Improved PEGylation of Gold Nanoparticles for Quantitative Off-Axis Holography-based Detection of Biological Nanoparticles

Licentiate thesis, 2025

A central aspect of modern diagnostics is the detection of biomarkers, which calls for both sensitive and specific biosensing technologies. Biosensors are devices which based on various physicochemical principles are able to detect biomarkers by incorporating target-specific biological components. Due to their unique optical properties, gold nanoparticles (AuNPs) are often a core component of such techniques. The aim of this thesis is to investigate the use of twilight off-axis holographic microscopy combined with surface-modified AuNPs to specifically detect optically faint biological nanoparticles. In Paper I, twilight off-axis holography is introduced and the ability to verify detection based on material properties is investigated. It is demonstrated that by analyzing the real and imaginary parts of the complex-valued optical field, metallic particles can be discerned from dielectric material. Moreover, it is shown that dielectric particles below the detection limit can, through complex formation with functionalized AuNPs, be detected and quantified based on refractive index. In Paper II, this approach was further developed for the use of twilight off-axis holography for specific detection of biotin-modified lipid vesicles. To form specific complexes between AuNPs and biotinylated lipid vesicles, AuNPs were surface modified with streptavidin via biotinylated PEG attached to the AuNPs. Using holographic microscopy, waveguide scattering microscopy and surface plasmon resonance, it was demonstrated that surrounding non-biotinylated PEG may sterically hinder streptavidin-biotin interaction, requiring a thousandfold excess of streptavidin. By decreasing the molecular weight of non-biotinylated PEG, the biotin accessibility was significantly increased and streptavidin-modified AuNPs could be fabricated without the need for excessive protein consumption. Through complex formation with these AuNPs, lipid vesicles could be detected using twilight off-axis holographic microscopy with a sub-pM level of detection.