A single-wavelength excited NIR fluorescence probe for distinguishing GSH/H2S and Cys/Hcy in living cells and zebrafish through separated dual-channels

Biothiols and hydrogen sulfide, as critical sulfur-containing reactive substances, serve essential functions in various human pathological processes, making it challenging to simultaneously distinguish them due to their similar reactivity and structures (−SH). Here, we rationalized the development of a single-wavelength excitation near-infrared (NIR) fluorescence probe, FC-NBD, for distinguishing GSH/H2S and Cys/Hcy by separated fluorescence dual channels. In this probe, FC-NBD, composed of coumarin-benzopyrylium derivatives linked with nitro benzoxadiazole (NBD) via ether bonds, could quantitatively and selectively distinguish GSH/H2S and Cys/Hcy with a low limit of detection (LOD) of 0.199/0.177 μM and 0.106/0.076 μM, respectively. As expected, under single-wavelength excitation (470 nm), FC-NBD demonstrated distinctly separable green and NIR fluorescence emissions towards Cys/Hcy at 550 and 660 nm, but only exhibited a noticeable NIR fluorescence emission towards GSH/H2S at 660 nm. Moreover, FC-NBD could simultaneously visualize and discriminate GSH/H2S and Cys/Hcy in living cells as well as zebrafish through green and NIR channels at a single excitation wavelength.