Proteases play important role in various biological processes as well as in the dairy industry. In this work we were focused on detection of chymotrypsin which is an important protease in human digestion.
We performed comparative analysis of detection chymotrypsin activity by two methods - optical and acoustical. In the first case we used gold nanoparticles (AuNPs), modified with β-casein. We characterized the changes in absorption spectra after modification by β-casein and 1-mercaptohexanol (MCH). This modification protected AuNPs from aggregation. Addition of chymotrypsin resulted in cleavage of β-casein, which caused changes in absorption spectra due to formation of AuNPs aggregates. The absorption spectra were measured every 15 minutes up to one hour after chymotrypsin addition, which allowed us to monitor the kinetics of the β-casein cleavage. By measuring the absorption maximum, change of its position and change in absorbance, it was possible to construct calibration curve and to determine limit of detection (LOD). This value was 0.21 nM. In the second case we detected chymotrypsin by means of the acoustic shear mode method (TSM). β-casein was immobilized on the surface of the TSM transducer (AT cut piezo-crystal). Addition of chymotrypsin resulted in an increase of resonance frequency, f, and a decrease of motional resistance, Rm, indicating cleavage of short peptide fragments from β-casein. In this case the LOD = 3.1 nM. Thus, both methods represent effective tool for study of the chymotrypsin activity. The assay based on AuNPs, however, allowed for better sensitivity and easier operation.
Acknowledgement
A portion of this research was conducted at the Center for Nanophase Materials Sciences, project No. CNMS2018-293. This work was funded under European Union’s Horizon 2020 research and innovation program through the Marie Skłodowska-Curie grant agreement No 690898 and by Science Agency VEGA, project No. 1/0419/20.
