Application of Multiharmonic QCM-D for Detection of Plasmin at Hydrophobic Surfaces Modified by beta-Casein

Plasmin protease plays an important role in many processes in living systems, including milk. Monitoring plasmin activity is important for control of the nutritional quality of milk and other dairy products. We designed a biosensor to detect the proteolytic activity of plasmin, using multiharmonic quartz crystal microbalance with dissipation (QCM-D). The beta-casein immobilized on the hydrophobic surface of 1-dodecanethiol on the AT-cut quartz crystal was used to monitor plasmin activity. We demonstrated detection of plasmin in a concentration range of 0.1-20 nM, with the limit of detection about 0.13 +/- 0.01 nM. The analysis of viscoelastic properties of the beta-casein layer showed rapid changes of shear elasticity modulus, y, and coefficient of viscosity, eta, at plasmin sub-nanomolar concentrations, followed by modest changes at nanomolar concentrations, indicating multilayer architecture beta-casein. A comparative analysis of viscoelastic properties of beta-casein layers following plasmin and trypsin cleavage showed that the higher effect of trypsin was due to larger potential cleavage sites of beta-casein.