Development of novel bioanalytical assays with single-molecule readout for biomarker detection and drug candidate characterization

Sammanfattning: Bioanalytical assays with single-molecule readout for studying molecular interactions have in the past decades received increasing attention. The high sensitivity often offered by this readout scheme has for example enabled ultra-sensitive analyte detection, having important implications for monitoring early disease progression and the effects of drug treatment. In addition, single-molecule studies of molecular interactions with membrane protein receptors have proven useful for the development of new and more effective drugs. Ultra-sensitive detection as well as the possibility to unravel heterogeneities in molecular interactions, offered by single-molecule readout schemes, are both key components for the future of personalized health care and the discovery of new disease biomarkers.This thesis mainly focuses on the development of new bioanalytical assays with single-molecule readout, with the purpose to enable studies of molecular interactions with membrane protein receptors (an important class of drug targets) and to detect diagnostically relevant biomarkers and pathogens. Lipid assemblies, either in the form of liposomes or supported lipid bilayers, have been exploited for their compatibility and flexibility offered in the context of studying many essential biological interactions. In the first part of the thesis, two surface-based assays, both utilizing total internal fluorescence (TIRF) microscopy, were developed to study molecular interactions with a low-abundant and sensitive class of membrane proteins; G protein-coupled receptors (GPCRs). With the insights gained from that work, the focus was shifted towards solution-based detection schemes, based on a home-built dual-color fluorescence microscopy setup. Two detection schemes, based on Förster resonance energy transfer (FRET), for biomarker detection (phospholipase and miRNA), and a third scheme for detection of virus particles via induced colocalization of fluorescent liposomes, were developed.As for future perspectives the thesis puts emphasis on how the different bioanalytical assays can have implications for personalized health care and how the performance of the solution-based colocalization assay can be further improved to become a generic tool for biosensing purposes.