Biosensors% Bioektmnics 5 (1990) 461-471
Studies on Acetylcholine Sensor and its Analytical Application Based on the Inhibition of Cholinesterase C. Tran-Minh,
P. C. Pandey* & Satish Kumaran
Laboratoire de Biotechnologie,
Ecole des Mines, 42023, Saint-Etienne Cedex, France
ABSTRACT Acetylcholine esterase electrodes, based on glass, Pd/pdO and Ir/IrOz electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some otganophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine estemse. Reactivation of immobilized AChE afrer inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working bu#er and acetylcholine, whereas reactivation afrer inhibition with inwersible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be IO-‘M whereas for paraoxon, methyl parathion and malathion are found to be 10m9~ and 10-‘“M. Key words: enzyme potentiometry.
electrode,
enzyme
inhibition,
toxic
detection,
1 INTRODUCTION There have been numerous reports on the development of potentiometric biosensing probes during recent years (Mokhallati et al., 1985; Romette, *Permanent address: Department of Chemistry, Banaras Hindu University, Varanasi221 005, India. 461
Biosensors% Bioekctmnics O!X6-5663/!W/$O3.50 (Q1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
462
C Tran-Mrtfi et al.
1985; Tor & Freeman, 1986). The basic requirements for a reliable biosensing probe are its sensitivity, linearity, response time, reproducibility and stability. These parameters are usually controlled by immobilization procedures and the sensitivity of the base electrochemical sensors. Recently a new prepolymer (Tor & Freeman, 1986) has been described for the immobilization of enzymes with which -5Oym thin films of immobilized enzyme can be obtained at the active surface of the transducer. This prepolymer was prepared by the copolymerization of 30% methacrylamide and 70% acrylamide followed by controlled hydrazinolysis. An a~~lcholine sensor was designed (Tor & Freeman, 1986)using this prepolymer with high storage and operational stability, however the overall response of the sensor was not very impressive, i.e. < 30 mV at the substrate saturation. The objective of the present research investigation was to design an acetylcholine sensor suitable for the analysis of toxic chemicals since the activity of ace~lcholinesterase is inhibited in presence of certain toxic chemicals (Winter & Ferrari, 1964; Ott & Gunther, 1966; Voss and Sachase, 1970).In order to get a high detection range and reproducible data on toxic analysis, the acetylcholine sensor should have impressive response and high storage and operational stability. In this paper a~e~lcholine sensors with impressive response and high storage and operational stability have been designed based on glass, Pd/PdO and Ir/Ir@ electrodes as pH sensors (Grubb & King, 1980;Bordi d al., 1984) using acrylamide-methacrylamide hydrazides as membrane matrix and have been successfully exploited for the analysis of toxic chemicals. The detection of the toxic is based on the measu~ment of the percentage inhibition in presence of the toxic. The reactivation of enzyme activity depends on the type of inhibition (Trammel et al., 1984; Tran-Minh & Yamani, 1988). If the inhibition is reversible, the reactivation of the enzyme can be carried out by using a mixture of working buffer and substrate whereas if the inhibition is irreversible, the reactivation can be carried out by using a mixture of substrate and 2-pyridine aldoxime methiodide (PAM). Irreversible inhibition of AChE by organophosphorus compounds is accompanied by the formation of a covalent phosphorus-enzyme linkage at the active site serine which in contrast is relatively stable to hydrolysis. The reaction between the enzyme and the or8anophospho~s compound is similar to that with the a~e~l~holine and can be depicted as follows: E-OH + AX ‘Fzz= = = = & E-OI&AX ---+,-GA + X- + H+ (inhibited enzyme)
Acetylcholinesensor and its analyticalapplication
463
where E-OH represents the enzyme and OH is the serine OH in the active site, AX is an organophosphorus compound (the symbol A designates a dialkyl phosphoryl group and X is the leaving group which in the case of paraoxon isp-nitrophenol). The reactivation of the inhibited enzyme by the use of an oxime is due to the formation of phosphorylated oxime and restoring the enzyme activity and can be depicted as follows:
/NOH
-----3
E-OH +
//NOA
I
(ZPAM) In this paper we report data on the analysis of nicotine and fluoride ion as reversible inhibitors and paraoxon, malathion and parathion methyl as irreversible inhibitors. The detection limits for the nicotine and fluoride ion are found to be 10-5~ whereas for paraoxon, parathion methyl and malathion they are found to be 10-9~ and 10-lo~.
