Engineering a Single-Plasmid Dual-Pigment Escherichia coli Biosensor for Simultaneous Colorimetric Detection of Pb(II) and Hg(II)

Abstract 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.