Quantum dot encoded magnetic beads for multiplexed fluorescence biosensing

摘要

In recent years, the use of encoded beads has received considerable attention due to their potential for measuring multiple analytes in solution.(1-4) This can be achieved without the need for knowledge of their spatial position, as in the case of microarray technology. Encoded bead technology also relies on the solution kinetics rather than diffusion to a fixed surface as in the case of microarray technology, offering the possibility of developing rapid high throughput screening methods. This thesis describes the production, characterisation and application of quantum dot encoded beads prepared using layer-by-layer assembly of different colour quantum dots around a magnetic bead. To achieve this, two different strategies were used to make “coloured” barcodes. The first strategy used thiol chemistry to immobilise quantum dots in a layer-by-layer assembly onto magnetic beads whereas the second strategy uses the interaction between quantum dot-biotin and quantum dot-streptavidin conjugates to create constructs on the magnetic bead surface. The development of both of these immobilisation strategies was characterisation using X-ray photoelectron spectroscopy and fluorescence spectroscopy of immobilised quantum dot structures onto a plain glass substrate. After the preparation of encoded beads, they were characterised using single bead fluorescence spectroscopy. It was found that attempts to prepare barcodes by layer-by-layer assembly of CdSe/ZnS quantum dots using thiol chemistry onto magnetic beads did not comply with the necessary barcode characteristics i.e., different colour coded beads could not be distinguished from each other. However, the encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads gave distinct multicolour coded bead spectra. These barcodes were characterised in terms of different spectral responses, stability at raised temperatures, stability in biotin solutions, and long-term stability after storage. Encoded beads prepared using layer-by-layer assembly of quantum dot-biotin and quantum dot-streptavidin conjugates onto streptavidin coated magnetic beads were then used to develop multiplexed immunoassays. Four different barcodes were prepared and used to perform model multiplexed immunoassays. The barcodes were identified upon the basis of different spectral response measured using single bead fluorescence spectroscopy. Finally, a quantitative immunoassay for human IgG was performed using these barcodes, which showed that different concentrations of human IgG can be determined in solution.