Multiparametric Optical Characterization of Biological Nanoparticles using Evanescent Field Sensing

Doctoral thesis, 2022

In light of the increasingly realized dependence of many biological functions on nanoscopic supramolecular assemblies, also including novel biotechnological applications, there is a need for advanced analysis methods capable of accurately quantifying different characteristics of these elusive entities. The prime aim of this thesis is the development and utilization of surface-based bioanalytical sensing methods for quantitative characterization of biological nanoparticles. The possibility to construct and use a waveguide-based evanescent light scattering microscopy instrument for investigation of various nanoparticle properties is explored through the study of liposomes and mRNA-containing lipid nanoparticles as well as polystyrene and silica nanoparticles. It is shown that through analysis of scattered light from such particles, single-particle-resolved information on their size, refractive index and interactions with surrounding protein solutions is obtainable, thus providing multiparametric characterization beyond the ensemble average. Additionally, this is combined with information gained from fluorescent labeling of certain biomolecular components, allowing nanoparticle content to be correlated with the other particle properties. The aforementioned systems were additionally investigated using a range of complementary methods, including nanoparticle tracking analysis, 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 simultaneous evanescent light fluorescence microscopy measurements, thus providing new insights regarding nanoparticle heterogeneity.