Food Control 27 (2012) 94e99

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Comparison of four commercial enzymatic assay kits for the analysis of organophosphate and carbamate insecticides in vegetables
Ting Xu a, Jun Wang b, Xintong Wang a, Richard Slawecki c, Fernando Rubio c, Ji Li a, *, Qing X. Li b, **
a

College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East-West Rd., Honolulu, HI 96822, USA c Abraxis LLC, 54 Steam whistle Drive, Warminster, PA 18974, USA
b

a r t i c l e i n f o
Article history: Received 30 November 2011 Received in revised form 29 February 2012 Accepted 2 March 2012 Keywords: Cholinesterase Organophosphate and carbamate (OP/C) Insecticide Food safety Insecticide exposure

a b s t r a c t
Contamination of organophosphate and carbamate (OP/C) insecticides in vegetables is a serious problem in China. An increasing number of acute poisoning accidents caused by OP/C residues in vegetables have brought great concerns on food safety. Four OP/C commercial enzymatic assay kits were compared on their sensitivity (half maximum inhibition concentration, IC50), matrix interference (i.e., background noise), accuracy (i.e., recovery) and precision (i.e., standard deviations), assay stability, simplicity of analysis, and cost. This work is not to advocate one companys products over others. Research intention is only to provide some useful information to potential users such as various characteristics on their performance and their particular applications of the four kits. The results showed that the four kit tests have various advantages. In general, kit 1 exhibited better precision, while kit 4 had better sensitivity. Kit 1 exhibited better recovery data for the organophosphates and kit 4 had better recoveries for the carbamates than the other kits. Kit 1, kit 3 and kit 4 were more stable than kit 2 under the same temperature condition. In addition, the operation of kit 4 was the easiest. According to the performance comparison results and the cost-effective factor, kit 1 and kit 4 were more suitable for the local market. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Use of pesticides undoubtedly has contributed to pest control and increase in crop yields. However, extensive use of pesticides has caused many toxicological and environmental problems such as unbalancing the equilibrium of ecological systems, contamination of farm products, leading to adverse effects on human health (Bempah, Donkor, Yeboah, Dubey, & Osei-Fosu, 2011; Feola, Rahn, & Binder, 2011; Panuwet et al., 2012; Pasmavathip, Prabhavathi, & Reddy, 2000; Wang, Zhang, Li, & Xing, 2002). Organophosphate and carbamate (OP/C) insecticides have been heavily used during the past decades because of their high efcacy on numerous pests (Liu, 2005; Wang et al., 2012). The mode of action of OP/C insecticides is mainly based on the inhibition of acetylcholinesterase (AChE) that catalyzes the hydrolysis of the neurotransmitter acetylcholine (Mileson, Chambers, & Chen, 1998). The AChE is

Abbreviations: AChE, Acetylcholinesterase; OP/C, Organophosphate and Carbamate Insecticides; OPs, Organophosphate Insecticides; IC50, Half Maximum Inhibition Concentration; LOD, Limit of Detection. * Corresponding author. Tel./fax: 86 10 62732017. ** Corresponding author. Tel.: 1 808 956 2011; fax: 1 808 956 3542. E-mail addresses: liji@cau.edu.cn (J. Li), qingl@hawaii.edu (Q.X. Li). 0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.03.002

present in vertebrates and insects, and its inhibition disrupts the transmission of nerve impulse (Moreira et al., 2010; Zaheer-ul-Haq, Halim, Uddin, & Madura, 2010). Although the production and usage of some highly toxic organophosphate insecticides, such as methamidophos, parathion, methyl-parathion, phosphamidon and monocrotophos, have been gradually prohibited in some countries, the worldwide incidence of OP/C poisoning is still high because of the easy availability and misuse (especially in developing countries) of these insecticides. Pesticide poisoning, especially OP/C, has caused an estimated 175,000 deaths per year in China (Eddleston & Phillops, 2004). Due to the increasing public concern over pesticide contamination in foods and the environment, there is an increasing demand of sensitive, reproducible, robust, and high-throughput screening assays for pesticide monitoring. Regulatory agencies are criticized for monitoring too few samples and low efciency, which is mainly the result of complicated and time-consuming sample preparation procedures involved with current chromatographic methods and high analytical cost. Class-specic screening of pesticides by an enzymatic assay prior to chromatographic conrmation could be an attractive approach for broader pesticide monitoring. If the total quantity of a class of pesticides in a sample can be determined and their concentration determined to be less than the maximum

