Development of a Rapid-Response Fluorescent Probe for H₂S: Mechanism Elucidation and Biological Applications

Hydrogen sulfide (H₂S) is an important signaling molecule involved in various physiological and pathological processes, making its accurate detection in biological systems highly desirable. In this study, two fluorescent probes (M1 and M2) based on 1,8-naphthalimide were developed for H₂S detection via a nucleophilic aromatic substitution. M1 demonstrated high sensitivity and selectivity for H₂S in aqueous media, with a detection limit of 0.64 µM and a strong linear fluorescence response in the range of 0-22 µM of NaHS. The reaction kinetics revealed a rapid response, with a reaction rate constant of 7.56 × 10² M^-1 s^-1, and M1 was most effective in the pH range of 6-10. Mechanism studies using ¹H NMR titration confirmed the formation of 4-hydroxyphenyl-1,8-naphthalimide as the product of H₂S-triggered nucleophilic substitution. M1 was applied in MDA-MB-231 cells for cell imaging, in which M1 provided significant fluorescence enhancement upon NaHS treatment, confirming its applicability for detecting H₂S in biological environments. In comparison, M2, designed with extended conjugation for red-shifted emission, exhibited weaker sensitivity due to the reduced stability of its naphtholate product and lower solubility. These results demonstrate that M1 is a highly effective and selective fluorescent probe for detecting H₂S, providing a valuable resource for investigating the biological roles of H₂S in health and disease.