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Type: Thesis
Title: Spectroscopic Imaging of Multiplex Bioassays Encoded by Raman and SERS Tags
Author: Azhar, Umar
Issue Date: 2019
School/Discipline: School of Chemical Engineering & Advanced Materials
Abstract: Suspension microsphere multiplex immunoassays are rapidly gaining recognition in immunoglobulin G (IgG) identification. Detection of multiple analytes from a single sample is critical in modern bioanalytical technique, which always requires complex encoding. However, traditional fluorescent technology has various limitations in such multiplex encoding systems. The aim of this study is to use Raman and surface enhanced Raman scattering (SERS) signatures as novel encoding elements in immunoassays to overcome various problems associated with fluorescence labels. In addition to the amplified capacity of Raman/SERS encoding elements, the use of Raman imaging aimed at reinforcement of qualitative analysis has been demonstrated for the first time. This holds great promise in biomedical applications. In this thesis, a series of IgGs were selected as model proteins, and gold nanoparticles (AuNPs) served as the “hotspot” of SERS-active substrates. Three different Raman-active molecules namely, 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), and 3- mercaptopropionic acid (3-MPA) can be easily self-assembled on the AuNPs to form functional SERS tags. Various polymer microbeads (prepared by dispersion polymerization) were utilized as the immune-solid supports together with providing Raman signatures. Additionally, focus was laid on the fabrication of different SERS nanotags and Raman spectroscopic-encoded polymer microbeads for the multiplex, specific and selective detection of biomarkers in dual encoded immunoassay systems. Raman imaging of different uniform polymer microbeads were evaluated. Polymers are long chain molecules containing many repeating monomer units, which give rise to the strong Raman signals of these monomers. In addition, different monomers can be polymerised together to produce co-polymer microbeads to provide different Raman signatures. The development of polymer microbeads not only improves the detection sensitivity significantly, but also makes these microbeads to be more multiplexed in lieu of specific post-reaction labelling. Four Raman spectroscopic-encoded copolymer microbeads were fabricated by dispersion polymerization with their average diameters of 1.1 to 1.7 μm. These synthesised microbeads namely, poly(Sty-co-AA), poly(4tBS-co-AA), poly(4MS-co-AA) and poly(GMAco- AA) revealed narrow size distribution and unique Raman fingerprints, which rendered them to be suitable for Raman imaging and immunoassay analysis. Furthermore, microbeads combining the Raman and SERS signals were successfully fabricated by conjugating a SERS nanotag (4-ATP on the surface of AuNPs) to two Raman encoded polymer microbeads of poly(Sty-co-AA) and poly(4tBS-co-AA). The spectroscopic and imaging results reinforce the suitability of such dual coding systems for immunoassays, which further expands the possibility of Raman/SERS multiplex systems for biological analysis. Finally, a practical demonstration of multiplex IgG immunoassay system based on carboxylated Raman encoded polymer microbeads and SERS nanotags was developed. Antibodies (donkey anti-goat IgG & donkey anti-rabbit IgG) were conjugated to polymer microbeads by EDC coupling chemistry. Two different batches of SERS nanotags comprising of Raman-active molecules (4-MBA & 3-MPA) with AuNPs were synthesised. Moreover, antigens (goat anti-human IgG and rabbit anti-human IgG) were conjugated on SERS tags to form SERS reporters. The immunoassays were performed by mixing the protein conjugated polymer microbeads and SERS reporters. Due to the specific recognition between antibody and antigen, SERS nanotags attached on the surface of their specific antibody polymer microbeads. The results were positively verified from both Raman imaging and spectroscopic analysis. In summary, a series of SERS nanotags and Raman spectroscopic-encoded copolymer microbeads were successfully synthesised. The thesis further demonstrates Raman imaging analysis as a new strategy for qualitative analysis of complicated multiplex immunoassay with high sensitivity and specificity.
Advisor: Dai, Sheng
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Chemical Engineering & Advanced Materials, 2019
Keywords: Raman
polymer micro-beads
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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