Cyanine-scaffold Fluorogenic Probes for Visual Detection of Nitroreductase in Living Bacteria

Abstract

We developed a novel nitro-containing fluorescent probe, Cy5-NO2, for the real-time monitoring of bacterial nitroreductase (NTR) activity. The synthesis of Cy5-NO2 was highly efficient, yielding a stable compound confirmed by X-ray diffraction and spectroscopic methods. This "turn-on" probe exhibits no fluorescence in its native state but shows a significant 30-fold fluorescence enhancement at 620 nm upon reduction by NTR, with a detection limit of 10 ng/mL. Time-dependent density functional theory (TDDFT) calculations elucidated the mechanism underlying this fluorescence change. The probe demonstrated high specificity towards NTR and optimal performance under physiological conditions. Importantly, Cy5-NO2 successfully detected and imaged live bacterial cells such as E. coli and S. aureus using confocal laser scanning microscopy (CLSM), confirming its robust response to bacterial NTR activity. These findings highlight the potential of Cy5-NO2 as a promising tool for sensitive and specific bacterial detection and imaging, advancing microbiological research and clinical diagnostics.