Adsorption of Bovine Serum Albumin (BSA) at the Oil/Water Interface: A Neutron Reflection Study

Abstract

The structure of the adsorbed protein layer at the oil/water interface is essential to the understanding of the role of proteins in emulsion stabilization, and it is important to glean the mechanistic events of protein adsorption at such buried interfaces. This article reports on a novel experimental methodology for probing protein adsorption at the buried oil/water interface. Neutron reflectivity was used with a carefully selected set of isotopic contrasts to study the adsorption of bovine serum albumin (BSA) at the hexadecane/water interface, and the results were compared to those for the air/water interface. The adsorption isotherm was determined at the isoelectric point, and the results showed that a higher degree of adsorption could be achieved at the more hydrophobic interface. The adsorbed BSA molecules formed a monolayer on the aqueous side of the interface. The molecules in this layer were partially denatured by the presence of oil, and once released from the spatial constraint by the globular framework they were free to establish more favorable interactions with the hydrophobic medium. Thus, a loose layer extending toward the oil phase was clearly observed, resulting in an overall broader interface. By analogy to the air/water interface, as the concentration of BSA increased to 1.0 mg mL(-1) a secondary layer extending toward the aqueous phase was observed, possibly resulting from the steric repulsion upon the saturation of the primary monolayer. Results clearly indicate a more compact arrangement of molecules at the oil/water interface: this must be caused by the loss of the globular structure as a consequence of the denaturing action of the hexadecane.

Reflectometry Reveals Accumulation of Surfactant Impurities at Bare Oil/Water Interfaces

“…Water/fluid interfaces are structurally simpler and therefore particularly well suited to give unambiguous results. NR studies on liquid/liquid interfaces involving thin water or oil films on planar solid substrates [48,49,50,51,52,53,54,55] usually used partially deuterated oils and/or H2O/D2O mixtures, were aimed at characterizing organic interfacial layers rather than the bare interfaces, and will be discussed further below.…”

Langmuir films at the oil/water interface revisited

“…The variety of studies found in literature include not only monolayers of surfactants of different chemical nature (e.g. lipids 1–3 , asphaltenes 4 , proteins 5,6 and polymers 7 ) but also of solid particles (nanoparticles, particles in the colloidal size range and larger) 8,9 . While one of the bulk liquid is usually water the other liquid phase can be volatile solvents (e.g.…”

Antibody adsorption on the surface of water studied by neutron reflection

“…17). Neutron reflection has enabled molecular models of protein adsorption to be constructed, 18 has been used to determine the orientation of antibodies non-specifically adsorbed at the solid/liquid interface, 19 and has yielded the structural characterization of membrane proteins in bilayer models resting on a water-filled layer. 20 Current approaches to mitigate chemical and physical instabilities require the addition of buffers and excipients to mAb formulations.…”