H ₂ O ₂ /Viscosity Tandem-Locked Fluorescent Probes Based on an In Situ Fluorophore Synthesis Strategy for Colitis Imaging and Diagnosis

Unimolecular dual-locked activatable fluorescent probes have emerged as promising tools for biomedical applications. However, the majority of dual-locked probes are designed by introducing biomarker-recognition moieties as fluorescence quenchers into classical fluorophores, a strategy that often suffers from persistent background fluorescence and nonspecific activation, ultimately resulting in limited signal activation and increased risk of false positives. Herein, to address this issue, we report tandem-locked activatable fluorescent probe NPB based on an in situ fluorophore synthesis strategy for colitis imaging and diagnosis. Probe NPB exhibited a remarkable fluorescence enhancement (420.0-fold) upon activation by H 2 O 2 /viscosity associated with colitis, wherein H 2 O 2 triggers the fluorogenic reaction to form dye NPH, and the subsequently elevated viscosity markedly enhances its fluorescence. Moreover, the probe enables in situ monitoring of pathological oxidative stress and microviscosity elevation with high specificity and good biocompatibility in living cells. More importantly, near-infrared (NIR) fluorescence imaging with probe NPB allows for the accurate visualization and diagnosis of dextran sulfate sodium (DSS)-induced acute colitis in both zebrafish and mouse models.This work not only develops a H 2 O 2 /viscosity tandem-locked fluorescent probe for colitis imaging and diagnosis but also provides a valuable in situ fluorophore synthesis strategy for the rational design of next-generation dual-locked probes.