Mechanistic Investigation of Fluorescence Quenching in the Benzydamine–Hg 2+ System by Combined Experimental and Computational Studies

ABSTRACT The development of fluorescent probes capable of operating in fully aqueous media under physiologically relevant conditions remains an important challenge for Hg 2+ detection. In this study, benzydamine hydrochloride (BNZ), a commercially available anti‐inflammatory drug, was investigated as a fluorescent turn‐off probe for Hg 2+ in neutral aqueous solution. Fluorescence titration demonstrated a sensitive response toward Hg 2+ , providing a linear detection range of 1.0–10.0 μM, a limit of detection (LOD) of 0.20 μM, and a limit of quantification (LOQ) of 0.67 μM, together with excellent selectivity over a broad range of competing inorganic ions and biologically relevant species. To elucidate the sensing mechanism, comprehensive spectroscopic investigations were combined with density functional theory (DFT), natural bond orbital (NBO), and time‐dependent density functional theory (TD‐DFT) calculations. Experimental results demonstrated that the fluorescence quenching originates from intrinsic BNZ–Hg 2+ complexation rather than from aggregation, precipitation, inner‐filter effects, structural transformation, or temperature‐dependent artifacts. DFT calculations indicated that Hg 2+ interacts with BNZ through ligand‐exchange processes involving hydrated Hg 2+ species, while NBO analysis confirmed the formation of N→Hg coordination interactions. Furthermore, TD‐DFT calculations performed on both ground‐state and optimized excited‐state geometries revealed that Hg 2+ complexation substantially reduces the oscillator strength of the emissive transition, providing a molecular‐level explanation for the experimentally observed fluorescence quenching. Overall, this work demonstrates the analytical applicability of BNZ as a simple, inexpensive, and readily available fluorescent probe for Hg 2+ detection in aqueous media. More importantly, it establishes a comprehensive strategy for elucidating fluorescence‐quenching mechanisms through the integration of spectroscopic investigations with quantum‐chemical calculations, which may facilitate the rational design and mechanistic understanding of future fluorescent probes operating under physiologically relevant conditions.