Reflectivity studies of human serum albumin adsorption onto well-defined surfaces

Neutron and X-ray reflectivity (NR and XR) have been widely used for the investigation of the structure of thin organic films. This work demonstrates how these sensitive techniques were applied, for the first time, in studies of Human Serum Albumin (HSA) blood protein adsorption to well-characterized self-assembled monolayers (SAMs) with different surface properties. NR was used for in situ studies, while XR provided complementary information on the initial surfaces and dried layers measured in air after the protein was adsorbed. Information regarding the kinetics of protein adsorption on the self-assembled monolayer was obtained using Total Internal Reflection Fluorescence through a scientific collaboration. Self-assembled monolayers were prepared from Cl₃Si(CH₂)₁₅CH₃, Cl₃Si(CD₂)₁₅CD₃ and Cl₃Si(CH₂)₁₁NH₂ alkyltrichlorosilanes. In situ measurements of the adsorption of human serum albumin onto hydrophilic (NH₂ terminated) and hydrophobic (CH₃ and CD₃ terminated) monolayers clearly showed the presence of a thin, monomolecular layer of adsorbed protein. Differences in structure of the adsorbed protein layers on SAMs with different hydrophobicity have been observed. It was found that surfaces with higher hydrophobicity induce bigger conformational changes in the adsorbed albumin molecules. These findings were corroborated by kinetic Total Internal Reflection Fluorescence (TIRF) measurements, where evidence of desorption of protein due to structural rearrangements of irreversibly adsorbed molecules on hydrophobic substrates was found. The influence of the monolayer ordering on protein adsorption tenacity on hydrophobic self-assembled monolayers has been also studied. Monolayers with different thicknesses and electron densities were prepared using different deposition times. The tenacity of protein adsorption was tested by elution of adsorbed albumin molecules with a solution of sodium dodecyl sulfate surfactant. It was found by means of X-ray reflectivity that the tenacity of albumin adsorption was significantly higher on surfaces with less dense, more disordered alkyl chains. In situ neutron measurements of albumin adsorption onto SAMs with different density of hydrocarbon chains provided insight into the possible nature of the effect of increased adsorption tenacity. Deuterated self-assembled monolayers were used to increase the sensitivity of the neutron reflectivity experiment. It was found that interpenetration of the adsorbed proteins into the hydrocarbon chain region occurs for 'incomplete' SAMs that had a less dense packing of alkyl chains. No such penetration was observed on dense, extended-chain 'complete' monolayers. This penetration effect was specific for the albumin protein, as was shown by TIRF measurements. Differences in adsorption kinetics on 'complete' vs. 'incomplete' monolayers were observed only for the albumin. Another common blood protein, gamma-globulin, showed no significant difference upon adsorption onto 'complete' and 'incomplete' SAMs. The experimental data for albumin adsorption onto surfaces of SAMs indicates, though indirectly, that there is a strong possibility that specific interactions exist between the alkyl chains in 'incomplete' monolayers and albumin fatty acid binding sites. The concept of 'incomplete' monolayers that utilize such specific interactions can be successfully used in the design of new biomaterials and medical implants.