Simple and sensitive fluorescence detection of trypsin with Cu-Bovine serum albumin complex as a peroxidase mimic.

Trypsin is a serine protease playing a key role in regulating pancreatic exocrine function and can be applied as a marker for the diagnosis of pancreatitis. In this work, a convenient and sensitive fluorescent assay was developed toward trypsin. Hydrogen peroxide slowly oxidized a non-fluorescent o-phenylenediamine (OPD) into a fluorescent product 2,3-diaminophenothiazine (DAP) under the catalytic from copper ions. After the introduction of bovine serum albumin (BSA), the combination of BSA with copper ions formed a peroxidase mimic and significantly accelerated the reaction rate. As an efficient protease, trypsin cleaved the lysine and arginine residues in BSA. This destroyed the binding between Cu and BSA, and brought in a reduction of the catalytic effect. The accompanying decrease in fluorescence provided a response to trypsin in the range of 0.01-600 ng/mL, with a detection limit of 0.007 ng/mL. The scheme had a good selectivity and was successfully applied to the detection of real samples.