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Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents
Talanta 57 (2002) 169– 176 www.elsevier.com/locate/talanta
Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents Silvana Andreescu a,b, Thierry Noguer a, Vasile Magearu b, Jean-Louis Marty a,* a
Centre de Phytopharmacie, Uni6ersite´ de Perpignan-UMR CNRS 5054, 52, A6enue de Villeneu6e, 66860 Perpignan Cedex, France b Uni6ersity of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, 90 -92 Panduri, 76234, Bucharest, Romania Received 15 June 2001; received in revised form 17 December 2001; accepted 18 December 2001
Abstract A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91 × 10 − 8 M paraoxon and 1.24 × 10 − 9 M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholinesterase; Organic phase biosensor; Pesticide; Disposable; Screen-printed electrodes; Inhibition
1. Introduction Pesticides are very toxic compounds, which have been shown to be responsible for many ecological problems and damages to human health. Among the many methods reported for pesticides detection, chromatographic methods such as HPLC and GC are often used as reference * Corresponding author. E-mail address: [email protected] (J.-L. Marty).
methods. Despite their high sensitivities, these techniques are expensive, time-consuming and require highly trained personnel; furthermore they are not adapted for in situ and real time detection of pollutants. At the same time, they are not able to give any information concerning the toxicity of the sample. Biochemical sensors appear as a reliable alternative to classical methods for the rapid and simple detection of pesticides. Various systems for the detection of insecticides are described in literature. Biological sensors
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based on organophosphate hydrolase [1– 3] which catalyse the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent into a direct detectable compound (p-nitrophenol) have been used for the determination of pesticides, leading to the detection of 0.2 and 1 mM paraoxon and methyl parathion respectively [2]. Immunological sensors coupled with different transduction modes have also been reported to detect pesticides with a very low detection limits [4,5]. For instance 0.3 ppb parathion was detected with a optic fiber immunosensor [5]. Other studies report the determination of pesticides based on their inhibitory effect on the enzyme activity. In these studies, the decrease of enzyme activity caused by inhibition is correlated to the concentration of pesticide in the sample or their metabolites concentration in the sample. Tyrosinase-based sensors have been developed for the detection of enzymes inhibitors with a detection limit in the range of micromolar concentrations [6,7]. These sensors suffer from a poor specificity because of possible interferences caused by the presence of various possible substrates and inhibitors of tyrosinase. Most traditional methods refer to the use of cholinesterases (ChE), acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) which are the real biological target of main organophosphorus and carbamate insecticides, representing 40% of the world market of this class of compounds. Various electrode configurations based on different transduction systems have been reported for ChE sensors [8–17]. The activity of ChE is usually determined by using esters of choline which are hydrophilic substrates. In a biosensor configuration, acetyl- or butyryl-choline can be used as substrate if the cholinesterase is associated with a choline oxidase (ChO) [13–15]. In this case, two detection methods are possible, by monitoring either oxygen consumption using a Clark-type electrode [16] or hydrogen peroxide production by oxidation at 650 mV vs. Ag/AgCl [8,13]. Such a complicated device can be simplified to a mono-enzyme system using acetyl- or butyryl-thiocholine as substrate; in this case the detection is based on the oxidation of thiocholine produced upon enzyme reaction. An important advantage of such a system is re-
lated to low overpotential necessary for thiocholine oxidation (410 mV vs. Ag/AgCl, or 100 mV vs. Ag/AgCl using TCNQ as mediator [12,17] or cobalt phtalocyanine [18,19]) as mediator. An alternative substrate, p-aminophenyl acetate has been used as AChE substrate for amperometric sensors, the detection being based on the oxidation of p-aminophenol at 250 mV vs. SCE [20,21]. Using this system, a detection of 4 nM paraoxon and 13 nM carbaryl was reported. Pesticide detection is generally performed in aqueous solutions. However, these compounds are generally characterized by a low solubility in water and a high solubility in organic solvents. Extraction and concentration of pesticides from solid matrices (fruits, vegetables,…) are thus commonly carried out in such solvents. The ability of enzymes to work in non-aqueous solvents has been proved for many years [22–25]. Depending on the nature and the amount of the solvent, the enzyme activity can be strongly affected when experiments are performed in these media. Some enzymes like glucose oxidase or tyrosinase work in various organic solvents as well as in water, but in all cases a minimal amount of water is required to retain catalytic activity [22–25]. ChE seems to be more sensitive to organic solvents than the previously named enzymes. The influence of organic solvents on free AChE activity [26] has been reported and few publications refer to the detection of pesticides with immobilized AChE in these media [27–30]. In a recent paper, we have reported the influence of acetonitrile, ethanol and DMSO on a cholinesterase sensor using acetylthiocholine as substrate [29]. An increase of the output current was noticed when working in 5% acetonitrile and 10% ethanol. The sensor was used to detect chlorpyrifos ethyl oxon. The detection of dichlorvos, diazinon and fenthion in the presence of ethanol was also reported with an amperometric AChE biosensor based on thiocholine-hexacyanoferrate reaction [30] and the bi-enzyme AChE/ChO system [28] allowed to detect 0.2 ppb paraoxon in the presence of 5% cyclohexane. p-aminophenyl acetate, which was already been used as substrate for AChE sensors is characterized by a good solubility in organic solvents such as acetonitrile. This
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feature suggests the possibility to extend the application field of AChE biosensors to non-aqueous media. The aim of this work was to study the effect of water-miscible organic solvents on the catalytic activity of immobilised AChE by using paminophenyl acetate as substrate. The biosensor was applied to the detection of organophosphorus insecticides (paraoxon and chlorpyrifos ethyl oxon) in the presence of acetonitrile and ethanol. Low cost screen-printed disposable electrodes have been used for tests, eliminating the need to reactivate the enzyme after inhibition measurement. These single use sensors also avoid the problems related to the possible fouling of the electrode which generally involve a chemical or electrochemical activation of the working electrode surface. To our knowledge, this is the first study referring to the use of p-aminophenyl acetate substrate to study the effect of organic solvents on immobilised AChE. These developed screen-printed biosensors have been applied to the detection of pesticides in the presence of such media.
