β-Galactosidase-activated bioluminescent probe for in vivo tumor imaging†

β-Galactosidase (GAL) is a lysosomal protease implicated in cellular senescence and various malignant cancers. However, bioluminescent probes for specific GAL detection remain scarce, and their applications are largely limited to transgenic models expressing firefly luciferase (fLuc). Herein, we report a bioluminescent probe, , for detecting GAL activity in nontransgenic cells and tumor-bearing mice. exhibited high selectivity toward GAL, with a limit of detection of 8.5 mU mL⁻¹. Combined with fLuc mRNA-loaded lipid nanoparticles, was able to sensitively monitor GAL activity in vitro and in vivo. Based on this strategy, intestinal metastatic tumors were precisely delineated, showing 25.2-fold higher bioluminescence intensity in non-transgenic tumor-bearing mice.