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A novel immobilized cholinesterase for on-site screening of organophosphate and carbamate compounds
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 86 (2006) 162–166 www.elsevier.com/locate/ypest
A novel immobilized cholinesterase for on-site screening of organophosphate and carbamate compounds Ming-Qiang Zou
a,* a
, Rui Yang b, Da-Ning Wang a, Jin-Feng Li a, Qin-Han Jin
b
Chinese Academy of Inspection and Quarantine, Beijing 100025, PR China b College of Chemistry, Jilin University, Changchun 130023, PR China Received 15 August 2005; accepted 3 March 2006 Available online 19 April 2006
Abstract Acetylcholinesterase was physically adsorbed on polyvinylpyrrolidone K30 to prepare a novel immobilized cholinesterase, which was described for the first time. The immobilized enzyme was tested for its reactivity, enzyme activity stability, the solubility, the influence of pH and buffer, and characteristic reactivity with inhibitors, e.g., organophosphate and carbamate compounds. The result revealed that the immobilized cholinesterase held its original activity in solution, stable enough, easy to dissolve for convenient operation, and this immobilized cholinesterase could be well used for rapid, on-site screening of organophosphate and carbamate compounds, together with a portable pesticide analyzer. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Immobilized cholinesterase; Polyvinylpyrrolidone K30 (PVP); On-site screening; Organophosphate and carbamate compounds
1. Introduction Organophosphates (OPs), such as parathion, malathion, fenitrothion, phosalone, dichlorvos, and chlorpyrifos, and carbamates (CBs), such as carbaryl, carbofuran, and propoxur, are widely used as insecticides in agriculture and as biocides in household products since 1970s. Although these compounds, being quickly decomposed, are relatively nonpersistent in the environment, they have been characterized as being highly acutely toxic by strongly inhibiting the activity of acetylcholinesterase (AChE) [1]. Therefore, an effective analytical method for rapid screening of OPs and CBs residues is urgently needed. Though traditional analytical methods, such as chromatography/mass spectrometry(GC/MS) and liquid chromatography/mass spectrometry(LC/MS) can precisely determine the residues of OPs and CBs, these methods need professional operators, expensive instruments, and complicated pretreatment pro-
*
Corresponding author. Fax: +86 10 85745897. E-mail address: [email protected] (M.-Q. Zou).
0048-3575/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.03.003
cedures, which limit their application for on-site screening of these compounds. Moreover, particularly in some undeveloped countries where dispersed and individual agricultural systems are carried out, it is more necessary to find a rapid analytical method available to operate in fields to screen toxic pesticide residues. Biomarkers are sensitive and cost-effective tools for screening, monitoring, and identifying environmental risk on organisms. AChE is considered as a specific biomarker for screening the organophosphorus and carbamate pesticides and has been the fastest growing one among methodologies for rapid screening the residues in environmental samples [2,3]. AChE, which plays an important role in nervous system function by reducing the concentration of acetylcholine (ACh) in the junction through AChE-catalyzed hydrolysis of ACh, is poisoned by a variety of OPs and CBs [4,5], and thus can serve as an effective biomarker indicating OPs and CBs residue levels. The residue level can be monitored, by simply comparing the activity of the non-inhibited enzyme with the inhibited one. Most biochemical assays in the literature [1–5] follow the classical colorimetric method described by Ellman et al. [6], which uses
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
acetylthiocholine (ACTC) as a specific substrate. Ellman method is based on the increase of yellow colour nitrobenzoate (TNB) ion produced from thiocholine when it reacts with dithiobisnitrobenzoate (DTNB) ion (Eq. 1, below). AChE
Acetylthiocholine ! Thiocholine þ Acetic acid Thiocholine þ DTNB!Formation of TNB ½yellow colourð412 nmÞ
163
in solution reaction. Therefore, OPs and CBs could conveniently be monitored on-site and real-time by using a pesticide analyzer together with the immobilized AChE. 2. Materials and methods 2.1. Materials
ð1Þ
