Atomic Force Microscopy and X-ray Reflectivity Studies of Albumin Adsorbed onto Self-Assembled Monolayers of Hexadecyltrichlorosilane

Abstract

Atomic force microscopy (AFM) and X-ray reflectivity (XR) have been used together to provide a detailed and direct look at the structure of human serum albumin protein adsorbed onto well-characterized self-assembled monolayer (SAM) surfaces at several protein concentrations. The duration of SAM deposition was also varied to investigate the influence of the density of hydrocarbon chains in the SAM on protein binding tenacity. Concurrent study of adsorption to bare silicon wafers with native oxide surfaces provided a comparison with a hydrophilic surface similar to widely studied glass and quartz surfaces. Both AFM and XR measurements showed that after adsorption, rinsing, and drying, the surfaces of all substrates were covered with no more than a single layer of adsorbed protein. Thin dense protein layers were seen for the substrates exposed to protein concentrations of 0.1 and 0.5 mg/mL. Partial surface coverage by protein aggregates having larger thicknesses was seen for substrates exposed to lower concentrations. The tenacity of the protein adsorption on different substrates was tested by eluting the adsorbed protein with a 1% solution of sodium dodecyl sulfate surfactant. This treatment removed almost all protein from the bare silicon surface and from the fully formed, dense SAMs. A significant amount of adsorbed protein remained on the surface of the less dense, “incomplete” monolayers, suggesting that protein adsorbed more tenaciously on that surface.