Multi-signal fluorescent probe for simultaneous differentiation and imaging of Hg2+, Cys, Hcy, and GSH in living cells and zebrafish

The Mercury (II) ion (Hg²⁺) is a toxic heavy metal that threatens biological systems by inducing oxidative stress and disrupting the redox balance. Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are critical in maintaining redox homeostasis and are implicated in numerous physiological and pathological processes. Understanding the complex interactions between Hg²⁺ and biothiols requires molecular tools capable of simultaneous detection. Herein, we report a novel multi-signal fluorescent probe, DPC, designed with four specific recognition sites and rhodamine and coumarin fluorophores. The DPC probe enables the selective and sensitive differentiation of Mercury (II) ion (Hg²⁺), cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), with distinct fluorescence signals. DPC can detect mercury (II) ions (Hg²⁺) in water samples with a high recovery rate ranging from 90.44 % to 112.27 %. DPC was also successfully applied for real-time imaging of these species in living cells and zebrafish, revealing that Hg²⁺ induces oxidative stress, reducing cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) levels. Furthermore, detoxifying agents such as sodium selenite (Na₂SeO₃), 2,3-dimercaptopropan-1-ol (DMPC), and L-selenocysteine (Sec) restored biothiol levels, counteracting Mercury (II) ion (Hg²⁺) toxicity. These results highlight the exceptional performance of DPC for the simultaneous imaging of Mercury (II) ions (Hg²⁺) and biothiols, providing insights into their interactions and proposing a chemobiological framework for studying heavy metal toxicity and developing detoxification strategies.The Mercury (II) ion (Hg²⁺) is a toxic heavy metal that threatens biological systems by inducing oxidative stress and disrupting the redox balance. Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are critical in maintaining redox homeostasis and are implicated in numerous physiological and pathological processes. Understanding the complex interactions between Hg²⁺ and biothiols requires molecular tools capable of simultaneous detection. Herein, we report a novel multi-signal fluorescent probe, DPC, designed with four specific recognition sites and rhodamine and coumarin fluorophores. The DPC probe enables the selective and sensitive differentiation of Mercury (II) ion (Hg²⁺), cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), with distinct fluorescence signals. DPC can detect mercury (II) ions (Hg²⁺) in water