However, for the purpose of on-site detection as well as popular application, there are at least two key technical problems to be resolved. One is an inexpensive portable pesticide analyzer, which was successfully developed by us and reported in [7]. The other is a suitable immobilized enzyme with good enzyme activity stability at room temperature for on-site operation convenience. Since the poor stability of enzyme in solution, it is difficult to realize a real-time, on-site detection by the above-mentioned biochemical assay. In another words, an immobilized cholinesterase with good activity stability, which meets the requirements of practical operation, is of great significance. There are several methods for enzyme immobilization available. They can be mainly divided into two classes: covalent bonding and physical adsorbing between matrix and protein molecules. Generally, the former will cause a remarkable decrease of enzyme activity but with good stability, and also need a rather complicated procedure for immobilization. While the latter will be of much more advantages over the former in above-mentioned aspects except stability, however, it is difficult to find suitable matrixes. Herein, in order to find the very matrixes suitable for on-site detection of pesticide residues, the following factors should be taken into consideration: a. The enzyme activity should be stable for long enough to permit on-site operation. b. The matrix to be loaded into should be of better solubility. c. The matrix within the quantity used should not affect the measurement of inhibition of enzyme. d. The immobilized enzyme should retain higher activity, without too much activity decrease than in solution. According to these considerations, matrix, polyvinylpyrrolidone K30 (PVP) was finally found after a great deal of tests. In the present study, a novel immobilized acetylcholinesterase with PVP was successfully developed. The AChE was shown to be active enough and stable at room temperature for at least 20 days, at 4 °C for at least 40 days, and at 20 °C it was still stable on the 90th day after the immobilization. This immobilized acetylcholinesterase tends to be well dissolved in water within a given range of concentration and thus can meet the requirement of on-site operation. Little matrix interference was found and the immobilized enzyme was sensitive to various OPs and CBs with inhibition patterns similar to those obtained
Acetylcholinesterase (AChE), 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB), acetylcholine iodide (ACTC) were all purchased from Sigma Chemicals (US. Louis, MO, USA). P-buffer (0.1 M phosphate, pH 8.0) and P-buffer (0.1 M phosphate, pH 7.0) were prepared by dissolving phosphates (G.R. Tianjin Chemical Plant, China). H2SO4 (G.R., Beijing Chemical Plant, China). Tris (hydromethyl)-aminomethane (99.5%) was acquired from RiedeldeHaen (Seelze, Germany). Tris–H2SO4-buffer (0.1 M, pH 8.0). The acetylcholinesterase was diluted to 1.5:1000 with P-buffer (pH 8.0) for comparison, yielding a solution of around 1.5 U/mL; and was diluted to 0.5:1000 with Tris-buffer (0.1 M, pH 8.00) for immobilization onto polyvinylpyrrolidone K30 (PVP) (average molecular weight is 30,000 g/mol, T.T.R.C., China), to 0.75:1000 for immobilization onto sodium citrate (Na3H6O7Æ2H2O, 294.11 g/mol, A.R., Beijing Chemical Plant, China), to 0.3:1000 for immobilization onto potassium sulfate (K2SO4, 174.25 g/ mol, G.R., Beijing Chemical Plant, China) and to 0.4:1000 for immobilization onto sodium sulfate (Na2SO4, 142.04 g/mol, A.R. Tianjin Chemical Plant, China), respectively. The solution was stored at 20 °C until use. Handheld pesticide analyzer (GDYN-201S) was from Changchun Jilin University-Little Swan Instruments Co. Ltd.(Changchun, PR China). Freezing-dryer (Alpha 1-2) were from Chaist (Germany). 2.2. AChE immobilization on matrixes The immobilized acetylcholinesterases were made by a two-step procedure. The first step was the dissolution of matrixes (polyvinylpyrrolidone, sodium citrate, potassium sulfate, and sodium sulfate) with acetylcholinesterase dilutions in Tris–H2SO4-buffer (0.1 M, pH 8.0), respectively. The mixtures were usually used for the second step after reaction for some time (more than 1 h at least). The second step was the freezing-dry of the mixtures. All immobilization procedures are similar to the following example: weight 6.0 g sample, e.g., polyvinylpyrrolidone, in 50 mL beaker, dissolve it carefully with 30 mL Tris-buffer solution (0.1 M, pH 8.00) of acetylcholinesterase, crushing and stirring it with a glass rod until a clear solution is obtained. Allow to react for over 1 h. Transfer the solution into a 100 mL freezing-dry flask. Spread it as equably as possible onto the inner-wall of the flask in a low-temperature ethanol bath (20 °C below). Fix the flask to a freezing-dryer over night until dry powder is formed on the inner-wall of the flask. Take the flask out of the freezing-dryer, scrape the powder off and transfer it into a small bottle with cover,
164
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and store it at 4 °C. For the convenience of comparison, equal quantity of four different matrixes loaded equal units of acetylcholinesterases to make four kinds of immobilized acetylcholinesterases for this study.
where At and A0 are the absorbency values after and before the measurement, respectively.
2.3. Enzymatic reaction measurements
3.1. Activities of AChEs in different forms
The enzyme reaction was photometrically evaluated based on the Ellman method [6]. P-buffer (4.0 mL, pH 8.0), 30 mg immobilized AChE (or 50 lL of AChE in solution at 5.0 U/mL), 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into sample cells. Fifty microlitres of ACTC in double-distilled water (2.0 mM) was used as a substrate to determine enzyme activity. AChE-catalytic ACTC hydrolysis reaction was measured based on an increase in A407 in 3 min time intervals [7]. Operation was carried out at room temperature.
