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

Abstract

Rapid, low-cost trace inorganic Hg(ii) detection in environmental waters remains a critical public-health challenge. Here, we engineered Escherichia coli into a naked-eye whole-cell biosensor by coupling a redesigned MerR-Pmer element to the pyomelanin biosynthetic pathway. Three 4-hydroxyphenylpyruvate dioxygenase (HppD) homologs from Aeromonas media WS, Aeromonas hydrophila 4AK4, and Pseudomonas aeruginosa 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.

Fig. 1. Heterologous expression of HppD homologs in E. coli BL21(DE3). (A) Schematic representation of pET-21a-derived expression cassettes for 4AK4, PAO1, and WS HppD homologs. (B) Representative 10% SDS-PAGE analysis (lanes left to right): 1, uninduced total protein; 2, IPTG-induced total protein; 3, post-sonication lysate; 4, soluble fraction; 5, insoluble pellet; M, protein marker. (C) Absorption spectra (left) and photographs of the supernatant (right) of BL21(DE3)/pET-4AK4, BL21(DE3)/pET-PAO1, and BL21(DE3)/pET-WS after IPTG induction. The experiment was repeated three times, and this is a representative result. (D) Absorption spectra of the BL21(DE3)/pET-PAO1 supernatant at three different temperatures over 0–16 h after IPTG induction.

Fig. 2. Visual whole-cell Hg(ii) biosensor design and kinetic response. (A) Schematic of the MerR-Pmer-controlled pyomelanin pathway in E. coli. (B–D) Time-course A₄₀₀ (top) and corresponding culture supernatant photographs (bottom) for TOP10/pHg-4AK4 (B), TOP10/pHg-PAO1 (C), and TOP10/pHg-WS (D) exposed to 0–156.3 nM Hg(ii). Data are means ± SD (n = 3).

Fig. 3. Dose-dependent pyomelanin production in response to Hg(ii). (A) A₄₀₀ of culture supernatants versus Hg(ii) concentration (mean ± SD, n = 3). (B) Non-linear regression curves for each biosensor. (C) In the representative photographs of supernatants after 6 h induction, shaded boxes indicate the quantifiable windows. (D) Representative visible-light absorption spectra of culture supernatant. A red dashed line highlights the 400 nm region.

Fig. 4. Metal selectivity and anti-interference performance of TOP10/pHg-PAO1. (A) Pyomelanin production after exposure to 1.25, 2.5, and 5 μM of individual metal ions. The horizontal line represents the metal-free control (A₄₀₀). (B) Co-exposure to 50 nM Hg(ii) plus 2.5 or 5 μM of the indicated metals; bars show mean ± SD (n = 3), ***P < 0.001. Representative photographs of supernatants are shown below each panel.

Fig. 5. Quantitative Hg(ii) detection in environmental waters by TOP10/pHg-PAO1. (A) A₄₀₀ after 6 h exposure to increasing Hg(ii) in deionized water, tap water, surface water, and seawater (mean ± SD, n = 3). (B) Corresponding non-linear regression curves. (C) Representative photographs illustrating color gradients across matrices.