2 EXPERIMENTAL 2.1 Materials Puritied AChE (EC 3.1.1.7), acetylcholine chloride, hydrazine hydrate, nicotine and Zpyridine aldoxime methiodide were obtained from the Sigma Chemical Company; glyoxal and methacrylamide were obtained from the Aldrich Chemical Co.; acrylamide, paraoxon, malathion and parathion methyl were obtained from Riedel de Haen AG. All other chemicals employed were of analytical grade.
2.2 Synthesis of the prepolymer The acrylamide-methacrylamide hydrazide prepolymer was synthesized according to the procedure of Tor & Freeman (1986), however the polymerization of the monomers was carried out for 2 h instead of 1 h. Further, after hydrazinolysis, the derivative of acrylamide-methacrylamide hydrazide was precipitated in cold methanol and the precipitate was collected by centrifugation at 4°C and stored at - 18°C in a tightly closed vessel.
464
C. 7ban-Minh et al.
2.3 Preparation of enzyme-prepolymer solutions (a) Co-polymer solutions: a 30% (w/v) solution was made by adding the dry prepolymer to the distilled water and stirring magnetically at room temperature until the solution became homogeneous. (b) Enzyme solution: 1000 E.units/ml acetylcholine esterase was dissolved in O-1 M phosphate buffer pH 8.0 containing 0.01% gelatin. (c) Enzyme-copolymer solution: into 1 ml of 30% copolymer solution, 65 ~1 of 2 M phosphate buffer, pH 7.5 was added followed by the addition of 100 ~1 enzyme stock solution, 10 ~1 of 1% gelatin, and 25 ~1 of distilled water which resulted in a final enzyme concentration 83 EU/ml, and 25% final copolymer concentration. 2.4 Fabrication of AChE sensors AChE sensor employing a glass electrode as a pH sensor: a new glass electrode (Tacussel G 202 B) was treated with standard buffer, pH 7.0, thoroughly washed with double distilled water, and dried with lens paper. The enzyme-copolymer solution was applied to the tip of the glass electrode, dried and crosslinked according to the procedure of Tor & Freeman (1986). AChE sensor employing Pd/PdO and Ir/IrQ electrodes as pH sensor: palladium wire (1.0 mm diameter) and iridium wire (0.5 mm diameter) were obtained from Good Fellow Metal Advance Materials, England. Lengths of 1 cm of these wires were fused with platinum wire (0.5 mm diameter, 5 cm long) and oxidized according to the previously described publications (Grubb & Ring, 1980; Bordi et al., 1984). The wires were then placed in distilled water for 3 days and mounted into teflon tubing leaving an equal surface area. The slopes of the electrodes obtained in this manner were found to be -59 mV. The electrodes were then dried under a stream of nitrogen, coated with enzyme-copolymer solution, dried, and crosslinked according to the procedure of Tor & Freeman, (1986). 2.5 Measurement of the potential All potential measurements were made in a cell with a working volume of 15 ml, equipped with magnetic stirrer and thermostated at 25°C using an Ultra thermostat K5 Colora Messtechnik, GMBH LORCH/WURTT. The potential was recorded with a Radiometer PHM 64 pH meter connected to a SEFRAM SRD No. 429 recorder. HEPES buffer containing 20 mM MgC&, 100 mM NaCl and 0.01% gelatin (w/v) was
Acetylcholinesensor and its analyticalapplication
465
found to be a more suitable working buffer. The AChE electrode was placed in the reaction cell containing 10 ml of working buffer and the base line potential was recorded. Following the establishment of the steady base line potential, varying concentrations of the substrate were added and the steady-state potential was recorded. 2.6 Measurement of the inhibition For the measurement of the AChE inhibition, first the appropriate concentration of the substrate was selected from the calibration curve such that the rate of non-inhibited reaction can be considered unchanged and could be used as a reference. Then AChE inhibition was measured in two different ways depending on the type of inhibitor. For reversible inhibitors, i.e. nicotine and fluoride ion, varying concentrations of the inhibitor were added to the reaction cell at the steady-state responses of the selected substrate concentration and the new steadystate potential was recorded. From the difference of the two steady-state potentials, the percentage of AChE inhibition was calculated. The reactivation of the AChE sensor was done by placing the inhibited sensor inside the reaction cell containing 10 ml working buffer and the initial steady-state potential was re-established by adding the selected concentration of the substrate. However, for irreversible inhibitors, i.e. paraoxon, malathion and parathion methyl, the AChE sensor was first incubated with the varying concentrations of the inhibitors for 1 h and the steady-state potential was recorded by adding the selected concentration of the substrate. From the difference of the two steady states, i.e. before and after inhibition, the percentage of AChE inhibition was calculated. The reactivation of the AChE sensor was done by adding the 1 mM pyridine-Zaldoxime methiodide (PAM) and selected concentration of the substrate. Methyl parathion and malathion were converted into their oxygen analogues by treating with bromine water since these chemicals as such do not show maximum toxicity.