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Paraoxon ethyl and chlorpyrifos ethyl oxon were purchased from Dr Ehrenstorfer GmbH (Germany) and Dow Elanco (AGR 203674). All other reagents used in the tests were analytical grade. The supporting electrolytes used in this work contained 0.1 M KCl. All measurements were performed at 25 °C.
2.2. Apparatus A DEK 248 screen-printing system (DEK, UK) was used to fabricate the electrodes. Amperometry experiments were carried out with a simplified electrochemical system, consisting in two electrodes made by screen-printing, a 641VA (Metrohm 64 VA, Swiss) potentiostat and a BD40 (Kipp & Zonen, Holland) recorder. Cyclic voltammetric studies were carried out with a 362 EG & G (Princeton Applied Research, USA) potentiostat connected to a LY 1400 x–y recorder. In these experiences a platinum electrode was used as counter electrode.
2.3. Methods 2. Experimental
2.1. Materials AChE from Drosophila melanogaster was provided from Prof. D. Fournier (University Paul Sabatier, Toulouse, France). p-nitrophenyl acetate and p-aminophenol were obtained from Sigma. Photocrosslinkable poly(vinyl alcohol) with styrylpyridinium groups polymer (PVASbQ), polymerization degree 1700, was provided by Toyo Gosei Co. (Chiba, Japan). Hydroxyethylcellulose (HEC) was supplied by Fluka. Printing pastes (Electrodag PF-410, 423SS, 6037SS) were obtained from Acheson, France. Graphite Timrex T15 was supplied by Timcal (Switzerland). The insulating layer was a commercial Astral paint (France). Clear PVC sheets (200×100 mm2, 0.5 mm thick, supplied by SKK, Germany) were used as support for the screenprinting electrodes.
2.3.1. Synthesis of p-aminophenyl acetate substrate p-aminophenyl acetate substrate was synthesized by catalytic reduction of p-nitrophenyl acetate in presence of SnCl2 under nitrogen atmosphere according to Pariente et al. [20]. The product was extracted in ethyl acetate and then treated with NaOH. After extraction, the organic phase was dried with MgSO4 and concentrated. The residue was purified by two successive silica gel chromatography (CH2Cl2/MeOH 98:2) and after evaporation the product was dissolved in ether and recrystallised in hexane. The purity was checked by thin layer chromatography, proton NMR spectrum and UV–VIS spectroscopy. White crystals of p-aminophenyl acetate were obtained. The substrate was stored at a temperature below 0 °C under nitrogen atmosphere. Before assays, p-aminophenyl acetate solutions were prepared using pure organic solvent (acetonitrile or ethanol).
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2.3.2. Electrochemical measurements of p-aminophenol in organic sol6ents AChE catalyses the hydrolysis of paminophenyl acetate into p-aminophenol. The electrochemical behaviour of p-aminophenol was studied by cyclic voltammetry in a three electrode system consisting in a screen-printed Ag/AgCl pseudo-reference electrode, a graphite screenprinted working electrode and a platinum auxiliary electrode. Experiments were performed in 0.1 M phosphate buffer containing 0.1 M KCl, pH 8 in the presence of different concentrations of organic solvent. The potential scan was carried out between −500 and 700 mV at a scan rate of 50 mV/sec. 2.3.3. Biosensor preparation The sensor system consisted of two electrodes: an Ag/AgCl reference electrode and a graphite working electrode, both of them made by screenprinting technology. The fabrication technique was based on consecutive depositions of several layers on a plastic sheet: a silver conducting layer for the electrical connections, a carbon pad, a reference electrode (Ag/AgCl), an insulating layer and a graphite ink (made of graphite powder and HEC 4%) to cover the working electrodes surface. Between each deposition the electrodes were dried at 60 °C for 30 min. The sensing layer containing the enzyme was then deposed on the working electrode surface. AChE immobilisation was made by entrapment in a photocrosslinkable PVA-SbQ polymer. A 3 ml homogeneous mixture of enzyme solution in 0.1 M phosphate buffer, pH 8 and PVA-SbQ polymer (30% v/v) was deposed on the working electrode surface. The amount of enzyme immobilised on each electrode was calculated to be 1 mU. The electrodes were then placed for three hours at 4 °C under neon lamp to allow polymerization. They are stored at 4 °C and can be used one day after preparation. 2.3.4. Amperometric measurements The amperometric measurements were carried out in a batch system by measuring the current corresponding to the oxidation of p-aminophenol, product of the enzymatic reaction. The two elec-
trodes system (working and reference electrodes) was immersed in a cell containing 3 ml 0.1 M phosphate buffer containing 0.1 M KCl pH 8, or the same buffer containing a given percentage of organic solvent. A potential of 100 mV vs. Ag/ AgCl reference electrode was applied between the two electrodes and the current obtained upon injection of 1 mM p-aminophenyl acetate was recorded. A non-enzymatic response due to the chemical hydrolysis of p-aminophenyl acetate was observed. For this reason, the response obtained with an enzyme electrode was compared with the response obtained with the same electrode after a complete inactivation of the enzyme in a very concentrated solution of pesticide. The influence of organic solvents on the catalytic activity of immobilised AChE was studied by measuring the response of the sensor in phosphate buffer containing water miscible organic solvent (acetonitrile and ethanol) in a concentration ranging from 1 to 25%. Operational stability was evaluated by carrying out consecutive injections of substrate at a final concentration of 1 mM, the cell being rinsed out with phosphate buffer solution between assays. To perform inhibition tests, the biosensor was incubated during a defined period in the cell containing phosphate buffer/organic solvent and a given pesticide concentration diluted in organic solvent. The reaction was started by addition of p-aminophenyl acetate substrate at a final concentration of 1 mM and the inhibition rate was determined according to the relation: I%= (I1 − I2)/I1 × 100, I1 and I2 being respectively the sensor response before and after inhibition. The measurement of enzyme activity and its inhibition is thus performed in the same reaction cell. The current before inhibition (I1) was measured according to the same procedure in the absence of pesticide. 3. Results and discussion
