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Title: Measuring zeta potential using tunable resistive pulse sensing : applications in biosensing
Author: Blundell, Emma L. C. J.
ISNI:       0000 0004 6350 2032
Awarding Body: Loughborough University
Current Institution: Loughborough University
Date of Award: 2017
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The aim of this PhD was to develop and optimise an analytical method that incorporated zeta potential measurements within tunable resistive pulse sensing (TRPS) for biosensing. Modern society is dependent upon the accurate and rapid quantification of biological analytes within solution (biological or environmental) and on materials (clothing, skin, food). If the characterisation of particles within biological samples such as blood, plasma and serum is done simply by optical methods such as light scattering or microscopy, the various particulates and molecules, many of which are similar in size may not be able to be identified. TRPS is a label-free, non-optical based technique that can complete size, concentration, and more recently aided by the work in this thesis, zeta potential measurements in real time. Zeta potential could be a powerful analytical tool, as it is relative to the charge on an analyte and can be measured by monitoring the velocities of analytes as they traverse a nanopore in an electric field. Monitoring translocation velocities through the pore and thus zeta potentials could allow for an extra signal to help characterise analytes. Following a literature review in chapter 1 which focuses on the use of nanoparticles and their characterisation within bioassays, a general theory chapter (chapter 2) covers common theory and experimental setup used throughout the research. Chapter 3 contains theory specific to zeta potential measurements using TRPS developed with an industrial sponsor to which chapter 4 is the application of this theory. It contains details on applying the method of inferring zeta potential from particle velocities to measure the change in zeta potential of nanoparticles as their surfaces are functionalised with DNA of varying packing density, length, structure, and hybridisation times to also determine the sensitivity of the method. As described the zeta potential is determined via the particle velocities as they traverse a pore that are determined from the signal produced using a TRPS measurement, a blockade. The blockade gives information on the particle velocities at relative positions within the pore as well as information on the size and charge of the particle. TRPS is an evolving analytical platform that can differentiate samples of similar and the same size by their charge in a range of electrolyte solutions. This is important for whole blood and biological samples, for example, as there will always be other biomolecules or contaminants present, of similar size that may not be the target of interest. A large part of this PhD was the incorporation of DNA aptamers onto nanoparticles as recognition elements to a specific target. They were of particular interest as aptamers are ssDNA (single-stranded DNA) strands of high affinity and specificity to a target analyte. Nanoparticles can be functionalised with DNA aptamers or proteins as a means to capture a target analyte. TRPS was used to monitor the binding of DNA aptamers to their target proteins, aided by zeta potential measurements. The results showed that a smaller zeta potential value was observed when a target protein was bound to the aptamer-modified particles. As well as protein detection and quantification, a new assay using nanoparticles as tags was investigated, chapter 5. TRPS was used to monitor controlled particle aggregation in the presence of target bioparticles mimicking a streptavidin-biotin assay at first. It was found that when two differently sized particles, one functionalised with biotin and the other streptavidin (70 nm and 115 nm at a 10:1 ratio), the particles in excess saturated the larger particles resulting in a large change in size and zeta potential that could be monitored using the tunable pores. This method was then applied to nanoparticles in complex biological media, including plasma, serum, and biological buffers used to suspend bacteriophage samples, two examples are given in the thesis; the first in chapter 5 and second in chapter 7. In chapter 5, as well as sub 150 nm particles, bacteriophages of similar sizes were investigated to test the technique to biologically relevant particles. State of the art methods of counting bacteriophage via optical techniques have proven difficult, or inconsistent. In preliminary work shown in chapter 6, the characterisation of phage samples in their respective media is demonstrated. TRPS has overcome some of these challenges and preliminary data has been obtained for the size and charge characteristics of different phage types including Salmonella phage and coliphage. The study has also progressed to the size and concentration analysis of Clostridium difficile phage that has gained interest in recent decades due to their uses in therapeutics. As an alternative to nanoparticle based assays, the pores themselves were modified with DNA aptamers, see chapter 6, for direct detection of a target analyte without the need for a particle label . Pore surface modifications have been completed to enable pores to be easily functionalised with DNA and this work has enabled current rectification properties of conically shaped pores to be explored. Limits of detection for DNA-modified pores were found to be similar to that of a particle-based assay (5 pM and 18 pM, respectively) but the particle assays are more versatile and may be used in future for multiplexing experiments. Finally, in chapter 7, the technique and methodology were able to monitor changes in the behaviour of nanoparticles as they were immersed in protein rich solutions, to mimic an in vivo environment. Here the protein corona around the nanoparticles was investigated as a function of temperature (25oC and 37oC). The kinetics and binding mechanism of high and low affinity proteins forming a protein corona could be monitored in real time as well as displacement reactions between various proteins, showing the advantages of TRPS technology. In summary, from working with a commercial partner and collaborating with other institutions, we have delivered 4 papers (plus one JoVE paper) including a review of applications of TRPS technology and work detailed in this thesis, presented at 14 conferences and user meetings, and facilitated the development and implementation of zeta potential into bioassays.
Supervisor: Not available Sponsor: Loughborough University ; Izon Science Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available