摘要
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.
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.