3.1. Electrochemical studies of p-aminophenol in the presence of organic sol6ent Fig. 1 shows the cyclic voltammograms performed with a screen-printed electrode in the pres-
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ence and in the absence of 1 mM p-aminophenol in buffer solution containing 5% acetonitrile. paminophenol shows a well-defined oxidation peak starting from 50 mV with a maximum current peak at around 100 mV, while p-aminophenyl acetate shows only an oxidation peak starting from 550 mV with a maximum current peak at 690 mV. Thus, the reaction product, p-aminophenol can be easily detected at + 100 mV without interference due to the substrate. The influence of working potential on the screen-printed electrode response was also studied by amperometric measurements with p-aminophenyl acetate substrate in the presence of 1 and 5% acetonitrile (Fig. 2). As can be seen, the recorded current increases for potentials ranging between 0 and 100 mV, while no significant variation is observed for potentials over 100 mV. Consequently, amperometric measurements were performed at a potential of 100 mV vs. Ag/AgCl screen-printed reference electrode.
Fig. 1. Cyclic voltammograms on the screen-printed electrode in buffer containing 5% acetonitrile in the presence and in the absence of 1 mM p-aminophenyl acetate (p-APA) and 1 mM p-aminophenol (p-AP). Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8. Three electrode system: Ag/AgCl pseudo-reference screen-printed electrode, Pt auxiliary electrode and graphite screen-printed working electrode. Scan potential between − 0.5 and 0.7 V; scan rate: 50 mV /s − 1.
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Fig. 2. Influence of the working potential on the amperometric response of AChE screen-printed electrode in 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; 1 mM p-aminophenyl phosphate.
3.2. The effect of acetonitrile and ethanol on the immobilised AChE The presence of organic solvent has a strong influence on the enzyme activity. Generally, the enzymes are much more inactivated in hydrophilic solvents than in hydrophobic solvents, probably because water-miscible solvents remove the essential water layer more easily than hydrophobic solvents. The behaviour of AChE in various ethanol and acetonitrile concentrations was studied. Usually, extraction of pesticides from different matrices is carried out with ethyl acetate, but this solvent is not compatible with screen-printed electrode material. Extraction can be also realised with miscible solvents as acetonitrile and ethanol [31]. An additional advantage of using these solvents is that both, p-aminophenyl acetate substrate and pesticides are highly soluble in acetonitrile and ethanol. Previous works have reported the protective effect induced by entrapment of AChE in a PVA-SbQ matrix [27]. It was shown that PVA-SbQ-immobilized cholinesterase retains 50% of initial activity in acetonitrile while the free enzyme loses its activity in the same conditions. Based on this results, this immobilization method was used in these experiments.
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The percentage of organic solvents to be used for inhibition studies was chosen in order to save more than 80% of the initial enzyme activity. The effect of various amounts of solvent on the sensor response was evaluated taking as 100% control the response in presence of 1% of organic solvent (Fig. 3). The presence of more than 20% organic solvent induces a complete and irreversible inactivation of the enzyme, while 80% of the activity is conserved using 1– 5% of organic solvent. The electrode showed a stable response for more than 12 assays when experiments were performed in less than 5% of organic solvent. The response was shown to be unstable using solvent ratios over 5%. Solvent percentages higher than 20% does not allow to record any current. The calibration curves obtained for paminophenyl acetate substrate in buffer containing 1 and 5% acetonitrile are presented in Fig. 4. The apparent Michaelis– Menten constants were found to be 2.39 mM and 1.16 mM using respectively 1 and 5% acetonitrile. No significant difference was obtained when working in 1 and 5% organic solvent, the same linear range (1× 10 − 6 to 1 ×10 − 3 M) and sensitivity (0.190.05
Fig. 4. Calibration curves of p-aminophenyl acetate (p-APA) substrate with AChE screen-printed electrode in the presence of 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8, applied potential: 100 mV vs. Ag:AgCl reference electrode.
mA M − 1) being obtained in both cases. Other studies based on the use of acetylthiocholine as substrate show an activation of cholinesterase using acetonitrile and ethanol ranging between 5 and 15% [26]. This feature was not observed using p-aminophenyl acetate as substrate and the system presents the same characteristics in the presence of 5 or 1% acetonitrile.