The first set experiments were designed to understand whether the immobilized AChEs were active in various entrapped matrix forms. Four different immobilized esterases, polyvinylpyrrolidone-entrapped AChE, sodium citrate-entrapped AChE, potassium sulfate-entrapped AChE, and sodium sulfate-entrapped AChE, as well as AChE solution were tested at various AChE concentrations (ranging from 2.5 to 10 U/g for immobilized AChE and 1.5 U/mL for AChE solution). The quantities of all AChEs were accurately sampled to ensure the final units of AChE in reaction systems to be equal for the convenience of comparison. The curves shown in Fig. 1 [representing the activity with only one of all the tested concentrations (2.5 U/g for immobilized AChE and 1.5 U/g for AChE solution)] showed that except potassium sulfate-entrapped AChE with few activity, the other three immobilized esterases, polyvinylpyrrolidone-entrapped AChE, sodium citrate-entrapped AChE and sodium sulfate-entrapped AChE were all alive, but with remarkably different activities. The activity of polyvinylpyrrolidone-entrapped AChE was almost the same as that of the solution. The reaction rate of sodium citrate-entrapped AChE is around one third of that of the solution, and sodium sulfate-entrapped AChE with only about one fifth of the solution. Similar differences in activities were found at all tested AChE concentrations: 5.0, 7.5, and 10.0 U/g of immobilized esterases (data not shown). Therefore, polyvinylpyrrolidone-entrapped AChE was chosen to catalyze the biochemical assay for on-site application.
2.4. Enzyme inhibition measurements P-buffer (4.0 mL, pH 8.0), 30 mg immobilized AChE, 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into sample cells. Inhibition of esterase was carried out by application of 20 lL of anti-cholinesterase compounds diluted to an appropriate concentration. The reaction system was incubated for 10 min at room temperature (20–25 °C). Enzyme reaction measurement was carried out as above-mentioned after the addition of 50 lL of ACTC in double-distilled water (2.0 mM). The extent of inhibition, I, was calculated as a relative decrease of A407 (in 3 min time intervals, with a handheld pesticide analyzer) by Ið%)= At-A0At 100 ,
3. Results and discussion
0. 600
Activity (A407)
0. 500 0. 400
0. 300
0. 200 0. 100
0. 000 0. 000
0. 500
1. 000
1. 500
2. 000
2. 500
3. 000
3. 500
Reaction time (min)
AChE solution Sodium citrate-entrapped AChE
PVP-entrapped AChE Sodium sulfate-entrapped AChE
Potassium sulfate-entrapped AChE
Fig. 1. Activities of AChEs in different forms.
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
3.2. Activity stability of immobilized acetylcholinesterases Thiry milligrams of polyvinylpyrrolidone-entrapped AChE was sampled to examine the activity stability, the measurement being carried out under the conditions described above. The activities of the immobilized AChE stored at 20, 4 °C, and room temperature, respectively, were measured on the day of immobilization, and on the 2nd day, 9th day, 20th day, 40th day, 65th day, and 90th day after the immobilization and it was shown in Fig. 2. The result was described as follows. For the case of storage at 20 °C, no decrease of enzymatic activity was found until the 90th day after the immobilization. For storage at 4 °C, the situation was similar before the 40th day after the immobilization, and about 10% decrease of enzymatic activity was observed on the 65th day after the immobilization, then the activity was found to decrease slightly up to approximate 15% decrease on the 90th day after the immobilization. For the case of storage at room temperature, less than 5% decrease of enzymatic activity was found on the 20th day after the immobilization, and about 20% decrease of enzymatic activity was detected on the 40th day after the immobilization, then the activity went gradually down to around 40% decrease on the 90th day after the immobilization. Considering 5% decrease of enzymatic activity is still acceptable to exhibit the reaction, the polyvinylpyrrolidone-entrapped AChE stored at room temperature is still useful within 20 days after the immobilization, with a satisfactory application performance for on-site determination. 3.3. Effect of pH on measured reaction rate in the assay The reaction rates catalyzed by AChE in solution and immobilized AChE were compared at various pH values ranging from 5.5 to 9.0. To make the compare, the following reagents were added to each sample cells sequentially: 30 mg immobilized AChEs (or 50 lL of AChE in solution at 5.0 U/mL), 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), 4.0 mL 0.02 M
Activity (A407/30 mg PVP-entrapped AChEs)
0. 550 0. 500 0. 450 0. 400 0. 350 0. 300
165
phosphate buffer with PH of 5.5, 6.0, 7.0, 8.0, 8.5 or 9.0; and 50 lL of ACTC in double-distilled water (2.0 mM) was used as substrate for determining the reaction rate. The result showed that P-buffer of pH 8.0, 8.5, and 9.0 gave the highest reaction rates, and about one-third activity dropping at pH 7.0, and almost to zero value at pH 6.0 (Table 1). The type of the buffer used for the preparation of immobilized AChE was also examined. A comparison of the results obtained with P-buffer (0.1 M phosphate, pH 8.0) to Tris–H2SO4-buffer (0.1 M, pH 8.0) revealed that much better stability was obtained using Tris–H2SO4-buffer than using P-buffer (data not shown). The reason for this is probably due to the influences of buffer molecules themselves on the hydrolysis during the reaction of biochemical assays. 3.4. Solubility of PVP The solubility of immobilized enzyme is another important parameter for practical on-site operation. With a volume of 4.0 mL, more than 100 mg PVP was difficult to dissolve while less than 50 mg was found to rapidly dissolve only with soft shaking. The test result shown that PVP was easy to be dissolved in water and is then well qualified for use as a matrix of immobilized AChE for on-site analysis. 3.5. Effect of PVP quantity on enzyme activity The chosen PVP matrix concentration was further tested by looking at the effect of PVP quantity in 4.0 mL final volume of reaction solution on reaction rate of ACTC hydrolysis. 0, 10, 20, 25, 30, 50, and 70 mg of PVP were added into a series of sample cells, respectively. Fifty microlitres of AChEs in solution at 5.0 U/mL, 50 lL of Ellman reagents containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into each sample cell, diluting contents in every cells with P-buffers (0.1 M phosphate, pH 8.0) to 4.0 mL. Using 50 lL of ACTC in double-distilled water (2.0 mM) as a substrate to examine enzyme activities in each cell. AChE-catalytic ACTC hydrolyses were measured by measuring A407 after 3 min reaction time intervals, using a handheld pesticide analyzer. Operation was carried out at room temperature. Within the range of 0–30 mg PVP, the highest reaction rates were observed. When PVP is over 40 mg, the negative matrix effects were obvious, and the more the quantity increasing, the greater the activity decreasing. When the quantity reached 70 mg an activity dropping to about one-third of initial value was observed (Fig. 3). In our study, 30 mg PVP in 4 mL final volume was selected taking
0. 250 0
20
40
60
80
100
Time (days after the immobilization) −
−4
room temperature
Fig. 2. Stability of immobilized AChEs at different storage temperatures.
