CellBio  Vol.2 No.3 , September 2013
Screening of Total Organophosphate Pesticides in Agricultural Products with a Cellular Biosensor

Organophosphates belong to the most important pesticides used in agricultural practice worldwide. Although their analytical determinations are quite feasible with various conventional methods, there is a lack of efficient screening methods, which will facilitate the rapid, high-throughput detection of organophosphates in different food commodities. This study presents the construction of a rapid and sensitive cellular biosensor test based on the measurement of changes of the cell membrane potential of immobilized cells, according to the working principle of the Bioelectric Recognition Assay (BERA). Two different cell types were used, derived either by animal (neuroblastoma) or plant cells (tobacco protoplasts). The sensor was applied for the detection of a mixture of two organophosphate pesticides, diazinon and chlorpyrifos in two different substrates (tomato, orange). The pesticides in the samples inhibited the activity of cell membrane-bound acetylcholinesterase (AChE), thus causing a measurable membrane depolarization in the presence of achetylcholine (Ach). Based on the observed patterns of response, we demonstrate that the sensor can be used for the qualitative and, in some concentrations, quantitative detection of organophosphates in different substrates with satisfactory reproducibility and sensitivity, with a limit of detection at least equal to the official Limit of Detection (LOQ). The assay is rapid with a total duration of 3 min at a competitive cost. The sensitivity of the biosensor can be further increased either by incorporating more AChE-bearing cells per test reaction unit or by using cells engineered with more potent AChE isoforms. Standardization of cultured cell parameters, such as age of the cells and subculture history prior to cell immobilization, combined with use of planar electrodes, can further increase the reproducibility of the novel test.

Cite this paper
K. Lokka, P. Skandamis and S. Kintzios, "Screening of Total Organophosphate Pesticides in Agricultural Products with a Cellular Biosensor," CellBio, Vol. 2 No. 3, 2013, pp. 131-137. doi: 10.4236/cellbio.2013.23015.
[1]   FAOSTAT, “Database on Pesticide Consumption. Rome: Food and Agriculture Organization of the United Na- tions,” Statistical Analysis Service, 2009.

[2]   M. Pohanka, K. Musilek and K. Kuca, “Progress of Biosensors Based on Cholinesterase Inhibition,” Current Medicinal Chemistry, Vol. 16, No. 14, 2009, pp. 1790-1798. doi:10.2174/092986709788186129

[3]   R. A. Maselli and B. C. Soliven, “Analysis of the Or- ganophosphate-Induced Electromyographic Response to Repetitive Nerve Stimulation: Paradoxical Response to Edrophonium and D-Tubocurarine,” Muscle & Nerve, Vol. 14, No. 12, 1991, pp. 1182-1188. doi:10.1002/mus.880141207

[4]   B. Eskenazi and N. A. Maizlish, “Effects of Occupational Exposure to Chemicals on Neurobehavioral Functioning,” In: R. E. Tarter, D. H. V. Thiel and K. L. Edwards, Eds., Medical Neuropsychology: The Impact of Disease on Behavior, New York, 1988, pp. 409-419. doi:10.1007/978-1-4757-1165-3_9

[5]   L. Rosenstock, M. Keifer and W. E. Daniell, “Chronic Central Nervous System Effects of Acute Organophosphate Pesticide Intoxication. The Pesticide Health Effects Study Group,” Lancet, Vol. 338, No. 8761, 1991, pp. 223- 227. doi:10.1016/0140-6736(91)90356-T

[6]   M. Eddieston, N. A. Buckley, P. Eyer and A. H. Dawson, “Medical Management of Acute Organophosphorus Pesticide Self-Poisoning,” Lancet, Vol. 371, No. 9612, 2008, pp. 597-607. doi:10.1016/S0140-6736(07)61202-1

[7]   J. Haib, I. Hofer and J. M. Renaud, “Analysis of Multiple Pesticide Residues in Tobacco Using Pressurized Liquid Extraction, Automated Solid-Phase Extraction Clean-Up and Gas Chromatography-Tandem Mass Spectrometry,” Journal of Chromatography A, Vol. 1020, No. 2, 2003, pp. 173-187. doi:10.1016/j.chroma.2003.08.049

[8]   J. Sherma, “Review of Advances in the Thin Layer Chromatography of Pesticides: 2004-2006,” Journal of Environmental Science & Health B, Vol. 42, No. 4, 2007, pp. 429-440. doi:10.1080/03601230701316440

[9]   R. T. Andres and R. Narayanaswamy, “Fibre-Optic Pesticide Biosensor Based on Covalently Immobilized Acetylcholinesterase and Thymol Blue,” Talanta, Vol. 44, No. 8, 1997, pp. 1335-1352. doi:10.1016/S0039-9140(96)02071-1

