Development of a highly sensitive PbrR-based biosensor via directed evolution and its application for lead detection.

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.