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Title: Droplet interface bilayers : on the theory and application of the small molecule passive membrane permeability assay
Author: Barlow, Nathan
ISNI:       0000 0004 7229 0772
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Investigations into characterising and measuring bio-membrane permeability have been ongoing for over a century. Driven by both industry and academia, a variety of in vivo, in vitro, and in silico techniques have been developed and employed to understand the mechanisms and thermodynamics of drug and agrochemical transport in living systems. Unfortunately, many of the techniques suffer from a lack of high-throughput implementation or suffer physical restrictions such as bulk diffusion limitations. Moreover, it is apparent that membrane permeability data is lacking, particularly with respect to the agricultural industry and non-mammalian biological sciences. With the invention of the droplet interface bilayer (DIB) a decade ago, there have been several breakthroughs in membrane technologies for electro-physiology, membrane protein reconstitution, and membrane permeability assay methods. In this thesis, I have studied DIBs as a possible candidate for a rapid and high-throughput membrane permeability assay that can be used to measure rates for speci c agrochemicals in varying lipid systems in situ. The rst output of this research includes device engineering, design and fabrication techniques of novel micro uidic chips for improved DIB formation, droplet rendering, and manipulation. More speci cally, on demand and high through-put DIB manufacture has been achieved for the rst time, the results of which has been published in the peer reviewed journal, Lab on a Chip. Furthermore, to prove that the application of DIBs are not categorically limited to a small subset of lipid types, it has been proven that DIBs can be formed with a variety of lipids, including some plant lipid extracts. For the rst time, DIB model membranes have been formed to mimic soy, Arabidopsis, tobacco, and oat plasma membranes. A successful permeability assay was performed with these DIBs, and the results were published in the peer reviewed journal, Biomicro uidics. A serious challenge of measuring membrane permeability in DIBs is the limitation of bulk diffusion, which often leads to underestimates in intrinsic membrane permeability rates. To further the understanding of this limitation, the uid dynamics of coupled advection-diffusion in stirred droplets has been investigated experimentally and computationally. As a result, a novel micro uidic device has been developed to induce shear stress along the membrane to disrupt the effects of the bulk uid stagnation in the permeability assay, which allows for more accurate measurements of the intrinsic membrane permeability. To the best of my knowledge, this is the most accurate technique available, and is a breakthrough tool for future applications, such as supplying permeability data to systems transport models. The results of the intrinsic membrane permeability of various lipid types have been published in the journal, Nature Scienti c Reports. Furthermore, the physical properties of DIBs have been investigated including surface energy driven morphology, formation dynamics, and bilayer surface tension measurements. For the rst time, the effect of membrane curvature in DIBs has been thoroughly scrutinized, and new insights into DIB behaviour have been established.
Supervisor: Ces, Oscar ; Barter, Laura ; Brooks, Nick ; Flemming, Anthony Sponsor: European Commission
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral