Ultrasensitive Fluorescent Detection of Urinary Trypsin for Early Pancreatic Disease Diagnosis: Toward...

Early detection of pancreatic disease requires non-invasive, highly sensitive biomarker detection. Here, we report a fluorescence-based method for rapid, ultrasensitive detection of urinary trypsin without sample pretreatment. A π-extended pyrene fluorescent probe functionalized with four carboxylate groups was designed to facilitate selective interaction and micellar aggregation with protamine, a trypsin-cleavable substrate. Protamine-induced aggregation results in amplified fluorescence quenching, which is efficiently reversed by trypsin-mediated proteolysis. Trypsin cleavage reduces the arginine-rich positive charge density and length of protamine, disassembling the compound 1-protamine aggregates and recovering monomeric fluorescence, enabling highly sensitive detection. The sensing platform achieved a limit of detection (LOD) of 2.0 ng/mL in 10% human urine, highlighting its strong analytical performance. The developed sensor exhibited excellent selectivity and enabled reliable quantification of trypsin at sub-microgram levels (0.03-0.7 µg/mL) in 10% diluted human urine, making it suitable for early-stage pancreatic disease screening. Its clinical applicability was further validated in the presence of acute pancreatitis (AP)-inducing factors, including aspirin, ibuprofen, dexibuprofen, acetaminophen, 6-mercaptopurine, as well as ethanol and its metabolites (acetaldehyde and acetate). In addition, urine samples were successfully analyzed using a smartphone-integrated fluorescence detection system, highlighting its practicality and clinical potential for early pancreatic disease diagnosis.