Quantum Dot Bioconjugate Platforms for Analysis of Enzyme Activity

摘要

Quantum dots are semiconductor nanocrystals with size-dependent optical propertiesudthat result from their nanoscale dimensions. These materials are emerging as simplerudand more sensitive alternatives to traditional fluorescent small molecules andudradioactive reporters in biomarker assays. Quantum dot emission can be modulatedudvia proximal binding of organic dyes or gold nanoparticles, which can form the basisudof a sensor. Their multivalency and ease of functionalisation allow for the attachmentudof multiple biosensing ligands, boosting the detection sensitivity. Quantum dots withuddifferent emission wavelengths can be excited simultaneously and distinguished fromudone another spectrally, a property which can be used for multiplexing.udEnzymes mediate the chemical modification of proteins thereby controlling theudsignalling cascades that regulate cell behaviour. Hence, aberrant activity of enzymesudcan be associated with the onset of disease. Clinical tests typically determine the totaludconcentration of enzyme in a sample without regard to quantification of activity. Inudthis thesis, the development of generic activity-dependent tests for acetyltransferases,udkinases and proteases is described.udEnzyme activity is reported via decoration of quantum dots with enzyme substrateudpeptides and subsequent binding of FRET acceptor dye-labelled antibodies, whichudmediate changes in quantum dot emission spectra. Using this platform, p300 histoneudacetyltransferase was detected with a limit of detection comparable to that ofudradiolabelling assays. Modifications of the platform to detect serine and tyrosineudphosphorylation were investigated. The phosphotyrosine assay was combined with audgold nanoparticle-quantum dot assay for the detection of a kinase/protease biomarkerudpair relevant for the determination of breast cancer prognosis. The modular nature ofudthis assay design allowed for the detection of different classes of enzymes singly andudsimultaneously, representing a generic platform for high-throughput enzymeudscreening in rapid disease diagnosis and drug discovery.