Botulinum neurotoxins are one of the most potent toxins known to man; they work upon the nervous system blocking the release of chemical neurotransmitters causing flaccid paralysis. Despite their toxicity when administered in low doses they have therapeutic benefits and are used to treat conditions such as muscle spasms and twitches. Due to their extreme toxicity methods of determining the concentration of botulinum neurotoxins in pharmaceutical products must be highly sensitive and accurate. Currently the mouse bioassay is used to monitor the activity of the toxin however this assay is lengthy and has both cost and ethical issues due to the use of live animals. Replacement assays would have a large potential market with a number of users benefiting from them. These include the pharmaceutical industry, clinical environments for point of care sensors in suspected cases of botulism, the food industry for quality control steps and the military for detecting botulinum toxins being used as biowarfare agents. This project details the development of different biosensors for the detection of botulinum neurotoxin all based on measuring the changes to a monolayer of the toxin specific protein SNAP-25. SNAP-25 is selectively cleaved by botulinum neurotoxin types A, C and E; this project focusses on type A as this is used by IPSEN Biopharm in their product Dysport@. In the assays presented in this thesis SNAP-25 is immobilised on gold substrates through its four, naturally occurring, cysteine amino acids which are clustered together in the middle of the protein. The changes to this protein are then monitored electrochemically by cyclic voltammetry or electrochemical impedance spectroscopy and through colourimetry using a UV-visible spectrometer and a microplate reader. These assays have been successful in identifying botulinum neurotoxin with the most sensitive the electrochemical impedance assay, detecting down to 500 ag/ml and the quickest, the microplate reader, only taking seven minutes to perform. The results also show clear differences between the toxin product and the placebo, which contained the excipients RSA and lactose, thus proving that the assays are detecting active botulinum neurotoxin.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:664505 |
Date | January 2014 |
Creators | Halliwell, Jennifer Helen |
Publisher | Bangor University |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
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