Biosensors & Bioelectronics 7 (1992) 335-343

Sensitive detection,of pesticides using amperometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilized cholinesterases Petr Skladal* Dipartimento

& Marco Mascini

di Sanita Pubblica, Epidemiologia e Chimica Analitica Ambientale, via G. Capponi 9,50121 Florence, Italy

Sezione di Chimica Analitica,

(Received 16 July 1991; revised version received 3 October 1991; accepted 6 October 1991)

Abstract: The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes coupled with acetylcholinesterase or butyrylcholinesterase. Covalent immobilization of enzymes on Immobilon membranes or nylon nets was examined; the highest sensitivity to inhibitors was found for the nylon net containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetyl- or butyrylthiocholine as substrates; thiocholine produced by hydrolysis in the enzyme membrane was electrochemically oxidized at +300 mV (vs. Ag/AgCl reference). The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, 1.5 and 8.4 pg 1-r ofparaoxon and heptenophos, respectively, can be detected in less than 3 min. These detection limits are similar as those obtained when analyses were performed using free cholinesterase and 10 min incubation with inhibitor. Keywords: biosensor, enzyme electrode, acetylcholinesterase, butyrylcholinesterase, cobalt phthalocyanine, composite electrode, paraoxon, carbatyl, heptenophos, malathion.

INTRODUCTION Organophosphate and carbamate pesticides are widely used in agriculture because of their low persistence. However, a high acute toxicity of *On leave from Department of Biochemistry, Masaryk University,

Kotlarska

2,61137 Bmo, Czechoslovakia.

0965-5663/92/$05.00 Q 1992 Elsevier Science Publishers

these compounds creates a need for fastresponding detection systems in order to protect human health during manufacturing and application processes and subsequently sensitive systems for reliable control of food products and environment compounds Ltd.

pollution. were produced

similar Because as possible nerve 335

P, Skladal, M. Mascini

poisons, a further area of application is in the military. The mode of action of these pesticides is based on irreversible inhibition of acetylcholinesterase (Eto, 1974; Froede &Wilson, 1971) and the same principle is utilized for analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are widely used for construction of various biosensing devices monitoring the decrease of enzyme activity in the presence of inhibitor. According to the method selected for measurement of enzyme activity, a variety of spectrophotometric (Leon-Gonzales & Townshend, 1990; Kindervater et al., 1990; Wolfbeis & Koller, 1989), fluorometric (Guilbault & Kramer, 1965) piezoelectric (Guilbault & Ngwainbi, 1989) potentiometric (Goodson & Jacobs, 1976) pH (Iran-Minh et al., 1990; Durand &Thomas, 1984) and voltammetric (Medyantseva et al., 1990) devices was developed, but recently attention has been directed especially to amperometric sensors. Two amperometric approaches were studied, depending on the substrate being utilized. If acetylcholine is the choice, the cholinesterase system is coupled with a choline probe based on a choline oxidase and hydrogen peroxidemeasuring platinum anode (Marty et al., 1990; Bernabei er al., 1991). The other utilized substrates are acetylthiocholine (ATCh) and butyrylthiocholine (BTCh). These are hydrolysed by the action of cholinesterase to the corresponding organic acid and thiocholine, which can be oxidized electrochemically. The platinum anode is not convenient for this purpose since a large overvoltage is necessary (Halbert & Baldwin, 1985). Similar problems in detection of other sulphydryl compounds were overcome by use of a variety of carbon-based electrodes chemically modified with cobalt phthalocyanine (CoPC) (Halbert & Baldwin, 1985; Qi etal., 1991; Wring et al., 1989, 1991), tetracyano-p-quinodimethane (TCNQ), tetrathiafulvalene and l,l’-dimethylferrocene (Kulys & Drungiliene, 1991). Recently, two systems for detection of pesticides based on BChE coupled to a TCNQ-modified graphite (Kulys & D’Costa, 1991) and CoPC-modified carbon paste electrode (Skladal, 1991) have been described. In this work, sensitive and fast-responding sensors based on a CoPC-modified carbon epoxy composite electrode are reported and two modes of operation, using soluble and immobilized cholinesterases, are compared. 336

