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Title: Retention and enhancement of biomolecule activity on quantum dots
Author: Gupta, Manish
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2010
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Quantum dots (QDs) are semiconductor nanoparticles which have emerged as powerful fluorescent probes for biological imaging applications due to their unique size-dependent optical and electrical properties. QDs have several advantages over small organic dyes and fluorescent proteins such as size-tunable photoluminescence, wide excitation-narrow emission properties, improved brightness and high resistance to photobleaching and degradation. So far QDs have been used to track individual biomolecules, but for this application a widespread concern is that biomolecules can lose activity when they are attached to QDs because these are multivalent and large. Thus, recent attention has turned toward labelling strategies which enable site-specific recognition and controlling the number of molecules that can be attached to a single QD down to a single molecule with retention of activity. Apart from showing ability to recognise appropriate biological partners, relatively little is known about the biological activity of biomolecules attached to QDs. In this thesis various strategies for preserving and enhancing the activity of biomolecules on QDs were developed to address and investigate these aspects and to extend the biological applications of QDs. Nitrilotriacetic acid (NTA)-modified QDs were used for site specific labelling of a hexahistidine (His6)-tagged Glutathione-S-Transferase (GST). GSTs catalyse nucleophilic substitution reactions between glutathione and a wide range of endogenous and xenobiotic electrophiles, which makes them important detoxifying enzymes and anticancer targets. The hydrophobic CdSe-ZnS (core-shell) QDs were made water soluble by ligand exchange with dihydrolipoic acid and coupled to NTA-Ni via an amide bond. Ni-NTA capped QD were capable of binding recombinant S. japonicum His6-GST selectively. As a result of the His6 tag's ability to provide a docking site for the QD away from the active site, the GST molecules bound to these QDs retained their catalytic activity. In contrast, the non specific binding which takes place in the absence of the His6 tag leads to loss of catalytic activity. Hydrophobic interactions were used to functionalize CdSe-ZnS QDs with Kdo2-lipid A -the lipopolysaccahride (LPS) present in the outer membrane of E.coli. These constructs were used as pathogen models to investigate how pathogens and pathogen associated molecular patterns (e.g. LPS) interact and are processed by the immune system. The ability of QDs to enhance the biological activity of a biomolecule was demonstrated in vitro and in vivo for the first time. QD-LPS micelles were able to induce stronger production of cytokines in macrophages and dendritic cells in vitro and a model antigen (DNP-OVA) in vivo than control LPS. Also presented in this thesis is the first attempt to exploit the multivalency and site specific labelling properties of NTA-Ni-decorated QDs to mimic the surface of a parasite. The focus here was on the Plasmodium falciparum malaria merozoite, which has MSP 1 as major component of its surface. Conjugation of a recombinant form of His6-MSP-l hybrid to three different types of NTA-Ni-decorated QDs was accomplished. Morever, by changing the linker units separating the QDs and Ni-NTA complexes it was possible to control the number of MSP 1 molecules attached to each QD.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available