砷全细胞生物传感器的优化

Optimization of a whole-cell bosensor for heavy metal arsenic

砷全细胞生物传感器具有专一性强、操作简单、费用低和不依赖大型检测仪器的优点,已被用于环境砷污染的监测中.但目前砷全细胞生物传感器的灵敏度低,尤其是信噪比低,是阻碍其实际应用的主要因素之一.本研究利用砷抗性操纵子ars R和报告基因gfp构建砷全细胞生物传感器,通过优化组成型启动子强度、ars R DNA结合序列(ArsR binding site,ABS)及检测条件等对该传感器的检测性能进行改进,并通过对加标的天然水样检测,验证了其实际应用性.结果表明:优化后的砷全细胞生物传感器在0~300μg·L^(-1)和7.5~75μg·L^(-1)的亚砷酸钠浓度范围分别表现出良好的剂量依赖效应和线性响应,对砷检测限达到1.83μg·L^(-1);新增ABS和中等强度组成型启动子J114能够使生物传感器背景荧光降低并提高其对砷的响应强度;结合对宿主菌种类、生长状态和起始细菌数量等检测条件的优化,生物传感器荧光信号响应得到显著性提高.在6和75μg·L^(-1)亚砷酸钠诱导下,信噪比分别提高2.6倍和6.1倍.此外,该生物传感器对天然水样检测结果与真实值接近,这表明这些优化方法可以提高砷全细胞生物传感器性能,有利于传感器的实际应用.

Taking advantages of high specificity,simplicity of operation,cost-effectiveness and independence on large detection instruments,arsenic whole-cell biosensors have been used in monitoring of environmental arsenic contamination.However,the low sensitivity and signal-to-noise ratio of current arsenic whole-cell biosensors is one of the main factors hindering their practical application.In this study,we constructed an arsenic whole-cell biosensor using the arsenic resistance operon arsR and the reporter gene gfp;improved the detection performance of the sensor by optimizing the constitutive promoter strength,arsR DNA binding site(ABS)and detection conditions;and verified its practical applicability by testing spiked natural water samples.The results showed that the optimized arsenic whole-cell biosensor exhibited good dose-dependent and linear responses in the concentration ranges of 0~300μg·L^(-1)and 7.5~75μg·L^(-1)for sodium arsenite,respectively.The detection limit of the optimized arsenic whole-cell biosensor is 1.83μg·L^(-1).By addition of ABS and the moderate intensity constitutive promoter J114,the background fluorescence of the biosensor was significantly reduced and the response intensity to sodium arsenite was greatly increased.The fluorescence induction was significantly improved by combining the optimization of detection conditions,such as bacterial species,growth state and the number of initial bacteria.The signal-to-noise ratio was improved by 2.6-fold and 6.1-fold under the induction of 6 and 75μg·L^(-1)sodium arsenite,respectively.In addition,the results of the biosensor for natural water samples detection are close to the real value,indicating that these optimized methods can improve the performance of arsenic whole-cell biosensors and facilitate their practical application.