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Title: Quantum dot bioconjugate platforms for analysis of enzyme activity
Author: Lowe, Stuart Bhimsen
ISNI:       0000 0004 2724 3790
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Quantum dots are semiconductor nanocrystals with size-dependent optical properties that result from their nanoscale dimensions. These materials are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. Quantum dot emission can be modulated via proximal binding of organic dyes or gold nanoparticles, which can form the basis of a sensor. Their multivalency and ease of functionalisation allow for the attachment of multiple biosensing ligands, boosting the detection sensitivity. Quantum dots with different emission wavelengths can be excited simultaneously and distinguished from one another spectrally, a property which can be used for multiplexing. Enzymes mediate the chemical modification of proteins thereby controlling the signalling cascades that regulate cell behaviour. Hence, aberrant activity of enzymes can be associated with the onset of disease. Clinical tests typically determine the total concentration of enzyme in a sample without regard to quantification of activity. In this thesis, the development of generic activity-dependent tests for acetyltransferases, kinases and proteases is described. Enzyme activity is reported via decoration of quantum dots with enzyme substrate peptides and subsequent binding of FRET acceptor dye-labelled antibodies, which mediate changes in quantum dot emission spectra. Using this platform, p300 histone acetyltransferase was detected with a limit of detection comparable to that of radiolabelling assays. Modifications of the platform to detect serine and tyrosine phosphorylation were investigated. The phosphotyrosine assay was combined with a gold nanoparticle-quantum dot assay for the detection of a kinase/protease biomarker pair relevant for the determination of breast cancer prognosis. The modular nature of this assay design allowed for the detection of different classes of enzymes singly and simultaneously, representing a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery.
Supervisor: Stevens, Molly Sponsor: Engineering and Physical Sciences Research Council ; Institute of Materials, Minerals, and Mining ; Imperial College London ; Royal Academy of Engineering
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral