The biological significance of protease for regulating various cellular events accelerates the in-depth investigation into the structural/functional relationships of proteases. To this end, smart and facile methods for the rapid and efficient analysis of dynamic interactions between enzymes, substrates and inhibitors especially with the respect to the structure-sensitive inhibition kinetics are extensively focused. Among these, protease inhibition, which turns off the catalytic activity, plays key roles in the regulation mechanism.
By the integration of quartz crystal microbalance (QCM) and flow injection analysis (FIA) technique, a continuous-flow mass biosensor system was fabricated for on-line analysis of protease inhibition. The interactions between trypsin inhibitor (TI) and three proteases, trypsin, chymotrypsin and pepsin, were chosen as the model system. Benefiting from the label-free and sensitive nature of the QCM sensor, binding kinetics between proteases and inhibitor were facilely monitored in real-time. The flow injection analysis technique provides the advantage to supply the crystal interface with always-fresh analytes and thus may better mimic the enzymatic process in vivo. It is also interesting to find that the dynamic sensing processes are responsive to the conformation features of different proteases. The association/dissociation kinetics can sensitively reflect subtle differences in the chemical properties and the spatial distribution of structural elements in the active binding sites. Owing to such conformation-dependent dynamic sensing, the FIA-QCM biosensor is potential in the structural/functional analysis of proteases, which is appealing for the design and selection of novel peptide inhibitors for proteases.