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residue limits (MRLs), the samples can be eliminated from further inspection for these pesticides, since the vast majority of food and environmental samples turn out to be under the MRL of pesticides (Ma, 2006; Sun et al., 2003; Zhang & Zhou, 2007), the time and cost saved by this approach can be tremendous. The savings would be greater in the case of the major class of pesticides such as OP/C insecticides. Over the past decades, there have been numerous reports on the development of class-specic determination of OP/C insecticides based on inhibition of AChE (Arduini et al., 2006; Du, Chen, Cai, & Zhang, 2008; Eyer, Szinicz, Thiermann, Worek, & Zilker, 2007; Han et al., 2012; Laschi, Ogonczyk, Palchetti, & Mascini, 2007; Li, Ji, & Sun, 2006; Mishra, Dominguez, Bhand, Muoz, & Marty, 2012; Nagatani et al., 2007; Ni, Cao, & Kokot, 2007; Vamvakaki & Chaniotakis, 2007; ). The inhibition of AChE activity is dependent on the concentration of the insecticide in contact with the enzyme. These methods have been developed in the form of kits, biosensors and dipsticks, all of which allow the qualitative and semiquantitative detection of pesticides in a laboratory setting or onsite. AChE-based test kits for OP/C have already been commercialized in China and worldwide. These test kits have been developed by several companies. However, these commercial test kits have not been compared for their important features for the detection of OP/ C in the same sample. Therefore, it is suggested that users know the characteristics of the test kits prior to their use. To help the potential users to choose commercial kits to meet their needs, the detection performances of four commercial AChE-based test kits, three produced by Chinese companies and one from an American company, have herein been evaluated by comparing their performance characteristics which involve testing commercial vegetable samples containing different OP/C insecticides (ve OPs and two carbamates). 2. Experimental 2.1. Chemicals and test kits All reagents were of analytical grade unless specied otherwise. OP/C insecticide standards were purchased from the Institute for the Control of Agrochemicals, Ministry of Agriculture, Beijing, China. Stock solutions of the OP/C were prepared in methanol. The concentrations of phosphamidon, monocrotophos, parathion, parathion-methyl, carbofuran, and aldicarb were each 10 ppm and the concentration of methamidophos was 100 ppm. The four AChEbased test kits used in this study are as follows: kit 1, purchased from Shanghai Jiemen & Baolinmai Bioengineering, Co., Ltd (Shanghai, China); kit 2, purchased from Shijiazhuang David Biotech, Co., Ltd (Shijiazhuang, Hebei, China); kit 3, purchased from Shandong Jingpeng Bio-Research, Co., Ltd (Yantai, Shandong, China); and kit 4, kindly provided by Abraxis LLC. (Warminster, PA, USA). 2.2. Test kit assay procedures All reagents in the kits were allowed to come to ambient temperature prior to use. Stock solutions of AChE, chromogen and substrate were prepared in phosphate buffer (30 mg/ mL Na2HP4O$12H2O, 3.18 mg/mL KH2PO4, pH 7.5) according to the manufacturers recommendation. Assay procedures were performed as follows. (1) Assay procedure of kit 1, kit 2 and kit 3: A 100-mL aliquot of AChE and 100 mL of chromogen were added to a test tube containing 2.5 mL phosphate buffer containing varying concentrations of a target insecticide (one insecticide at a time). The tube was swirled moderately by hand for 30 s and incubated for 15 min at 37  C. After adding 100 mL of substrate

