构建全细胞生物传感器测定农田土壤中有机磷农药甲基对硫磷含量

Development of a whole-cell biosensor for detecting organophosphorus pesticide methyl parathion in the farmland soil

摘要:

土壤中污染物的生物有效性评估对于准确评价环境污染风险至关重要，而全细胞生物传感器是此类评估的重要工具之一。本研究旨在使用新型全细胞生物传感器建立土壤中甲基对硫磷（methyl parathion，MP）的检测方法。首先，使用筛选出的甲基对硫磷水解酶基因（methyl parathion degrading gene，mpd）和pUC19质粒骨架以及已有的特异性诱导元件pobR为材料，构建全细胞生物传感器。然后，以96孔的酶标板为载体和以5种全细胞生物传感器为指示细胞，建立了土壤提取液样品中甲基对硫磷的分析方法，并应用于实际测试和田间土壤样品中甲基对硫磷的检测。以检测性能的最佳大肠杆菌（Escherichia coli） DH5α/pMP-AmilCP为例，其检测限为6.21—6.66 μg/L，线性范围为10—10 000μg/L。E. coliDH5α/pMP-RFP和E. coliDH5α/pMP-AmilCP的方法用于分析土壤提取液样品中甲基对硫磷的浓度，具有较好的检测性能。这种全细胞生物传感器方法有助于快速评估土壤中甲基对硫磷的生物有效性强弱，从而有效判断有机磷农药甲基对硫磷对土壤的污染风险。

Abstract:

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21−6.66 µg/L and a linear range of 10−10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.