A Viscoelasticity Evaluating System of Amyloid-β Peptide Fibrillation on Electrodeless Quartz Crystal Microbalance Biosensor

Alzheimer's disease, one of the most devastating dementia, is an elderly suffered illness which involves neurodegenerative disorder. The disease pathogenesis is recently found that it is related to a structural-changing phenomenon: Amyloid b (Ab) aggregation. In order to clarify the pathogenesis, researchers have made intensive efforts for studying the Ab aggregation both in vitro and in vivo (or clinical). However, most of the previous in vitro studies were held in bulk solutions and which are different from the process in reality, where the aggregations take place on the cell membrane. In the present thesis, the aim is to develop an in vitro Ab aggregation detection system on a surface, which more resembles to the in vivo Ab aggregation, and to analyze the aggregation process from mechanical viewpoint. The measurement is based on an electrodeless quartz-crystal-microbalance (QCM) biosensor. Here, Ab aggregations is taken place on surfaces of the quartz crystal, which is similar to those of the actual Alzheimer's disease. Since sizes of amorphous-like Ab aggregates were reported to be about 100 nm or less, a nanoscale sensing QCM should be established. To this end, a high frequency quartz crystal, whose fundamental resonant frequency is 58 MHz, is used and the resonance overtones up to 7th modes is measured simultaneously. The quartz crystal is sealed in a package which is equipped with a small chamber to hold the quartz lightly in preventing energy loss by attachment. By connecting a microchannel embedded in the package, Ab monomer solution can be introduced to the quartz crystal. Consequently, the aggregation can be monitored via acoustic overtones of QCM. Present experimental studies revealed that the overtone frequencies are shifted as progress of the Ab aggregation, and that the aggregation process is classified into three stages. First is the binding stage where the frequencies have dropped as soon as the solution is injected. Second is the lag stage where frequencies are relatively stable for a long time after the binding stage. Finally, we have noticed the frequency ramp stage, where increasing in frequencies are found after the lag stage at around 20 hours. In order to analyze the experimental results obtained by QCM, a two-dimensional viscoelastic model is introduced. It is consisted of three acoustic layers including quartz crystal, viscoelastic Ab and surrounding liquid. This model relates the acoustic overtone frequencies to mechanical properties of Ab aggregates on the quartz surface, i.e., shear stiffness, viscosity, and thickness. This analysis revealed that the mechanical properties change with the stages in frequency response. In the binding stage, the three properties, shear stiffness, viscosity, and thickness, increased simultaneously due to the growth of the viscoelastic layer of Ab. In the lag stage, the thickness decreases while other properties remained stable. It indicates that condensation of the monomers to Ab aggregates are taken place on the QCM surface. In the frequency ramp stage, the stiffness increases with decreasing of thickness. In this stage, we have found the transformation of amorphous-like Ab aggregates into the crystal-like fibrils. In addition to the above mentioned QCM measurements, we conducted another observation of morphology of Ab aggregates formed on the QCM by atomic force microscopy (AFM). In this experiment, QCM monitoring are interrupted in a preassigned stage and the surface of QCM is observed by AFM to clarify the relation between the mechanical properties and morphologies of Ab. After the binding stage, small Ab aggregates are confirmed on the surface of QCM. The aggregates are consisting of Ab monomers and which cause the increasing in the three properties. After the lag stage, we confirmed the growth of the aggregates due to condensation and finally, after the frequency ramp stage, the rod-like fibrils, i.e., fibrillation is verified. From the experimental and theoretical analysis, we conclude that the present viscoelasticity evaluation system can detect the sequential structural change of Ab aggregates on QCM surfaces. Consequently, the thesis revealed the mechanical properties of Ab in entire aggregation process. The system has promising usage in further applications such as discovering inhibitory medicines in drug development.