into the tube, the reaction proceeded at ambient temperature for 3 min. The absorbance (412 nm) was read at 0 min and 3 min and the difference in absorbance was recorded as DAt. Meanwhile, phosphate buffer in the absence of insecticides was employed as the test control and the absorbance difference was recorded as DA0. Inhibition was calculated according to the following formula. Inhibition (%) [(DA0 DAt)/DA0] 100%. (2) Assay procedure of kit 4: A 100-mL aliquot of the appropriate control or sample was added to the designated assay tube, by the supplied pipette. The tube was then swirled moderately by hand for 30 s. After adding 2 drops of oxidizer, the tube was swirled briey and incubated for 5 min at 25  C. After that, 2 drops of neutralizer and 2 drops of AChE were added into the tube. The tube was swirled again and incubated for 5 min at 25  C. Then, 2 drops of substrate-ATC and 2 drops of chromogen were added into the tube. The tube was swirled and incubated for 5 min at 25  C. After adding 2 drops of stopping solution, the tube was read at 450 nm to record the absorbance as DAn. The absorbance of negative control was recorded as DAm. Inhibition was calculated according to the following formula. Inhibition (%) [DAn/ DAm] 100%. The doseeresponse curve of each kit was generated in SigmaPlot 10. The half-maximum inhibition concentration (IC50), an expression of the assay sensitivity, and the limit of detection (LOD) dened as the IC20 values were obtained from the t of the data with a four-parameter logistic equation. 2.3. Sample preparation Chinese cabbage, cucumber and cherry tomato were purchased from local supermarkets in Beijing, China. Four OP/Cs, phosphamidon, methamidophos, carbofuran and aldicarb, were employed in the recovery study. Chinese cabbage and cucumber were chopped before fortication. Each insecticide stock solution was diluted and spiked into the above vegetables based on the practical detection ranges of each kit. The spiked vegetables were stored at 4  C for 1 h before extraction. A sample (10 g), intact cherry tomato or chopped cabbage/cucumber, was mixed with 10 mL phosphate buffer and swirled moderately for 1.5 min. After passing through a lter paper, the ltrate was diluted with phosphate buffer prior to assay. Matrix interference was estimated prior to the recovery study. Phosphamidon was prepared in phosphate buffer containing various percentages of the extracts from vegetables being free of OP/C. The inhibition curves and IC50 values were evaluated to determine the matrix interference on the enzymatic assay. 3. Results and discussion 3.1. Assay sensitivity and specicity Seven commonly used insecticides (ve OPs and two carbamates) were applied to evaluate the sensitivity of AChE to individual OP/C based on the inhibition potency. Fig. 1 shows inhibition curves of the four kits responding to the seven insecticides. The inhibition curves of kit 1 (except carbafuran) and kit 3 (except methamidophos), particularly kit 3 are clustered closely, which is a good property for monitoring different OP/Cs as a class. The AChE used in kit 3 is less selective to different OP/Cs. Most slopes of the inhibition curves of kit 4 are very similar and steep. The shape similarity indicates that these OP/Cs act on AChE in a similar way, while the steep slopes indicate high measurement accuracy. The wide dispersion of the curves of kit 4 suggests that this kit is more suitable for selected OP/Cs rather than as an OP/C class specic assay. Table 1 shows the sensitivities and detection ranges of the

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100 Phosphamidon Methamidophos Monocrotophos Parathion Parathion-methyl Carbafuran Aldicarb

T. Xu et al. / Food Control 27 (2012) 94e99

100 Phosphamidon Methamidophos Monocrotophos Parathion Parathion-methyl Carbafuran Aldicarb

80

80

Inhibition (%)

Inhibition (%)
Kit1

60

60

40

40

20

20

Kit2
0 100 101 102 103 104 105

0 10-2

10-1

100

101

102
-1

103

104

Concentration (ngmL )
100
100

Concentration (ng/mL)

80

Kit3
Inhibition (%)

80

Inhibition (%)

60 Phosphamidon Methamidophos Monocrotophos Parathion Parathion-methyl Carbafuran Aldicarb 101 102 103 104

60

Phosphamidon Methamidophos Monocrotophos Parathion Parathion-methyl Carbafuran Aldicarb

40

40

20

20

Kit4
0 10-2 10-1 100 101 102 103 104

0 100

Concentration (ng/mL)

Concentration (ng/mL)

Fig. 1. Inhibition curves of the insecticides in PBS determined with the four OP/C kits. The data are mean of four replicates.

