Engineering a Single-Plasmid Dual-Pigment Escherichia coli Biosensor for Simultaneous Colorimetric Detection of...

This work presents an engineered single-plasmid, dual-pigment whole-cell biosensor for simultaneous colorimetric detection of Pb-(II) and Hg-(II). To address the challenge of signal interference in cocontaminated environmental samples, a Pb-(II)-responsive PbrR regulatory module driving indigo biosynthesis (blue, 620 nm) and an Hg-(II)-responsive MerR module driving deoxyviolacein biosynthesis (purple, 570 nm) were integrated into Escherichia coli TOP10. The orthogonal decoupling of the two pigment pathways, combined with a unified 10 h induction protocol, enables independent chromogenic readout of both metals from a single cultivation. Dose-response assays afforded a broad linear dynamic range for Pb-(II) of 0.977-2000 μM (R² = 0.9503) with a limit of detection (LOD) of 0.977 μM, alongside nanomolar sensitivity for Hg-(II) with a linear range of 0.244-250 nM (R² = 0.9800) and an LOD of 0.244 nM. A two-dimensional cross-concentration matrix experiment further demonstrated that the two detection channels maintain distinguishable dose-dependent responses under coexposure conditions, with crosstalk remaining within an acceptable range. Robust performance was retained in spiked surface water and seawater matrices, yielding Pb-(II) LODs of 0.244 μM (surface water) and 0.488 μM (seawater), and Hg-(II) LODs of 0.488 nM (surface water) and 0.977 nM (seawater). These detection limits approach or fall below relevant environmental regulatory thresholds, including the Class III surface-water standard for Hg-(II). This self-contained, instrument-free pigment-based approach enables naked-eye discrimination of both metals in a standard microplate assay, offering a low-cost, high-throughput screening platform that complements standard instrumental methods for on-site environmental monitoring and emergency pollution response.