Existing methodologies for biomolecular detection are limited in several key areas. Heterogeneous assays struggle with various wash steps which can prolong assay time, while other assays require costly reagents, lack mobility and can be highly complex in nature. This project demonstrates how a bio-nano particle in the form of M13 bacteriophage (M13) can be used for the basis of a novel homogeneous immunoassay which incorporates the use of linear dichroism spectroscopy (LD). M13 has a high aspect ratio which allows it to align easily in shear flow, this in turn generates a large LD signal. This property of M13 has been manipulated for use in a new in-vitro diagnostic technique. Existing M13 production yields are much lower than those required for this assay. A new method was developed which increased the yield 10 fold. Chemical modifications were made by covalently attaching chromophores, this enabled the M13 LD signal to be visualised in the visible region and develops the potential for multiplexing. By chemically modifying M13 with chromophores and antibodies it was possible to create an assay capable of detecting 10⁵ cells/mL of Escherichia coli O157. This is 100 times more sensitive than the M13 based assay developed by Pacheco-Gomez et al. (2012). The assay was reassembled to detect small molecules and was found to have a sensitivity of 0.01 mM. The assays presented form a sensitive, specific, fast diagnostic tool capable of detecting pathogens and small molecules. It offers significant improvements over existing methods, and could act as a platform in developing a multimodal detection system.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642379 |
| Date | January 2015 |
| Creators | Sandhu, Sandeep Kaur |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/5737/ |
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