Leucine Aminopeptidase-Responsive Bioluminescent Probe for Point-of-Care Diagnosis of Pancreatic Cancer in Blood and Urine

Early detection of pancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge due to its asymptomatic onset and the lack of noninvasive diagnostic tools. Leucine aminopeptidase (LAP) has emerged as a promising biomarker for pancreatic cancer progression, with significantly elevated levels observed in both blood and urine during tumorigenesis. Herein, we report the rational design and synthesis of two LAP-activatable bioluminescent probes, N-LAP-Luc and O-LAP-Luc, for sensitive detection of LAP activity in blood and urine. Compared to N-LAP-Luc, O-LAP-Luc, incorporating a self-immolative linker, exhibits superior sensitivity (LOD = 8.7 mU/L), excellent selectivity, and 13.5-fold higher luminescence signals. Mechanistic studies revealed that this enhancement originates from both lower probe–enzyme binding energy and brighter luciferin substrate released upon activation. In KPC-Luc cells and PDAC mouse models, O-LAP-Luc enabled highly sensitive imaging of endogenous LAP, with significantly prolonged signal persistence and enhanced signal-to-background ratios. Notably, O-LAP-Luc achieved sensitive monitoring of LAP levels in the urine and blood samples from orthotopic PDAC mice, as well as urine specimens from PDAC patients. Furthermore, we established a portable smartphone-based detection platform that allows visual urine testing within 5 min under 365 nm UV light, with quantitative RGB analysis achieving excellent linear correlation ( R 2 = 0.96). To our knowledge, O-LAP-Luc represents the first bioluminescent probe applied to point-of-care testing of urinary LAP, offering a promising strategy for noninvasive, rapid, and cost-effective diagnosis of PDAC.