Table 1 Effect of pH on measured reaction rate of immobilized AChE pH
5.5
6.0
7.0
8.0
8.5
9.0
Activity (A407)
0.0
0.06
0.28
0.41
0.43
0.40
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PVP-immobilized acetylcholinesterase were almost the same as the ones with AChE in solution.
0.550
0.500
Activity (A407)
4. Concluding remarks 0.450
0.400
0.350
0.300 0
20
40
60
80
PVP quantity (mg)
Acknowledgments
Fig. 3. Effect of PVP quantity on activity of AChE.
Table 2 PVP-entrapped AChE sensitivity to various OPs and CBs Compounds
Fenthion Malathion Dichlorvos Chlorpyrifos Carbaryl Carbofuron
I50 (lg/L)
A novel PVP-immobilized acetylcholinesterase was successfully developed for the first time in this study. The excellent enzyme activity, great stability, good solubility for convenience of operation, few interference, high sensitivity and validity of being inhibited by OPs and CBs provide a basis of efficient on-site and screening assay for these two groups insecticides.
I20 (lg/L)
PVP-entrapped AchE
AChE solution
PVP-entrapped AChE
90 47 99 53 104 2
88 48 96 55 101 2
35 20 40 26 29 1
I50 and I20 represent 50 and 20% inhibition, respectively.
both loading capability and less influence on reaction rate into consideration. 3.6. Characterization of the immobilized acetylcholinesterase In the present study, several typical inhibitors of OPs and CBs, such as fenthion, malathion, dichlorvos, chlorpyrifos, carbaryl, and carbofuran which have long been known to inhibit selectively cholinesterase, were used to characterize the immobilized acetylcholinesterase. I20 was used to determine limits of detection. It was found that the PVP-immobilized AChE was effectively inhibited by OPs and CBs, although different sensitivities were observed for different insecticides (Table 2). Comparison of I50 volumes indicated that the sensitivities obtained with the
The financial support for food safety control from a Strategic Tenth-five-year Scientific Research Grant from the Ministry of National Science and Technology of China (No. 2001BA804A11-8) and from a Scientific Development Grant from Jilin Province Science and Technology Council of China (No. 931008) are appreciated. References [1] H.C. Tsai, R.A. Doong, Optimization of sol–gel based fibre-optic cholinesterase biosensor for the determination of organophosphorus pesticides, Water Science and Technology 42 (7–8) (2000) 283–290. [2] G. Rodrigue-fuentes, G. Gold-Bouchot, Environmental monitoring using acetylcholinesterase inhibition in vireo: a case study in two Mexican Lagoons, Marine Environmental Research 50 (2000) 357–360. [3] T. Hamers, K.R.J. Molin, J.H. Koeman, A.J. Murk, A small-volume bioassay for quantification of the esterase inhibiting potency of mixtures of organophosphate and carbamate insecticide in rainwater: development and optimization, Toxicological Science 58 (2000) 60–67. [4] M. Altstein, G. Segev, N. Aharinson, O. Ben-Aziz, A. Turniansky, D. Avnir, Journal of Agricultural and Food Chemistry 46 (1998) 3318–3324. [5] T.R. Fukato, Mechanism of action of organophosphorus and carbamate insecticides, Environmental Health Perspectives 87 (1990) 245–254. [6] G.L. Ellman, K.O. Coutney, V. Andrres, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical Pharmacology 7 (1961) 88–99. [7] M.Q. Zou, R. Yang, L.L. Zhao, A.M. Yu, Q.H. Jin, Rapid on-site determination of the toxicity of organophosphates and carbamates in vegetables by enzyme catalytic dynamic photometry with handheld pesticide analyzer, Chemical Journal of Chinese Universities 24 (6) (2003) 1016–1018.