Biosensors & Bioelectronics

EXPERIMENTAL Materials AChE (EC 3.1.1.7,950 IU mg-‘, from electric eel), BChE (EC 3.1.1.8, 900 IU mg-‘, from horse serum), ATCh chloride, BTCh chloride, glutaraldehyde (25%) and paraoxon were purchased from Sigma Chemical Co. (St Louis, MO, USA). Malathion was from Supelco (Bellefonte, PA, USA); heptenophos and carbaryl were kindly provided by Dr B. Safar, Institute of Analytical Chemistry, Brno, Czechoslovakia. CoPC was from Aldrich Chemical Co. (Milwaukee, WI, USA), and dimethylsulphate was supplied by Fluka (Buchs, Switzerland). Immobilon AV affinity membranes were obtained from Millipore (Bedford, MA, USA), nylon net (120 threads cm-‘, 1OOpm thickness) was from A. Bozzone (Appian0 Gentile, Italy); cellulose acetate dialysis membrane was obtained from Carlo Erba (Milan, Italy). Epoxy resin was supplied by Lachema (Bmo, Czechoslovakia). All other chemicals were of reagent grade. Preparation of CoPC-modified composite electrodes Equal amounts (w/w) of epoxy resin and carbon black containing 7% of CoPC were thoroughly mixed for 10 min and then packed into glass tubes (2 mm i.d.). The electrical contact was realized by pushing a wire down through the tube into the back of mixture. The electrodes were allowed to cure for 24 h at room temperature. The hardened surface was then polished with a fine emery paper and washed with water. Immobilization of cholinesterases Two different supports were used for immobilization of enzymes, nylon nets and Immobilon membranes. The activation of nylon net was carried out as described elsewhere (Mascini ec al., 1983). In order to obtain membranes varying in cholinesterase content, the discs of nylon net (13 mm diameter) activated with glutaraldehyde were washed and then incubated in 0.7 ml of 50 mM phosphate buffer, pH 7.3, containing increasing amounts of activity. Incubation proceeded for 2 h at room temperature and then overnight at 4°C. After washing, membranes were stored dry.

Biosensors & Bioelectronics

The binding of BChE to Immobilon was performed by dot immobilization as recommended by the manufacturer. Ten microlitres of 100 mM phosphate, pH 7.3, containing increasing amounts of BChE (2, 4, 7, 15 or 40 IU) were applied to 13 mm diameter discs of Immobilon. After 1 h of incubation at room temperature, the remaining binding sites were saturated in 2 M glycine buffer for 2 h. Membranes were washed and stored dry. Electrochemical measurements A three-electrode system consisting of the CoPCmodified carbon composite working, stainlesssteel auxiliary and Ag/AgC1/3 M KC1 reference electrodes was used together with a model 3001 Amperometric Biosensor Detector (Universal Sensors, Metairie, LA, USA). Output signal was recorded on a model 868 recorder (Amel, Milan, Italy). Phosphate buffer (50 mM, pH 7-3) was used throughout and experiments were carried out in a 5 ml vessel thermostated to 37°C and equipped with a stirring bar. When measuring the activity of free cholinesterase, the working electrode was covered by a dialysis membrane in order to minimize disturbances from stirring and to avoid surface fouling. The activity of free enzyme was expressed as the time current change after the addition of substrate. The cholinesterase biosensors were constructed prior to use by placing an enzyme membrane disc (3 mm diameter) before the working electrode, covering by a piece of nylon net and fixing by an o-ring.

Sensitive detection of pesticides

current (I,,) corresponding to the enzyme hydrolysis of substrate (O-5 mM ATCh or BTCh) was recorded. The addition of inhibitor followed and current decrease (dildt) was measured.

