Nitroreductase-Activated Fluorescence Probe for Detection of Bacterial Infections

Nitroreductase (NTR), found in various bacteria, plays a crucial role in the detoxification of nitro-containing compounds, making it a valuable target for bacterial detection. In this study, we developed an NTR-responsive fluorescent probe (<b>IND-NO₂</b> ) for the selective detection of NTR activity in microorganisms. In preliminary tests, Escherichia coli NTR (<i>Ec</i>NfsB) was used as a model enzyme, and it effectively converted the probe's nitro group to an amino group in the presence of reduced nicotinamide adenine dinucleotide (NADH), resulting in a significant increase in fluorescence at 564 nm. To confirm the product identity, <b>IND-OH</b>, the reaction was analyzed by fluorescence spectroscopy and high-performance liquid chromatography (HPLC). Kinetic parameters for probe reduction were determined, revealing a limit of detection of 6.21 nM (0.16 μg/mL). The probe demonstrated high specificity for NTR, with the control experiments showing no reduction by various biological reductants. Furthermore, the probe successfully detected NTR activities in the bacteria E. coli TISTR780, Pseudomonas aeruginosa TISTR781, and Staphylococcus aureus TISTR1466, representing ESKAPE pathogens, by fluorescence spectroscopy and the naked eye, demonstrating its potential for real-time bacterial detection.