Laboratory of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of Athens, Athens, Greece.
Talanta. 2010 Mar 15;80(5):1799-804. doi: 10.1016/j.talanta.2009.10.026. Epub 2009 Nov 5.
Two of the most important categories of pesticides used in agricultural practice are organophosphates and dithiocarbamates. Their extensive and inappropriate use has rendered their reliable monitoring at trace levels more and more necessary. 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). The cells were immobilized by entrapment in a sodium alginate bead and directly applied in different pesticide dilutions and agricultural samples. The pesticides used were the organophosphate insecticide diazinon and the dithiocarbamate fungicide propineb. Two different cell types, N2a (neuroblastoma) and Vero (fibroblast) were used as the biosensory elements in order to investigate their differential response against the pesticides. In this way, we hoped to increase the selectivity of the assay. 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 the pesticides with a high degree of reproducibility. The lowest detected concentration was 3nM. Finally, for the investigation of the effects of different pesticides on the accumulation of cytosolic Ca(2+), we conducted a fluorescent assay on N2a cells treated with tomato sample extracts, which were replicates of the E.U. proficiency test sample. The tomato samples were either organically grown or contained 14 different pesticides. The experimental results showed a higher increase of the intracellular Ca(2+) concentration in cells treated with non-organic samples compared to the cells treated with organic samples.
在农业实践中使用的两种最重要的农药类别是有机磷农药和二硫代氨基甲酸盐。它们的广泛和不适当使用使得在痕量水平上对其进行可靠监测变得越来越必要。本研究根据生物电识别分析（BERA）的工作原理，提出了一种基于固定化细胞细胞膜电位变化测量的快速灵敏细胞生物传感器测试的构建。细胞通过包埋在海藻酸钠珠中被固定化，并直接应用于不同的农药稀释液和农业样品中。所用的农药是有机磷杀虫剂二嗪磷和二硫代氨基甲酸盐杀菌剂丙森锌。为了研究它们对农药的不同反应，我们使用了两种不同的细胞类型，N2a（神经母细胞瘤）和 Vero（成纤维细胞）作为生物传感元件。通过观察到的反应模式，我们证明该传感器可用于定性检测，并且在某些浓度下，可用于定量检测农药，具有高度的重现性。检测到的最低浓度为 3nM。最后，为了研究不同农药对细胞质 Ca(2+)积累的影响，我们对用番茄样品提取物处理的 N2a 细胞进行了荧光分析，这些提取物是欧盟能力验证样品的复制品。番茄样品要么是有机种植的，要么含有 14 种不同的农药。实验结果表明，与用有机样品处理的细胞相比，用非有机样品处理的细胞中细胞内 Ca(2+)浓度的增加更高。
