Precision Screening and Surgical Resection of Pan‐Cancer Using a Tandem‐Locked NIR‐II Fluorescent Probe with Optimized Activation Efficiency

Abstract The development of fluorescent probes for cancer detection and imaging that balance high sensitivity, precision, and broad applicability remains a significant challenge. Although “dual‐locked” probes have been devised to enhance diagnostic accuracy via two biomarkers, most fall short in sensitivity, response time, and generalizability for pan‐cancer use. We address these gaps with ACy‐H‐NTR , a cascaded‐activation, doubly quenched NIR‐II probe. Engineered to respond to hypoxia and acidity—universal tumor hallmarks—it offers fast response, high sensitivity, and specificity for pan‐cancer screening and imaging. In contrast to existing probes, its tandem‐locked design ensures robust activation exclusively within pan‐tumor microenvironments, effectively reducing false positives and delineating precise diagnostic boundaries. Its NIR‐II emission and “activation–retention” mechanism enhance tumor imaging efficacy and effectively tackle issues of rapid clearance and background noise, achieving 4.9‐fold higher tumor‐to‐normal (T/N) ratio than ICG and retaining tumor specificity for over 2 days. In pan‐cancer models, it enabled high‐contrast imaging (T/N ∼7.8) and precise resection with sub‐2 mm margins. Crucially, it differentiates human carcinoma from adjacent tissues with sharp boundaries, confirming clinical potential. By integrating a tandem‐locked and doubly quenched design that simultaneously optimizes activation efficiency, NIR‐II imaging capabilities, and activation‐retention mechanisms, this probe overcomes current limitations to enable precise pan‐cancer identification and surgical navigation.