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Genosensors: design and characterisation

Williams, Edna P. (2002) Genosensors: design and characterisation. PhD thesis, Dublin City University.

Abstract
A sensor capable of detecting specific sequence of DNA was designed by bulk modification of a graphite epoxy composite electrode with either avidin or streptavidin (2% w/w). Avidin or streptavidin are used to capture a biotinylated DNA strand and immobilize this capture DNA onto the surface of the electrode. Subsequent hybridisation is achieved between this biotin DNA strand and a target DNA sequence. The rapid binding kinetics of avidin/ streptavidin-biotin allow a one step immobilization and hybridisation procedure. Detection of this DNA duplex is achieved through use of a small antibody labelled DNA strand. Immersion of DNA electrode in a solution containing a horseradish peroxidase labelled antigen allows electrochemical detection to be achieved in the presence of H2O2 and an electron mediator hydroquinone. Optimization of the sensor design, percentage modifier, immobilization and hybridisation times, was achieved using a simple nucleotide sequence and detection limits of 150 picomolar are reported. Regeneration of the sensor is achieved by using a simple polishing procedure, and shows good reproducibility. A two percent avidin-graphite epoxy composite gave the best performance, lowest non-specific adsorption, greater reproducibility and stability. Subsequently a genosensor was prepared that was capable of detecting a gene, meek, specific to Methicillin Resistant Staphylococcus aureus (MRSA). A characterization of these graphite epoxy composite electrode is also reported. This characterization involved an electrochemical and microscopic study. Results indicate that these composite electrodes exhibit properties similar to a random assembly of microelectrodes, as they possess characteristics such as generation of steady state current at short times, capability of being use in highly resistive media. Scanning electron microscopy illustrates that these electrodes are heterogeneous in nature. Resistance measurements indicate these electrodes have a resistance of 11.45 Cl that is comparable with glassy carbon electrode, 10.18 Q. The RC time constant was calculated to be 0.07 s. Using the Nicholson-Shain model for the measurement of the rate of heterogeneous electron transfer show that these graphite epoxy composite electrodes posses a sluggish rate of electron transfer. 0.35 x 10"3 cm s'1 and scan rates no greater than 20 Vs'1 can be employed. To study the influence various physiological buffers exert on the electrochemical response polymer modified electrodes (PMEs) were prepared using a glassy carbon electrode by drop-casting 40 jul of a metallopolymer, [Os(dime(bpy)2PVIioCl]PF6,(Os-PVIio) where dime(bpy) is 2’2 dimethyl-bipyridyl and PVI in poly-N-vinylimidazole. This metallopolymer was synthesized from the [Os(dime(bpy)2Cl]2+/3+ and PVI. Characterisation of the synthesised product was achieved using HPLC, UC-visible and elemental analysis and indicate good purity. To investigate the influence of a biocomponent progesterone was incorporated within the PME. Electrochemical characterization involved cyclic voltammetry to determine the value of the diffusion coefficient and to compare the values with those obtained from chronoamperometry. Results indicate that the rate of charge transport is slower in buffered systems but shows the best response in phosphate buffer. An investigation into the rate of heterogeneous electron transfer for Os-PVIio and progesterone modified show rates of 1.55 x 105 and 0.51 x 105 cm s’1 respectively.
Metadata
Item Type:Thesis (PhD)
Date of Award:2002
Refereed:No
Supervisor(s):Forster, Robert J.
Uncontrolled Keywords:Biosensors; Design and construction
Subjects:Physical Sciences > Chemistry
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Chemical Sciences
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:18278
Deposited On:27 May 2013 14:57 by Celine Campbell . Last Modified 07 Oct 2013 12:28
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