3 RESULTS AND DISCUSSION Figure 1 shows the calibration curve of the new acetylcholine sensors made of glass, Pd/PdO and Ir/IrO, electrodes as pH sensors in 5 mM HEPES buffer pH 8.0. The data show that the detection range of acetylcholine using the present AChE sensors is 10-+~-4 X 10-3~ in 5 mM HEPES buffer pH 8-O containing the salts and gelatin as stabilizing agent. The response time of the present AChE sensors (Fig. 2) is greater
466
C. iVan-Minh
Fig. 1. Responses of the AChE electrodes at different substrate concentrations in 5 mM HEPES buffer pH 8.0 at 25°C by using three pH sensors: (1) glass electrode; (2) Pd/PdO electrode; (3) Ir/IrQ electrode.
et al.
L i
Subs
cont.
CM/ 11
60-
2. Response curves for AChE sensors in 5 mM HEPES buffer, with 1.5 mM acetylcholine, pH 8.0 at 25°C by using three pH sensors: (1) glass electrode; (2) Pd/PdO electrode; (3) Ir/It@ electrode. Fii.
0
I 0
I 3
I 6
I
Time
0
(min)
(5-6 min for glass electrode and 6-7 min for Pd/PdO and Ir/IrO* electrodes) than the response time of the earlier AChE sensor devised by Tor & Freeman (1986). This is due to the increased copolymer concentration for the immobilization of enzyme in order to get the maximum response from the electrodes, since response time does not contribute significantly as compared to the overall response of the sensor for the measurement of the AChE inhibition. Consequently, all the present AChE sensors show very high response (> 100 mV) at the appropriate concentration of the substrate as compared to the AChE sensor devised by Tor & Freeman (1986) (< 30 mv). The sensitivity of the present AChE sensors to the chemical nature and the buffer capacity of the buffer is critical for getting high response and wide range linearity. The maximum response, keeping the same pH and buffer capacity for HEPES, T&acetate and phosphate buffer, is obtained in HEPES buffer. The response is high at low buffer concen-
467
,,l--i_-J 6
7
8 VW
9
,.
Fig. 3. Effect of the pH on the response of the AChE sensor by using glass electrode in 2.5 mM HEPES buffer with 2 mM acetylcholine at 25°C.
tration (1 mM) and decreases as the concentration of the buffer is increased. However, at low buffer concentration (1 mM), the pH of the buffer was changed during the measurement, In 2-S mM HEPES buffer, no appreciable variation in the pH was observed during the measu~men~ hence, this buffer concen~tio~ was selected for the analysis of the toxic chemicals. Figure 3 shows the effect of the pH on the response of the AChE sensor in 25 mM HEPES with 1.5 mM acetylcholine at 25°C. The optimum pH for the present sensor is 8-O.pH dependence of the sensor response is due to the pH dependence of the AChE activity. Determination of toxic chemicals is based on the measurement of percentage inhibition (I) which is equal to the potential difference given by the sensor with and without the inhibitor for the same substrate concentration and can be depicted as follows: I=(Eo-E1)X 100 where E0 is the potential difference given by the sensor in absence of the inhibitor and El is the potential difference in presence of the inhibitor at the same substrate concentration. Usually the substrate concentration is large enough so that the rate of the non-inhibited reaction can be considered unchanged and could be used as a reference, In the present case the substrate concentration used for the analysis is 5 mM. A typical response of the sensor with and without the inhibitor is given in Fig. 4 for the reversible inhibitors (fluoride ion). Data on the quantitative analysis of fluoride ion and nicotine are recorded in Figs 5 and 6 in terms of percentage inhibition as a function of inhibitor ~ncen~tion for a fmed concentration of substrate (5 mM acetylcholine). Figure 7 shows a typical response of the sensor on the addition of 5 mM acetylcholine: (a) in absence of the inhibitor and (b) after incubation with the sensor at a particular concentration of the irreversible inhibitor for 1 h. Reactivation of the enzyme is facilitated by adding 1 mM 2-PAM. Data on the
468
C i9wd4inh
et al.
