A selective and sensitive dual-mode Hg²⁺ biosensing platform based on thermostable phycocyanin biosynthesis with...

Phycocyanin has been proposed as a good biosensor for heavy metals. However, the extraction of phycocyanin from cyanobacteria is both time-consuming and labor-intensive. In this study, we heterologously biosynthesized the C-phycocyanin β subunit (CpcB) from Thermosynechococcus elongatus in Escherichia coli, achieving exceptional chromophore binding efficiency (A₆₂₀/A₂₈₀ = 2.13) and strong red fluorescence with a high fluorescence quantum yield (Φ_F = 0.38) due to an optimized genetic design and intrinsic chromophore-protein interactions. Compared with CpcB derived from other cyanobacteria, CpcB acquired from thermophilic cyanobacteria demonstrated superior Hg²⁺-selective quenching, concentration-dependent fluorescence responsivity and thermostability. The recombinant CpcB exhibited a dual-mode response to Hg²⁺, integrating both fluorometric quantification and colorimetric analysis capabilities. In the fluorometric mode, the limits of detection (LODs) were determined to be 0.43 nM under red light excitation and 2.71 nM under UV excitation with a large Stokes shift (288 nm). The fluorescence-based colorimetric mode achieved an LOD of 1.73 nM through a smartphone-assisted analysis. We propose a hybrid response mechanism that involves dynamic quenching via surface C153-Hg²⁺ coordination at low concentrations, which transitions to static quenching through C82 binding at elevated Hg²⁺ levels. The CpcB sensor demonstrated good recovery and color changes that were detectable by the naked eye in real samples. Our study reveals a thermostable recombinant phycocyanin that can serve as a robust platform for developing Hg²⁺ biosensors.