Simple and sensitive fluorescence detection of trypsin with Cu²+-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 Cu2+ 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.