?-77x-7 limo
(min)
Fig. 4. A typical response of the AChE sensor with and without the reversible inhibitor (fluoride ion) in 2.5 mu HEPES buffer with 5 mM acetylcholine
Fluoride
pH 8.0 at 25°C.
ion [MJ
Fig. 5. Determination of fluoride ion with the AChE sensor (2.5 mM HEPES buffer, pH 8.0, temperature 25°C and 5 nw acetylcholine as substrate).
quantitative analysis of paraoxon, malathion and methyl parathion are recorded in terms of percentage inhibition as a function of inhibitor concentration for a fured concentration of the substrate in Figs 8-10 respectively. The lowest detection limit for malathion is 10-r”~ and for paraoxon and methyl parathion is 10-9~.
Acetylcholine sensor and its analytical application
469
Fig. 6. Determination
* Nicotine [Id]
of nicotine with the AChE sensor (2.5 mM HEPES buffer, pH 8.0, temperature 25°C and 5 mM acetylcholine as substrate).
D Time (mid
Fig. 7. A typical response of the AChE sensor with and without the irreversible inhibitor (paraoxon) in 2.5 mu HEPES buffer, with 5 mM acetylcholine, pH 8-O at 25°C: (a) in the absence of the inhibitor; (b) after incubating the sensor with the inhibitor for 1 h. loo60 E a .c c 40 s 4
20 60 0I $0
, .,,..,A_ 169
$6 Porooxon
16'
CM]
166
1
5
Pig. 8. Determination of paraoxon with the AChE sensor (2.5 mM HEPES buffer, pH 8-0, at 25°C incubation time 1 h, and 5 mM acetylcholine as substrate).
C. Tran-Minh et al.
Fii. 9. Determination of malathion with the AChE sensor (25 mM HEPES buffer, pH 8.0, at 25”C, incubation time 1 h, and 5 mMacetylcholine as substrate).
Fig. 10. Determination of methyl parathion with the AChE sensor (2.5 mMHEPES buffer, pH 8.0, at 25% incubation time 1 h, and 5 mu acetylcholine as substrate).
Malathion
Methyl
CM]
Parathion
[M]
REFERENCES Bordi, S., Carla, M. & Papeschi, G. (1984). Iridium/iridium oxide electrode for potentiometric determination of proton activity in hydroorganic solutions at sub-zero temperature. Anal. Chem., 56, 317-18. Grubb, W. T. & Ring, L. H. (1980). Palladium and palladium oxide pH electrodes. Anal. Chem., 52,270-73. Mokhallati, A. O., Instudor, A, Virtosu, D. & Lucas, C. (1985). Use of M/MO type pH sensor for the determination of carbon dioxide in an air gap electrode. Roum. Chim., 30(l), 31-4. Ott, D. E. & Gunther, F. A. (1966). Analysis of tech. grade parathion in water plants by an anticholinesterase. Anal. Chem., 49, 669-71. Romette, J. L. (1985). InMethods ofEnzymatic Analysis,VU.8,3rd edn (Bergmeyer, H. U., ed.), VCH Publishers, New York, pp. 393-409. Tor, R. & Freeman, A. (1986). New enzyme membrane for enzyme electrodes. Anal. Chem., 58, 1042-6. Trammel, A. M., Simmons, J. E. & Borchardt, R. T. (1984). An efficient in vitro assay for acetylcholinesterase reactivators using immobilized enzyme. Phurm. Rex, 3, 115-20. Tran-Minh, C. & Yamani, H. El. (1988). Enzyme sensors for determination of toxic chemicals in environmental samples. In Junrer ElectrochemicalDetection Techniques in the AppZied Biosciences, Sec. 1.3, Ellis Horwood Publishers, Halsted Press, pp. 131-41.
