Label-Free Characterization of Amyloids and Alpha-SynucleinPolymorphs by Exploiting Their Intrinsic Fluorescence Property br

Conventional in vitro aggregation assays ofteninvolve tagging with extrinsicfluorophores, which can interferewith aggregation. We propose the use of intrinsic amyloidfluorescence lifetime probed using two-photon excitation andrepresented by model-free phasor plots as a label-free assay tocharacterize the amyloid structure. Intrinsic amyloidfluorescencearises from the structured packing of beta-sheets in amyloids and isindependent of aromatic-basedfluorescence. We show thatdifferent amyloids [i.e.,alpha-Synuclein (alpha S),beta-Lactoglobulin(beta LG), and TasA] and different polymorphic populations of alpha S(induced by aggregation in salt-free and salt buffers mimicking theintra-/extracellular environments) can be differentiated by their uniquefluorescence lifetimes. Moreover, we observe thatdisaggregation of the preformedfibrils of alpha S and beta LG leads to increasedfluorescence lifetimes, distinct from those of theirfibrillarcounterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latterof which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.