S.J. Lehotay, N. Aharonson, E. Pfeil, M.A. Ibrahim, J. AOAC 78 (1995) 831–840. [11] G.S. Nunes, I.A. Toscano, D. Barceló, Trends Anal. Chem. 17 (1998) 79–87. [12] G.S. Nunes, M.P. Marco, D. Barceló, M.L. Ribeiro, J. Chromatogr. A 823 (1998) 109–120. [13] G.S. Nunes, M.-P. Marco, M. Ferrer, D. Barceló, Anal. Chim. Acta 387 (1999) 245–253. [14] I.A. Toscano, G.S. Nunes, D. Barceló, Food Technol. Biotechnol. 36 (1998) 245–255. [15] P. Skládal, G.S. Nunes, H. Yamanaka, M.L. Ribeiro, Electroanalysis 9 (1997) 1083–1087. [16] G.S. Nunes, P. Skládal, H. Yamanaka, D. Barceló, Anal. Chim. Acta 362 (1998) 59–68. [17] G.S. Nunes, D. Barceló, Analusis 26 (1998) R156–R159. [18] G.S. Nunes, D. Barceló, B.S. Grabaric, J.M. D´ıaz-Cruz, M.L. Ribeiro, Anal. Chim. Acta 399 (1999) 37–49. [19] A. Avramescu, V. Magearu, J.-L. Marty, Anal. Chim. Acta, 2001, in press. [20] T. Montesinos, S. Perez-Munguia, F. Valdes, J.-L. Marty, Anal. Chim. Acta, 2001, in press. [21] T. Noguer, B. Leca, G. Jeanty, J.-L. Marty, Field Anal. Chem. Technol. 3 (1999) 171–178. [22] T.T. Bachman, R.D. Schmid, Anal. Chim. Acta 401 (1999) 95–103. [23] R.D. O’Brien, B.D. Hilton, L. Gilmour, Mol. Pharmacol. 2 (1966) 593–605. [24] G.L. Ellman, K.D. Courtney, K.D. Andres, R.M. Featherstone, Biochem. Pharmacol. 7 (1961) 88–95. [25] I.H. Segel, Enzyme Kinetics, Wiley/Interscience, New York, 1975. [26] P. Skládal, M. Mascini, Biosens. Bioelectron. 7 (1992) 335– 343.