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Title: Design and synthesis of linkers for conjugation of DNA to SE(R)RS active nanoparticles
Author: Wrzesien, Joanna
ISNI:       0000 0004 2739 0539
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2011
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Surface enhanced resonance Raman scattering (SERRS) can be used to detect specific DNA sequences by methods based on hybridisation of oligonucleotide functionalised nanoparticles to a complementary DNA strand. In order to obtain a strong SERRS signal the analyte has to adsorb onto a suitable, roughened metal surface and a visible chromophore, which is resonant with the excitation frequency of the light and plasmon of the metal surface, has to be present in the molecule of interest. The problem which has to be overcome to use this technique for the detection of specific DNA sequences is that DNA is not SERRS active due to the lack of a visible chromophore and presence of the highly negatively charged phosphate backbone. To obtain SERRS active DNA a label containing a surface seeking group, to allow adsorption of DNA on to a metal surface, and a visible chromophore has to be attached to the DNA strand. This thesis reports the synthesis of four linkers containing a: fluorescent (aminofluorescein, fluorescein, TAMRA) or nou-fluorescent (BHQ) Raman tag, a surface complexing group (cyclic disulphide-thioctic acid) and a chemical functionality for the attachment of DNA (carboxyl group). Each of the linkers also contained poly(ethylene glycol) (PEG) (3 or 41 mer) which reduces the non-specific adsorption of molecules to nanoparticles surface and provides colloidal stability. The synthesised linkers were used to functionalize the following type's M metallic nanoparticles: Au citrate stabilised (18 and 50 nm), Ag citrate stabilised (40nm) and Ag EDTA stabilised (35nm). All prepared conjugates gave good SERRS responses at laser excitation frequencies of 633 nm and also exhibit high stability- they could be stored at room temperature for several weeks without any changes observed. In order to conjugate the prepared linker functionalized nanoparticles to oligonucleotides, the linker terminal COOH groups were reacted with amino-modified single stranded DNA in the presence of one of the coupling agents (N-(3- dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride-EDCHCl with N- hydroxysulfosuccinimide or 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium chloride -DMT MM). This produced probes with a permanent SERRS signal which were then used for the detection of specific DNA sequences in a sandwich assay format. Surface enhanced Raman scattering (SERS) can also be a useful tool capable of providing information regarding changes to the chemical environment inside living cells, which would allow better understanding of the biochemistry of diseases. For example, it was reported that acidification of endosomes play a central role in a number of pathologies, including cystic fibrosis, kidney diseases and certain types of cancer. It has been reported that metallic nanoparticles, when incubated with the cells, are taken up by the cells, then trapped in endosomes. Formation of SERS active, pH sensitive metallic nanoparticle probes would allow the detection of changes of endosomal pH. In order to prepare such probes Au (citrate stabilised, 18 nm) and Ag (citrate and EDTA stabilised) nanoparticles were functionalised with two different pH sensitive molecules: 4-mercaptopyridine and 2-aminothiophenol. Ag EDTA nanoparticles functionalized with 4-mercaptopyridine gave very strong SERS signals and were found to be the most sensitive among all the pH sensing probe prepared. This probe was used to detect intracellular pH inside macrophages and HeLa cells. Because it was reported that the attachment of cell penetrating peptide or nuclear localization sequences (NLS) to nanoparticle surfaces makes it possible to deliver nanoparticles to other than endosomes components of a cell, a mixed monolayer of pH sensitive molecule (4-mercaptopyridine) and multifunctionallinker, able to attach to metal surface and to react with biomolecules of interest, was formed on gold and silver nanoparticles surface. It was found that pH sensitive probes prepared in such a way are the source of strong SERS signals and have the same sensitivity as metallic nanoparticles functionalised with 4-mercaptopyridine only. The prepared pH sensitive probe can be conjugated to NLS and used to detect pH inside the cell nucleus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available