3.3. Inhibition measurements with paraoxon and chlorpyrifos ethyl oxon
Fig. 3. Influence of the percentage of acetonitrile and ethanol on the residual activity of AChE biosensor; Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag:AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
Inhibition experiments using the AChE screenprinted electrodes have been performed with paraoxon and chlorpyrifos ethyl oxon. In order to preserve the enzyme activity and the stability of the sensor, a buffer containing less than 5% acetonitrile was selected for the assays. The influence of incubation time on the inhibition of AChE biosensor by 3× 10 − 8 M chlorpyrifos ethyl oxon is presented in Fig. 5. Experiments were carried out in phosphate buffer solution containing 1 or 5% acetonitrile. As expected, the percentage of AChE inhibition increased when increasing the incubation time. The inhibition rate was shown to be slighly higher when working in 5% acetonitrile. In order to obtain a better sensitivity, an incuba-
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Fig. 5. The influence of incubation time on the inhibition degree of AChE biosensor by 3 ×10 − 8 M chlorpyrifos ethyl oxon in 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
Fig. 7. Inhibition curves of AChE biosensor by paraoxon (Px) and chlorpyrifos ethyl oxon (Cp-ox) in 5% acetonitrile after 10 min. incubation. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8: applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
tion time of 10 min was selected for subsequent inhibition studies. Inhibition curves of immobilized AChE by
chlorpyrifos ethyl oxon in the presence of 1 or 5% acetonitrile are shown in Fig. 6. Each reported value is calculated as the mean of the responses of three electrodes prepared in the same conditions. The detection limits calculated at 20% inhibition (I20) were found to be 1.769 0.66×10 − 9 M using 1% acetonitrile and respectively 1.249 0.95× 10 − 9 M using 5% acetonitrile. The chlorpyrifos ethyl oxon is the most powerful AChE-inhibiting pesticide. A comparison of inhibition curves obtained in 5% acetonitrile using paraoxon and chlorpyrifos ethyl oxon is presented in Fig. 7. As low as 1.919 0.44×10 − 8 M paraoxon can be detected in 5% acetonitrile. The sensitivity of the enzyme towards the two pesticides follows the same variation as previously reported, chlorpyrifos ethyl oxon being much more toxic for AChE than paraoxon. The inhibition constant rate (ki) measured by spectrophotometric measurement with free enzyme were 1.6× 10 − 6 M − 1 min − 1 and 1.2× 10 − 9 −1 −1 M min for paraoxon and respectively for chlorpyrifos ethyl oxon. Very similar detection limits have been found when experiments are carried out using ethanol as solvent.
Fig. 6. Inhibition curves of AChE biosensor by chlorpyrifos ethyl oxon in 1 and 5% acetonitrile after 10 min. incubation. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
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4. Conclusions This work shows the possibility to use paminophenyl acetate as AChE substrate to detect pesticides in the presence of water-miscible organic solvents. The main advantage of using this substrate instead of the classically used acetylthiocholine relies on its high solubility in organic solvents. The results show that analytical characteristics of the electrode are very similar when working in either 5 or 1% acetonitrile. The presence of organic solvents does not enhance the enzyme activity as reported using acetylthiocholine. It was demonstrated that the presence of a concentration of 5% acetonitrile does not affect the detection limit and the selectivity of the AChE biosensor towards insecticides. The sensor shows good characteristics and it appears to be suitable for the detection of pesticides in the presence of small amount of organic solvent. In a first approach, these sensors could be used as alarm systems which are able to provide an estimation of the global toxicity index of food products or environmental samples. They could be use as a complementary technique to classical methods, the determination procedure being extremely simple and rapid (a complete test is realised in 25 min). The disposable sensor offers several advantages including low cost, simple handling, mass production and is suitable for miniaturisation as a portable device. On the other hand, the single use of these sensors avoids the need to reactivate the enzyme after each analyse but also eliminates the activation step often required in the case of multiple use. This feature is particular important for this system, as the product of the electrochemically reaction is unstable and could be easily adsorbed on the working surface inducing the fouling of the electrode. Further experiments will be focused on the detection of pesticides extracted from real samples and on the stabilization of AChE in higher concentrations of organic solvents, using bioengineered ChE.