Pesticide Biochemistry and Physiology 86 (2006) 162–166 www.elsevier.com/locate/ypest
A novel immobilized cholinesterase for on-site screening of organophosphate and carbamate compounds Ming-Qiang Zou
a,* a
, Rui Yang b, Da-Ning Wang a, Jin-Feng Li a, Qin-Han Jin
b
Chinese Academy of Inspection and Quarantine, Beijing 100025, PR China b College of Chemistry, Jilin University, Changchun 130023, PR China Received 15 August 2005; accepted 3 March 2006 Available online 19 April 2006
Abstract Acetylcholinesterase was physically adsorbed on polyvinylpyrrolidone K30 to prepare a novel immobilized cholinesterase, which was described for the first time. The immobilized enzyme was tested for its reactivity, enzyme activity stability, the solubility, the influence of pH and buffer, and characteristic reactivity with inhibitors, e.g., organophosphate and carbamate compounds. The result revealed that the immobilized cholinesterase held its original activity in solution, stable enough, easy to dissolve for convenient operation, and this immobilized cholinesterase could be well used for rapid, on-site screening of organophosphate and carbamate compounds, together with a portable pesticide analyzer. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Immobilized cholinesterase; Polyvinylpyrrolidone K30 (PVP); On-site screening; Organophosphate and carbamate compounds
1. Introduction Organophosphates (OPs), such as parathion, malathion, fenitrothion, phosalone, dichlorvos, and chlorpyrifos, and carbamates (CBs), such as carbaryl, carbofuran, and propoxur, are widely used as insecticides in agriculture and as biocides in household products since 1970s. Although these compounds, being quickly decomposed, are relatively nonpersistent in the environment, they have been characterized as being highly acutely toxic by strongly inhibiting the activity of acetylcholinesterase (AChE) [1]. Therefore, an effective analytical method for rapid screening of OPs and CBs residues is urgently needed. Though traditional analytical methods, such as chromatography/mass spectrometry(GC/MS) and liquid chromatography/mass spectrometry(LC/MS) can precisely determine the residues of OPs and CBs, these methods need professional operators, expensive instruments, and complicated pretreatment pro-
*
Corresponding author. Fax: +86 10 85745897. E-mail address: [email protected] (M.-Q. Zou).
0048-3575/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2006.03.003
cedures, which limit their application for on-site screening of these compounds. Moreover, particularly in some undeveloped countries where dispersed and individual agricultural systems are carried out, it is more necessary to find a rapid analytical method available to operate in fields to screen toxic pesticide residues. Biomarkers are sensitive and cost-effective tools for screening, monitoring, and identifying environmental risk on organisms. AChE is considered as a specific biomarker for screening the organophosphorus and carbamate pesticides and has been the fastest growing one among methodologies for rapid screening the residues in environmental samples [2,3]. AChE, which plays an important role in nervous system function by reducing the concentration of acetylcholine (ACh) in the junction through AChE-catalyzed hydrolysis of ACh, is poisoned by a variety of OPs and CBs [4,5], and thus can serve as an effective biomarker indicating OPs and CBs residue levels. The residue level can be monitored, by simply comparing the activity of the non-inhibited enzyme with the inhibited one. Most biochemical assays in the literature [1–5] follow the classical colorimetric method described by Ellman et al. [6], which uses
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
acetylthiocholine (ACTC) as a specific substrate. Ellman method is based on the increase of yellow colour nitrobenzoate (TNB) ion produced from thiocholine when it reacts with dithiobisnitrobenzoate (DTNB) ion (Eq. 1, below). AChE
Acetylthiocholine ! Thiocholine þ Acetic acid Thiocholine þ DTNB!Formation of TNB ½yellow colourð412 nmÞ
163
in solution reaction. Therefore, OPs and CBs could conveniently be monitored on-site and real-time by using a pesticide analyzer together with the immobilized AChE. 2. Materials and methods 2.1. Materials
ð1Þ
However, for the purpose of on-site detection as well as popular application, there are at least two key technical problems to be resolved. One is an inexpensive portable pesticide analyzer, which was successfully developed by us and reported in [7]. The other is a suitable immobilized enzyme with good enzyme activity stability at room temperature for on-site operation convenience. Since the poor stability of enzyme in solution, it is difficult to realize a real-time, on-site detection by the above-mentioned biochemical assay. In another words, an immobilized cholinesterase with good activity stability, which meets the requirements of practical operation, is of great significance. There are several methods for enzyme immobilization available. They can be mainly divided into two classes: covalent bonding and physical adsorbing between matrix and protein molecules. Generally, the former will cause a remarkable decrease of enzyme activity but with good stability, and also need a rather complicated procedure for immobilization. While the latter will be of much more advantages over the former in above-mentioned aspects except stability, however, it is difficult to find suitable matrixes. Herein, in order to find the very matrixes suitable for on-site detection of pesticide residues, the following factors should be taken into consideration: a. The enzyme activity should be stable for long enough to permit on-site operation. b. The matrix to be loaded into should be of better solubility. c. The matrix within the quantity used should not affect the measurement of inhibition of enzyme. d. The immobilized enzyme should retain higher activity, without too much activity decrease than in solution. According to these considerations, matrix, polyvinylpyrrolidone K30 (PVP) was finally found after a great deal of tests. In the present study, a novel immobilized acetylcholinesterase with PVP was successfully developed. The AChE was shown to be active enough and stable at room temperature for at least 20 days, at 4 °C for at least 40 days, and at 20 °C it was still stable on the 90th day after the immobilization. This immobilized acetylcholinesterase tends to be well dissolved in water within a given range of concentration and thus can meet the requirement of on-site operation. Little matrix interference was found and the immobilized enzyme was sensitive to various OPs and CBs with inhibition patterns similar to those obtained
