Microfluidic self-assembly and fibrillation of amyloid peptides for high-throughput analysis

Abstract

Recently, many researchers have been interested in the study of natural building blocks, such as peptides, nucleic acids, phospholipids, for the fabrication of novel bio-inspired materials by self-assembly process, a spontaneous organization of nanoscopic or macroscopic materials into ordered structures. In particular, the formation of amyloid fibrils that consist of mainly beta-sheet secondary structure is one of well-known examples of biomaterials formed through the self-assembly of peptides. The amyloid aggregation in the brain is a hallmark of Alzheimer`s disease, but the precise mechanism leading to the self-assembly of soluble amyloid monomers into insoluble amyloid fibrils has not been resolved yet. Therefore, a rapid and convenient method that enables the high-throughput analysis of environmental conditions affecting the self-assembly of amyloid fibril formation would have a substantial impact. In this thesis study, a microfluidics-based system for the analysis of amyloid fibril formation in vitro has been developed for high-throughput screening, low-consumption of reagents, high sensitivity, and short reaction time. We used insulin and $A\beta$ 42 as model amyloidogenic peptides for amyloid self-assembly and fibril growth within microchannels. Multi-microchannels were immobilized by monomeric amyloid peptides via N-hydroxysuccinimide ester activated and subsequently incubated with factors associated with the formation of amyloid aggregates using a continuous flow injection of fresh amyloid monomer solution. In this work, we investigated (1) temporal evolution of insulin amyloid aggregation within microchannels by using monomeric insulin peptides in the continuous shear flow, (2) high-throughput evaluation of inhibition activity of 12 small molecules known to bind amyloid peptides, and (3) effects of shear flow and metal ions ($Fe^{3+}, Cu^{2+}, Zn^{2+}, Al^{3+}$) on $A\bata$ fibrillogenesis by using ThT-induced fluorescence microscopy and ex situ atomi...