Browsing by Author "Thiollet, Sarah"
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Item Open Access Application of multiple response optimization design to quantum dot-encoded microsphere bioconjugates hybridization assay(Elsevier Science B.V., Amsterdam, 2011-07-01T00:00:00Z) Thiollet, Sarah; Bessant, Conrad M.; Morgan, Sarah L.The optimization of DNA hybridization for genotyping assays is a complex experimental problem that depends on multiple factors such as assay formats, fluorescent probes, target sequence, experimental conditions, and data analysis. Quantum dot-doped particle bioconjugates have been previously described as fluorescent probes to identify single nucleotide polymorphisms even though this advanced fluorescent material has shown structural instability in aqueous environments. To achieve the optimization of DNA hybridization to quantum dot-doped particle bioconjugates in suspension while maximizing the stability of the probe materials, a nonsequential optimization approach was evaluated. The design of experiment with response surface methodology and multiple optimization response was used to maximize the recovery of fluorescent probe at the end of the assay simultaneously with the optimization of target-probe binding. Hybridization efficiency was evaluated by the attachment of fluorescent oligonucleotides to the fluorescent probe through continuous flow cytometry detection. Optimal conditions were predicted with the model and tested for the identification of single nucleotide polymorphisms. The design of experiment has been shown to significantly improve biochemistry and biotechnology optimization processes. Here we demonstrate the potential of this statistical approach to facilitate the optimization of experimental protocol that involves material science and molecular biology.Item Open Access Development of a quantum dot-encoded microsphere suspension assay for the genotyping of single nucleotide polymorphisms(Cranfield University, 2009-03) Thiollet, Sarah; Morgan, Sarah; White, NicolaThis thesis describes the investigation of quantum dot-doped particle fluorescent technology commercially available for its application to analyte profiling in suspension. The first part of the thesis described the characterisation of the quantum dot-encoded microspheres, QDEMs, developed by Crystalplex (PA, USA). The multiple fluorescence signatures of QDEMs were analysed using microscopy and flow cytometry technology which provided high-content measurements with a single excitation sources and multiple emission wavelength detectors. The sensitivity and stability of the materials was evaluated under typical biomedical conditions encounter in multiple analyte suspension assays. Novel analytical parameters were defined to study QDEM stability and confocal microscopy detection system was used to provide structural and fluorescent imagines of the fluorescent microspheres under various conditions. Composition of the aqueous environment, temperature and physical forces applied to QDEM induced changes in their fluorescent codes and structural properties. Optimal conditions were then defined for the application of the material to biomedical assays. In a second stage, a conjugation method was developed to produce optimised QDEM bioconjugates for the detection of single strand DNA in suspension. The impact of the conjugation buffer, the concentration and the structure of oligonucleotides was evaluated to optimise QDEM bioconjugates. Then, a novel approach was investigated to optimise the hybridisation of ssDNA to QDEM bioconjugates. Experimental design with response surface methodology determined optimum conditions for the hybridisation of oligonucleotides to QDEM surface in suspension array. Finally, the specific hybridisation of ssDNA to QDEM bioconjugates in a small liquid format adapted to single nucleotide polymorphism detection was demonstrated. The work presented here shows the potential of QDEM bioconjugates for suspension array technology and DNA genotyping. Further, this report highlights the challenges that remain for QDEM fluorescent technology to be reliable for biomedical and suspension array applications.