RESULTS AND DISCUSSION Optimization of the cholinesterase biosensor The CoPC-modified carbon paste electrodebased biosensor was used for determination of pesticides previously (Skladal, 1991). This type of composite electrode is quite suitable for preliminary experiments, but it is necessary to prepare a new electrode surface before using a new enzyme membrane. For this work, mechanically more robust epoxy composite electrodes were prepared and used for the construction of the cholinesterase biosensor. The hydrodynamic voltammogram for ATCh using a CoPC epoxy composite electrode covered by the nylon net with immobilized AChE is shown in Fig. 1 together with the background current. A potential of 300 mV was selected for further amperometric experiments. The signal/background current ratio is quite good and this potential is low enough to minimize possible interference.

Inhibition of cholinesterase by pesticides All solutions of pesticides were prepared in methanol fresh every day. When measuring the inhibition of free cholinesterase, the reaction was started by the addition of enzyme (0.16 IU ml-’ final concentration) into the buffer solution containing the required concentration of pesticide. After exactly 10 min, the substrate (1 mM ATCh or BTCh) was added and the remaining enzyme activity (ai) was determined. Incubation of enzyme in the absence of any inhibitor was used as a control (a,). The percentage inhibition was calculated as 100 X (a, - ai)/a, and was used for further evaluations. A different approach was used for determination of pesticides using immobilized enzymes. At the beginning, the steady-state

0.0

0.2

0.4

0.6

E (V) vs. Ag/AgCI

Fig. I. Hydrodynamic voltammogram for the CoPCmod@ed carbon epoxy composite electrode covered by a nylon net with immobilized AChE. The enzyme was prepared using 40 IUofAChE; measurements were made in 50m~ phosphate bu$er, pH 7.3, at 37°C. The cuwent increases determined after addition of ATCh to 1 mbtfinal concentration (0) and the background current (V) are shown. 337

I? Skladal. M. Mascini

Biosensors & Bioelectronics

A similar shape of hydrodynamic voltammograms was observed also for cysteine oxidation using bare CoPC-modified carbon electrodes (Wring er al., 1989). It was assumed that electrooxidation of Co’+ Co” occurs in this region of potentials, Co’ being produced by chemical reaction between thiocompound and CoPC. It seems that the same reaction sequence is valid also for thiocholine. According to Fig. 1, higher responses can be achieved at more positive potentials, but the increase of baseline current becomes unacceptable. Two different- supports were investigated for immobilization of BChE: nylon net and Immobilon membranes. In both cases, membranes containing increasing loadings of activity were prepared in order to find the optimal composition for detection of inhibitors. For nylon nets, various loadings of activity were achieved by incubation of activated nets in buffer solutions containing various activity of BChE (Fig. 2). In the case of Immobilon, different amounts of activity were directly applied on membrane discs (Fig. 3). For both supports, the maximal response to inhibitor (heptenophos) was obtained for membranes containing the lowest BChE activity. This is different from results obtained with BChE cross-linked by glutaraldehyde; the optimal response to pesticides

0.0

L 0

1

I

20

40 BChE

I

60

I

80

100

(NJ)

Fig 2. Ejhect of BChE loading in the nylon net on sensor responses to substrate (0) and inhibitor( A). The horizontal axis (BChE activity) is used-for of the enzyme _ preparation _ membrane (see text for details). Response to substrate is expressed as the slope of the calibration curve for BTCh; sensitivity for inhibitor is the relative inhibition measured for 4 mg I-’ heptenophos with 0.5 mMBTCh as substrate. 338

0.0 -0

10

20 BChE

30

40

0.0

(IU)

Fig. 3. Effect of BChE loading in the Immobilon membrane on sensor responses to substrate (0) and inhibitor (A). The horizontal axis is BChE activity applied to a disc of Immobilon (13 mm diameter). Conditions of measurements are the same as for Fig. 2.

