A Ratiometric genetically encoded fluorescent probe for ultrasensitive and self-calibrated detection of Hg²

Mercury ion (Hg²⁺) pollution poses severe environmental and health risks, necessitating the development of highly sensitive and reliable detection methods. Although ratiometric and genetically encoded fluorescent probes each offer distinct advantages, their integration for Hg²⁺ sensing remains largely unexplored. Herein, we report the rational design of a novel ratiometric genetically encoded fluorescent probe through the fusion of a circularly permuted green fluorescent protein (cpEGFP), a mercury-binding domain (MerBD), and a large Stokes shift reference fluorescent protein (LSSmOrange). This probe enables self-calibrated Hg²⁺ quantification by measuring the fluorescence intensity ratio of the response channel (F₅₁₂) to the stable reference channel (F₅₇₂), which effectively minimizes interferences from probe concentration, environmental fluctuations, and instrumental variations. The probe exhibits an ultra-high affinity for Hg²⁺ (apparent K_d' = 2.71 × 10^-13 M) and a detection limit of 4 nM, alongside long-term stability and high selectivity against most common metal ions. Competitive titration, circular dichroism, and fluorescence lifetime analyses reveal that the probe operates through a cooperative conformational-change mechanism, which translates picomolar-level binding into a nanomolar-level fluorescence response. This work not only addresses a critical gap in ratiometric genetically encoded probes for Hg²⁺ but also provides a robust and versatile platform for accurate Hg²⁺ monitoring in environmental and biological systems.