Acetylcholinesensor and its analyticalapplication
471
Voss, G. & Sachase, K. (1970). Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetylcholine/DTNB [5-5_dithiobis-2-nitrobenzoic acid] procedure. Toxicol. Appl. Phatmacol.. 16, 764-7.
Winter, G. D. & Ferrari, A (1964). Automatic wet them. analysis as applied to pesticides residue. Residue Rev., 5, 139-44.
Studies on Acetylcholine Sensor and its Analytical Application Based on the Inhibition of Cholinesterase C. Tran-Minh,
P. C. Pandey* & Satish Kumaran
Laboratoire de Biotechnologie,
Ecole des Mines, 42023, Saint-Etienne Cedex, France
ABSTRACT Acetylcholine esterase electrodes, based on glass, Pd/pdO and Ir/IrOz electrodes as pH sensor, using the immobilized acetylcholine esterase in acrylamide-methacrylamide hydrazides prepolymer are reported and compared. New data on the analysis of nicotine, fluoride ion, and some otganophosphorus compounds are reported using the present AChE sensor based on the inhibition of the immobilized acetylcholine estemse. Reactivation of immobilized AChE afrer inhibition with reversible inhibitor, i.e. nicotine and fluoride ion is carried out using a mixture of working bu#er and acetylcholine, whereas reactivation afrer inhibition with inwersible inhibitor, i.e. organophosphorus compounds is carried out using a mixture of acetylcholine and pyridine-2-aldoxime methiodide (PAM). The detection limits for the nicotine and fluoride ion are found to be IO-‘M whereas for paraoxon, methyl parathion and malathion are found to be 10m9~ and 10-‘“M. Key words: enzyme potentiometry.
electrode,
enzyme
inhibition,
toxic
detection,
1 INTRODUCTION There have been numerous reports on the development of potentiometric biosensing probes during recent years (Mokhallati et al., 1985; Romette, *Permanent address: Department of Chemistry, Banaras Hindu University, Varanasi221 005, India. 461
Biosensors% Bioekctmnics O!X6-5663/!W/$O3.50 (Q1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain.
462
C Tran-Mrtfi et al.
1985; Tor & Freeman, 1986). The basic requirements for a reliable biosensing probe are its sensitivity, linearity, response time, reproducibility and stability. These parameters are usually controlled by immobilization procedures and the sensitivity of the base electrochemical sensors. Recently a new prepolymer (Tor & Freeman, 1986) has been described for the immobilization of enzymes with which -5Oym thin films of immobilized enzyme can be obtained at the active surface of the transducer. This prepolymer was prepared by the copolymerization of 30% methacrylamide and 70% acrylamide followed by controlled hydrazinolysis. An a~~lcholine sensor was designed (Tor & Freeman, 1986)using this prepolymer with high storage and operational stability, however the overall response of the sensor was not very impressive, i.e. < 30 mV at the substrate saturation. The objective of the present research investigation was to design an acetylcholine sensor suitable for the analysis of toxic chemicals since the activity of ace~lcholinesterase is inhibited in presence of certain toxic chemicals (Winter & Ferrari, 1964; Ott & Gunther, 1966; Voss and Sachase, 1970).In order to get a high detection range and reproducible data on toxic analysis, the acetylcholine sensor should have impressive response and high storage and operational stability. In this paper a~e~lcholine sensors with impressive response and high storage and operational stability have been designed based on glass, Pd/PdO and Ir/Ir@ electrodes as pH sensors (Grubb & King, 1980;Bordi d al., 1984) using acrylamide-methacrylamide hydrazides as membrane matrix and have been successfully exploited for the analysis of toxic chemicals. The detection of the toxic is based on the measu~ment of the percentage inhibition in presence of the toxic. The reactivation of enzyme activity depends on the type of inhibition (Trammel et al., 1984; Tran-Minh & Yamani, 1988). If the inhibition is reversible, the reactivation of the enzyme can be carried out by using a mixture of working buffer and substrate whereas if the inhibition is irreversible, the reactivation can be carried out by using a mixture of substrate and 2-pyridine aldoxime methiodide (PAM). Irreversible inhibition of AChE by organophosphorus compounds is accompanied by the formation of a covalent phosphorus-enzyme linkage at the active site serine which in contrast is relatively stable to hydrolysis. The reaction between the enzyme and the or8anophospho~s compound is similar to that with the a~e~l~holine and can be depicted as follows: E-OH + AX ‘Fzz= = = = & E-OI&AX ---+,-GA + X- + H+ (inhibited enzyme)
Acetylcholinesensor and its analyticalapplication
463
where E-OH represents the enzyme and OH is the serine OH in the active site, AX is an organophosphorus compound (the symbol A designates a dialkyl phosphoryl group and X is the leaving group which in the case of paraoxon isp-nitrophenol). The reactivation of the inhibited enzyme by the use of an oxime is due to the formation of phosphorylated oxime and restoring the enzyme activity and can be depicted as follows:
/NOH
-----3
E-OH +
//NOA
I
(ZPAM) In this paper we report data on the analysis of nicotine and fluoride ion as reversible inhibitors and paraoxon, malathion and parathion methyl as irreversible inhibitors. The detection limits for the nicotine and fluoride ion are found to be 10-5~ whereas for paraoxon, parathion methyl and malathion they are found to be 10-9~ and 10-lo~.
