Measurement of the thickness of ultra-thin adsorbed globular protein layers with dual-polarisation interferometry: a comparison with neutron reflectivity

Abstract

Dual-polarisation interferometry (DPI) was used to investigate the effectiveness of the random sequential adsorption (RSA) model to describe the adsorption of the globular proteins bovine serum albumin (BSA) and transferrin at the silicon/water interface. DPI provided an almost real-time view of dynamic protein adsorption. The results showed that initial BSA adsorption followed the Langmuir kinetic model below a protein concentration of 0.5 mg ml⁻¹ and the random sequential adsorption kinetic model of particle deposition at and above 0.5 mg ml⁻¹. At long adsorption times the predicted t^−1/2 scaling law for the dependence of the surface coverage on time as the globular particles approached the 2-D jamming limit was observed for BSA over 3 logarithms in time and for transferrin over 4 logarithms. Furthermore, BSA adsorption was used as a model system to compare measurements of surface coverage and layer thickness as measured by DPI and neutron reflectivity (NR) at a pulsed spallation source (SURF at ISIS). The analysis of the DPI data based on a simplified single-layer model was capable of accurately determining the overall protein layer thicknesses over a large concentration range for these ultra-thin adsorbed layers (thickness ≤7 nm, surface coverage 0–3.5 mg m⁻²). NR required a two-layer model to cover the same concentration range. NR data modelling showed non-uniform density distributions of adsorbed BSA over the high surface coverage range, clearly indicating changes of protein conformation with concentration. Differences in equilibrium thicknesses between the DPI and NR techniques are discussed.