Detecting and Tracking β‑Amyloid Oligomeric\nForms and Dynamics In Vitro by a High-Sensitivity Fluorescent-Based\nAssay

Aggregation of β-amyloid\nprotein is a hallmark pathology\nof the neurodegenerative disorder Alzheimer’s disease and proceeds\nfrom monomers to insoluble misfolded fibril forms via soluble and\nhighly toxic oligomeric intermediates. Given the dual feature of being\nthe most toxic form of the Aβ aggregate proteome and an early\nmarker of pathogenesis, there is a need for sensitive methods that\ncan be used to detect Aβ oligomers and investigate the dynamics\nof aggregation. Herein, we describe a method based on the application\nof an oligomer-sensitive fluorescent chemical probe pTP-TFE combined\nwith the use of a QIAD (Quantitative determination of Interference\nwith Aβ Aggregate Size Distribution) assay to correctly identify\nAβ oligomers in high sensitivity. pTP-TFE was evaluated and\ncompared to thioflavin T and pFTAA, the two most widely used amyloid\nfibril dyes, and shown to be the only probe capable of detecting significant\ndifferences across all oligomeric species of β-amyloid. Furthermore,\nby observing changes in pTP-TFE fluorescence emission over time, we\ncould track the dynamics of oligomer populations and thereby obtain\nkinetic information on the Aβ42 dynamic aggregation model. Therefore,\nwe have established a highly sensitive, readily available, and simple\nmethod for studying β-amyloid protein aggregation dynamics.