Dynamics of protein aggregation at the oil-water interface characterized by single molecule TIRF microscopy

Single molecule total internal reflectance fluorescence microscopy was used to observe the dynamic mechanisms of bovine serum albumin layer formation at the silicone oil-water interface. After an initial induction period, the mean diffusion coefficient of protein objects was observed to decrease as a function of exposure time. Simultaneously, the total adsorption rate was observed to decrease; both observations are consistent with protein layer formation. Importantly, the distribution of diffusion coefficients broadened systematically with exposure time, suggesting the presence of surface objects with a wide range of hydrodynamic radii ranging from a few nm (consistent with monomers) to similar to 180 nm. The spatial distribution of the adsorption rate, which was initially uniform, gradually became inhomogeneous, also consistent with the existence of large aggregates. The kinetics of protein layer formation was dependent on the bulk protein concentration in the aqueous phase, and a kinetic population balance model was used to explain the nonlinear concentration dependence.