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Title: Engineered ferritin nanocages for use in single molecule biosensors
Author: Byford, Rebekah Ellen
ISNI:       0000 0004 8504 8299
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2019
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Within modern healthcare personalised medicine is an important goal. A huge part of this goal is the early detection of disease, providing personalised treatment and eradication through targeted therapies. Single molecule detection offers the possibility to uncover rare hidden events from bulk populations, promising early detection of disease. Biosensors using nanopores or nanowells are an example of this. Whilst highly sensitive, they are often large in comparison to the analyte of interest and require additives to attract the particle to the nanopore. This thesis explored the creation of a nanowell based lab-on-chip device for the early detection of cancer, enabling personalised targeted treatment. Initially this was focused on cholangiocarcinoma (CCA), liver cancer of the bile ducts. To attract the CCA cells into the wells a novel engineered ferritin nanocage would be used. The high adaptability of ferritin proteins and self-assembly of the 24 subunit nanocage in maxi ferritins, make them an attractive targeting scaffold within bio-nanotechnology. Due to instability of the original bacterioferritin nanocage, this thesis describes the development of a stable fluorescent engineered ferritin nanocage through three alternative routes. Chemical labelling and fusing ferritin with Green Fluorescent Protein alone proved unsuccessful. Combining a smaller fluorophore, Improved Light, Oxygen or Voltage domain of the blue light receptor phototropin (iLOV), produced a stable iLOV bacterioferritin protein and Au-nanocage complex. Single molecule studies on the engineered ferritin nanocages showed high polydispersity within the solutions, temporarily improved upon filtration. Transition Electron Microscopy provided the only experimental evidence that gold encapsulated nanocages were formed with the iLOV bacterioferritin Au-nanocage complex. Early proof-of-concept antibody studies demonstrated the potential of using engineered ferritin nanocages in a single molecule biosensor. It is hoped that other researchers can expand on this work, using the novel stable iLOV bacterioferritin Au-nanocage complex for complete integration into a nanowell based lab-on-chip device, for the early detection of cancer.
Supervisor: Baldwin, Geoff ; Edel, Joshua ; Cass, Tony Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral