Enhanced response speed and selectivity of fluorescein-based H₂S probe via the cleavage of nitrobenzene sulfonyl ester assisted by ortho aldehyde groups

In this work, we developed three fluorescent probes (F-1, F-2, and F-3) based on fluorescein, mono-formylated fluorescein, and bis-formylated fluorescein for hydrogen sulfide (H₂S) detection. The probe F-3, which bears two aldehyde groups, exhibited the fastest response. This fast response is attributed to the ortho effect of the aldehyde group, which enables fast nucleophilic addition of H₂S to an aldehyde group and subsequent intramolecular thiolysis of dinitrophenyl ether. In addition, the aldehyde groups on F-3 react with biothiols (e.g., cysteine, homocysteine) to form thiazolidine diastereomers, which suppress the fluorescence of fluorescein. The introduction of two aldehyde groups also resulted in high selectivity of F-3 towards H₂S. Furthermore, good linearity was observed between F-3 fluorescence intensity at 510 nm and H₂S concentration in the range of 0–10 µM. F-3 exhibited a detection limit as low as 0.024 μM. Confocal laser scanning micrographs of HeLa cells incubated with F-3 confirmed that F-3 is cell-permeable and can successfully detect H₂S in living cells.