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.