Hydrogen sulfide (H₂S) is an important gasotransmitter, but only a few methods are available for real-time detection. Fluorescent probes are attractive tools for biological applications because of their high sensitivity, convenience, rapid implementation, noninvasive monitoring capability, and simplicity in fluorescent imaging of living cells and tissues. Herein, we report on a pro-fluorescent probe, NAP-Py-N₃ based on naphthalimide derivative, which was found to show high selectivity toward H₂S over various other analytes, including biothiols, making it feasible to detect H₂S. After reaction with H₂S, this probe showed rapid and significant turn-on green fluorescent enhancement at 553 nm (about 54-fold, k₂ = 9.62 M⁻¹s⁻¹), high sensitivity (LOD: 15.5 nM), significant Stokes shift (118 nm), and it was found that the fluorescence quantum yield of fluorescence product can reach 0.36. Moreover, the probe has also been successfully applied to detect the gaseous H₂S and to confirm the presence of H₂S released from modern organic donors, which in recent years have been commonly used to investigate the role of H₂S in biological systems. All the results indicate that this probe is excellent and highly valuable.

Hydrogen sulfide (H₂S) and its bioderivatives analogs, such as L-cysteine (L-Cys) and glutathione (GSH), are ubiquitous biological thiols in the physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Although several NBD-based fluorescence probes have been developed to detect biological thiols, the HPLC-detection of H₂S, GSH, L-Cys, and N-acetylcysteine-specific products has not been described. Herein, a novel NBD-derived pro-coumarin probe has been synthesized and used to develop a new strategy for the triple mode detection of H₂S and such thiols as GSH, L-Cys, and NAC. Hydrogen sulfide and those biothiols at physiological pH release fluorescent coumarin from the probe and cause a significant fluorescence enhancement at 473 nm. The appropriate NBD-derived product for H₂S, L-Cys, GSH, and NAC has a different color and retention time that allows distinguishing these biological thiols meaning the probe has a great possibility in the biological application. Fluorescent imaging combined with colorimetric and HPLC detection of H₂S/biothiol-specific product(s) brings a potential tool for confirming the presence of biological thiols and determining concentrations in various aqueous biological samples.

Hypochlorous acid (HOCl) has been implicated in numerous pathologies associated with an inflammatory component, but its selective and sensitive detection in biological settings remains a challenge. In this report, imaging of HOCl was realized with a thiomorpholine-based probe as derivative of nitrobenzothiadiazole (NBD-S-TM). The fluorescence is based on photoinduced electron transfer by using nitrobenzothiadiazole core as a donor and thiomorpholine substituent as an acceptor. NBD-S-TM showed high sensitivity and a fast response to HOCl k = (2.6 ± 0.2) × 10⁷ M⁻¹s⁻¹ with a 1:1 stoichiometry. The detection limit for HOCl was determined to be 60 nM. Furthermore, the desirable features of NBD-S-TM for the detection of HOCl in aqueous solutions, such as its reliability at physiological pH, rapid fluorescence response, and biocompatibility, enabled its application in the detection of HOCl in myeloperoxidase enzymatic system. Moreover, NBD-S-TM exhibited excellent selectivity and sensitivity for HOCl over other biologically relevant species, such as hydrogen peroxide and peroxynitrite. The fluorescent S-oxidized product (NBD-S-TSO) is only formed in the presence of HOCl. Probing with NBD-S-TM may be helpful to further the development of high throughput screening assays to monitor the activity of myeloperoxidase.