A Biotin-Guided Near-Infrared Fluorescent Probe for Nitroreductase Activated Tumor Imaging

Abstract Tumor hypoxia-associated nitroreductase (NTR) is a promising biomarker for cancer diagnosis, yet conventional probes often lack active tumor targeting and sustained retention. Herein, we developed a biotin-functionalized near-infrared fluorescent probe, DHX-Bio-NO2, for dual-recognition tumor imaging via biotin receptor recognition and NTR activation. The probe shows a 48-fold fluorescence turn-on at 715 nm upon NTR reduction, with a detection limit of 12.8 ng/mL, excellent selectivity over interferents, and stable performance at pH 3−9. Cellular imaging studies confirm that biotin mediates active uptake, while hypoxia-induced NTR upregulation triggers strong fluorescence, which is suppressed by the inhibitor dicoumarol. Probe DHX-Bio-NO2 also can clearly distinguish cancer cells (HeLa, A549, HepG2) from normal BEAS-2B cells. In vivo, intravenous injection into 4T1 tumor-bearing mice produces bright, tumor-specific fluorescence that persists for 24 h, whereas a biotin-free control shows weak and rapidly fading signals. Ex vivo imaging verifies predominant accumulation in tumor tissues with minimal organ background. These results demonstrate that biotin modification enhances both tumor accumulation and retention, enabling sensitive, long-lasting NTR imaging. The probe offers a reliable tool for real-time NTR monitoring with clear advantages in sensitivity, selectivity, and translational feasibility.