A multi-signal fluorescent probe was engineered for simultaneously distinguishing and sequentially sensing cysteine/homocysteine, glutathione, and hydrogen sulfide in living cells.

Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a crucial role in many physiological processes. Cys production and metabolism is closely connected with Hcy and GSH; meanwhile, the dynamic antioxidant defenses network by Cys is independent of the GSH system, and Cys can serve as a more effective biomarker of oxidative stress. Hence, it is significant and urgent to develop an efficient method for specific detection of Cys over other biothiols (Hcy/GSH). However, most of the present Cys-specific fluorescent probes distinguished Cys from Hcy through response time, which would suffer from an unavoidable interference from Hcy in long-time detection. In this work, in order to improve the selectivity, we employed an improved aromatic substitution-rearrangement strategy to develop a ratiometric Cys-specific fluorescent probe (Cou-SBD-Cl) based on a new fluorescence resonance energy transfer (FRET) coumarin-sulfonyl benzoxadiazole (Cou-SBD) platform for discrimination of Hcy and GSH. Response of Cou-SBD-Cl to Cys would switch FRET on and generate a new yellow fluorescence emission with a 56.1-fold enhancement of ratio signal and a 99 nm emission shift. The desirable dual-color ratiometric imaging was achieved in living cells and normal zebrafish. In addition, probe Cou-SBD-Cl was also applied to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish.

A novel multifunctional logic gate based on a triple-chromophore (coumarin-NBD-flavylium, CNF) fluorescent biothiol probe with diverse fluorescence signal patterns was rationally designed and synthetized. On the new triad CNF, diverse logic operations such as OR, TRANSFER, INH, NOT, and YES logic gates were achieved by using biothiols and fluorescence signal patterns as the multiple inputs and outputs, respectively.

The mitochondria-targeted turn-on fluorescent probe (Mito-FMP) based on a benzoxadiazole platform was developed for detection of malondialdehyde (MDA). Mito-FMP performed with large enhancement of the optical signal (774-fold) in response to MDA in an aqueous system and has the capability of monitoring endogenous MDA in HeLa cells and onion tissues.

A H2O2-responsive fluorescent chemosensor (CNBE) with a ratiometric emission signal was elaborately designed and synthesized. The ratio signal of the chemosensor was manipulated by an interplaying ICT-activated FRET mechanism. The ratiometric fluorescence imaging was successfully applied to detect H2O2 using CNBE in living cells and zebrafish.