Acetylcholinesterase (AChE), 5,5 0 -dithiobis(2-nitrobenzoic acid) (DTNB), acetylcholine iodide (ACTC) were all purchased from Sigma Chemicals (US. Louis, MO, USA). P-buffer (0.1 M phosphate, pH 8.0) and P-buffer (0.1 M phosphate, pH 7.0) were prepared by dissolving phosphates (G.R. Tianjin Chemical Plant, China). H2SO4 (G.R., Beijing Chemical Plant, China). Tris (hydromethyl)-aminomethane (99.5%) was acquired from RiedeldeHaen (Seelze, Germany). Tris–H2SO4-buffer (0.1 M, pH 8.0). The acetylcholinesterase was diluted to 1.5:1000 with P-buffer (pH 8.0) for comparison, yielding a solution of around 1.5 U/mL; and was diluted to 0.5:1000 with Tris-buffer (0.1 M, pH 8.00) for immobilization onto polyvinylpyrrolidone K30 (PVP) (average molecular weight is 30,000 g/mol, T.T.R.C., China), to 0.75:1000 for immobilization onto sodium citrate (Na3H6O7Æ2H2O, 294.11 g/mol, A.R., Beijing Chemical Plant, China), to 0.3:1000 for immobilization onto potassium sulfate (K2SO4, 174.25 g/ mol, G.R., Beijing Chemical Plant, China) and to 0.4:1000 for immobilization onto sodium sulfate (Na2SO4, 142.04 g/mol, A.R. Tianjin Chemical Plant, China), respectively. The solution was stored at 20 °C until use. Handheld pesticide analyzer (GDYN-201S) was from Changchun Jilin University-Little Swan Instruments Co. Ltd.(Changchun, PR China). Freezing-dryer (Alpha 1-2) were from Chaist (Germany). 2.2. AChE immobilization on matrixes The immobilized acetylcholinesterases were made by a two-step procedure. The first step was the dissolution of matrixes (polyvinylpyrrolidone, sodium citrate, potassium sulfate, and sodium sulfate) with acetylcholinesterase dilutions in Tris–H2SO4-buffer (0.1 M, pH 8.0), respectively. The mixtures were usually used for the second step after reaction for some time (more than 1 h at least). The second step was the freezing-dry of the mixtures. All immobilization procedures are similar to the following example: weight 6.0 g sample, e.g., polyvinylpyrrolidone, in 50 mL beaker, dissolve it carefully with 30 mL Tris-buffer solution (0.1 M, pH 8.00) of acetylcholinesterase, crushing and stirring it with a glass rod until a clear solution is obtained. Allow to react for over 1 h. Transfer the solution into a 100 mL freezing-dry flask. Spread it as equably as possible onto the inner-wall of the flask in a low-temperature ethanol bath (20 °C below). Fix the flask to a freezing-dryer over night until dry powder is formed on the inner-wall of the flask. Take the flask out of the freezing-dryer, scrape the powder off and transfer it into a small bottle with cover,
164
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
and store it at 4 °C. For the convenience of comparison, equal quantity of four different matrixes loaded equal units of acetylcholinesterases to make four kinds of immobilized acetylcholinesterases for this study.
where At and A0 are the absorbency values after and before the measurement, respectively.
2.3. Enzymatic reaction measurements
3.1. Activities of AChEs in different forms
The enzyme reaction was photometrically evaluated based on the Ellman method [6]. P-buffer (4.0 mL, pH 8.0), 30 mg immobilized AChE (or 50 lL of AChE in solution at 5.0 U/mL), 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into sample cells. Fifty microlitres of ACTC in double-distilled water (2.0 mM) was used as a substrate to determine enzyme activity. AChE-catalytic ACTC hydrolysis reaction was measured based on an increase in A407 in 3 min time intervals [7]. Operation was carried out at room temperature.
The first set experiments were designed to understand whether the immobilized AChEs were active in various entrapped matrix forms. Four different immobilized esterases, polyvinylpyrrolidone-entrapped AChE, sodium citrate-entrapped AChE, potassium sulfate-entrapped AChE, and sodium sulfate-entrapped AChE, as well as AChE solution were tested at various AChE concentrations (ranging from 2.5 to 10 U/g for immobilized AChE and 1.5 U/mL for AChE solution). The quantities of all AChEs were accurately sampled to ensure the final units of AChE in reaction systems to be equal for the convenience of comparison. The curves shown in Fig. 1 [representing the activity with only one of all the tested concentrations (2.5 U/g for immobilized AChE and 1.5 U/g for AChE solution)] showed that except potassium sulfate-entrapped AChE with few activity, the other three immobilized esterases, polyvinylpyrrolidone-entrapped AChE, sodium citrate-entrapped AChE and sodium sulfate-entrapped AChE were all alive, but with remarkably different activities. The activity of polyvinylpyrrolidone-entrapped AChE was almost the same as that of the solution. The reaction rate of sodium citrate-entrapped AChE is around one third of that of the solution, and sodium sulfate-entrapped AChE with only about one fifth of the solution. Similar differences in activities were found at all tested AChE concentrations: 5.0, 7.5, and 10.0 U/g of immobilized esterases (data not shown). Therefore, polyvinylpyrrolidone-entrapped AChE was chosen to catalyze the biochemical assay for on-site application.
