A Cascade-Responsive AND-Logic-Activatable Nanoprobe for Intraoperative Fluorescence Imaging of Colorectal Cancer

Precise intraoperative fluorescence (FL) imaging is crucial for improving cancer surgical outcomes. However, currently approved FL probes are consistently “always-on” fluorescent and often accumulate nonspecifically, which leads to suboptimal tumor-to-background ratios (TBR) and hampers an accurate margin assessment. We present duNP-DA, a cascade-responsive AND-logic activatable near-infrared (NIR) FL nanoprobe designed for precise intraoperative imaging of colorectal cancer (CRC). duNP-DA leverages the acidic tumor microenvironment (TME) to trigger a charge inversion and utilizes a unique AND-logic activation mechanism mediated by endogenous hydrogen sulfide (H2S) and lysosomal pH, resulting in significant tumoral uptake and a remarkable NIR FL enhancement at 800 nm (>100-fold). Notably, duNP-DA holds fast secondary reaction kinetics (k2 = 6050 ± 43 M–1 s–1) toward H2S following preincubation at lysosomal pH, enabling rapid and specific activation within CRC cells. Postintravenous injection, duNP-DA effectively minimizes background signals, accumulating predominantly in primary and peritoneal metastatic colorectal tumors and achieving a TBR of up to 8.2. Noninvasive NIR FL imaging delineates lesions as small as 2 mm, aiding in the surgical resection of deep-seated peritoneal metastases that are otherwise undetectable through direct visual inspection. In clinical evaluations, duNP-DA effectively delineates primary, occult lymph node metastases and omental metastatic lesions. Furthermore, duNP-DA demonstrates 100% diagnostic sensitivity and specificity in distinguishing tumors from normal tissues, with FL intensity showing strong correlation with artificial intelligent-quantified tumor cellularity (Pearson’s r = 0.9103). Our work establishes duNP-DA as a promising translational platform for NIR FL-guided CRC surgery and intraoperative margin assessment.