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Having previously developed methods to study interfacial diffusion of small molecule surfactants and proteins at the liquid/liquid interface, we have now applied these methods to investigate the dynamics of bovine serum albumin at the silicone oil/water interface and of phospholipids at the hydrocarbon oil/water interface.

Single-molecule total internal reflectance fluorescence microscopy was used to observe the dynamic behavior of >4000 bovine serum albumin objects at the silicone oil/water interface. The surface residence time distribution indicated the presence of three populations at the interface. Each population had a characteristic fluorescence intensity and distinctive interfacial diffusion behavior. Larger fluorescence intensity correlated with longer residence times and slower diffusion. These combined observations of fluorescence intensity, surface residence time, and interfacial diffusion suggested that the three populations represent monomers, dimers, and trimers respectively. In the case of oligomers, multiple diffusive modes were observed, suggesting that the oligomers could adopt more than one conformation or orientation at the interface.

Fluorescence recovery after photobleaching was used to characterize the diffusion of fluorescently-labeled phospholipids at the oil/water interface for oil viscosities that varied over four orders of magnitude. Measurements were performed over a range of surface concentrations corresponding to molecular areas of 40-130 square Angstroms/molecule. As expected, the interfacial diffusion coefficient increased with molecular area, saturating at an area of ~100 square Angstroms/molecule. At molecular areas below about 80 square Angstroms/molecule macroscopic domains of a condensed monolayer phase were observed; the diffusion of these domains was characterized by direct tracking and trajectory analysis. For oils with viscosity <1500 cP, the diffusion coefficients of both individual probe molecules and condensed domains were consistent with a mechanism where the objects moved within the interface, experiencing drag from the adjacent bulk phases. Since this drag was dominated by the oil viscosity, the diffusion coefficients decreased proportionally to the inverse of the oil viscosity. However, for oils with higher viscosity, the diffusion coefficient of individual probe molecules decreased much more slowly. These observations suggested that two diffusive mechanisms are involved Ð one where surfactant molecules move within the interface and one that is analogous to the activated "hopping" processes that occur at the solid/liquid interface. This latter mode becomes significant only for very viscous oil phases.