four kits to an individual insecticide. Kit 4 is more sensitive to most of the tested OP/Cs than the other kits, showing the lowest IC50 value for OPs/Cs except parathion-methyl. IC50 values of kit 4 for phosphamidon, methamidophos, parathion, carbofuran and aldicarb were 29, 774, 30, 0.9 and 18 ng/mL, respectively. All kits have very high IC50 values for methamidophos, the lowest concentration being 742 ng/mL obtained by kit 3 and the highest concentration 27,674 ng/mL by kit 2. The result may suggest that all the kits may not be suitable for analysis of methamidophos in vegetables. An insensitive assay would likely produce false negative or positive results when it is used for the determination of these target analytes in real world samples. Both specicity and sensitivity of AChE to individual OP/C are important factors to evaluate the performance of the test kits. Such information, however, is often not available from kit manufacturers. Therefore, it is important to know the detection characteristic of test kits prior to their uses. These kits were applied to ve commonly used pyrethroid insecticides (cypermethrin, permethrin, deltamethrin, bifenthrin and cyuthrin) to evaluate the specicity of the AChE. Differing from OP/Cs, pyrethroids cause paralysis by keeping the sodium channels open in the neuronal membranes of an organism rather than direct action on the AChE. As expected, all kits showed high IC50 values

(!20 ppm) for the pyrethroid insecticides, suggesting the specicity of the AChE to OP/Cs. 3.2. Matrix effects AChE is susceptible to sample matrix effects, which could be minimized by means of cleaning up or diluting extracts. When an AChE-based kit is used for screening, dilution of sample extracts is often an effective way to minimize matrix effects. The insecticide standards were prepared in phosphate buffer-diluted vegetable extracts and the inhibition was determined with the four kits. Fig. 2 shows the inuence of cabbage matrix on the detection of phosphamidon by the four OP/C kits. To minimize the matrix effect of cabbage, 4-fold dilution of the extract is needed for kit 1 and 8-fold dilution for both kit 2 and kit 3.Kit 4 was least affected by cabbage matrix as no signicant differences were observed between the inhibition curve constructed in phosphate buffer and those constructed in the extracts. The inuence of cucumber and cherry tomato matrices on the four OP/C kits was also evaluated in the same way as that for cabbage. It is noticed that tomato showed the least matrix effect on the four kits compared to cabbage and cucumber, which may be due to the least matrix extracted out from

Table 1 The IC50 values and the detection ranges of the four OP/C kits for the analysis of ve OPs and two carbamate insecticides in phosphate buffer.a Insecticides IC50 values (detection range, IC20eIC80) (ng/mL) Kit 1 Phosphamidon Methamidophos Monocrotophos Parathion Parathion-methyl Carbofuran Aldicarb
a

Kit 2 277 27674 78 91 116 16 2419 (87e858) (3714e96155) (33e226) (18e330) (32e280) (1.4e212) (454e8596)

Kit 3 67 742 37 32 40 25 26 (16e278) (286e2064) (16e81) (9e127) (14e126) (6e138) (9e91)

Kit 4 29 774 21 30 463 0.9 18 (10e198) (282e3128) (9e78) (11e63) (741e1357) (0.3e10) (7e112)

109 921 24 387 45 1.1 239

(42e250) (145e3415) (9e63) (63e1595) (21e93) (0.5e2.8) (35e1574)

The average coefcient of variation (%) of the standard curves of kit 1, kit 2, kit 3 and kit 4 was 5, 7, 7 and 4, respectively.

T. Xu et al. / Food Control 27 (2012) 94e99

100 100

97

Kit1
80 80

Buffer, IC50=276.6ng/mL oringinal extract, IC50=448.39ng/mL 2-fold, IC50=372.87ng/mL 4-fold, IC50=339.36ng/mL 40 8-fold, IC50=283.69ng/mL

Inhibition (%)

Inhibition (%)

60

Buffer, IC50=108.5ng/mL oringinal extract, IC50=71.0ng/mL

60

40

2-fold, IC50=89.9ng/mL 4-fold, IC50=107.3ng/mL 8-fold, IC50=109.7ng/mL 101 102 103 104

20

20

Kit2

0 100

0 100

101

102

103

104

Concentration of Phosphamidon (ng/mL)

100
100

Concentration of Phosphamidon (ng/mL)

Kit4
80

Kit3
Buffer, IC50=66.9ng/mL oringinal extract, IC50=102.9ng/mL

80

Inhibition (%)

Inhibition (%)

60

60

40

2-fold, IC50=76.9ng/mL 4-fold, IC50=99.4ng/mL 8-fold, IC50=60.3ng/mL

40

buffer, IC50=29.27ng/mL oringinal extract, IC50=27.34ng/mL

20

20

0 100

101

102

103

104

0 100

101

102

103

Concentration of Phosphamidon (ng/mL)

