A Multivalent Targeting Strategy for Developing Reactive Oxygen Species‐Activated Tumor‐Seeking Probe to Guide Precise Surgical Resection

Abstract Optical molecular imaging with near‐infrared (NIR) dyes provides a promising strategy for the visual detection of tumor tissues. However, conventional molecular probes often suffer from poor targeting specificity and low signal contrast during tumor detection. In this work, we developed a reactive oxygen species (ROS)‐activated tumor‐seeking probe (termed MB‐PEG‐Bio2 ) based on a multivalent biotin‐targeting strategy. A bivalent biotin‐targeting ligand was bonded to an ROS‐responsive molecular scaffold to maximize receptor–ligand interactions, resulting in significantly stronger tumor affinity and selectivity of the probe. The optical signal of the proposed probe was initially masked and could be selectively activated by overexpressed ROS levels in the tumor microenvironment. This activation led to the specific release of the clinical dye methylene blue (MB), offering high‐contrast NIR fluorescence and photoacoustic imaging signals for precise tumor visualization. MB‐PEG‐Bio2 demonstrated strong tumor selectivity, enabling the detection of tumors in different mice models, including accurate identification of small tumors (∼3 mm). Consequently, the probe could delineate tumor boundaries during fluorescence‐guided surgery, significantly reducing postoperative tumor recurrence.