Two-photon small molecular fluorogenic probe visualizing biothiols and sulfides in living cells, mice brain slices and zebrafish

Biothiols and sulfides, especially glutathione (GSH), cysteine (Cys), homocysteine (Hcy) and hydrogen sulfide (H₂S) play important roles in an extensive range of biological processes and functions. Due to their reductivity, they are deemed as second messengers of oxidative stress, and the imbalance of these biothiols are closely associated to a variety of diseases. Herein, we report a two-photon (TP) turn-on probe DT-01 that can detect biothiols and sulfides both in vitro and in vivo. The probe was synthesized by nitrobenzoxadiazole (NBD) derivative (NBD-Cl) and pyrimidine-based TP fluorophore (FL-01), the fluorescence of DT-01 is quenched due to the photoinduced charge transfer (PET) from FL-01 moiety to NBD moiety. Upon substitution reaction by nucleophilic biothiols, TP fluorophore released and exhibited turn-on fluorescence at 550 nm. We further demonstrated that DT-01 can detect biothiols and H₂S in living cells, mice brain slices and zebrafish. More importantly, by real-time detecting the total content of biothiols and H₂S, we have successively measured the changes of oxidative stress levels in HeLa cells.