Gold nanoparticles-enhanced biosensors for study of protease activity

This work is focused on the preparation and improvement of biosensors for monitoring protease activity. Proteases play an important role in various biological processes as well as in the dairy industry. Milk proteases like plasmin or trypsin play important role in shelf-life of milk where the inactivation of these proteases is essential for improving the shelf-life of milk. In the case of cheese products, however, the activity of plasmin is essential for the correct manufacturing of curd [1]. In dairy industry there is still a lack of universal method for determination of protease activity in milk that would be fast, cheap and sensitive [2].

In this work, we focused on the possibility of spectrophotometric methods for monitoring the activity of one of the milk proteases: trypsin. To monitor the activity we used gold nanoparticles, which we modified with milk protein: β-casein. We characterized the change in absorption spectra after casein modification. Subsequently, it was necessary to further modify these nanoparticles with 1-mercaptohexanol (MCH), which made it possible to measure the aggregation of these gold nanoparticles by changing their absorption spectra after adding different concentrations of trypsin [3]. We measured absorption spectra every 15 minutes up to one hour after trypsin addition. By monitoring the absorption maximum, its change of position and change in absorbance, we made a calibration curve and then calculated the detection limit (LOD = 0,05 nM).

We also focused on chymotrypsin detection using the acoustic shear method (TSM). In this case, β-casein was immobilized on the surface of the TSM transducer - piezo-crystal. The addition of chymotrypsin resulted in an increase in resonance frequency, f, and a decrease in dynamic resistance, R, indicating cleavage of short peptide fragments from β-casein and demonstrates protease activity. We demonstrated that the modification of the substrate, β-casein, with gold nanoparticles increased the sensitivity of detection at least twice. Both methods could represent a novel approach for detection of proteases in dairy industry.