Ciccone, Jonah Louis; (2023) Using Nanodiscs to Insert Nanopores into Membranes for Bio-Analysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nanopore sensing technologies have enabled rapid single-molecule sequencing of nucleic acids, advancing scientific research and clinical diagnostics. The utility of nanopore based nucleic acid analysis has demonstrated the need to extend the technique to other biologically relevant an- alytes, such as proteins. Shape based interrogation of proteins would be a boon to the field of proteomics and advance difficult to study fields such as post-translational modifications. Further, the need for portable sensing of antibody proteins has been demonstrated unequivocally during the pandemic of the 2020’s. The development of nanopore sensors for protein analytes is limited by lack of readily available biological nanopores with wide apertures to accommodate intact proteins. DNA nanotechnology holds a ready solution to this problem, and has provided researchers with tools to reliably and precisely generate nanopores of customisable size. However, poly-anionic DNA nanopores struggle to puncture hydrophobic membranes, particularly robust polymeric membranes used inside commercial nanopore apparatus. Difficulties associated with the hydrophilicity of DNA are compounded by structural motifs that excel at membrane puncturing often being incompatible with structural motifs that excel as detectors in a nanopore analysis platform. This thesis demonstrates the use of delivery agents to transport nanopores into membranes otherwise difficult to puncture. Dry lipids are rehydrated with hydrophobically modified nanopores, assembling vesicles in a nanopore accommodating conformation. Lipid vesicles populated with transmembrane nanopores are then solubilised into nanoscale discoidal lipid patches using amphipathic polymers, termed lipid nanodiscs. Lipid nanodiscs with embedded nanopores are then demonstrated to deliver nanopores into both lipid and polymer membranes. This work lays the foundation for the generation of protein conductive nanopore sensing apparatus.

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