2 EXPERIMENTAL 2.1 Materials Puritied AChE (EC 3.1.1.7), acetylcholine chloride, hydrazine hydrate, nicotine and Zpyridine aldoxime methiodide were obtained from the Sigma Chemical Company; glyoxal and methacrylamide were obtained from the Aldrich Chemical Co.; acrylamide, paraoxon, malathion and parathion methyl were obtained from Riedel de Haen AG. All other chemicals employed were of analytical grade.
2.2 Synthesis of the prepolymer The acrylamide-methacrylamide hydrazide prepolymer was synthesized according to the procedure of Tor & Freeman (1986), however the polymerization of the monomers was carried out for 2 h instead of 1 h. Further, after hydrazinolysis, the derivative of acrylamide-methacrylamide hydrazide was precipitated in cold methanol and the precipitate was collected by centrifugation at 4°C and stored at - 18°C in a tightly closed vessel.
464
C. 7ban-Minh et al.
2.3 Preparation of enzyme-prepolymer solutions (a) Co-polymer solutions: a 30% (w/v) solution was made by adding the dry prepolymer to the distilled water and stirring magnetically at room temperature until the solution became homogeneous. (b) Enzyme solution: 1000 E.units/ml acetylcholine esterase was dissolved in O-1 M phosphate buffer pH 8.0 containing 0.01% gelatin. (c) Enzyme-copolymer solution: into 1 ml of 30% copolymer solution, 65 ~1 of 2 M phosphate buffer, pH 7.5 was added followed by the addition of 100 ~1 enzyme stock solution, 10 ~1 of 1% gelatin, and 25 ~1 of distilled water which resulted in a final enzyme concentration 83 EU/ml, and 25% final copolymer concentration. 2.4 Fabrication of AChE sensors AChE sensor employing a glass electrode as a pH sensor: a new glass electrode (Tacussel G 202 B) was treated with standard buffer, pH 7.0, thoroughly washed with double distilled water, and dried with lens paper. The enzyme-copolymer solution was applied to the tip of the glass electrode, dried and crosslinked according to the procedure of Tor & Freeman (1986). AChE sensor employing Pd/PdO and Ir/IrQ electrodes as pH sensor: palladium wire (1.0 mm diameter) and iridium wire (0.5 mm diameter) were obtained from Good Fellow Metal Advance Materials, England. Lengths of 1 cm of these wires were fused with platinum wire (0.5 mm diameter, 5 cm long) and oxidized according to the previously described publications (Grubb & Ring, 1980; Bordi et al., 1984). The wires were then placed in distilled water for 3 days and mounted into teflon tubing leaving an equal surface area. The slopes of the electrodes obtained in this manner were found to be -59 mV. The electrodes were then dried under a stream of nitrogen, coated with enzyme-copolymer solution, dried, and crosslinked according to the procedure of Tor & Freeman, (1986). 2.5 Measurement of the potential All potential measurements were made in a cell with a working volume of 15 ml, equipped with magnetic stirrer and thermostated at 25°C using an Ultra thermostat K5 Colora Messtechnik, GMBH LORCH/WURTT. The potential was recorded with a Radiometer PHM 64 pH meter connected to a SEFRAM SRD No. 429 recorder. HEPES buffer containing 20 mM MgC&, 100 mM NaCl and 0.01% gelatin (w/v) was
Acetylcholinesensor and its analyticalapplication
465