Concentration of Phosphamidon (ng/mL)

Fig. 2. Inuence of cabbage matrix on the detection of phosphamidon by the four OP/C kits. The data are mean of triplicate.

the intact tomatoes. Although direct dilution of extracts into phosphate buffer could minimize the matrix effect on AChE-based kits, it will cause an increase of limits of detection (LODs). Table 2 shows the LODs of the kits for the insecticides in the vegetable matrices at the least dilution needed to minimize the matrix effect. 3.3. Recoveries of OP/Cs spiked in vegetable samples Varying concentrations of insecticides spiked into vegetables were based on the detection range of different kits and the matrix effect. Each sample extract at an optimal dilution was directly detected by the kits. The results were then multiplied by the
Table 2 Limits of detection of the four test kits for the four insecticides in cabbage, cucumber and tomato extracts. Vegetable Insecticide IC20 (ng/mL) Kit 1a Cabbage Phosphamidon Methamidophos Carbofuran Aldicarb Phosphamidon Methamidophos Carbofuran Aldicarb Phosphamidon Methamidophos Carbofuran Aldicarb 170 582 2 141 170 582 2 141 42 145 0.5 35 Kit 2b 698 >2 104 11 3632 349 >1.4 104 6 1816 87 3714 1 454 Kit 3c 127 2286 50 74 16 286 6 9 16 286 6 9 Kit 4d 11 282 0.3 7 11 282 0.3 7 11 282 0.3 7

dilution factors. The four kits have shown good recoveries for the insecticides from the spiked samples. The average recoveries of the OPs phosphamidon and methamidophos measured by kit 1, kit 2, kit 3 and kit 4 were in a range of 89e90%, 89e110%, 70e78% and 65e79%, respectively. For the carbamates carbofuran and aldicarb, the average recoveries by kit 1, kit 2, kit 3 and kit 4 were in a range of 74e79%, 82e91%, 70e89% and 93e105%, respectively (Table 3). The low coefcients of variation (4e10%) indicate that all kits were reproducible (Table 3). 3.4. Stability AChE solution should be preserved at 20  C. It is known that many freezing and thawing cycles can destroy the enzyme activity. In order to determine the stability of the kits during storage at 4  C, the enzyme activity was evaluated daily for 2 weeks by testing the inhibition of a constant concentration of phosphamidon on the enzyme activity. Kit 1, kit 3 and kit 4 showed good stability during the rst three days (Fig. 3). The inhibitions of phosphamidon, methamidophos, carbofuran and aldicarb at approximate concentrations causing 50% inhibition remained quite constant. Kit 1, kit 3 and kit 4 after storage of ve days or longer and kit 2 after storage of 2 days or longer showed large uctuations and a general trend of decline in inhibitions at the approximate pesticide concentrations causing 50% inhibition. AChEs used in the four test kits are different. Differences of AChEs and potential OP/C degradation and/or oxidation may cause the inhibition changes at a constant concentration over time. The OP/C solutions we used here were prepared every day at the same concentration. The results indicated that kit 2 was not as stable as the other three kits at 4  C. 3.5. Analysis time The four test kits have had different procedures instructed in the kit manuals, and, therefore, the analysis time required varied

Cucumber

Tomato

a Dilution factors for the matrix of cabbage, cucumber and tomato were 4, 4 and 1, respectively. b Dilution factors for the matrix of cabbage, cucumber and tomato were 8, 4 and 1, respectively. c Dilution factors for the matrix of cabbage, cucumber and tomato were 8, 1 and 1, respectively. d Each vegetable matrix was analyzed directly without any dilutions.

