Tinker-Mill, Claire and Allsop, David and Kolosov, Oleg (2015) Nanoscale imaging and characterisation of Amyloid-β. PhD thesis, Lancaster University.
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Abstract
In this work several novel Scanning Probe Microscopy (SPM) methods have been applied to the study of the amyloid peptide implicated in the pathogenesis of Alzheimer’s disease (AD). Amyloid-β (Aβ) undergoes a hierarchy of aggregation following a structural transition making it an ideal subject of studying with SPM. The application of SPM based techniques to biological samples has become increasingly common place. However, these techniques are not always immediately suitable for imaging delicate samples of proteins and adaptions must be made before imaging can be considered successful. AD is the most common form of dementia worldwide, and a growing concern for health authorities. As a result it has attracted the attention of a wide range of disciplines. There has been much work conducted which combines the main pathogenic peptide, Aβ , with Atomic Force Microscopy (AFM) in order to elucidate more about its aggregation behaviour, however these techniques offer little more than structural comments, with only the most advanced forms of cryo-Electron Microscopy (EM) providing more details on the nanoscale. Presented here is a method for reliably and robustly producing samples of Aβ by capturing them at various stages of aggregation, as well as the results of subsequent imaging by various methods of AFM. Each of the AFM techniques studied provides additional “added value” to the data which can typically be collected by AFM; either nanomechanical, elastic, thermal or spectroscopical. By imaging samples of Aβ with Ultrasonic Force Microscopy, a detailed substructure to the morphology could be seen, which correlates well with the most advanced cryo-EM work. In addition this technique was ideal for detecting the most toxic from of Aβ, early aggregates, in a sensitive and non-destructive fashion robustly differentiating them from the underlying layer of another peptide (poly-L-Lysine) that was designed to reliably capture the Aβ aggregates. Early work investigating the potential for combining an established method of thermal AFM with a mid-IR laser system also shows promise for detecting the response of the protein. It was also the focus of this work to study the aggregation of Aβ using Dynamic Light Scattering (DLS), in order to confirm whether the technique could identify differences between populations throughout the aggregation process. This was applied in conjunction with potential therapeutics which target the early aggregates to prevent their accumulation, as well as block formation of fibrils. Ultimately this work aims to shows with care to the initial protocols used, physical techniques such as AFM and DLS can be added to the existing methods of monitoring aggregation. Synergistic use of these techniques can generate a clearer overall picture of the effect of metal ions/developing therapeutics on Aβ aggregation and provide more detail than classical biological techniques alone.