Shen Liang, Chen Yiwen, Pan Jiajie, Yu Xin, Zhang Yubo, Guo Bingxin, Wang Jiaqi, Liu Ying, Xiao Xiang, Chen Shaopeng, Bao Lingzhi
School of Public Health, Wannan Medical College, Wuhu 241002, China; Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China.
School of Public Health, Wannan Medical College, Wuhu 241002, China.
J Hazard Mater. 2025 May 5;488:137489. doi: 10.1016/j.jhazmat.2025.137489. Epub 2025 Feb 3.
The Whole-cell biosensor (WCB) is a convenient and practical assay that can monitor bioavailable lead (Pb) contamination. However, existing Pb-responsive WCB struggle to meet practical detection needs due to the lack of sensitivity, specificity, and stability. In this study, we developed a Pb WCB using the Pb resistance transcriptional regulatory factor (PbrR) and green fluorescent protein (GFP), and improved its performance by directed evolution in conjunction with fluorescence-activated cell sorting (FACS). After 3 rounds of screening, we acquired a biosensor mutant (PbrR-E3). The evolved biosensor exhibited an approximately 11-fold increase in maximum fluorescence output signal compared to the non-evolved biosensor, resulting in an improvement of its sensitivity and specificity. This biosensor demonstrated a limit of detection (LOD) of 0.045 μg/L. Furthermore, the evolved biosensor showcased outstanding performance in the detection of Pb(II) in tea infusion and also demonstrated good stability in tests with spiked real water samples. These results highlight the potential of the evolved WCB as a viable approach for monitoring Pb.
全细胞生物传感器（WCB）是一种方便实用的检测方法，可监测生物可利用的铅（Pb）污染。然而，现有的铅响应型WCB由于缺乏灵敏度、特异性和稳定性，难以满足实际检测需求。在本研究中，我们利用抗铅转录调节因子（PbrR）和绿色荧光蛋白（GFP）开发了一种铅WCB，并通过结合荧光激活细胞分选（FACS）的定向进化提高了其性能。经过三轮筛选，我们获得了一种生物传感器突变体（PbrR-E3）。与未进化的生物传感器相比，进化后的生物传感器最大荧光输出信号增加了约11倍，从而提高了其灵敏度和特异性。该生物传感器的检测限（LOD）为0.045μg/L。此外，进化后的生物传感器在茶浸出液中铅（II）的检测中表现出优异的性能，并且在加标真实水样的测试中也表现出良好的稳定性。这些结果突出了进化后的WCB作为监测铅的可行方法的潜力。
