Development of a point-of-care biomedical imaging diagnostics for cancer screening
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Date
2019
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University of Zululand
Abstract
Despite our improved understanding of cancer biology, this disease remains a global health phenomenon, affecting the populations in developed and developing countries, of every socioeconomic strata. It is the second leading cause of death worldwide with over 10 million new cases and over 5 million deaths annually. The point-of-care diagnostic approach holds important promise for the early detection of cancer since most cases are treatable at an early stage of the disease. Research has shown that over 90 per cent of cancer cells express some forms of proteins, which can serve as biomarkers for early detection, thereby increasing survival rate in cancer patients. Recently, nanotechnology has been used to develop nanoparticles like a quantum dot, known for their unique optical properties and sizes and applicable for diagnosis in cancer cells research. In this study, colloidal cadmium telluride quantum dots were synthesised, capped and stabilised with Gum Arabic polymer. These particles were characterised using UV absorption, Photoluminescence (PL), X-Ray diffraction (XRD), High-Resolution Transmission Electron Microscopy (HRTEM), Fourier transform infrared (FTIR) analysis and zeta potential to determine their stability in different media. These quantum dots were then used for in-vitro cytotoxicity studies on 4 different cancer cell lines (HeLa, MCF-7, PC-3 and U87) in a dose-dependent manner. Results: cells were found to have over 50% viability for almost all cell line. Binding studies were evaluated in-vitro. Chlorotoxin peptide labelled with FITC at the C and N-terminals respectively were used for this study while fluorescence intensity was used to determine the binding of the cell to the peptides and expressed in percentage. Results show that cells bind more to chlorotoxin peptide with FITC attached to the N-terminal of the CTX than CTX peptide with FITC at the C- terminal and this binding was dose-dependent. Chlorotoxin (HIS-CTX-GST tagged) was genetically recombined and expressed in Escherichia coli (E. coli) BL21 cells, purified using nickel beads. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was
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used to analyse the protein and results show noticeable bands at 30 kDa which corresponding to the HIS-GST-CTX protein. Block studies experiment was carried out using CTX-FITC labelled peptide and recombinantly expressed CTX to determine the binding effect of both peptides to the cells that is if they are specific or receptor-dependent. The results revealed that the binding of the CTX-FITC was receptor dependent. Biotinylated chlorotoxin was conjugated to quantum dots using streptavidin-biotin chemistry and cytotoxicity of this conjugates was carried out using WST-1 cell viability assay. Cell binding and uptake were also analysed. These studies will serve as a point-of-care diagnosis for cancer treatment as early diagnosis a key to surviving the disease. Also the in vivo studies cell uptake can be used to improve surgical procedure by ensuring total tumour removal, and this can prevent tumour reoccurrence.
Description
Thesis submitted to the Department of Biochemistry and Microbiology in fulfilment of the requirements for the award of Degree Doctor of Philosophy (PhD), Faculty of Science and Agriculture at the University of Zululand, 2019.
Keywords
Cancer screening, Cancer biology