Sensitivity Increase of Enzimatic Qcm Sensor for Trypsin Activity Determination by Nanoparicles Aplication

Introduction. Trypsin (EC 3.4.21.4) is a protein degrading enzyme of the group of the serine proteases having many applications: in food and beverage industries for protein hydrolysates production, in cold stabilization of beer etc. It is employed in biochemistry for protein identification through peptide sequencing techniques [1]. The activity of trypsin serves as a reliable diagnostic test of pancreatic function and its alteration [2]. The industrial, biotechnological, and biochemical importance of trypsin, as well as its clinical significance make important the development of methods for quantification of its activity. Many methods already have been developed mainly radioimmunoassay-based and spectrophotometric techniques [3]. Methods based on measurement of the change of temperature, pressure [4] and humidity [5] due to the action of trypsin on gelatin films were recently developed as well. Unfortunately the known sensors and methods are very complicated and slow. A new approach for trypsin activity determination based on QCM application allowing significant LOD improvement and sensitivity increase is the subject of this work. The main point of this approach is the application of Ag nanoparticles loaded gelatin, deposited on the QCM crystal surface, serving as trypsin substrate. During the substrate enzymatic degradation by trypsin, the heavier nanoparticles also leave it together with the substrate degradation products, provoking thus a greater mass change, compared with the no loaded substrate. As a result the QCM frequency response depending on the total QCM crystal mass increases, improving thus the LOD and the sensitivity of the trypsin determination.