Visualizing biothiols in vivo using a dual-channel sensitive fluorescent probe

Biothiols are essential for maintaining appropriate redox status in organisms, and developing fluorescent probes for highly effective detection of biothiols in the living system is of great importance. In this work, we designed and synthesized a dual-channel sensitive fluorescent probe (TMN-NBD) for the highly effective detection of biothiols in vitro and in vivo. The probe was constructed by conjugating NBD chloride (NBD-Cl) moiety to a dicyanoisophorone derivative (TMN-OH) as the recognition group and fluorophore. TMN-NBD shows negligible fluorescence due to the strong electron-withdrawing property of NBD. Upon exposure to Cys/Hcy, the fluorescence was dramatically increased in the green and red channels. While for GSH, the fluorescence was only enhanced in the red channel. Therefore, a dual-channel sensitive fluorescent probe was constructed. TMN-NBD performs well and features high sensitivity and good selectivity to biothiols. Furthermore, due to the low cytotoxicity, good cell, and tissue-permeable, TMN-NBD was successfully used for tracking biothiols in living cancer cells and the zebrafish model. This scaffold of the dual-channel sensitive fluorescent probe provides a new tool for the study of biothiols in the living organism, which is also helpful for the clinical research of thiol-related diseases.