Proximity-Catalyzed In Situ Anchoring Strategy for Fluorescence-Guided Precise Delineation of Minimal Residual...

Minimal residual disease (MRD) after breast cancer surgery frequently triggers tumor recurrence and metastasis, creating an urgent unmet clinical need for accurate intraoperative MRD visualization. Conventional approaches, such as intraoperative histopathology and preoperative imaging, fail to provide adequate real-time surgical guidance. Their utility is primarily hindered by protracted procedures, insufficient spatial resolution, and anatomical shifts between preoperative scans and the intraoperative environment. To overcome these limitations, we developed a two-probe in situ catalytic anchoring system that enables precise MRD visualization through cooperative targeting and localized signal amplification. Using breast cancer as a representative disease model and HER2 as a prototypical membrane biomarker for proof-of-concept validation, this strategy establishes a generalizable framework for tumor-specific surface labeling. The system employs a pretargeted catalytic probe ("molecular hook") composed of a HER2 aptamer fused to a peroxidase-mimicking DNAzyme core. It binds selectively to tumor cells and generates reactive oxygen species (ROS) in a spatially restricted region. A responsive optical probe ("molecular bait") is administered afterward. It contains a tyramine moiety for covalent anchoring and a ROS-activatable fluorophore for fluorescence turn-on. Upon catalytic activation, the two components operate through orthogonal yet coordinated chemistries: the tyramine group undergoes radical-mediated cross-linking with membrane proteins to ensure localized deposition, while the fluorophore is specifically activated by ROS to produce confined signal output. Decoupling tumor targeting and proximity labeling into independent functional modules, this design overcomes the stoichiometric constraints that usually restrict proximity labeling efficiency, thereby enabling multisite deposition and catalytic signal amplification. Validation in coculture systems and murine tumor models demonstrated a cancer-to-normal signal ratio of 11.6 in mixed cell populations and a tumor-to-normal signal ratio of 7.4 for accurate surgical margin delineation. Owing to its modular targeting design, this catalytic anchoring platform can be readily adapted to diverse membrane biomarkers and tumor types, providing a versatile and clinically compatible solution for intraoperative MRD detection and advancing the technological paradigm of precision oncologic surgery.