was found with enzyme loadings higher than the minimal being tested (Skladal, 1991). The value of relative inhibition obtained for nylon net was 35 times greater than for Immobilon (Figs 2 and 3). The maximal responses to substrate (BTCh) were obtained for membranes with higher enzyme loading and were similar for both supports: 0.200 and 0.257 CAM-’ for nylon net and Immobilon, respectively. For comparison of long-term stability of both types of enzyme membranes, they were stored dry at room temperature. For nylon net membrane (Fig. 2, 10 IU membrane), the relative inhibition rate increased to O+KW s-’ after one month storage, while for Immobilon (Fig. 3, 2 IU membrane) an increase only to OW15 s-’ was determined. The sensitivity for substrate was decreased twice during this period. This means that further decrease of the enzyme loading in membrane will probably result in a shortened lifetime of membranes. When stored for one month at 4°C the parameters ofboth membranes remained practically constant. The response time to substrate (95% of steady-state current) is 50-70 s for_ nylon net and 2 min for Immobilon-based biosensor. For further experiments, the immobilization of AChE or BChE on nylon nets using 10 IU of activity was selected as the best.

Biosensors & Bioelectronics

Sensitive detection of pesticides

600

0

2

4 [substrate]

6

a

(mM)

Fig. 4. Substrate calibration curvesfor biosensors based on CoFC-modtjied carbon epoxy composite electrodes covered by nylon membranes with immobilized AChE (V. 1) or BChE (0, a), prepared using 40 IU of cholinesterase. The open symbols show calibration curvesforATCh;Jilledones correspond to BTCh. The enlarged initial parts of calibrations are shown in the inset graph.

Biosensor response to substrate

TABLE 1 Kinetic parameters of free and immobilized cholinesterases on two substrates Acetylthiocholine &I (app) (mW Free AChE BChE Immobilized AChE BChE

oTM54 0.616 5.36 6.54

:Zs-l)

0.419 0.357 1370 1660

Determination of pesticides The determination of pesticides is usually carried out by the incubation of free or immobilized cholinesterase in the presence of inhibitor. These approaches are based on the equation for percentage inhibition (a): lOg(lOO/U) = ki[I]t

Calibration curves for biosensors based on AChE and BChE for substrates ATCh and BTCh are shown in Fig. 4. The apparent K, and I,,, values are presented in Table 1. For comparison, corresponding constants were determined also for soluble enzymes under the same conditions (50 mM phosphate, pH 7*3,37”C, CoPC-modified composite electrode as a detector). K, and V,,,,, (I,,,) constants were calculated by fitting experimental data to the Michaelis-Menten equation using the non-linear regression curvefitting procedure provided in SigmaPlot version

Enzyme

4-Osoftware (Jandel Scientific, Corte Madera, CA, USA). For BChE, the K, constants were increased after immobilization 10 and 20 times for ATCh and BTCh, respectively. The biosensor sensitivities were similar for both these substrates; slopes of calibration curves O-227 f 0.003 (ATCh) and 0.216 + 0.002 mA M-’ (BTCh) were determined. In the case of AChE, a sharp decrease of affinity for ATCh occurred, but K, for BTCh decreased twice. This indicates that the strong preference for ATCh found for soluble enzyme is affected by immobilization. Nevertheless, ATCh is still preferred as a substrate; sensitivities of 0.228 + 0.005 (ATCh) and O-0238 + OWl mA M-’ (BTCh) were measured.

Butyrylthiocholine

?pp) (mW

2.43 0.433 1.04 7.74

$Ys-l)

0.0143 0.392 34.1 1840

(1)

where ki is the bimolecular inhibition constant (Eto, 1974), [I] the concentration of inhibitor and t the incubation time. As the extent of inhibition is proportional to time, long incubation periods (lo-60 min) are used to achieve high sensitivity. Another period of time is necessary for the measurement of the remaining enzyme activity. It is clear that this approach is not very convenient for fast monitoring. The time required for one analysis can be reduced if the inhibition and measurement of enzyme activity are carried out simultaneously (Leon-Gonzales & Townshend, 1990; Goodson & Jacobs, 1976; Skladal, 1991). The equation for the rate of cholinesterase inhibition d[EI]/dt

= ki[E] [I]

(2)

is a theoretical basis for these kinetic approaches, where [E] and [EI] are concentrations of active and inhibited forms of cholinesterase, respectively. Usually, the response for inhibitor is obtained in about 1 min. Because the binding of inhibitor occurs at the same site as substrate, competition for these sites exists and sensitivity may be decreased. Both of the above-mentioned approaches were compared in this work. At first, analysis was 339

