Tesi di laurea magistrale
STUDY OF DNA HYBRIDISATION ON OLIGONUCLEOTIDE-NANOPARTICLES CONJUGATES USING 12 NEW RAMAN ACTIVE DYES FOR SERS DETECTION
CHIMICA E CHIMICA INDUSTRIALE
Corso di studi
relatore Prof.ssa Chiellini, Federica
- DNA targeting
- bio-functionalised silver nanoparticles
- Raman active dyes
Data inizio appello
Data di rilascio
The use of bio-functionalised metallic nanoparticles (MNPs) with oligonucleotide probes in order to detect DNA targets is highly important for diagnostic purposes. Surface Enhancement Raman Scattering (SERS) is a fast, highly sensitive and extremely effective technique for the DNA detection when MNPs are used. Here we report the functionalization of head-to-tail 12-mer and tail-to-tail 18-mer oligonucleotide-silver NPs conjugates with 12 new Raman active monomethine and trimethine chalcogenopyrylium and benzochalcogenopyrylium derivatives. Each dye contains sulphur and selenium atoms together with phenyl, 2-thienyl, or 2-selenophenyl substituents. The target hybridisation on the 12-mer oligonucleotide-NP conjugates have been explored with kinetic and melting experiments using UV-Vis spectroscopy, showing fast and effective hybridisation. The intensity of SERS from each dye upon conjugate hybridization with a complementary single stranded piece of DNA was also explored. Differing concentrations of each dye (1000, 3000, 5000 and 7000 equivalents per NP-DNA conjugate) were used to understand the effects of Raman reporter coating on the overall Raman intensity. It was found that the dye concentration did not affect the target/control ratio, which remained relatively constant and it was also discovered that a lower concentration of Raman reporter avoids non-specific aggregation. Finally a relationship between the dye structure and Raman intensity was found. Methine dyes containing S and Se in the backbone and at least 2 phenyls as substituents give the highest SERS signal following DNA-induced aggregation. Principal Component Analysis (PCA) was performed on the data to show differentiation and highlight possible future multiplexing capabilities of the 12 investigated dyes. The discovery of 12 new Raman active dyes to be used with bio-conjugates silver NPs is an important step in the nanotechnology for diagnostic purposes. Some of the dyes (2, 3 and 5) were tested on 18-mer oligonucleotide-NP conjugates sequences which were the less conservative sequences portions of some fungal species: Candida spp, C. krusei, C. glabrata, Aspergillus fumigatus and A. terreus. The assays were designed to have a tail-to-tail configuration and it was observed instability of the samples and lower on/off SERS ratios compared to the 12-mer head-to-tail models. Kinetics experiments were performed with UV-Vis spectroscopy showing a slower and uncompleted target hybridisation. The problems associated with the 18-mer conjugates are possibly due to the different orientation of the assay and to the shorter spacer of the 3’ sequences. Further investigation will be aimed to exploit the multiplexing capability of the studied dyes with oligonucleotide sequences as portions of the real DNA of different fungal species. The system should be increasingly complicated in order to deal with every possible issue and find the best methodology to be used to simultaneously differentiate real fungal DNA samples.
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