A single-colour spectroscopic sensor for monitoring detergent- and protein- induced vesicle damage

Detergent-induced membrane solubilisation is important for several biotechnological applications including cell lysis, virus inactivation and membrane protein extraction. Despite its significance, the mechanistic details remain largely underexplored, owing in part to a lack of suitable technologies that can investigate solubilisation events on the nanoscale. In this work, the use of intramembrane fluorescence de-quenching is presented as a sensitive technique for monitoring the interaction between detergent- and protein-vesicle interactions. The adaptability of this assay is showcased across a range of membrane-bound dye concentrations, surfactant conditions and model membrane vesicles of various sizes and compositions and its utility as a spectroscopic nanoruler is highlighted for a wide variety of biotechnological applications. Using a combination of time correlated single photon counting and fluorescence spectroscopy techniques, this thesis reveals that the widely used detergent Triton X-100 triggers the structural remodelling of lipid vesicles, even at concentrations below its critical micellar concentration, and confirms that vesicle swelling occurs before complete micellization. The study demonstrates that the progressive addition of Triton X-100, results in an enhancement of DiI’s fluorescence lifetime, and that this parameter can be used to quantify morphological changes and membrane restructuring events. These conformational changes were also probed using single-vesicle imaging techniques, which confirm the expansion and highlight the formation of toroids before complete solubilisation. The sensitivity of the de-quenching approach can therefore provide a platform for exploring a wide variety of membrane perturbations with important biomedical significance. In this context, preliminary investigations highlight that the fluorescence de-quenching technique can also be utilised to probe the disturbance of lipid vesicles by Amyloid-beta (1-42) aggregates, which are major hallmarks of Alzheimer’s Disease. Overall, the single-colour fluorescence de-quenching approach is a robust, sensitive and generalizable tool for detecting nanoscale membrane remodelling events and can be used to resolve detergent- and protein-induced membrane intermediates.