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Microfluidic diffusional sizing
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Secondary nucleation and elongation occur at different sites on Alzheimer’s amyloid-β aggregates

Procedure

The fabrication and the operation of the microfluidic diffusion device used in the present studies have been described in previous papers (33, 48). Briefly, the microfluidic chips were fabricated by using standard soft lithography. The sample to be analyzed and the buffer were introduced into the system through reservoirs connected to the inlets, and the flow rate in the channel was controlled by applying a negative pressure at the outlet by a syringe pump (CETONI neMESYS, Korbussen, Germany) at typical flow rates in the range from 60 to 90 μl/hour. Lateral diffusion profiles were recorded at 12 different positions (3.5, 5.3, 8.6, 10.3, 18.6, 20.3, 28.6, 30.4, 58.7, 60.4, 88.7, and 90.5 mm) by standard epifluorescence microscopy using a cooled charge-coupled device camera (Photometrics Evolve 512, Tucson, USA). The diffusion profiles were fitted to model simulations on the basis of advection-diffusion equations assuming a bimodal Gaussian distribution (48). From the area under the curves of the two Gaussian populations, the concentrations of the bound and free molecular chaperones were evaluated. The dissociation constant KD was calculated by direct nonlinear regression on the basis of the Langmuir binding isotherm (49, 50)$[clusterinbound]=[clusterinfree]⋅[M]KD+[clusterinfree]$(3)where M is the total concentration of binding sites available on the Aβ(M1-42) fibrils, representing the maximum concentration of bound molecular chaperone, while [clusterinbound] and [clusterinfree] are the concentrations of bound and free molecular chaperone, respectively.

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