Enzyme-Activated Dual-Locked Probes for Detecting Nitroreductase and Carboxylesterase

Lung cancer, the leading cause of global cancer mortality, demands precise diagnostic tools that target the tumor microenvironment. Hypoxia-driven nitroreductase (NTR) and carboxylesterase (CarE) overexpression serve as promising biomarkers in lung cancer, yet fluorescent probes that can simultaneously detect these markers are not available. Here, we present a series of fluorescein-based probes, with Flu-NitroCa-4 emerging as the optimal dual-locked probe. Flu-NitroCa-4 exhibits a 48-fold fluorescence enhancement at 515 nm upon activation by CarE and NTR, with rapid response kinetics (<30 min) and low detection limits (LOD = 0.18 μg/mL for NTR and 0.032 U/mL for CarE). In vitro, Flu-NitroCa-4 accurately visualizes the activity of CarE and NTR in both A549 cells and patient-derived lung cancer organoids under hypoxic conditions, as validated by inhibitor studies; in vivo, it delineates tumors in subcutaneous and orthotopic murine lung tumors models. Additionally, Flu-NitroCa-4 can be effectively utilized to distinguish human lung cancer samples from adjacent tissues via NTR/CarE-triggered fluorescence. The dual-locked fluorescent probe offers a rapid, low-radiation, and high-sensitivity tool for intraoperative tumor margin mapping and clinical diagnosis.