This work describes the successful design and synthesis of a new fluorescence resonance energy transfer (FRET)-based sensor, denoted as RD1. This sensor incorporates a robust dual-fluorophore design, which combines a rhodamine and a dansyl derivative, functionalized with a thiosemicarbazide group that acts as Hg(II) specific recognition site. A synthetic pathway was developed that allowed the efficient synthesis of RD1 with a remarkable overall yield of 44% over four steps, through microwave-assisted protocols. The influence of ethyl, benzyl and phenyl substituents of isothiocyanate in the preparation of the thiosemicarbazide moiety was studied, revealing a crucial dependence of the nature of the isothiocyanate in the formation of the recognition site.Owing to its characteristic ratiometric detection, RD1 exhibited remarkable robustness to external parameters such as pH and solvent composition. The sensor demonstrated a hybrid two-stage response to Hg(II), with an initial quenching of fluorescence followed by an enhancement of emission through a FRET mechanism, both stages being corroborated by DFT (density functional theory) calculations. In vitro studies demonstrated that RD1 presents excellent cytocompatibility and capacity to permeate cellular membranes and be effectively internalized by L929 cell line. Importantly, RD1 retained its sensory ability in a complex cellular environment, affirming its efficacy as a fluorescent sensor for the in vitro detection of bioaccumulated mercury species. These results suggest the potential of RD1 for the detection of toxic bioaccumulated mercury species, aiding in environmental and biomedical research.
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