2.4. Enzyme inhibition measurements P-buffer (4.0 mL, pH 8.0), 30 mg immobilized AChE, 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into sample cells. Inhibition of esterase was carried out by application of 20 lL of anti-cholinesterase compounds diluted to an appropriate concentration. The reaction system was incubated for 10 min at room temperature (20–25 °C). Enzyme reaction measurement was carried out as above-mentioned after the addition of 50 lL of ACTC in double-distilled water (2.0 mM). The extent of inhibition, I, was calculated as a relative decrease of A407 (in 3 min time intervals, with a handheld pesticide analyzer) by Ið%)= At-A0At 100 ,
3. Results and discussion
0. 600
Activity (A407)
0. 500 0. 400
0. 300
0. 200 0. 100
0. 000 0. 000
0. 500
1. 000
1. 500
2. 000
2. 500
3. 000
3. 500
Reaction time (min)
AChE solution Sodium citrate-entrapped AChE
PVP-entrapped AChE Sodium sulfate-entrapped AChE
Potassium sulfate-entrapped AChE
Fig. 1. Activities of AChEs in different forms.
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
3.2. Activity stability of immobilized acetylcholinesterases Thiry milligrams of polyvinylpyrrolidone-entrapped AChE was sampled to examine the activity stability, the measurement being carried out under the conditions described above. The activities of the immobilized AChE stored at 20, 4 °C, and room temperature, respectively, were measured on the day of immobilization, and on the 2nd day, 9th day, 20th day, 40th day, 65th day, and 90th day after the immobilization and it was shown in Fig. 2. The result was described as follows. For the case of storage at 20 °C, no decrease of enzymatic activity was found until the 90th day after the immobilization. For storage at 4 °C, the situation was similar before the 40th day after the immobilization, and about 10% decrease of enzymatic activity was observed on the 65th day after the immobilization, then the activity was found to decrease slightly up to approximate 15% decrease on the 90th day after the immobilization. For the case of storage at room temperature, less than 5% decrease of enzymatic activity was found on the 20th day after the immobilization, and about 20% decrease of enzymatic activity was detected on the 40th day after the immobilization, then the activity went gradually down to around 40% decrease on the 90th day after the immobilization. Considering 5% decrease of enzymatic activity is still acceptable to exhibit the reaction, the polyvinylpyrrolidone-entrapped AChE stored at room temperature is still useful within 20 days after the immobilization, with a satisfactory application performance for on-site determination. 3.3. Effect of pH on measured reaction rate in the assay The reaction rates catalyzed by AChE in solution and immobilized AChE were compared at various pH values ranging from 5.5 to 9.0. To make the compare, the following reagents were added to each sample cells sequentially: 30 mg immobilized AChEs (or 50 lL of AChE in solution at 5.0 U/mL), 50 lL of Ellman reagent containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), 4.0 mL 0.02 M
Activity (A407/30 mg PVP-entrapped AChEs)
0. 550 0. 500 0. 450 0. 400 0. 350 0. 300
165
phosphate buffer with PH of 5.5, 6.0, 7.0, 8.0, 8.5 or 9.0; and 50 lL of ACTC in double-distilled water (2.0 mM) was used as substrate for determining the reaction rate. The result showed that P-buffer of pH 8.0, 8.5, and 9.0 gave the highest reaction rates, and about one-third activity dropping at pH 7.0, and almost to zero value at pH 6.0 (Table 1). The type of the buffer used for the preparation of immobilized AChE was also examined. A comparison of the results obtained with P-buffer (0.1 M phosphate, pH 8.0) to Tris–H2SO4-buffer (0.1 M, pH 8.0) revealed that much better stability was obtained using Tris–H2SO4-buffer than using P-buffer (data not shown). The reason for this is probably due to the influences of buffer molecules themselves on the hydrolysis during the reaction of biochemical assays. 3.4. Solubility of PVP The solubility of immobilized enzyme is another important parameter for practical on-site operation. With a volume of 4.0 mL, more than 100 mg PVP was difficult to dissolve while less than 50 mg was found to rapidly dissolve only with soft shaking. The test result shown that PVP was easy to be dissolved in water and is then well qualified for use as a matrix of immobilized AChE for on-site analysis. 3.5. Effect of PVP quantity on enzyme activity The chosen PVP matrix concentration was further tested by looking at the effect of PVP quantity in 4.0 mL final volume of reaction solution on reaction rate of ACTC hydrolysis. 0, 10, 20, 25, 30, 50, and 70 mg of PVP were added into a series of sample cells, respectively. Fifty microlitres of AChEs in solution at 5.0 U/mL, 50 lL of Ellman reagents containing 2.5 mM DTNB in pH 7.0 phosphate buffer (0.1 M), were added in sequence, into each sample cell, diluting contents in every cells with P-buffers (0.1 M phosphate, pH 8.0) to 4.0 mL. Using 50 lL of ACTC in double-distilled water (2.0 mM) as a substrate to examine enzyme activities in each cell. AChE-catalytic ACTC hydrolyses were measured by measuring A407 after 3 min reaction time intervals, using a handheld pesticide analyzer. Operation was carried out at room temperature. Within the range of 0–30 mg PVP, the highest reaction rates were observed. When PVP is over 40 mg, the negative matrix effects were obvious, and the more the quantity increasing, the greater the activity decreasing. When the quantity reached 70 mg an activity dropping to about one-third of initial value was observed (Fig. 3). In our study, 30 mg PVP in 4 mL final volume was selected taking
0. 250 0
20
40
60
80
100
Time (days after the immobilization) −
−4
room temperature
Fig. 2. Stability of immobilized AChEs at different storage temperatures.
