Development of a quantum dot-encoded microsphere suspension assay for the genotyping of single nucleotide polymorphisms

摘要

This thesis describes the investigation of quantum dot-doped particle fluorescenttechnology commercially available for its application to analyte profiling in suspension.The first part of the thesis described the characterisation of the quantum dot-encodedmicrospheres, QDEMs, developed by Crystalplex (PA, USA). The multiple fluorescencesignatures of QDEMs were analysed using microscopy and flow cytometry technologywhich provided high-content measurements with a single excitation sources and multipleemission wavelength detectors. The sensitivity and stability of the materials was evaluatedunder typical biomedical conditions encounter in multiple analyte suspension assays. Novelanalytical parameters were defined to study QDEM stability and confocal microscopydetection system was used to provide structural and fluorescent imagines of the fluorescentmicrospheres under various conditions. Composition of the aqueous environment,temperature and physical forces applied to QDEM induced changes in their fluorescentcodes and structural properties. Optimal conditions were then defined for the application ofthe material to biomedical assays. In a second stage, a conjugation method was developedto produce optimised QDEM bioconjugates for the detection of single strand DNA insuspension. The impact of the conjugation buffer, the concentration and the structure ofoligonucleotides was evaluated to optimise QDEM bioconjugates. Then, a novel approachwas investigated to optimise the hybridisation of ssDNA to QDEM bioconjugates.Experimental design with response surface methodology determined optimum conditionsfor the hybridisation of oligonucleotides to QDEM surface in suspension array. Finally, thespecific hybridisation of ssDNA to QDEM bioconjugates in a small liquid format adaptedto single nucleotide polymorphism detection was demonstrated.The work presented here shows the potential of QDEM bioconjugates for suspension arraytechnology and DNA genotyping. Further, this report highlights the challenges that remainfor QDEM fluorescent technology to be reliable for biomedical and suspension arrayapplications.