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Novel surfaces to improve oligonucleotide immobilisation and hybridisation for breast cancer assay development

McKenna, Milena (2016) Novel surfaces to improve oligonucleotide immobilisation and hybridisation for breast cancer assay development. PhD thesis, Dublin City University.

This thesis mainly focuses on development of suitable films and imobilisation methods for bio conjugation of the biomolecules to solid supports with future application as biosensors for early breast cancer (BC) detection. Point-of-care (POC) device in biosensor format are recognised as a valuable tool in diagnostics field. However there are number of limitations such as non-specific binding, sensitivity, cost, and fabrication challenges. Hence, new formats of POC platforms are required to improve overall performance of the devices. The fabrication and evaluation of surfaces including carboxylic acid and amino deposition by both wet chemistry and Plasma Enhanced Chemical Vapour Depositions (PECVD) are discussed in detail in this thesis. The recipes and production methods as well as morphological, structural and functional characterisation of deposited films are investigated using a panel of different techniques. This thesis presents that through appropriate surface treatment, poly(methyl methacrylate) (PMMA) can be oxidised using simple methods to form a carboxylic acid surface to enable the covalent attachment of biomolecules for bioassay development. Although spin coating PMMA is a relatively well-known process, to date there has been no direct comparison of the different oxidation methods to activate the surface, nor has oxidised spin coated PMMA been used as a platform for DNA hybridisation or immunoassays. Here, a comparison of the stability, functionality and fabrication process of spin coating and surface activating a thin film of PMMA on a variety of underlying substrates is described. This research reports on the conditions under which amino-modified ssDNA immobilisation onto carboxylic acid surfaces using 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) linkers leads to binding at multiple anchoring sites, i.e. back bone (BB) binding; the effects of such binding on overall hybridisation efficiency are described. An alternative conjugation method, click chemistry (CC), is shown to improve the quantity and quality of target binding by enabling direct covalent attachment of probe oligonucleotides to the surface without BB binding. EDC and CC approaches are compared in terms of hybridisation efficiency in a direct and sandwich DNA hybridisation experiments. This thesis includes the investigation of the orientation of miR16 DNA probe upon hybridisation with miR16 DNA target and miR195 DNA target on various substrates
Item Type:Thesis (PhD)
Date of Award:November 2016
Supervisor(s):Daniels, Stephen
Uncontrolled Keywords:miR16 DNA; breast cancer; detection
Subjects:Physical Sciences > Plasmas
Physical Sciences > Chemistry
Engineering > Biomedical engineering
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Engineering and Computing > School of Electronic Engineering
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
Funders:Science Foundation Ireland
ID Code:21255
Deposited On:21 Nov 2016 15:21 by Stephen Daniels . Last Modified 19 Jul 2018 15:08

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