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Title: Exploiting resistive pulse sensing for the detection of biomarkers
Author: Healey, Matthew
ISNI:       0000 0004 9349 0730
Awarding Body: Loughborough University
Current Institution: Loughborough University
Date of Award: 2020
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Having a robust and reliable assay that can be used by clinicians inside or outside the hospital laboratory is vital for diagnosing and monitoring the health of a population. The aim of this PhD was to build a foundation of knowledge that could be used to utilise resistive pulse sensing (RPS) coupled with nanoparticles as a platform for biomarker detection. The target of interest, and the overall goal of this thesis, was the prion protein in human blood,which is used for the detection of variant Creutzfeldt–Jakob disease (vCJD). There is no standard detection method for this disease, instead doctors rely on a series of post-mortem tests to determine a diagnosis of an individual. A few assays have been developed, but due to their selectivity issues, and the fact they can take many hours, or days, they are not currently used. This PhD combines the use of RPS with deoxyribonucleic acid (DNA) modified particles as a label free sensor that allows the characterisation of ananalyte bound to DNA modified particles. Namely, it is the 'surface charge' of the particle that is monitored using RPS, which could be a useful clinical tool for the detection of other biomarkers and not just vCJD described here. Altering and optimising the bound ligand to the particle's surface would make this possible. Here, only DNA, either as a capture probe or aptamer is used, but the ligand could be inorganic or organic molecules, peptides or RNA. Monitoring of a particle's relative velocity as it translocates through a pore gives information about its surface charge, which can be used to determine its zeta potential. The literature review in chapter 1 concentrates on highlighting the range of diseases caused by the conformational change of the human cellular prion protein (PrPc ) to the infectious 'scrapie' form (PrPsc), known collectively as prion diseases. The chapter moves on to the cause of the outbreak of vCJD in the United Kingdom (UK) in the 1990s and the extrapolated prevalence in the population. Finally, some of the most recent developments in the detection of Creutzfeldt–Jakob diseases (CJD) is given. The thesis moves to the theory of RPS, introducing the operation of the RPS platform called tunable resistive pulse sensing (TRPS), theory of transport in the system, particle velocities and zeta potential. Theory of aptamers and magnetic particles are also discussed. Before development of an assay for vCJD, the use of nanoparticlesin biological media needed to be investigated, and work at characterising the formation of the protein corona is demonstrated in chapter 4. This study used blood proteins, as well as human plasma and serum. Understanding how the formation of a protein corona around a particle is essential to understand its new biological identity. Carboxylated polystyrene nanoparticles were incubated as a function of protein, blood fraction and temperature; offering an insight into the effects. The subsequent zeta potential values could be used in later work in the assay's development. The surface of nanoparticles can be easily modified with a ligand(s), and in chapter 5 a complementary DNA sequence of the Rassf1a promoter region is immobilised onto the surface. Since the charge of a particle is related to its translocation velocity through a pore, the varying concentration of bound DNA could be detected. Being confident in RPS's ability to monitor this change, a site assessment study was conducted to determine hyper methylation of the Rassf1a sequence. Here, it was possible to assess if the sequence was single or double methylated, also the site of the methylation was obtained. The dose response of the method was investigated and determined to be more sensitive when half the binding capacity is used. The final assay for vCJD aims to use a DNA aptamer, so with the knowledge from previous chapters the foundations for an assay for the always fatal prion disease could be developed. The methods here determined the protocol's limit of detection without pre-concentration, the selectiveness of the aptamer in 3 abundant blood proteins and the first stages of optimising the assay through a pre-concentration magnetic separation. To summarise, the work from this thesis has delivered three published journal papers, as well as being presented at numerous conferences, including the 22nd Congress of the European Hematology Association in Madrid, Biosensors 2018 in Miami and the House of Common's STEM for Britain event.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Aptamers ; Biomarkers ; Resistive pulse sensing ; prion diseases