Escherichia coli O157:H7 contamination is a major hazard in the water supply, causing outbreaks of disease. Conventional methods of E. coli O157:H7 detection usually takes 1-2 days and require hands-on preparation. There is a need to develop a rapid, inexpensive means of detecting the organism. The amperometric biosensor technology has achieved success in the area of metabolite detection. In this study, a bench scale amperometric biosensor was investigated to rapidly detect Escherichia coli O157:H7. The amperometric biosensor consisted of a power source, Clark electrode, autoranging picoammeter, and fabricated polyvinyl chloride (PVC) outer insert with nitrocellulose membrane and attached horseradish peroxidase labeled E. coli antibodies. The interaction of horseradish peroxidase and hydrogen peroxide produced dissolved oxygen which is thought to be altered by the binding of the antigen to the antibody. After submerging the amperometric biosensor in the samples containing various concentrations of heat sterilized E. coli O157:H7 cells, as little as 10 cells/ml of E. coli O157:H7 were detected. The time for detection for the final system was approximately 20 minutes. There was a need to use a custom conjugated antibody to control and increase the molar concentration of conjugated HRP. The minimum concentration of HRP needed for this system was 6 X 10-8M HRP. The system showed optimal performance at pH values 6-8 and showed no response in acidic environments with pH values less than 5. The sensor also showed good performance between 10-30°C. The results indicated that change in dissolved oxygen response was able to distinguish between 0 and 10-5000 cells/ml by maximum increases in dissolved oxygen of 3.53mg/L ± 0.26mg/L when bacterial cells were present and increases in the order of 6.26 ± 0.64mg/L when no cells were present. Despite satisfactory performance as an indicator method, the amperometric biosensor failed to quantify the organism. Further optimization experiments of the amperometric biosensor may be necessary for quantification. The amperometric biosensor with the use of a sandwich assay evaluated in this study offered a reliable means of quantification of the organism. Overall, the amperometric biosensor technology offered an efficient means of detection because of its ease of use and inexpensive, portable instrumentation.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-04062006-134604 |
| Date | 10 April 2006 |
| Creators | Small, Danyelle DeNise |
| Contributors | Marybeth Lima, W. Todd Monroe, Chandra Theegala |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-04062006-134604/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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