Tethered Lipid Vesicles as Nanoscale Sensor Elements
Abstract: The primary aim of this thesis work has been to explore and advance the potential of surface tethered lipid vesicles as a platform for studying in particular the following three biomembrane-related processes: trans-membrane transport, membrane-peptide interactions and the activity of membrane active enzymes. First, a method based on surface plasmon resonance (SPR) spectroscopy was developed for studying passive diffusion processes as well as the membrane-protein mediated transport of small non electrolyte molecules across lipid bilayers. The applicability of the method was demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite. A surface-based platform is of particular interest for this purpose as it allows screening of multiple reactions sequentially. Moreover, comprehensive experimental data sets examining, for instance, the transport kinetics at various temperatures allowed us to compare the activation energies for facilitated diffusion of different sugar alcohols. In addition a complementary assay based on osmotic-induced volume changes in tethered vesicles loaded with self-quenching fluorophores was developed with the aim to study fast transport events in the millisecond time scale. A Single-vesicle analysis revealed a surprisingly large heterogeneity in the osmotic permeability of both pure and human aquaporin (AQP5) containing vesicles. Our data suggest that the membrane-protein incorporation efficiency depends on vesicle size, with higher incorporation efficiency for larger vesicles. In a second project the focus was put on the design and optimization of a surface-based tethered vesicle assay to investigate how an antiviral amphipathic α-helical peptide (AH peptide) interacts with lipid membranes. Utilizing total internal reflection fluorescence (TIRF) microscopy, both peptide mediated pore formation and membrane destabilization could be monitored on the level of single vesicles. The main finding here was that new insights regarding the ability of this AH peptide to preferentially ruptures lipid envelops with sub-100 nm diameters could be obtained. Such information could certainly have a high impact on the field of virology, in which this AH-peptide could be a potential antiviral drug candidate with high specificity. Based on a theoretical analysis, we were able to estimate that pore formation is initiated when four peptides have nucleated. The key for this analysis was the combination of various experimental data from different analytical techniques including SPR and TIRFM. Additionally, the combination of a high sensitivity and the statistical power of the single vesicle analysis allowed analyzing the activity of the peptide at concentration as low as 10 nM, which is more than two orders of magnitude lower than the detection limit of previous works. Finally, arrays of surface tethered lipid vesicles were used to develop a novel assay for the detection of the phospholipase A2 (PLA2) activity in complex physiological samples. In particular, PLA2 is a potential biomarker for the early detection of neurodegenerative diseases such as Alzheimer disease and has therefore triggered a high interest in clinical research areas. Here, time-resolved TIRF imaging was applied to monitor the hydrolytic activity of PLA2, as single enzymes acted on individual vesicles. In contrast to classical ensemble measurements this method allows for the simultaneous evaluation of both the enzyme concentration and the specific enzyme activity in a complex biological sample such as cerebrospinal fluid (CSF). This achievement might be of particular interest for revealing whether the disease states correlate with changes in specific enzymatic activity or enzyme expression level, i.e. the concentration of active enzyme, or a combination of both. The assays presented in this thesis work stress the potential of tethered vesicle platform for membrane biosensing as well as the advantaged offered by surface based approaches, such as low sample consumption, integration with the microfluidic systems for automatic liquid handling as well as efficient and information-rich single vesicle data generation.
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