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Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents Silvana Andreescu a,b, Thierry Noguer a, Vasile Magearu b, Jean-Louis Marty a,* a
Centre de Phytopharmacie, Uni6ersite´ de Perpignan-UMR CNRS 5054, 52, A6enue de Villeneu6e, 66860 Perpignan Cedex, France b Uni6ersity of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, 90 -92 Panduri, 76234, Bucharest, Romania Received 15 June 2001; received in revised form 17 December 2001; accepted 18 December 2001
Abstract A screen-printed biosensor for the detection of pesticides in water miscible organic solvents is described based on the use of p-aminophenyl acetate as acetylcholinesterase substrate. The oxidation of p-aminophenol, product of the enzymatic reaction was monitored at 100 mV vs. Ag/AgCl screen-printed reference electrode. Miscible organic solvents as ethanol and acetonitrile were tested. The acetylcholinesterase (AChE) was immobilised on a screen-printed electrode surface by entrapment in a PVA-SbQ polymer and the catalytic activity of immobilised AChE was studied in the presence of different percentages of organic solvents in buffer solution. The sensor shows good characteristics when experiments were performed in concentrations of organic solvents below 10%. No significant differences were observed when working with 1 and 5% acetonitrile in the reaction media. Detection limits as low as 1.91 × 10 − 8 M paraoxon and 1.24 × 10 − 9 M chlorpyrifos ethyl oxon were obtained when experiments are carried out in 5% acetonitrile. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholinesterase; Organic phase biosensor; Pesticide; Disposable; Screen-printed electrodes; Inhibition
1. Introduction Pesticides are very toxic compounds, which have been shown to be responsible for many ecological problems and damages to human health. Among the many methods reported for pesticides detection, chromatographic methods such as HPLC and GC are often used as reference * Corresponding author. E-mail address: [email protected] (J.-L. Marty).
methods. Despite their high sensitivities, these techniques are expensive, time-consuming and require highly trained personnel; furthermore they are not adapted for in situ and real time detection of pollutants. At the same time, they are not able to give any information concerning the toxicity of the sample. Biochemical sensors appear as a reliable alternative to classical methods for the rapid and simple detection of pesticides. Various systems for the detection of insecticides are described in literature. Biological sensors
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based on organophosphate hydrolase [1– 3] which catalyse the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent into a direct detectable compound (p-nitrophenol) have been used for the determination of pesticides, leading to the detection of 0.2 and 1 mM paraoxon and methyl parathion respectively [2]. Immunological sensors coupled with different transduction modes have also been reported to detect pesticides with a very low detection limits [4,5]. For instance 0.3 ppb parathion was detected with a optic fiber immunosensor [5]. Other studies report the determination of pesticides based on their inhibitory effect on the enzyme activity. In these studies, the decrease of enzyme activity caused by inhibition is correlated to the concentration of pesticide in the sample or their metabolites concentration in the sample. Tyrosinase-based sensors have been developed for the detection of enzymes inhibitors with a detection limit in the range of micromolar concentrations [6,7]. These sensors suffer from a poor specificity because of possible interferences caused by the presence of various possible substrates and inhibitors of tyrosinase. Most traditional methods refer to the use of cholinesterases (ChE), acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) which are the real biological target of main organophosphorus and carbamate insecticides, representing 40% of the world market of this class of compounds. Various electrode configurations based on different transduction systems have been reported for ChE sensors [8–17]. The activity of ChE is usually determined by using esters of choline which are hydrophilic substrates. In a biosensor configuration, acetyl- or butyryl-choline can be used as substrate if the cholinesterase is associated with a choline oxidase (ChO) [13–15]. In this case, two detection methods are possible, by monitoring either oxygen consumption using a Clark-type electrode [16] or hydrogen peroxide production by oxidation at 650 mV vs. Ag/AgCl [8,13]. Such a complicated device can be simplified to a mono-enzyme system using acetyl- or butyryl-thiocholine as substrate; in this case the detection is based on the oxidation of thiocholine produced upon enzyme reaction. An important advantage of such a system is re-
lated to low overpotential necessary for thiocholine oxidation (410 mV vs. Ag/AgCl, or 100 mV vs. Ag/AgCl using TCNQ as mediator [12,17] or cobalt phtalocyanine [18,19]) as mediator. An alternative substrate, p-aminophenyl acetate has been used as AChE substrate for amperometric sensors, the detection being based on the oxidation of p-aminophenol at 250 mV vs. SCE [20,21]. Using this system, a detection of 4 nM paraoxon and 13 nM carbaryl was reported. Pesticide detection is generally performed in aqueous solutions. However, these compounds are generally characterized by a low solubility in water and a high solubility in organic solvents. Extraction and concentration of pesticides from solid matrices (fruits, vegetables,…) are thus commonly carried out in such solvents. The ability of enzymes to work in non-aqueous solvents has been proved for many years [22–25]. Depending on the nature and the amount of the solvent, the enzyme activity can be strongly affected when experiments are performed in these media. Some enzymes like glucose oxidase or tyrosinase work in various organic solvents as well as in water, but in all cases a minimal amount of water is required to retain catalytic activity [22–25]. ChE seems to be more sensitive to organic solvents than the previously named enzymes. The influence of organic solvents on free AChE activity [26] has been reported and few publications refer to the detection of pesticides with immobilized AChE in these media [27–30]. In a recent paper, we have reported the influence of acetonitrile, ethanol and DMSO on a cholinesterase sensor using acetylthiocholine as substrate [29]. An increase of the output current was noticed when working in 5% acetonitrile and 10% ethanol. The sensor was used to detect chlorpyrifos ethyl oxon. The detection of dichlorvos, diazinon and fenthion in the presence of ethanol was also reported with an amperometric AChE biosensor based on thiocholine-hexacyanoferrate reaction [30] and the bi-enzyme AChE/ChO system [28] allowed to detect 0.2 ppb paraoxon in the presence of 5% cyclohexane. p-aminophenyl acetate, which was already been used as substrate for AChE sensors is characterized by a good solubility in organic solvents such as acetonitrile. This
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feature suggests the possibility to extend the application field of AChE biosensors to non-aqueous media. The aim of this work was to study the effect of water-miscible organic solvents on the catalytic activity of immobilised AChE by using paminophenyl acetate as substrate. The biosensor was applied to the detection of organophosphorus insecticides (paraoxon and chlorpyrifos ethyl oxon) in the presence of acetonitrile and ethanol. Low cost screen-printed disposable electrodes have been used for tests, eliminating the need to reactivate the enzyme after inhibition measurement. These single use sensors also avoid the problems related to the possible fouling of the electrode which generally involve a chemical or electrochemical activation of the working electrode surface. To our knowledge, this is the first study referring to the use of p-aminophenyl acetate substrate to study the effect of organic solvents on immobilised AChE. These developed screen-printed biosensors have been applied to the detection of pesticides in the presence of such media.