found to be a more suitable working buffer. The AChE electrode was placed in the reaction cell containing 10 ml of working buffer and the base line potential was recorded. Following the establishment of the steady base line potential, varying concentrations of the substrate were added and the steady-state potential was recorded. 2.6 Measurement of the inhibition For the measurement of the AChE inhibition, first the appropriate concentration of the substrate was selected from the calibration curve such that the rate of non-inhibited reaction can be considered unchanged and could be used as a reference. Then AChE inhibition was measured in two different ways depending on the type of inhibitor. For reversible inhibitors, i.e. nicotine and fluoride ion, varying concentrations of the inhibitor were added to the reaction cell at the steady-state responses of the selected substrate concentration and the new steadystate potential was recorded. From the difference of the two steady-state potentials, the percentage of AChE inhibition was calculated. The reactivation of the AChE sensor was done by placing the inhibited sensor inside the reaction cell containing 10 ml working buffer and the initial steady-state potential was re-established by adding the selected concentration of the substrate. However, for irreversible inhibitors, i.e. paraoxon, malathion and parathion methyl, the AChE sensor was first incubated with the varying concentrations of the inhibitors for 1 h and the steady-state potential was recorded by adding the selected concentration of the substrate. From the difference of the two steady states, i.e. before and after inhibition, the percentage of AChE inhibition was calculated. The reactivation of the AChE sensor was done by adding the 1 mM pyridine-Zaldoxime methiodide (PAM) and selected concentration of the substrate. Methyl parathion and malathion were converted into their oxygen analogues by treating with bromine water since these chemicals as such do not show maximum toxicity.
3 RESULTS AND DISCUSSION Figure 1 shows the calibration curve of the new acetylcholine sensors made of glass, Pd/PdO and Ir/IrO, electrodes as pH sensors in 5 mM HEPES buffer pH 8.0. The data show that the detection range of acetylcholine using the present AChE sensors is 10-+~-4 X 10-3~ in 5 mM HEPES buffer pH 8-O containing the salts and gelatin as stabilizing agent. The response time of the present AChE sensors (Fig. 2) is greater
466
C. iVan-Minh
Fig. 1. Responses of the AChE electrodes at different substrate concentrations in 5 mM HEPES buffer pH 8.0 at 25°C by using three pH sensors: (1) glass electrode; (2) Pd/PdO electrode; (3) Ir/IrQ electrode.
et al.
L i
Subs
cont.
CM/ 11
60-
2. Response curves for AChE sensors in 5 mM HEPES buffer, with 1.5 mM acetylcholine, pH 8.0 at 25°C by using three pH sensors: (1) glass electrode; (2) Pd/PdO electrode; (3) Ir/It@ electrode. Fii.
0
I 0
I 3
I 6
I
Time
0
(min)
(5-6 min for glass electrode and 6-7 min for Pd/PdO and Ir/IrO* electrodes) than the response time of the earlier AChE sensor devised by Tor & Freeman (1986). This is due to the increased copolymer concentration for the immobilization of enzyme in order to get the maximum response from the electrodes, since response time does not contribute significantly as compared to the overall response of the sensor for the measurement of the AChE inhibition. Consequently, all the present AChE sensors show very high response (> 100 mV) at the appropriate concentration of the substrate as compared to the AChE sensor devised by Tor & Freeman (1986) (< 30 mv). The sensitivity of the present AChE sensors to the chemical nature and the buffer capacity of the buffer is critical for getting high response and wide range linearity. The maximum response, keeping the same pH and buffer capacity for HEPES, T&acetate and phosphate buffer, is obtained in HEPES buffer. The response is high at low buffer concen-
467
,,l--i_-J 6
7
8 VW
9
,.