[10]   J. W. Choi, Y. K. Kim, I. H. Lee, J. Min and W. H. Lee, “Optical Organophosphorus Biosensor Consisting Acetyl Cholinesterase/Viologen Hetero Langmuir-Blodjett Film,” Biosensors and Bioelectronics, Vol. 16, No. 9-12, 2001, pp. 937-943. doi:10.1016/S0956-5663(01)00213-5

[11]   V. G. Andreou and Y. D. Clonis, “A Portable Fiber-Optic Pesticide Biosensor Based on Immobilized Cholinesterase and Sol-Gel Entrapped Bromcresol Purple for In-Field Use,” Biosensors and Bioelectronics, Vol. 17, No. 1-2, 2002, pp. 61-69. doi:10.1016/S0956-5663(01)00261-5

[12]   S. Mavrikou, K. Flampouri, G. Moschopoulou, O. Mangana, A. Michaelides and S. Kintzios, “Assessment of Organophosphate and Carbamate Pesticide Residues in Cigarette Tobacco with a Novel Cell Biosensor,” Sensors, Vol. 8, No. 4, 2008, pp. 2818-2832. doi:10.3390/s8042818

[13]   E. Flampouri, S. Mavrikou, S. Kintzios, G. Miliadis and P. Aplada-Sarli, “Development and Validation of a Cellular Biosensor Detecting Pesticide Residues in Tomatoes,” Talanta, Vol. 80, No. 5, 2009, pp. 1799-1804. doi:10.1016/j.talanta.2009.10.026

[14]   J. Reinert and M. Yeoman, “Plant Cell Tissue Culture,” Springer-Verlag, Berlin, 1982. doi:10.1007/978-3-642-81784-7

[15]   M. Mizayawa, H. Tougo and M. Ishihara, “Inhibition of Acetylcholinesterase Activity by Essential Oil from Citrus paradise,” Natural Products Letters, Vol. 15, No. 3, 2001, pp. 205-210. doi:10.1080/10575630108041281

[16]   O. Aust, N. Ale-Agha, L. Zhang, H. Wollersen, H. Sies and W. Stahl, “Lycopene Oxidation Product Enhances Gap Junctional Communication,” Food Chemistry and Toxicology, Vol. 41, No. 10, 2003, pp. 1399-1407. doi:10.1016/S0278-6915(03)00148-0

[17]   J. Flaskos, W. G. McLean, M. J. Fowler and A. J. Hargreaves, “Tricresyl Phosphate Inhibits the Formation of Axon-Like Processes and Disrupts Neurofilaments in Cultured Mouse N2a and Rat PC12 Cells,” Neuroscience Letters, Vol. 242, No. 2, 1998, pp. 101-104. doi:10.1016/S0304-3940(98)00054-8

[18]   S. Madhavan, G. Sarath, B. H. Lee and R. S. Pegden, “Guard Cell Protoplasts Contain Acetylcholinesterase Activity,” Plant Science, Vol. 109, No. 2, 1991, pp. 119-127. doi:10.1016/0168-9452(95)04164-P

[19]   D. Ferentinos, C. P. Yialouris, P. Blouchos, G. Moschopoulou, V. Tsourou and S. Kintzios, “The Use of Artificial Neural Networks as a Component of a Cell-Based Biosensor Device for the Detection of Pesticides,” Procedia Engineering, Vol. 47, 2012, pp. 989-992. doi:10.1016/j.proeng.2012.09.313

[20]   E. Voumvouraki and S. Kintzios, “Differential Screening of the Neurotoxicity of Insecticides by Means of a Novel Electrophysiological Biosensor,” Procedia Engineering, Vol. 25, 2011, 964-967. doi:10.1016/j.proeng.2011.12.237

[21]   E. Flampouri and S. Kintzios, “Nafion and Polylysine Treated PEDOT Mammalian Cell Biosensor,” Procedia Engineering, Vol. 25, 2011, pp. 964-967.

[22]   V. Varelas, N. Sanvicens, M. P. Marco and S. Kintzios, “Development of a Cellular Biosensor for the Detection of 2, 4, 6-Trichloroanisole (TCA),” Talanta, Vol. 84, No. 3, 2010, pp. 936-940. doi:10.1016/j.talanta.2011.02.029

[23]   E. Larou, I. Yiakoumettis, G. Kaltsas, A. Petropoulos, P. Skandamis and S. Kintzios, “High Throughput Cellular Biosensor for the Ultra-Sensitive, Ultra-Rapid Detection of Aflatoxin M1,” Food Control, Vol. 29, No. 1, 2012, pp. 208-212. doi:10.1016/j.foodcont.2012.06.012