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Quantification of membrane transport rates using optofluidics 10.17863/CAM.93510 Fletcher, Marcus Keyser, Ulrich The transport of ions and small molecules across membranes is a ubiquitous process in biological systems. Effective studies of membrane transport phenomena require suitable model membrane systems, efficient methods to quantify transport kinetics and a robust theoretical framework to derive constitutive transport parameters. This thesis advances all of these aspects of transport measurements through a combination of experimentation and modelling. First, Octanol-assisted Liposome Assembly (OLA) - an existing microfluidic Giant Unilamellar lipid Vesicle (GUV) formation method – is refined by the integration with a novel Pinched Flow Fractionation (PFF) purification module. PFF removes formationassociated residues at a range of length-scales, from surfactant molecules to oil droplets, with high efficiency, leading to more homogeneous model systems for transport studies. The application of PFF to soft systems like GUVs reveals that PFF separates particles based on deformability and not merely on size as previously assumed. Integrating OLA and PFF allows for controlled fusion between oppositely charged vesicles on a single microfluidic device, enabling the sequential assembly of advanced membrane models directly on-chip. Next, we develop computational image processing algorithms to quantify transport in fluorescence experiments with single-vesicle resolution. We apply these techniques to the analysis of GUVs whose membranes are disrupted by membrano-lytic peptides, determining distributions of peptide-induced membrane disruption rates. By modelling the shapes of the extracted distributions, different modes of action by different peptides are revealed, providing insight into how these peptides function as membrane-permeabilizing agents. Finally, we combine microfluidic manipulation and DNA nanotechnology to develop a novel technique for the quantification of potassium ion transport at the single vesicle level. By theoretically modelling ion transfer across model cell membranes, we can quantify ion transport parameters, including potassium ion permeability and ionic selectivities. We apply this technique to the study of different membrane models, including lipid vesicles formed using different methodologies and membranes with embedded prototypical ion channels, building further complexity in our biomimetic system. These experiments demonstrate the versatility of our method for the quantification of transport in membranes with different compositions and physical properties. University of Cambridge 2022-08-01 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/346085 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f06d2a62-9823-4b1b-9f0d-f959562286ab/download 4a519a104ec4a1efa0bfaeff34d28ed2 https://www.rioxx.net/licenses/all-rights-reserved/ Membrane transport ion transport DNA aptamers G-quadruplex antimicrobial peptides Giant Unilamellar Vesicles microfluidics