Multispectral Nanoparticle Tracking Analysis for the Real-Time and Label-Free Characterization of Amyloid-β Self-Assembly In Vitro.

Abstract

The deposition of amyloid β (Aβ) plaques and fibrils in the brain parenchyma is a hallmark of Alzheimer's disease (AD), but a mechanistic understanding of the role Aβ plays in AD has remained unclear. One important reason could be the limitations of current tools to size and count Aβ fibrils in real time. Conventional techniques from molecular biology largely use ensemble averaging; some microscopy analyses have been reported but suffer from low throughput. Nanoparticle tracking analysis is an alternative approach developed in the past decade for sizing and counting particles according to their Brownian motion; however, it is limited in sensitivity to polydisperse solutions because it uses only one laser. More recently, multispectral nanoparticle tracking analysis (MNTA) was introduced to address this limitation; it uses three visible wavelengths to quantitate heterogeneous particle distributions. Here, we used MNTA as a label-free technique to characterize the in vitro kinetics of Aβ1-42 aggregation by measuring the size distributions of aggregates during self-assembly. Our results show that this technology can monitor the aggregation of 106-108 particles/mL with a temporal resolution between 15 and 30 min. We corroborated this method with the fluorescent Thioflavin-T assay and transmission electron microscopy (TEM), showing good agreement between the techniques (Pearson's r = 0.821, P < 0.0001). We also used fluorescent gating to examine the effect of ThT on the aggregate size distribution. Finally, the biological relevance was demonstrated via fibril modulation in the presence of a polyphenolic Aβ disruptor. In summary, this approach measures Aβ assembly similar to ensemble-type measurements but with per-fibril resolution.