Table 1 Effect of pH on measured reaction rate of immobilized AChE pH
5.5
6.0
7.0
8.0
8.5
9.0
Activity (A407)
0.0
0.06
0.28
0.41
0.43
0.40
166
M.-Q. Zou et al. / Pesticide Biochemistry and Physiology 86 (2006) 162–166
PVP-immobilized acetylcholinesterase were almost the same as the ones with AChE in solution.
0.550
0.500
Activity (A407)
4. Concluding remarks 0.450
0.400
0.350
0.300 0
20
40
60
80
PVP quantity (mg)
Acknowledgments
Fig. 3. Effect of PVP quantity on activity of AChE.
Table 2 PVP-entrapped AChE sensitivity to various OPs and CBs Compounds
Fenthion Malathion Dichlorvos Chlorpyrifos Carbaryl Carbofuron
I50 (lg/L)
A novel PVP-immobilized acetylcholinesterase was successfully developed for the first time in this study. The excellent enzyme activity, great stability, good solubility for convenience of operation, few interference, high sensitivity and validity of being inhibited by OPs and CBs provide a basis of efficient on-site and screening assay for these two groups insecticides.
I20 (lg/L)
PVP-entrapped AchE
AChE solution
PVP-entrapped AChE
90 47 99 53 104 2
88 48 96 55 101 2
35 20 40 26 29 1
I50 and I20 represent 50 and 20% inhibition, respectively.
both loading capability and less influence on reaction rate into consideration. 3.6. Characterization of the immobilized acetylcholinesterase In the present study, several typical inhibitors of OPs and CBs, such as fenthion, malathion, dichlorvos, chlorpyrifos, carbaryl, and carbofuran which have long been known to inhibit selectively cholinesterase, were used to characterize the immobilized acetylcholinesterase. I20 was used to determine limits of detection. It was found that the PVP-immobilized AChE was effectively inhibited by OPs and CBs, although different sensitivities were observed for different insecticides (Table 2). Comparison of I50 volumes indicated that the sensitivities obtained with the
The financial support for food safety control from a Strategic Tenth-five-year Scientific Research Grant from the Ministry of National Science and Technology of China (No. 2001BA804A11-8) and from a Scientific Development Grant from Jilin Province Science and Technology Council of China (No. 931008) are appreciated. References [1] H.C. Tsai, R.A. Doong, Optimization of sol–gel based fibre-optic cholinesterase biosensor for the determination of organophosphorus pesticides, Water Science and Technology 42 (7–8) (2000) 283–290. [2] G. Rodrigue-fuentes, G. Gold-Bouchot, Environmental monitoring using acetylcholinesterase inhibition in vireo: a case study in two Mexican Lagoons, Marine Environmental Research 50 (2000) 357–360. [3] T. Hamers, K.R.J. Molin, J.H. Koeman, A.J. Murk, A small-volume bioassay for quantification of the esterase inhibiting potency of mixtures of organophosphate and carbamate insecticide in rainwater: development and optimization, Toxicological Science 58 (2000) 60–67. [4] M. Altstein, G. Segev, N. Aharinson, O. Ben-Aziz, A. Turniansky, D. Avnir, Journal of Agricultural and Food Chemistry 46 (1998) 3318–3324. [5] T.R. Fukato, Mechanism of action of organophosphorus and carbamate insecticides, Environmental Health Perspectives 87 (1990) 245–254. [6] G.L. Ellman, K.O. Coutney, V. Andrres, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochemical Pharmacology 7 (1961) 88–99. [7] M.Q. Zou, R. Yang, L.L. Zhao, A.M. Yu, Q.H. Jin, Rapid on-site determination of the toxicity of organophosphates and carbamates in vegetables by enzyme catalytic dynamic photometry with handheld pesticide analyzer, Chemical Journal of Chinese Universities 24 (6) (2003) 1016–1018.