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Paraoxon ethyl and chlorpyrifos ethyl oxon were purchased from Dr Ehrenstorfer GmbH (Germany) and Dow Elanco (AGR 203674). All other reagents used in the tests were analytical grade. The supporting electrolytes used in this work contained 0.1 M KCl. All measurements were performed at 25 °C.
2.2. Apparatus A DEK 248 screen-printing system (DEK, UK) was used to fabricate the electrodes. Amperometry experiments were carried out with a simplified electrochemical system, consisting in two electrodes made by screen-printing, a 641VA (Metrohm 64 VA, Swiss) potentiostat and a BD40 (Kipp & Zonen, Holland) recorder. Cyclic voltammetric studies were carried out with a 362 EG & G (Princeton Applied Research, USA) potentiostat connected to a LY 1400 x–y recorder. In these experiences a platinum electrode was used as counter electrode.
2.3. Methods 2. Experimental
2.1. Materials AChE from Drosophila melanogaster was provided from Prof. D. Fournier (University Paul Sabatier, Toulouse, France). p-nitrophenyl acetate and p-aminophenol were obtained from Sigma. Photocrosslinkable poly(vinyl alcohol) with styrylpyridinium groups polymer (PVASbQ), polymerization degree 1700, was provided by Toyo Gosei Co. (Chiba, Japan). Hydroxyethylcellulose (HEC) was supplied by Fluka. Printing pastes (Electrodag PF-410, 423SS, 6037SS) were obtained from Acheson, France. Graphite Timrex T15 was supplied by Timcal (Switzerland). The insulating layer was a commercial Astral paint (France). Clear PVC sheets (200×100 mm2, 0.5 mm thick, supplied by SKK, Germany) were used as support for the screenprinting electrodes.
2.3.1. Synthesis of p-aminophenyl acetate substrate p-aminophenyl acetate substrate was synthesized by catalytic reduction of p-nitrophenyl acetate in presence of SnCl2 under nitrogen atmosphere according to Pariente et al. [20]. The product was extracted in ethyl acetate and then treated with NaOH. After extraction, the organic phase was dried with MgSO4 and concentrated. The residue was purified by two successive silica gel chromatography (CH2Cl2/MeOH 98:2) and after evaporation the product was dissolved in ether and recrystallised in hexane. The purity was checked by thin layer chromatography, proton NMR spectrum and UV–VIS spectroscopy. White crystals of p-aminophenyl acetate were obtained. The substrate was stored at a temperature below 0 °C under nitrogen atmosphere. Before assays, p-aminophenyl acetate solutions were prepared using pure organic solvent (acetonitrile or ethanol).
