Quantitative analysis of adsorbed proteins by X-ray photoelectron spectroscopy

Protein adsorption at solid surfaces is central to many phenomena of medical and technological interest. The determination of the amount of protein attached to the surface is a critical measurement performed by using a wide range of methods. X-ray photoelectron spectroscopy (XPS) is able to provide a straightforward quantitative analysis of the amount of protein adsorbed as an overlayer on a material surface. While XPS is commonly employed to assess qualitatively the amount of adsorbed protein, this is usually expressed in terms of the elemental fraction (or at. %) of nitrogen calculated using an assumption of depth homogeneity despite the fact that this does not linearly scale with the amount of protein. In this paper, we have shown that thicknesses derived from XPS data linearly correlated with spectroscopic ellipsometry data on the same samples with a scatter of 10%. A straightforward equation to convert the concentration of nitrogen from XPS into an equivalent thickness of a protein film is presented. We highlight some discrepancies in the absolute thicknesses determined by XPS and ellipsometry on dried films and quartz crystal microbalance on wet films, which appear likely to result from the inclusion of a contribution from water in the latter two techniques.