Transcription factor shielding yields a highly specific and sensitive whole-cell biosensor for Pb²⁺ detection

Whole-cell biosensors (WCBs) provide an economical and practical platform for the detection of environmental contaminants. However, their performance in complex samples is often compromised by limited specificity. In particular, sensing of lead ions (Pb²⁺) detection is frequently affected by interference from chemically similar metal ions such as mercury ions (Hg²⁺), resulting in non-specific signal responses. In this study, the wild-type Pb²⁺ WCB was constructed based on the Pb-resistance transcriptional regulatory factor from the plasmid pLVPK of Klebsiella pneumoniae CG43 (PbrR-kp). The sensitivity of the WCB was subsequently improved via directed evolution, followed by the implementation of a transcription factor (TF) shielding strategy to increase specificity. Negative/positive fluorescence-activated cell sorting (FACS) yielded the mutant WCB PbrR-3 with a limit of detection (LOD) of 0.08 nM but retained substantial responsiveness to Hg²⁺. To reduce Hg²⁺-induced interference, a mercury-resistant TF mutant MerR∗ was introduced into the biosensor circuit, and a ribosomal binding site (RBS) library was constructed to tune MerR∗ expression. Screened mutants suggested a potential positive correlation between MerR∗ translation initiation efficiency and specificity. The optimized WCB RBSM480 achieved an LOD of 0.03 nM and a 5-fold specificity improvement. Accurate detection of mixed Pb²⁺/Hg²⁺ real samples was validated against Inductively Coupled Plasma Mass Spectrometry (ICP-MS). This work presents a high-performance Pb²⁺ WCB and establishes a TF shielding strategy as a flexible approach for improving specificity in WCB engineering.