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2.3.2. Electrochemical measurements of p-aminophenol in organic sol6ents AChE catalyses the hydrolysis of paminophenyl acetate into p-aminophenol. The electrochemical behaviour of p-aminophenol was studied by cyclic voltammetry in a three electrode system consisting in a screen-printed Ag/AgCl pseudo-reference electrode, a graphite screenprinted working electrode and a platinum auxiliary electrode. Experiments were performed in 0.1 M phosphate buffer containing 0.1 M KCl, pH 8 in the presence of different concentrations of organic solvent. The potential scan was carried out between −500 and 700 mV at a scan rate of 50 mV/sec. 2.3.3. Biosensor preparation The sensor system consisted of two electrodes: an Ag/AgCl reference electrode and a graphite working electrode, both of them made by screenprinting technology. The fabrication technique was based on consecutive depositions of several layers on a plastic sheet: a silver conducting layer for the electrical connections, a carbon pad, a reference electrode (Ag/AgCl), an insulating layer and a graphite ink (made of graphite powder and HEC 4%) to cover the working electrodes surface. Between each deposition the electrodes were dried at 60 °C for 30 min. The sensing layer containing the enzyme was then deposed on the working electrode surface. AChE immobilisation was made by entrapment in a photocrosslinkable PVA-SbQ polymer. A 3 ml homogeneous mixture of enzyme solution in 0.1 M phosphate buffer, pH 8 and PVA-SbQ polymer (30% v/v) was deposed on the working electrode surface. The amount of enzyme immobilised on each electrode was calculated to be 1 mU. The electrodes were then placed for three hours at 4 °C under neon lamp to allow polymerization. They are stored at 4 °C and can be used one day after preparation. 2.3.4. Amperometric measurements The amperometric measurements were carried out in a batch system by measuring the current corresponding to the oxidation of p-aminophenol, product of the enzymatic reaction. The two elec-
trodes system (working and reference electrodes) was immersed in a cell containing 3 ml 0.1 M phosphate buffer containing 0.1 M KCl pH 8, or the same buffer containing a given percentage of organic solvent. A potential of 100 mV vs. Ag/ AgCl reference electrode was applied between the two electrodes and the current obtained upon injection of 1 mM p-aminophenyl acetate was recorded. A non-enzymatic response due to the chemical hydrolysis of p-aminophenyl acetate was observed. For this reason, the response obtained with an enzyme electrode was compared with the response obtained with the same electrode after a complete inactivation of the enzyme in a very concentrated solution of pesticide. The influence of organic solvents on the catalytic activity of immobilised AChE was studied by measuring the response of the sensor in phosphate buffer containing water miscible organic solvent (acetonitrile and ethanol) in a concentration ranging from 1 to 25%. Operational stability was evaluated by carrying out consecutive injections of substrate at a final concentration of 1 mM, the cell being rinsed out with phosphate buffer solution between assays. To perform inhibition tests, the biosensor was incubated during a defined period in the cell containing phosphate buffer/organic solvent and a given pesticide concentration diluted in organic solvent. The reaction was started by addition of p-aminophenyl acetate substrate at a final concentration of 1 mM and the inhibition rate was determined according to the relation: I%= (I1 − I2)/I1 × 100, I1 and I2 being respectively the sensor response before and after inhibition. The measurement of enzyme activity and its inhibition is thus performed in the same reaction cell. The current before inhibition (I1) was measured according to the same procedure in the absence of pesticide. 3. Results and discussion
3.1. Electrochemical studies of p-aminophenol in the presence of organic sol6ent Fig. 1 shows the cyclic voltammograms performed with a screen-printed electrode in the pres-
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ence and in the absence of 1 mM p-aminophenol in buffer solution containing 5% acetonitrile. paminophenol shows a well-defined oxidation peak starting from 50 mV with a maximum current peak at around 100 mV, while p-aminophenyl acetate shows only an oxidation peak starting from 550 mV with a maximum current peak at 690 mV. Thus, the reaction product, p-aminophenol can be easily detected at + 100 mV without interference due to the substrate. The influence of working potential on the screen-printed electrode response was also studied by amperometric measurements with p-aminophenyl acetate substrate in the presence of 1 and 5% acetonitrile (Fig. 2). As can be seen, the recorded current increases for potentials ranging between 0 and 100 mV, while no significant variation is observed for potentials over 100 mV. Consequently, amperometric measurements were performed at a potential of 100 mV vs. Ag/AgCl screen-printed reference electrode.
Fig. 1. Cyclic voltammograms on the screen-printed electrode in buffer containing 5% acetonitrile in the presence and in the absence of 1 mM p-aminophenyl acetate (p-APA) and 1 mM p-aminophenol (p-AP). Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8. Three electrode system: Ag/AgCl pseudo-reference screen-printed electrode, Pt auxiliary electrode and graphite screen-printed working electrode. Scan potential between − 0.5 and 0.7 V; scan rate: 50 mV /s − 1.
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Fig. 2. Influence of the working potential on the amperometric response of AChE screen-printed electrode in 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; 1 mM p-aminophenyl phosphate.
3.2. The effect of acetonitrile and ethanol on the immobilised AChE The presence of organic solvent has a strong influence on the enzyme activity. Generally, the enzymes are much more inactivated in hydrophilic solvents than in hydrophobic solvents, probably because water-miscible solvents remove the essential water layer more easily than hydrophobic solvents. The behaviour of AChE in various ethanol and acetonitrile concentrations was studied. Usually, extraction of pesticides from different matrices is carried out with ethyl acetate, but this solvent is not compatible with screen-printed electrode material. Extraction can be also realised with miscible solvents as acetonitrile and ethanol [31]. An additional advantage of using these solvents is that both, p-aminophenyl acetate substrate and pesticides are highly soluble in acetonitrile and ethanol. Previous works have reported the protective effect induced by entrapment of AChE in a PVA-SbQ matrix [27]. It was shown that PVA-SbQ-immobilized cholinesterase retains 50% of initial activity in acetonitrile while the free enzyme loses its activity in the same conditions. Based on this results, this immobilization method was used in these experiments.
