Capturing Conformation-Dependent Molecule–Surface Interactions When Surface Chemistry Is Heterogeneous

Molecular building blocks, such as carbon nanotubes and DNA origami, can be fully integrated into electronic and optical devices if they can be assembled on solid surfaces using biomolecular interactions. However, the conformation and functionality of biomolecules depend strongly on the local chemical environment, which is highly heterogeneous near a surface. To help realize the potential of biomolecular self-assembly, we introduce here a technique to spatially map molecular conformations and adsorption, based on single-molecule fluorescence microscopy. On a deliberately patterned surface, with regions of varying hydrophobicity, we characterized the conformations of adsorbed helicogenic alanine-lysine copeptides using Förster resonance energy transfer. The peptides adopted helical conformations on hydrophilic regions of the surface more often than on hydrophobic regions, consistent with previous ensemble-averaged observations of α-helix surface stability. Interestingly, this dependence on surface chemistry was not due to surface-induced unfolding, as the apparent folding and unfolding dynamics were usually much slower than desorption. The most significant effect of surface chemistry was on the adsorption rate of molecules as a function of their initial conformational state. In particular, regions with higher adsorption rates attracted more molecules in compact, disordered coil states, and this difference in adsorption rates dominated the average conformation of the ensemble. The correlation between adsorption rate and average conformation was also observed on nominally uniform surfaces. Spatial variations in the functional state of adsorbed molecules would strongly affect the success rates of surface-based molecular assembly and can be fully understood using the approach developed in this work

Single-Molecule Tracking of Polymer Surface Diffusion

The dynamics of polymers adsorbed to a solid surface are important in thin-film formation, adhesion phenomena, and biosensing applications, but they are still poorly understood. Here we present tracking data that follow the dynamics of isolated poly­(ethylene glycol) chains adsorbed at a hydrophobic solid–liquid interface. We found that molecules moved on the surface via a continuous-time random walk mechanism, where periods of immobilization were punctuated by desorption-mediated jumps. The dependence of the surface mobility on molecular weight (2, 5, 10, 20, and 40 kg/mol were investigated) suggested that surface-adsorbed polymers maintained effectively three-dimensional surface conformations. These results indicate that polymer surface diffusion, rather than occurring in the two dimensions of the interface, is dominated by a three-dimensional mechanism that leads to large surface displacements and significant bulk–surface coupling

Single-Molecule Resolution of Protein Dynamics on Polymeric Membrane Surfaces: The Roles of Spatial and Population Heterogeneity

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-<i>co</i>-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