An In Situ-Generated Near-Infrared Chemiluminescent Nanoprobe for High-Contrast Lymph Node Mapping and Tumor-Guided Surgery.

Chemiluminescent nanoprobes have attracted increasing attention in bioimaging owing to their excitation-free emission and high signal-to-background ratio. However, most reported self-sustaining chemiluminescent nanosystems rely on strong photosensitizers to generate large amounts of reactive oxygen species to initiate the emission process. Although this strategy may result in deep tissue penetration, the excessive ROS accumulation could raise potential phototoxicity concerns. In this work, we designed a bioimaging probe in which the ROS generation, ROS trapping, and near-infrared (760 nm) chemiluminescent emission units are integrated within one molecular framework. Unlike energy-transfer-based systems, our halogen-engineered probes enable direct NIR chemiluminescent emission at the single-molecule level. Halogen engineering modulates both the frontier molecular orbitals and excited-state properties. While the adjusted HOMO level contributes to the reactivity toward 1O2, the dominant enhancement arises from heavy-atom-induced effects, including strengthened spin-orbit coupling and promoted intersystem crossing, which facilitate efficient triplet-state formation and ultimately boost the chemiluminescent process. This design ensures high imaging sensitivity while minimizing excessive ROS-induced phototoxicity, aligning better with biological detection needs. Encapsulation using mPEG-DSPE2000 materials further improved the probe's in vivo stability and brightness, enabling clear visualization of lymph nodes and tumor-guided surgery. Overall, this study proposes a single-molecule strategy for direct NIR chemiluminescent emission that combines high safety with broad applicability, offering a promising approach for developing low-toxicity, high-reliability chemiluminescent imaging platforms.