Label-free, turn-on fluorescent sensor for trypsin activity assay and inhibitor screening

The development of new detection methods for proteases activity assay is important in clinical diagnostics and drug development. In this work, a simple, label-free, and turn-on fluorescent sensor was fabricated for trypsin, a protease produced in the pancreas. Cytochrome c, a natural substance of trypsin, could be selectively cleaved by trypsin into heme-peptide fragment. The produced heme-peptide fragment exhibited an intensive catalytic role on the H₂O₂-mediated the oxidation of thiamine to form strong fluorescent thiochrome. The fluorescence intensity was closely dependent on the amount of trypsin presented. The procedure allowed the measurement of trypsin over the range of 0.5-20.0μg/mL with a detection limit of 0.125μg/mL. The sensor showed better precision with a relative standard deviation of 1.6% for the measurement of 1.0μg/mL trypsin solution (n=11). This sensing system was applied to screen the inhibitor of trypsin, the IC₅₀ values were calculated to be 12.71ng/mL for the trypsin inhibitor from soybean and 2.0μg/mL for benzamidine hydrochloride, respectively, demonstrating its potential application in drug development and related diseases treatment.