Quantum dots offer a number of advantages over standard fluorescent dyes for monitoring biological systems including high luminescence, stability against photobleaching, and a wide range of fluorescence wavelengths from blue to infrared depending on the particle size. In this work, we investigated in using the protein cage apoferritin as a template for the synthesis of colloidal quantum dots. We obtained apoferritin after reductive dissolution of the ferritin iron core and showed that the protein structure was left intact during this process. We further studied the solubility of ferritin, apoferritin and cationized ferritin in organic and fluorinated solvents by hydrophobic ion pairing methodology in order to expand the possibility of using an apoferritin template for the synthesis of quantum dots in organic media. We then focused on the synthesis and fluorescence properties of PbS quantum dots in aqueous solution. PbS dots are thermally stable and emit in the range 1,100 to 1,300 nm depending on their size. We demonstrated the encapsulation of these PbS quantum dots within the cavity of the iron storage protein apoferritin using two routes: 1) the disassembly/reassembly of apoferritin subunits trapping previously synthesised PbS quantum dots; and 2) use of the channels present in the protein shell to allow the entrance of Pb2+ and S2- ions leading to formation of quantum dots in the apoferritin cavity. We show that PbS-apoferritin composites emit in the near infrared region which make them promising labels for biological applications. Furthermore, we demonstrated that PbS QDs can be excited via a bioluminescence resonance energy transfer (BRET) using luciferin from Luciola mingrelica which could be developed into a self-illuminating labelling system. Finally, in order to make PbS-apoferritin composites selectively attachable to biomolecules during labelling experiments, the apoferritin was modified by the incorporation of analogues of methionine introducing azido groups absent in the proteins. The azido groups can then be selectively modified in complex mixtures e.g. cell lysates using `bio-orthogonal' reactions such as the Cu(I) catalysed Staudinger ligation and Huisgen cycloaddition. This would allow highly selective addition of receptor targeting or cellular permeation of peptides to the outer surface of the apoferritin shell.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:493343 |
| Date | January 2008 |
| Creators | Hennequin, Barbara |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/11071/ |
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