Neutron and X-ray reflectivity studies of human serum albumin adsorption onto functionalized surfaces of self-assembled monolayers.

Neutron and X-ray reflectivity (NR and XR) have been widely used for the investigation of the structure of thin organic films. Here we demonstrate how these sensitive techniques may be applied to the study of protein adsorption to well-characterized self-assembled monolayers (SAMs) with different chemical functionalities. NR can be used for in situ study, while XR provides complementary information on the initial surfaces and dried layers measured in air after protein has been adsorbed. In situ measurements of adsorption of human serum albumin onto a hydrophilic NH2-terminated monolayer clearly show the presence of a thin layer of adsorbed protein next to the SAM. Adsorption of albumin onto a hydrophobic, deuterated, CD3-terminated SAM causes even bigger changes in the NR. Upon replacing the protein solution with protein-free buffer solution, the reflectivities from both kinds of monolayers do not change, demonstrating that the albumin adsorption is irreversible after several hours of contact with the protein solution. X-ray reflectivity measurements of dried substrates performed ex situ in air provide a lower bound estimate of the amount of protein which must be at the interface in situ. This combination of techniques provides a uniquely sensitive approach for studying changes that occur upon protein adsorption at an interface.