A great effort has been recently devoted to the development of new devices for the detection of specific sequences of DNA, due to increasing need of label - free, fast, cheap, and miniaturized analytical systems able to detect target sequences for screening purposes, especially in food industry for genetically modified organisms (GMOs).
In this study, development of a QCM - based DNA biosensor for the detection of the hybridisation of CaMV 35S promoter sequence (P35S) was investigated. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of probe sequences on the gold surface of the quartz crystal. Two immobilisation procedures were tested and compared considering the amount of the immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non - specific adsorption.
The two coating methods were based on the use of self - assembled monolayers. One of them employed the interaction between the thiol and gold for the immobilisation of a thiolated P35S probe, while the other employed formation of functionalised aldehyde groups by ethylenediamine plasma polymerization on the gold surface for the immobilisation of amined P35S probes through gluteraldehyde activation. Results indicated that immobilisation of a thiolated probe provides better immobilisation characteristic, higher sensitivity for the detection of the hybridisation reaction, absence of non - specific adsorption and a higher stability with respect to the regeneration step.
The optimised immobilisation procedure for the thiolated probe was used for the detection of P35S sequence in PCR - amplified DNAs and in real samples of pflp - gene inserted tobacco plants that produce ferrodoxin like protein additionally. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and sonication. The obtained results from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM - based DNA biosensor system.
The developed QCM-based DNA biosensor represented promising results for a real-time, label - free, direct detection of DNA samples for the screening of GMOs.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12608288/index.pdf |
| Date | 01 March 2007 |
| Creators | Karamollaoglu, Irem |
| Contributors | Oktem, Huseyin Avni |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | Ph.D. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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