Dual-Lock System\nfor High Sensitivity and Selectivity\nin Redox Enzyme Activation and Imaging

Reductase expression is a potential indicator of cellular\npathology.\nSingle-detection systems for reductases have been developed, however,\nthe development of dual-detection systems remain largely unexplored.\nWe rationally designed a dual-lock fluorescent probe that exhibited\na high signal-to-noise ratio with a fluorescence Off-On response exclusively\nfor the simultaneous activity of two reductases, NTR and hNQO1, which\nare overexpressed in cancer hypoxia. The system comprised a naphthalimide\nfluorophore with dual-lock control mediated by PET and ICT, a trimethyl-locked\nquinone group sensitive to hNQO1, and a nitrobenzyl carbamate group\nsensitive to NTR. This study employed a hypoxia model in HeLa cells\nto demonstrate that our developed dual-lock system detected hypoxia\nmore effectively than single-detection systems. Moreover, it enabled\nnoninvasive real-time monitoring of hypoxia in zebrafish embryos.\nConsequently, the dual-lock fluorescent probe, which strategically\nprovides a fluorescence response only when both NTR and NQO1 are active,\noffers a novel diagnostic platform for both in vitro and in vivo applications,\neffectively detecting hypoxia and monitoring various pathological\nstates.