Characterization of biological nanoparticles using evanescent field sensing

Sammanfattning: In light of the increasingly realized importance of nanoparticle-based biological processes, in biology as well as in technological applications, there is a need of analysis methods capable of accurately quantifying different characteristics and processes involving these elusive entities. The aim of this thesis is the development and utilization of surface-based bioanalytical sensing methods for quantitative characterization of biological nanoparticles, most of which being based on surface-confined evanescent-field illumination. The possibility to use waveguide-based evanescent scattering microscopy for quantitative analysis of protein binding to nanoparticles has been investigated through observation of binding of streptavidin and antibodies to biotinylated liposomes. This system was additionally investigated using a range of complementary measurement approaches, including nanoparticle tracking analysis, conventional and localized surface plasmon resonance sensing, and quartz crystal microbalance with dissipation monitoring. It was concluded that the waveguide microscopy method provides quantitative information in good agreement with established methods, but offers certain key advantages, such as the possibility to provide single-particle resolved label-free information on protein binding kinetics combined with the possibility of simultaneous total internal reflection fluorescence microscopy measurements. In addition, dual-wavelength surface plasmon resonance sensing was used to investigate how the lipid phase characteristics of liposomes affect their behaviour in relation to multivalent interactions with a supported lipid bilayer. This system mimics the initial processes of cellular uptake. Specifically, the interplay between lipid phase (i.e. gel or fluid), interaction valency and liposome deformation was studied. It was demonstrated that fluid-phase liposomes are more prone to deform than their gel-phase counterparts, and that the degree of deformation depends on the number of ligand-receptor pairs engaged in the binding.