Pyomelanin-powered whole-cell biosensor for ultrasensitive and selective detection of bioavailable Hg(<scp>ii</scp>)

Rapid, low-cost trace inorganic Hg(ii) detection in environmental waters remains a critical public-health challenge. Here, we engineered <i>Escherichia coli</i> into a naked-eye whole-cell biosensor by coupling a redesigned MerR-P<i>mer</i> element to the pyomelanin biosynthetic pathway. Three 4-hydroxyphenylpyruvate dioxygenase (HppD) homologs from <i>Aeromonas media</i> WS, <i>Aeromonas hydrophila</i> 4AK4, and <i>Pseudomonas aeruginosa</i> PAO1 were codon-optimized and functionally screened. The sensor strain of TOP10/pHg-PAO1 exhibited the broadest quantitative range (4.9-1250 nM) and the lowest detection limit (1.2 nM), outperforming most fluorescent counterparts. The water-soluble red-brown pyomelanin product was measured directly in the culture supernatants by absorbance at 400 nm, without requiring extraction. It remained chromogenically stable for at least 14 hours. The biosensor demonstrated absolute selectivity for Hg(ii) in the presence of other ions, including Mg(ii), Ca(ii), Cd(ii), Mn(ii), Cu(ii), Pb(ii), and Zn(ii), all at a concentration of 5 μM. It retained quantitative accuracy in tap, lake, and coastal seawater matrices. These features enable on-site, equipment-free screening of environmental waters and establish pyomelanin as a robust, user-friendly chromogenic reporter for next-generation whole-cell sensors.