A simple long-wavelength fluorescent probe for simultaneous discrimination of cysteine/homocysteine and glutathione/hydrogen sulfide with two separated fluorescence emission channels by single wavelength excitation

Small molecular biothiols, such as cysteine (Cys), homocysteine (Hcy), reduced glutathione (GSH), and hydrogen sulfide (H2S), play crucial parts in regulating the redox balance of life activities, regulating normal physiological activities and preventing various diseases. Quantitative analysis of these important small molecular substances is very important for revealing their diverse physiological and pathological effects. Although many fluorescent probes have been reported to detect biothiols in cells, it is still not sufficiently advanced to detect biothiols with separated fluorescence emission peak by same wavelength excitation. In our work, we designed a simple conjugate of Nile red and NBD (7-nitro-1,2,3-benzoxadiazole) as long-wavelength fluorescent probe NR-NBD for the simultaneous discrimination of these biothiols at single wavelength excitation. Probe NR-NBD could efficiently discriminate Cys/Hcy, GSH and H2S by two separated fluorescence emission channels and absorption spectra. Importantly, probe NR-NBD has excellent specificity and sensitivity towards the monitoring of endogenous/exogenous Cys/Hcy and GSH/H2S in living cells and zebrafish.