Dual‐Targeted Fluorogenic Probes for Precise In Vivo Imaging: Design Principles, Activation Mechanisms, and Applications†

Comprehensive Summary Fluorogenic probes with "off‐on" fluorescence signals have emerged as powerful tools for biosensing and bioimaging of biomolecules in living systems. Conventional single‐target probes, however, often suffer from false‐positive signals due to non‐specific activation in non‐target tissues or the diffusion of activated fluorescent products. To address these limitations, dual‐targeted fluorogenic probes (DTFPs) have been developed, which simultaneously target two biomarkers to enhance detection specificity and minimize false‐positive outcomes. DTFPs are designed to activate or retain fluorescence only when both biomarkers are present within a targeted region, enabling precise in vivo imaging of pathological conditions such as tumors and inflammation. This review highlights recent advances in DTFP development, focusing on their design principles, activation mechanisms, and applications in biosensing and bioimaging. We also discuss current challenges and future directions for DTFP research, aiming to inspire the design of next‐generation probes with improved accuracy and specificity. By providing a comprehensive overview of DTFPs, this review seeks to advance their potential for transformative applications in biomedical imaging and diagnostics. Key Scientists Dual‐activated fluorogenic probes have emerged as powerful tools in molecular imaging and precision diagnostics, with significant advancements across several subfields driven by key contributors. In the area of receptor‐targeting and biomarker‐activated probes, Jianghong Rao, Deju Ye, Mingyuan Gao, Zijuan Hai, and Sarah E. Bohndiek have developed highly selective and target‐specific systems, advancing the application of molecular probes in complex pathological environments. For combination‐activated probes responsive to two biomarkers, Tony James, Quli Fan, Yuezhong Xian, Zhihong Liu, Matthew Bogyo have explored multi‐responsive strategies that enhance diagnostic specificity and signal‐to‐noise ratio. In the domain of tandemly activated probes, Junrong Zheng, Xiaobing Zhang Caixia Yin, Wenying Zhong, and Kanyi Pu have focused on hierarchical activation mechanisms and signal integration, significantly improving imaging precision and biological adaptability. Collectively, these scientists have driven the evolution of dual‐activated fluorogenic probes from molecular design toward clinical potential.