The low-molecular weight biothiols like glutathione (GSH) and cysteine (Cys) play many important roles in various biological processes, the imbalance of biothiols level will lead to many diseases. However, methods that can selectively detect the GSH and Cys was rarely reported because of the similar reactivity and structure. Here, a fluorogenic method was presented to selectively detect the GSH and Cys in vitro using probe ML-FP based on fluorescent protein mimics. Probe ML-FP is a fluorescence turn on probe with lysosome targeting capacity. 2,4-dinitrobenzenesulfonyl serves as fluorescence quench and detection group in probe ML-FP. Differentiating the GSH and Cys was realized benefit from the different reaction time as well as fluorescence response between probe and target species. Low detection limit (4.98 nM for Cys and 4.39 nM for GSH) as well as fast response time was founded in this work. Probe ML-FP possess excellent biocompatibility due to fluorescent protein chromophore and was successfully used for bioimaging in living cells.