Biosensors & Bioelectronics

P Skladal, M. Mascini

-4

-5

-3

comparable with results published in the literature (Tran-Minh etal., 1990; Marty etal., 1990; Bernabei et al., 1991). In the case of malathion, the lowest detected concentration was substantially higher than expected (Tran-Minh et al., 1990). The inhibition by malathion is probably a slow process and the 10 min incubation used here is not enough (1 h was used by Tran-Minh). The measurements with cholinesterase biosensors were done as described previously (Skladal, 1991). At first, the steady-state currentl,, corresponding to O-5 mM substrate is measured, the addition of inhibitor follows and the time decrease of current (dildt) is determined. The relative inhibition (RI) is calculated from:

-1

-2

log c (g I-')

(a)

RI = (di/dt)/l,,

-6

@I

-5

-4

log

-3 c

-2

-1

0

(g 1-l)

Fig. 5. Inhibition of free AChE (a) and BChE (b) by pesticides. The decrease in enzyme activity is shown (0.16 IU ml-’ at the beginning) after 10 min incubation of paraoxon (0). carbatyl (A), heptenophos (0) or malathion (0). Inhibitions and measurements of activity (1 mMATCh (a) or BTCh (b) as substrate) were made in 50 mu phosphate, pH 7.3, at 37°C and CoFC-modified composite electrode protected by dialysis membrane was used as a detector

performed by incubation. Free AChE (Fig. 5(a)) and BChE (Fig. 5(b)) were inhibited in the presence ofparaoxon, carbaryl, heptenophos and malathion. An incubation time of 10 min was selected, which seems to be reasonable for possible practical utilization. CoPC-modilied electrodes were applied to the measurement of enzyme activities. The lowest detectable pesticide concentrations (defined as concentration required for 10% inhibition of cholinesterase activity) are 340

The relative inhibition obtained with biosensors consisting of AChE and BChE immobilized on nylon nets and CoPC-modified composite electrodes is shown in Fig. 6(a) and (b), respectively. Plots of log RI versus log c are used in order to cover several concentration ranges tested. In all cases, dependences of log RI versus log c are linear (for parameters see Table 2). In the case of cross-linked BChE membranes and CoPC-modified carbon paste electrode (CPE) (Skladad, 1991) slopes of similar plots were found to be close to 1, indicating linear dependency between RI and concentration of inhibitor. This can be expected, as the ratio (di/dt)/l,, seems to be analogous to (d[EI]/dt)/[E], equation (2) which linearly depends on the concentration of inhibitor [I]. Unfortunately, this simple model cannot be used for biosensors based on covalently immobilized cholinesterases (Table 2). Consequently, more complicated mathematical calculations are necessary for quantitative analyses. The lowest detected concentrations of pesticides determined by incubation and kinetic approaches are compared for both AChE and BChE in Table 3. Both methods exhibit quite similar sensitivities towards the pesticides being tested. Also the relationships among pesticides found with the immobilized forms of cholinesterases correspond to free forms. In the case of carbaryl, a great difference exists between AChE and BChE (Fig. 6(a) and (b)). It seems that for construction of cholinesterase-based biosensors the selection of the source of enzyme will be very important, as various cholinesterases exhibit different affinities

Biosensors & Bioelectronics

Sensitive detection of pesticides -2.0

-2.5 -2.5

/

-5 (a)

-4

-3 log c

-2

-1

--D

(g I-‘)

--3

--4

-2

-L

-1

log c (g I-?)

(b)

Fig. 6. Determination ofpesticida using CoPC-modtfied composite electrodes with AChE (a) and BChE (b) nylon net enzyme membranes. The relative inhibirions (RI) were measured as a decrease in steady-state currentfor substrate (0.5 muATCh for (a) and 0.5 mMBTCh for (b) a&r the addition ofpesticide uor symbols see Fig. 5). Calibration curve parameters arepresented in Table 2.