Fig. 3. Effect of the pH on the response of the AChE sensor by using glass electrode in 2.5 mM HEPES buffer with 2 mM acetylcholine at 25°C.
tration (1 mM) and decreases as the concentration of the buffer is increased. However, at low buffer concentration (1 mM), the pH of the buffer was changed during the measurement, In 2-S mM HEPES buffer, no appreciable variation in the pH was observed during the measu~men~ hence, this buffer concen~tio~ was selected for the analysis of the toxic chemicals. Figure 3 shows the effect of the pH on the response of the AChE sensor in 25 mM HEPES with 1.5 mM acetylcholine at 25°C. The optimum pH for the present sensor is 8-O.pH dependence of the sensor response is due to the pH dependence of the AChE activity. Determination of toxic chemicals is based on the measurement of percentage inhibition (I) which is equal to the potential difference given by the sensor with and without the inhibitor for the same substrate concentration and can be depicted as follows: I=(Eo-E1)X 100 where E0 is the potential difference given by the sensor in absence of the inhibitor and El is the potential difference in presence of the inhibitor at the same substrate concentration. Usually the substrate concentration is large enough so that the rate of the non-inhibited reaction can be considered unchanged and could be used as a reference, In the present case the substrate concentration used for the analysis is 5 mM. A typical response of the sensor with and without the inhibitor is given in Fig. 4 for the reversible inhibitors (fluoride ion). Data on the quantitative analysis of fluoride ion and nicotine are recorded in Figs 5 and 6 in terms of percentage inhibition as a function of inhibitor ~ncen~tion for a fmed concentration of substrate (5 mM acetylcholine). Figure 7 shows a typical response of the sensor on the addition of 5 mM acetylcholine: (a) in absence of the inhibitor and (b) after incubation with the sensor at a particular concentration of the irreversible inhibitor for 1 h. Reactivation of the enzyme is facilitated by adding 1 mM 2-PAM. Data on the
468
C i9wd4inh
et al.
?-77x-7 limo
(min)
Fig. 4. A typical response of the AChE sensor with and without the reversible inhibitor (fluoride ion) in 2.5 mu HEPES buffer with 5 mM acetylcholine
Fluoride
pH 8.0 at 25°C.
ion [MJ
Fig. 5. Determination of fluoride ion with the AChE sensor (2.5 mM HEPES buffer, pH 8.0, temperature 25°C and 5 nw acetylcholine as substrate).
quantitative analysis of paraoxon, malathion and methyl parathion are recorded in terms of percentage inhibition as a function of inhibitor concentration for a fured concentration of the substrate in Figs 8-10 respectively. The lowest detection limit for malathion is 10-r”~ and for paraoxon and methyl parathion is 10-9~.
Acetylcholine sensor and its analytical application
469
Fig. 6. Determination
* Nicotine [Id]
of nicotine with the AChE sensor (2.5 mM HEPES buffer, pH 8.0, temperature 25°C and 5 mM acetylcholine as substrate).
D Time (mid
Fig. 7. A typical response of the AChE sensor with and without the irreversible inhibitor (paraoxon) in 2.5 mu HEPES buffer, with 5 mM acetylcholine, pH 8-O at 25°C: (a) in the absence of the inhibitor; (b) after incubating the sensor with the inhibitor for 1 h. loo60 E a .c c 40 s 4
20 60 0I $0
, .,,..,A_ 169
$6 Porooxon
16'
CM]
166
1
5
Pig. 8. Determination of paraoxon with the AChE sensor (2.5 mM HEPES buffer, pH 8-0, at 25°C incubation time 1 h, and 5 mM acetylcholine as substrate).
C. Tran-Minh et al.
Fii. 9. Determination of malathion with the AChE sensor (25 mM HEPES buffer, pH 8.0, at 25”C, incubation time 1 h, and 5 mMacetylcholine as substrate).
Fig. 10. Determination of methyl parathion with the AChE sensor (2.5 mMHEPES buffer, pH 8.0, at 25% incubation time 1 h, and 5 mu acetylcholine as substrate).
Malathion
Methyl
CM]
Parathion
[M]
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Acetylcholinesensor and its analyticalapplication
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Voss, G. & Sachase, K. (1970). Red cell and plasma cholinesterase activities in microsamples of human and animal blood determined simultaneously by a modified acetylcholine/DTNB [5-5_dithiobis-2-nitrobenzoic acid] procedure. Toxicol. Appl. Phatmacol.. 16, 764-7.
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