In recent years there has been an increased drive towards point of care testing (POCT), in which assays are performed at the site of the patient. This has many benefits, critically; the time for a result to be obtained will be significantly reduced, allowing for greater and more effective decision making. Many currently used bioassay methods are not affordable in resource poor areas where infectious disease is most prevalent, in order to combat this issue many research groups are attempting to miniaturise equipment for portability and make assays more affordable and therefore more accessible. With the aims of generating a new assay platform which is highly portable and affordable, the work in this thesis presents the development of several generic methods utilising nano- and micro-scale beads coated with aptamer which are then monitored interacting with target proteins with Tunable Resistive Pulse Sensing (TRPS). Aptamers are short oligonucleotide sequences which are capable of binding to a wide range of targets with high selectivity and comparable affinity to antibodies while possessing greater stability and have begun to challenge the role of antibodies. When aptamers bind a target, they often undergo a conformational change. In the assays described herein, this conformational change is key to the observed signal changes. TRPS is a pore-based system in which beads moving through a pore cause a measurable increase in resistance which can be used to derive particle size, concentration, and mobility. During the course of this thesis several template TRPS aptasensors have been developed. TRPS was successfully used to confirm the successful coating of nano- and micro-scale beads with DNA aptamers by monitoring an increase in electrophoretic mobility when the negatively charged DNA is added to the surface. Following on from this, TRPS was used to monitor the interaction of aptamer tagged beads with thrombin protein enabling thrombin detection down to 1.4 nM and the comparison of several thrombin-aptamers with results comparable to previously published SPR data. Thrombin was postulated to shield the negative DNA, resulting in a decrease in mobility, and the magnitude of this charge shielding was found to depend upon the binding mechanism of the aptamer used. This effect is not thought to be specific to our system nor to thrombin, the principles outlined here may be applied to other RPS technologies, or by interchanging of the aptamer, different proteins. In later chapters, this method is expanded to include multiplexed detection of growth factors and a significant improvement in signal. vi Following on from this, the controlled aggregation of avidin coated beads in the presence of biotinylated-BSA was explored. Factors impacting upon this assay were discussed including magnetic separation, particle size and particle concentration, and different methods of data interpretation were presented. This aggregation study identified several key parameters in the use of TRPS in aggregation assays. Using the methods outlined by the study of aggregates, a dispersion assay was then designed in which the interaction of thrombin proteins with clusters of particles brought about the release of many small particles by the disruption of double stranded DNA linkages. This dispersion assay incorporated magnetic separation to simplify the read-out and relied on measuring particle concentration rather than mobility, enabling the use of additional pressure to increase speed and ease of use. Using this method, thrombin was able to be detected down to 100 fM, a significant advancement in TRPS aptasensors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:693028 |
| Date | January 2016 |
| Creators | Billinge, Emily R. |
| Publisher | Loughborough University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://dspace.lboro.ac.uk/2134/21468 |
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