TABLE 2 Parameters of calibration curves (log RI vs. log c plots) for pesticides using the biosensors based on AChE and BChE immobilized on the nylon net and the CoPC-modified composite electrodes

AChE Slope Intercept corr. CoeffP BChE Slope Intercept corr. coeff.

Paraoxon

Carbaryl

Heptenophos

Malathion

0*506 -1.19 0997(7)

0.506 -1.79 0998(7)

a940 -1.17 0997(4)

0313 -2.657 0998(6)

0.545 -0.686 0999(5)

0.247 -2.33 0993(5)

0643 -0.843 0993(5)

0.253 -2.82 0*999(4)

‘The number of points used for calculation is shown in parentheses. TABLE 3 Comparison of the lowest detected pesticide concentrations bg 1-l) using free and immobilized cholinesterases

Free AChE BChE Immobilized AChE BChE

Heptenophos

Malathion

Paraoxon

Carbaryl

11 1.7

56 340

76 10

1600 4700

12 1.5

19 250

1800 8.4

840 920

to pesticides (Guilbault et al,, 1970; Alfthan et al., 1989). The detection limits of biosensor systems are considerably improved when compared with the sensor based on cross-linked BChE (Skladal,

1991). For instance, the detection limit 0.3 mg 1-l for heptenophos was determined previously, while detection of a 10 pg 1-l concentration is now possible. An amount as low as l-5 pg 1-l of paraoxon can be measured with the immobilized 341

P Skladai, M. Mascini

BChE-based CoPC composite electrode sensor. This concentration is comparable with literature data (Tran-Minh et al., 1990; Marty et al., 1990; Bernabei et al., 1991) for this pesticide, but in all cases at least 30 min incubation was necessary, while only 3 min are sufftcient for reliable determination of RI when the approach proposed here is used. If only qualitative analysis is requested, results can be obtained even faster. One enzyme membrane can be used for live to ten analyses depending on the measured pesticide concentrations.

CONCLUSIONS It was demonstrated that CoPC-modified electrodes can be conveniently used for amperometric measurements of the activity of soluble cholinesterases. These electrodes are more stable and cheaper when compared with probes based on choline oxidase. Furthermore, they are suitable for the construction of amperometric cholines~mse biosensors for detection and determination of pesticides and other anticholinesterase agents. When the inhibition is performed in the presence of substrate, the sensitivity obtained is comparable with measurements based on pre-incubation with pesticide. The approach proposed here can be used for development of sensitive and fast-responding monitoring systems.

ACKNOWLEDGEMENTS The financial support from the Commission of European Communities for Petr Skladal (TEMPUS grant IMG-CZT-014590) is gratefully acknowledged. We also acknowledge the Target Project Chimica Fine of the National Council of Research for a research grant.

REFERENCES Alfthan, K., Kenttamaa, H. & Z&ale, T. (1989). Characterization and semiquantitative estimation of organophosphorous compounds based on inhibition of cholinesterases. Anal. Chim. Acta, 217,43-51. Bernabei, M., Cremisini, C., Mascini, M. & Palleschi, G. (1991). Determination of organophosphorus and carbamic pesticides with a choline and acetyl342

