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Title: DNA origami nanopores for protein biosensing
Author: Pugh, Genevieve Claire
ISNI:       0000 0004 7964 9995
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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There is an increasing demand in biomedicine for rapid diagnostic testing. This is fuelled by the improved knowledge of the proteome and genome and a drive towards personalised medicine. Furthermore, many new potential biomarkers for diseases are being identified. Portable, point-of-care biosensors can meet these demands and take advantage of the recent biomedical developments. In this thesis, we investigate the creation of a biosensor element, including a design that allows the detection of protein biomarkers via an electrical label-free method. The use of nanopores for single molecule sensing has led to the development of commercially viable, portable, label-free DNA sequencing devices.1 However, the use of nanopores for detection of protein analytes is yet to reach the same viability. A reason for this is the inability for current nanopore materials to combine both atomically precise structural definition and tuneable nanopore size of the widths needed to accommodate protein analytes. In this thesis's main project a route to overcome these limitations is described, by using the DNA origami technique. Multiple layers of DNA duplexes are interlinked to form a nanopore structure with a defined, predetermined central channel. The pore described can transport proteins with a higher fidelity than previously published work. Small DNA nanopores have shown promise for the transport of some small molecule analytes2,3,4. A secondary project looks at the use of a single loop of duplex associated with a lipid bilayer as a simplistic nanopore to induce ion transport through a membrane. Although consistent current steps were not demonstrated, the DNA loop was shown to associate with and cause some disruption and ion transport through the bilayer. Large DNA origami rings have been shown to template the formation of liposomes of a defined size5 . These rings, initially functionalised with lipid nucleation sites, were hypothesised to be adaptable for bilayer association and use in a nanopore sensing set up by replacing lipid nucleation sites with cholesterol molecules (lipid | 3 anchors) to induce bilayer association. Out of several anchor arrangements investigated one arrangement, with anchors located on the outside of the ring in the plane of the ring, was shown to be the most viable. By and large, only conductance values of a small magnitude were observed when single channel current recordings were conducted with the DNA origami rings in a Dphpc membrane. This suggested that association of the rings with the bilayer does not lead to the formation of a channel of the desired size. The origami funnel nanopore designed as the main aim of this thesis is of a form which is robust and versatile for further use. The DNA nanopore designed can be easily modified with additional functionalizations and is shown to associate with, and span, lipid bilayers. The nanopore can be used as a template from which further applications and advances in nanopore sensing research can be established.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available