The single-point active nonlinear microrheology of a colloidal suspension is measured using laser tweezers in the limit that the diameter of the probe particle approaches the diameter of the bath suspension particles. The microviscosity thins as the probe velocity (and corresponding microrheological Péclet number) increases. This thinning behavior correlates with the development of a nonequilibrium suspension microstructure surrounding the probe particle, in which a boundary layer forms on the upstream face of the probe and a wake depleted of bath particles trails the probe. The magnitude of the microviscosities and the thinning behavior are in good agreement with Brownian dynamics simulations reported by Carpen and Brady [J. Rheol. 49, 1483 (2005)]. The microviscosity increment collapses onto a single curve for all volume fractions when scaled by the contact distribution of bath particles around the probe. Scaling the microviscosity increment yields values lower than the dilute theory; furthermore, it plateaus at significantly higher Péclet numbers. The latter effect is corrected by rescaling the Péclet number with the suspension collective diffusion coefficient in place of the bath particle self-diffusivity. The magnitude of the microviscosity increment suggests the theory overestimates the frequency of bath-probe collisions. The presence and role of hydrodynamic interactions and the effect of the soft repulsive potential are discussed.

Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations, adsorbed proteins block strong sites from further protein adsorption. Importantly, this demonstrates that strong binding sites can be modified by changing solution conditions. Membrane surfaces are intrinsically heterogeneous; by employing single-molecule techniques, we have provided a new framework for understanding protein interactions with such surfaces.

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