A new GFP chromophore analogue for detection of viscosity and cysteine with AND logic-gate-based fluorescence readout

Due to the significant roles of cysteine and viscosity in living cells and related biological processes, the development of visualization tools for in situ detection is essential. Despite the fact that numerous fluorescent probes have been developed for real-time detection of cysteine or viscosity, there are still limited amounts of probes that could be used for dual detection of cysteine and viscosity, which will prevent deep insights into the biological roles of cysteine and viscosity in living cells, particularly their interrelationships. Herein, a new near-infrared (NIR) fluorescent probe (Cys-BTH) was designed and synthesized by conjugating thieno [3,2-b]thiophene-2,5-dicarbaldehyde to the green fluorescent protein (GFP) chromophore hydroxybenzylidene imidazolidinone (HBDI). Due to the intramolecular motion property and selective interaction of the carboxyl group, Cys-BTH exhibited "AND" logic-gate-based sensing performance in the presence of both Cys and high viscosity. Live-cell imaging assays further demonstrated the practical use of Cys-BTH in monitoring biological processes with overexpressed Cys and high viscosity.