Analysis of trypsin activity at β-casein layers formed on hydrophobic surfaces using a multiharmonic acoustic method

Proteolysis of milk proteins, such as caseins, caused by milk proteases, can change the organoleptic and nutritional characteristics of milk, and therefore it is essential to monitor this enzymatic activity. We used trypsin as a model protease because of its role as a biomarker for pancreatitis. The aim of this work was to demonstrate the detection of proteolysis of beta-casein by trypsin using a multiharmonic quartz crystal microbalance (QCM) biosensor. The beta-casein layer was deposited from a 0.1 mg mL(-1) solution on a hydrophobic surface consisting of a self-assembled monolayer of 1-dodecanethiol on the gold electrode of the QCM. The addition of an increasing concentration of trypsin leads to the removal of the casein layer due to proteolysis, and correlates with an increase in the resonant frequency of the QCM. We investigated the effect of trypsin concentrations (0.3-20 nM) on the kinetics of the proteolysis of beta-casein and demonstrated that the frequency increase is proportional to the protease concentration. Consequently, an inverse Michaelis-Menten model was used to estimate the Michaelis-Menten constant (K-M = 0.38 +/- 0.02 nM) and the limit of detection (LOD = 0.16 +/- 0.02 nM). The thickness, mass and viscoelastic properties of the protein adlayer after its formation and following the proteolytic cleavage were evaluated by means of multi-harmonic analysis. We found that beta-casein is preferably adsorbed on the hydrophobic surfaces as an asymmetrical double layer, of which the innermost layer was found to be denser and thinner (about 2.37 nm) and the outermost layer was found to be less tightly bound and thicker (about 3.5 nm).

Automated summary

The study demonstrated the detection of proteolysis of beta-casein by trypsin using a multiharmonic quartz crystal microbalance (QCM) biosensor. Increasing trypsin concentration led to removal of the casein layer, correlating with an increase in QCM resonant frequency. The study also investigated the kinetics of beta-casein proteolysis by trypsin and estimated the Michaelis-Menten constant and the limit of detection using an inverse Michaelis-Menten model.