Enzymatic Self-Immobilization Enhances Chemiluminescence for Selective Tumor Imaging and Image-Guided Surgery

Enzyme-activated chemiluminescence is emerging as a promising bioimaging modality, offering exceptional specificity, minimal background, and high sensitivity. To expand this paradigm for in vivo bioapplications, considerable efforts have been devoted to exploring effective chemiluminophores that exhibit high brightness under physiological conditions. However, only a limited number of chemiluminophores have been demonstrated to track analytes in living cells and cancerous tissues due to their insufficient light emission and low water solubility. Herein, we report an enzyme-triggered self-immobilizing strategy to enhance chemiluminescence for rapid tumor imaging and image-guided tumor surgery. The probe contains an ortho-fluoromethyl motif between the galactose enzymatic recognition site and the adamantylidene-dioxetane chemiluminescent unit. Upon β-galactosidase activation, the self-immolation cleavage of the fluoride group generates a highly reactive ortho-quinone methide species, which attaches to nucleophilic sites on proteins, including human serum albumin. This reaction results in an 8900-fold chemiluminescence enhancement, along with a signal-to-noise ratio exceeding 3220. The probe demonstrates a rapid and selective chemiluminescence increase in HepG2 liver tumor cells that overexpress β-galactosidase. The injection of this probe into HepG2 xenograft tumors activates chemiluminescence immediately with high brightness and long lifetime. Notably, chemiluminescence-guided surgical removal of orthotopic HepG2 liver tumors in mice has been demonstrated. This work presents an efficient enzymatic self-immobilizing scaffold with enhanced turn-on chemiluminescence for both in vitro and in vivo imaging of living cancer cells and malignant tumors. It provides a versatile chemiluminescent staining agent for image-guided tumor surgery and opens new avenues for biological sensing and therapeutic applications.