Biomolecules at Interfaces -Studied by Surface and Solution Techniques

Doctoral thesis, 2007

The interfacial behaviour of biomolecules in general and proteins in particular is interesting from a biochemical point of view since proteins are involved in processes such as complement activation, blood coagulation and atherosclerotic plaque formation, but also in the context of biomedical applications such as solid-phase diagnostics, therapeutic aphaeresis and biosensors. The objective of this thesis is to study biomolecular interactions using surface sensitive biosensor techniques such as surface plasmon resonance, SPR, in combination with bulk techniques. Lipopolysaccharide, LPS, is as a key mediator of sepsis mediated syndromes. Removal of circulating LPS by therapeutic aphaeresis, achieved by LPS-binding molecules that are anchored to a solid support, has been suggested as an adjuvant sepsis therapy. Anti-LPS peptides, designed from LPS-binding molecules, were covalently immobilized on model surfaces. The effect of immobilization on the LPS-binding ability of the peptides and the stability of the interaction was studied. Covalently attached peptides retained their activity although to varying extent. The LPS binding capacity of the peptides was in some cases dependent on the immobilization method. Furthermore, the solution behaviour of LPS in terms of aggregation onset and aggregate size was studied. It was found that LPS aggregated into spherical aggregates in a continuous manner at close to physiological conditions, possibly reflecting the polydispersity of the molecule. Suppression of non-specific protein adsorption is essential for biosensors applications in order to achieve sufficient selectivity and sensitivity and for blood contacting devices in order to avoid immune responses or blood coagulation. This can be accomplished with PEG-functionalized coatings. The effect of interfacial charge on the protein rejecting ability of two PEGylated copolymers was monitored. The results indicate that an adsorbed layer of PEGylated copolymers can be very efficient in preventing proteins from adsorbing at a surface provided there is charge matching between the polyelectrolyte and the underlying surface. Inadequate diagnosis contributes to the high mortality records of severe sepsis. Direct detection of elevated plasma levels of protein-bound isopeptides, from the disseminated intravascular coagulation that occurs in severe sepsis, could constitute a useful diagnostic marker. The interaction between an immobilized anti-isopeptide antibody and a protein-bound isopeptide was studied by SPR with the intention to develop a SPR based immunoassay for the diagnosis of sepsis. This first study shows that this direct assay has a high immunocapturing efficiency and detects protein-bound isopeptides in buffer and diluted plasma. Finally, the combined use of two surface sensitive biosensor techniques, SPR and quartz crystal microbalance with dissipation monitoring, QCM-D, was explored for the study of the interaction between low density lipoprotein, LDL and the proteoglycan decorin. LDL affinity to proteoglycans is a potential marker for atherogenic LDL that may be used in a clinical solid-phase or biosensor assay for atherosclerosis risk prediction.