Microfluidic confinement enables monitoring of oligomeric forms of 1-40 β-amyloid peptide in kinetic study by AFM.

Amyloid aggregates are hallmarks of a variety of pathologies. The amyloid beta (A-β), aggregates are critical actors in the pathophysiological process of Alzheimer's disease (AD). Understanding their mechanism of growth at the early stages, in terms of intermolecular and environmental factors, may help to identify critical compounds at the nanoscale and potentially new therapeutic targets. Mesoscopic confinement is a strong factor affecting the kinetics of nucleation and growth. Hence, we use microfluidics to mimic the confinement effects on nucleation kinetics and to control in space and time the concentration and the deposition rate of A-β 1-40 peptide. Atomic force microscopy (AFM) reveals the presence of four types of aggregates, classified by mean size and shape; without evidence of fibrils in the 24 h observation window, which instead appear when the substrate is incubated with the bulk solution. The distribution of aggregates on the mica surface changes with the incubation time and depends on the initial concentration of A-β 1-40. We model the kinetics of nucleation and aggregate growth assuming adsorption of a first monolayer and successive nucleation and growth by dynamic conversion between four different forms of aggregates. The concentration dependence of the total volume of amyloid aggregates exhibits a power law dependence whose exponent is approximately 4. This value translates into the smallest stable oligomer size made of four molecules. Molecular dynamics in aqueous solution further show enhanced intrinsic stabilization of the tetramer relative to smaller oligomers, associated with a nonlinear increase in hydrogen bonds and salt bridges. These results show that, in confined conditions, the smallest stable aggregate of A-β 1-40 is the tetramer, underlining the potential role of these small polymorphs in the neuropathological process and prompting them as targets for in-vitro studies.