Biosensors & Bioeiectronics choline ele~tr~hemical biosensor. Anal. Len., 24, 1317-31. Durand, P. & Thomas, D. (1984). Use of immobilized enzyme coupled with an electr~hemi~l sensor for the detection of organophosphate and carbamate pesticides. J. Environ. Pathol. Toxicol. Oncof., 5, 51-7. Eto, M. (1974). Olganophosphorus Pesticides, Otgunic and Biological Chemistry. CRC Press, Boca Raton, FL, pp. 123-57. Froede, H. C. & Wilson, I. B. (1971). Acetylcholinesterase. In The Enzymes, Vol. 5, ed. P. D. Boyer. Academic Press, New York p. 87. Goodson, L. H. &Jacobs, W. B. (1976). Monitoring of air and water for enzyme inhibitors. Meth. Enzymol., 44,647-58. Guilbault, G, G. & Kramer, D. N. (1%5). Fluorometric system employing immobilized cholinesterase for assaying anticholinesterase compounds. AnaL Chem., 37, 1675-80. Guilbault, G. G. & Ngwainbi, J. N. (1989). Use of protein coatings on piezoelectric crystals for assay of gaseous pollutants_ In NATO ASI Ser. C, Vol. 226, ed. G. G. Guilbault & M. Mascini. Reidel, Dordrecht, pp. 187-94. Guilbault, G. G., Sadar, M. H., Kuan, S. S. &Casey, D. (1970). Enzymatic methods of analysis: trace analysis of various pesticides with insect cholinesterases. Anal. Chim. Aeta, 52, 75-81. Halbert, M. K. & Baldwin, R P. (1985). Electrocatalytic and analytical response of cobalt phthalocyanine containing carbon paste electrodes toward sulphydryl compounds. Anal. Chem., 57, 591-5. Kindervater, R., Kunnecke, W. & Schmid, R D. (1990). Exchangeable immobilized enzyme reactor for enzyme inhibition tests in flow-injection analysis using a magnetic device. Anal. Chim. Acta, 234, 113-17. Kulys, J. & D’Costa, E. J. (1991). Printed amperometric sensor based on TCNQ and cholinesterase. Biosensors Bioelectronics, 6, 109- 15. Kulys, J. & Drungiliene, A. (1991). Chemically modified electrodes for the determination of sulphydryl compounds. Anal. Chim. Acta. 243,287-92. Leon-Gonzales, M. F. & Townshend, A (1990). Flowinjection determination of paraoxon by inhibition of immobilized acetylcholinesterase. Anal. Chim. Acta, 236,267-72. Marty, J. L., Rouillon, R, Sode, K. & Karube, I. (1990). Biosensors for carbamates and organophosphorus insecticides. B~osenso~ W, 2-4 May, Singapore, pp. 85-6. Mascini, M., Iannello, M. & Palleschi, G. (1983). Enzyme electrodes with improved mechanical and analytical characteristics obtained by binding enzymes to nylon nets. Anal. Chim. Acta, 146, 135-48. Medyantseva, E. P., Budnikov, G. K. & Babkina, S. S.

Biosensors & Bioelectronics 7 (1992) 000-000 (1990). Enzyme electrode based on immobilized cholinesterase for determination of potentional environmental pollutants. Zh. Anal. Khim.. 45, 1386-9. Qi, X, Baldwin, R, Li, H. & Guarr, T. F. (1991). Electrocatalytic amperometric detection at polymeric cobalt phthalocyanine electrodes. Electroanalysis, 3, 119-24. Skladal, P. (1991). Determination of organophosphate and carbamate pesticides using a cobalt phthalocyanine-modified carbon paste electrode and a cholinesterase enzyme membrane. Anal. Chim. Acta. 255, 11-15. Tran-Minh, C., Pandey, P. C. & Kumaran, S. (1990). Studies on acetylcholine sensor and its analytical application on the inhibition of cholinesterase.

Sensitive detection of pesticides Biosensors Bioelectronics, 5, 46 l-7 1. Wolfbeis, 0. & Koller, E. (1989). Fiber optic detection of pesticides in drinking water. In GBF Biosensor Workshop, ed. R D. Schmid & F. Scheller. GBF Monographs Vol. 13, VCH Publishers, New York, pp. 221-4. Wring, S. A., Hart, J. P. & Birch, B. J. (1989). Development of an improved carbon electrode chemically modified with cobalt phthalocyanine as a reusable sensor for glutathione. Analyst, 114, 1563-70. Wring, S. A., Hart J. P. & Birch, B. J. (1991). Voltambehavior of screen-printed carbon metric electrodes, chemically modified with selected mediators, and their application as sensors for the determination of reduced glutathione. Analyst, 116, 123-9.

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Sensitive detection of pesticides using amperometric sensors based on cobalt phthalocyanine-modified composite electrodes and immobilized cholinesterases.

The determination of organophosphate and carbamate pesticides was carried out using cobalt phthalocyanine-modified carbon epoxy composite electrodes c...
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