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The percentage of organic solvents to be used for inhibition studies was chosen in order to save more than 80% of the initial enzyme activity. The effect of various amounts of solvent on the sensor response was evaluated taking as 100% control the response in presence of 1% of organic solvent (Fig. 3). The presence of more than 20% organic solvent induces a complete and irreversible inactivation of the enzyme, while 80% of the activity is conserved using 1– 5% of organic solvent. The electrode showed a stable response for more than 12 assays when experiments were performed in less than 5% of organic solvent. The response was shown to be unstable using solvent ratios over 5%. Solvent percentages higher than 20% does not allow to record any current. The calibration curves obtained for paminophenyl acetate substrate in buffer containing 1 and 5% acetonitrile are presented in Fig. 4. The apparent Michaelis– Menten constants were found to be 2.39 mM and 1.16 mM using respectively 1 and 5% acetonitrile. No significant difference was obtained when working in 1 and 5% organic solvent, the same linear range (1× 10 − 6 to 1 ×10 − 3 M) and sensitivity (0.190.05
Fig. 4. Calibration curves of p-aminophenyl acetate (p-APA) substrate with AChE screen-printed electrode in the presence of 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8, applied potential: 100 mV vs. Ag:AgCl reference electrode.
mA M − 1) being obtained in both cases. Other studies based on the use of acetylthiocholine as substrate show an activation of cholinesterase using acetonitrile and ethanol ranging between 5 and 15% [26]. This feature was not observed using p-aminophenyl acetate as substrate and the system presents the same characteristics in the presence of 5 or 1% acetonitrile.
3.3. Inhibition measurements with paraoxon and chlorpyrifos ethyl oxon
Fig. 3. Influence of the percentage of acetonitrile and ethanol on the residual activity of AChE biosensor; Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag:AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
Inhibition experiments using the AChE screenprinted electrodes have been performed with paraoxon and chlorpyrifos ethyl oxon. In order to preserve the enzyme activity and the stability of the sensor, a buffer containing less than 5% acetonitrile was selected for the assays. The influence of incubation time on the inhibition of AChE biosensor by 3× 10 − 8 M chlorpyrifos ethyl oxon is presented in Fig. 5. Experiments were carried out in phosphate buffer solution containing 1 or 5% acetonitrile. As expected, the percentage of AChE inhibition increased when increasing the incubation time. The inhibition rate was shown to be slighly higher when working in 5% acetonitrile. In order to obtain a better sensitivity, an incuba-
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Fig. 5. The influence of incubation time on the inhibition degree of AChE biosensor by 3 ×10 − 8 M chlorpyrifos ethyl oxon in 1 and 5% acetonitrile. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
Fig. 7. Inhibition curves of AChE biosensor by paraoxon (Px) and chlorpyrifos ethyl oxon (Cp-ox) in 5% acetonitrile after 10 min. incubation. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8: applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
tion time of 10 min was selected for subsequent inhibition studies. Inhibition curves of immobilized AChE by
chlorpyrifos ethyl oxon in the presence of 1 or 5% acetonitrile are shown in Fig. 6. Each reported value is calculated as the mean of the responses of three electrodes prepared in the same conditions. The detection limits calculated at 20% inhibition (I20) were found to be 1.769 0.66×10 − 9 M using 1% acetonitrile and respectively 1.249 0.95× 10 − 9 M using 5% acetonitrile. The chlorpyrifos ethyl oxon is the most powerful AChE-inhibiting pesticide. A comparison of inhibition curves obtained in 5% acetonitrile using paraoxon and chlorpyrifos ethyl oxon is presented in Fig. 7. As low as 1.919 0.44×10 − 8 M paraoxon can be detected in 5% acetonitrile. The sensitivity of the enzyme towards the two pesticides follows the same variation as previously reported, chlorpyrifos ethyl oxon being much more toxic for AChE than paraoxon. The inhibition constant rate (ki) measured by spectrophotometric measurement with free enzyme were 1.6× 10 − 6 M − 1 min − 1 and 1.2× 10 − 9 −1 −1 M min for paraoxon and respectively for chlorpyrifos ethyl oxon. Very similar detection limits have been found when experiments are carried out using ethanol as solvent.
Fig. 6. Inhibition curves of AChE biosensor by chlorpyrifos ethyl oxon in 1 and 5% acetonitrile after 10 min. incubation. Experimental conditions: phosphate buffer solution 0.1 M/0.1 M KCl, pH 8; applied potential: 100 mV vs. Ag/AgCl reference electrode, 1 mM p-aminophenyl acetate substrate.
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4. Conclusions This work shows the possibility to use paminophenyl acetate as AChE substrate to detect pesticides in the presence of water-miscible organic solvents. The main advantage of using this substrate instead of the classically used acetylthiocholine relies on its high solubility in organic solvents. The results show that analytical characteristics of the electrode are very similar when working in either 5 or 1% acetonitrile. The presence of organic solvents does not enhance the enzyme activity as reported using acetylthiocholine. It was demonstrated that the presence of a concentration of 5% acetonitrile does not affect the detection limit and the selectivity of the AChE biosensor towards insecticides. The sensor shows good characteristics and it appears to be suitable for the detection of pesticides in the presence of small amount of organic solvent. In a first approach, these sensors could be used as alarm systems which are able to provide an estimation of the global toxicity index of food products or environmental samples. They could be use as a complementary technique to classical methods, the determination procedure being extremely simple and rapid (a complete test is realised in 25 min). The disposable sensor offers several advantages including low cost, simple handling, mass production and is suitable for miniaturisation as a portable device. On the other hand, the single use of these sensors avoids the need to reactivate the enzyme after each analyse but also eliminates the activation step often required in the case of multiple use. This feature is particular important for this system, as the product of the electrochemically reaction is unstable and could be easily adsorbed on the working surface inducing the fouling of the electrode. Further experiments will be focused on the detection of pesticides extracted from real samples and on the stabilization of AChE in higher concentrations of organic solvents, using bioengineered ChE.
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