A multisite-binding fluorescent probe for simultaneous monitoring of mitochondrial homocysteine, cysteine and glutathione in live cells and zebrafish

Homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) play crucial roles in redox homeostasis during mitochondria functions. Simultaneous differentiation and visualization of mitochondrial biothiols dynamics are significant for understanding cell metabolism and their related diseases. Herein, a multisite-binding fluorescent probe (MCP) was developed for simultaneous sensing of mitochondrial Cys, GSH and Hcy from three fluorescence channels for the first time. This novel probe exhibited rapid fluorescence turn-on, good water-solubility, high selectivity and large spectral separation for discriminating Cys, GSH and Hcy with 131-, 96-, 748-fold fluorescence increasement at 471, 520, 567 nm through different excitation wavelengths, respectively. Importantly, this probe was successfully applied to simultaneous monitoring of mitochondrial Cys, GSH, and Hcy in live cells and zebrafish from three fluorescence channels, promoting the understanding of the functions of Hcy, Cys and GSH. A multisite-binding fluorescent probe was rationally exploited for simultaneously differentiating and visualizing mitochondrial homocysteine, cysteine and glutathione in living cells and zebrafish.