Red and Near-Infrared Fluorescent Probe for Distinguishing Cysteine and Homocysteine through Single-Wavelength Excitation with Distinctly Dual Emissions

Small-molecule biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), participate in various pathological and physiological processes. It is still a challenge to simultaneously distinguish Cys and Hcy because of their similar structures and reactivities, as well as the interference from the high intramolecular concentration of GSH. Herein, a novel fluorescent probe, CySI, based on cyanine and thioester was developed to differentiate Cys and Hcy through a single-wavelength excitation and two distinctly separated emission channels. The probe exhibited a turn-on fluorescence response to Cys at both 625 nm (the red channel) and 740 nm (the near-infrared channel) but only showed fluorescence turn-on to Hcy at 740 nm (the near-infrared channel) and no fluorescent response to GSH. With the aid of built-in self-calibration of single excitation and dual emissions, simultaneous discriminative determinations of Cys and Hcy were realized through red and near-infrared channels. CySI exhibited excellent selectivity toward Cys and Hcy with a fast response. This probe was further exploited to visualize exogenous Cys and Hcy in cells through dual emission channels under one excitation. Moreover, it could efficiently target mitochondria and was applied to monitor the endogenous Cys fluctuations independently in mitochondria through the red emission channel.