Title: Monitoring of Alzheimer's Amyloid-β Peptide Aggregation via Fluorescence Correlation Spectroscopy and Total Internal Reflection Microscopy
Abstract: The aggregation mechanisms of amyloid-β peptide are complex. They have been investigated using a variety of different analytical techniques, including fluorescence spectroscopy and fluorescence correlation spectroscopy (A. Tiiman, J. Jarvet, A. Gräslund, and V. Vukojević, Biochemistry 2015 54, 7203-7211 and references therein). The benzothiazole salt Thioflavin T (ThT) fluorescence assay is a standard method for amyloid detection by fluorescence spectroscopy and ThT fluorescence could also be used for total internal reflection microscopy. It is known that amyloidogenic aggregates enriched with pleated β-sheet secondary structure readily bind ThT and significantly alter its spectral properties, shifting the absorption spectrum towards longer wavelengths and significantly increasing the fluorescence quantum yield. ThT acts as a smart dye becoming fluorescent only when bound to amyloid thereby strongly reducing the fluorescence background. Fluorescence correlation spectroscopy (FCS) is a time-resolved spectroscopic technique that can measure the concentration and size of fluorescently labeled particles. The method will benefit from the fact that many ThT molecules bind to a single amyloid aggregate. Total internal reflection microscopy (TIRF) combined with ThT fluorescence was used to follow the elongation of fibrils in time. We identified aggregates of different sizes with molecular weight from 260 kDa to more than 1 × 106 kDa and revealed the hitherto unobserved kinetic turnover of intermediates during amyloid-β aggregation.