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Title: Single molecule nanopore protein sensing
Author: Lin, Xiaoyan
ISNI:       0000 0004 9356 6643
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Nanopores have emerged as a powerful, label-free single molecule biosensor based on resistive pulse sensing. Nanopipettes, a sub-type of solid-state nanopores, exhibit several advantages, including easy and low-cost fabrication, good mechanical stability and easy routes for integration with other sensors. In this thesis, nanopipettes were used for single molecule sensing of proteins which play key roles in human diseases. In the first part, DNA-aptamer functionalised gold nanoparticles were used as carriers for single molecule protein sensing. High levels of selectivity were achieved, enabling effective detection of the target in a protein mixture. Furthermore, the larger dimension and decreased charge of the gold nanoparticle-aptamer/protein complex compared with the pure protein contributed to improved signal-to-noise ratios and enhanced event rates. Nanopores were subsequently employed to probe the heterogeneous protein aggregation, which is highly related to human neuro-degenerative diseases. The nanopore sensor was able to characterise the behaviours of different species of lysozyme aggregates during the seeded polymerisation, revealing the microscopic process occurring to 1% seed fibrils. Fragmentation of the seed fibrils was observed at a short time scale not previously reported, revealing the pathogenic mechanism of protein aggregations in human diseases. Attention was finally shifted to the subtle changes occurring to proteins during folding and drug binding. The effect of two actin-binding drugs was studied with the nanopore sensor, elucidating the different action modes that prevent the filamentation of actin. Furthermore, nanopipettes were employed, for the first time to our knowledge, to study the folding landscape of proteins, revealing the conformational states and transient intermediates of actin with urea incubation. In summary, different species of proteins were studied using nanopores with single molecule resolution. From selective protein sensing to macromolecular protein aggregation, subtle protein folding and drug binding, nanopores have proven their potential in early diagnostics, drug discovery and therapeutics.
Supervisor: Edel, Joshua Sponsor: Imperial College London
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral