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Automated biosensors utilising firefly bioluminescence

Murphy, Melanie Jane; (1999) Automated biosensors utilising firefly bioluminescence. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Abstract

A requirement exists for rapid, automated detectors capable of identifying the presence of Biological Warfare (BW) agents in the environment. The combination of optical biosensors with the bioluminescent reporter molecule, firefly luciferase, has great potential for meeting this need, and underpins the work described in this thesis. Two major approaches have been adopted. The first, which has proved highly appropriate for toxins, was the development of an optical fibre-based method in which the light emitted by an immobilised luciferase linked immunoassay was coupled evanescently into the fibre. This required the development of a method to enable antibodies to be directly labelled with firefly luciferase without excessive loss of enzyme activity. The larger size of bacteria limited the utility of luciferase as a direct label. The indirect use of luciferase (via its substrate adenosine-5-triphosphate, ATP) was considered to be more flexible. The ATP generating enzyme adenylate kinase (AK), was used to develop two different immunoassays for bacteria. In both cases, the AK was derived from the target bacterial cells (rather than added as a label via a secondary antibody), thereby simplifying assay design and improving the speed of response. The first indirect approach produced a specific assay capable of identifying fewer than 104 Salmonella cells ml-1 in 15 minutes. This is a significant improvement over conventional methods in terms of sensitivity and response time. It used magnetic micro-beads pre-coated with antibodies against Salmonella to selectively remove the organism from suspension, prior to chemical lysis. The second approach used bacteriophages to selectively lyse target organisms in mixed suspension, resulting in similar sensitivity but a longer assay time. Options for the automation of each assay and its use for BW detection in the field are discussed. Potential applications in other areas are explored using the food industry as an example.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Automated biosensors utilising firefly bioluminescence
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAIU642986; Applied sciences; Biosensors; Firefly luciferase
URI: https://discovery.ucl.ac.uk/id/eprint/10100769
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