A fluorescent probe for imaging nitroreductase with signal amplification in high-viscosity environments

Herein, we developed a fluorescent probe ENBT for in vitro detection of nitroreductase (NTR) as well as imaging intracellular NTR. ENBT itself is non-fluorescent and it could be catalyzed by NTR to generate a viscosity-sensitive fluorophore EBT. The fluorescence intensity of EBT could be further enhanced in cancer cells with relatively high viscosity due to the inhibition of the twisted intramolecular charge transfer effect. The probe ENBT has a good response to NTR with a detection limit of 36.8 ng mL^-1, and EBT has a good response to viscosity. Furthermore, different concentrations of NTR (0-1.4 μg mL^-1) were used to react with the probe and the reaction systems were subjected to different viscosity solutions, and the fluorescence signals of the products in the viscosity range of 45.86-163.60 cP were increased up to 1.69-fold. ENBT was successfully used to image NTR in cells under different hypoxic conditions as well as in Staphylococcus aureus. Finally, lipopolysaccharide was added to stimulate an increase in cellular viscosity after ENBT was catalyzed by intracellular NTR into EBT, and the fluorescence signals were observed to increase by 1.72-fold. The signal amplification capability gives ENBT higher sensitivity and immunity to interference. Moreover, it has the advantages of mitochondrial targeting, large Stokes shift (190 nm), high selectivity, and can be easily synthesized.