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Table 3 Average recoveries of OP/C insecticides spiked in cucumber, cabbage and tomato as determined with the four test kits. Insecticide Vegetable Average recovery and coefcient of variation (%), n 3 Kit 1a Rec Phosphamidon Cucumber Cabbage Tomato Average Cucumber Cabbage Tomato Average Cucumber Cabbage Tomato Average Cucumber Cabbage Tomato Average 69e95 74e105 92e102 89 75e98 73e97 97e98 90 74e89 75e89 99e112 74.1 66e86 55e85 85e101 79 CV 1e5 1e4 4e13 5 1e4 2e12 2e11 5 3e36 2e32 1e5 10 3e16 1e7 0e7 5 Kit 2b Rec 65e98 77e99 94e107 89 99e133 85e127 97e127 110 63e100 29e88 101e128 82 104e112 61e82 92e94 91 CV 1e5 2e4 4e13 5 1e4 2e12 2e11 5 3e36 2e32 1e5 10 5e16 3e7 1e3 6 Kit 3c Rec 64e101 56e85 67e94 78 40e70 55e64 93e107 70 25e94 15e74 96e120 70 67e98 57e92 100e111 89 CV 1e9 2e8 1e18 8 4e17 10e30 1e2 8 2e30 1e20 0e11 9 2e15 2e9 1e12 6 Kit 4d Rec 51e71 55e78 94e124 79 25e41 49e70 76e129 65 62e127 70e99 94e109 93 91e123 76e140 96e105 105 CV 3e14 2e12 1e5 6 5e14 1e27 1e8 8 1e11 2e7 1e12 5 0e13 1e43 0e28 10

Methamidophos

Carbofuran

Aldicarb

a Each of the insecticides, measured with kit 1, was fortied in each vegetable at a concentration of 250, 600 and 1250 ng/g of phosphamidon, 2, 3.75 and 7.5 mg/g of methamidophos, 2.5, 6 and 15 ng/g of carbofuran, or 0.25, 1.25 and 7.5 mg/g of aldicarb. b Each of the insecticides, measured with kit 2, was fortied in each vegetable at a concentration of 500, 1500 and 3500 ng/g of phosphamidon, 20, 150 and 400 mg/g of methamidophos, 7.5, 80 and 1000 ng/g of carbofuran, or 2.5 and 12.5 mg/g of aldicarb. c Each of the insecticides, measured with kit 3, was fortied in each vegetable at a concentration of 400 and 1000 ng/g of phosphamidon, 2, 3.75 and 7.5 mg/g of methamidophos, 50, 150 and 750 ng/g of carbofuran, or 50, 150 and 500 ng/g of aldicarb. d Each of the insecticides, measured with kit 4, was fortied in each vegetable at a concentration of 10, 30 and 180 ng/g of phosphamidon, 0.3, 1.0 and 3 mg/g of methamidophos, 0.3, 0.9 and 10 ng/g of carbofuran, or 7, 18 and 112 ng/g of aldicarb.

Fig. 3. Inuence of storage term on the stability of AChE in different kits in buffer solution. The data are mean of triplicate.

among them. The analysis time required for one sample was approximately 30 min for kit 1, kit 2 and kit 3, but approximately 50 min for kit 4.However, the analysis of time for 20 or more samples was approximately 2 h for kit 1, kit 2 and kit 3 whereas only 50 min were needed for kit 4 (because of the procedural differences among the kits). As a screening tool for a large number of samples, kit 4 is apparently more efcient than the others, while kit 1, kit 2 and kit 3 are more time-efcient for a few samples.

4. Conclusion The sensitivity (IC50 value) of each OP/C kit varied with different insecticides. All kits showed high IC50 values (!742 ng/mL) to methamidophos. When compared with other test kits, kit 4 had higher sensitivity (lower LOD) to the most tested insecticides. Matrix effects on the four kits could be minimized by direct dilution of the sample extracts into phosphate buffer. The least dilution factors needed to minimize matrix effects varied with different samples and

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test kits, kit 4 required no sample dilution, while kit 1, kit 2, and kit 3 required some sample dilution. Satisfactory recoveries of the insecticides from spiked samples were 65e110% for all kits. When AChE solutions of the kits were stored at 4  C, the AChE activity was gradually lost after 5 days for kit 1, kit 3 and kit 4 and 2 days for kit 2. To screen a large number of samples, kit 4 appeared to be more efcient than the others while kit 1, kit 2 and kit 3 were more efcient for the analysis of a few samples. When the criteria of sensitivity, accuracy, precision, assay stability and analysis time are considered, kit 1 and kit 4 appear to exhibit better performance than kit 2 and kit 3. Acknowledgments This project was supported in part by the Ministry of Environmental Protection of the Peoples Republic of China (No. 201109009), the US National Institute on Minority Health and Health Disparities (G12RR003061) and the State of Hawaii Department of Agriculture, USA. We thank Dr. Minghong Jia for helpful discussion. References
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