Abstract This thesis describes the design, fabrication and operation of a microfluidic device for the screening of biomolecule mixture surface mediated effects. The characterisation of a surface immobilisation strategy that will allow the robust attachment of candidate biomolecules on a substrate for use in cell culture applications. This is carried out in the form of a modified and optimised layer-by-layer surface immobilisation strategy and its subsequent thorough and robust characterisation. This was achieved by compiling and critically analysing large amounts of quartz crystal microbalance with dissipation (QCM-D) data and the model utilised to provide meaningful, physical data as an output. QCM-D data was combined with surface plasmon resonance (SPR) data to validate the assumptions used within the QCM-D model package. Further evidence demonstrating the presence of the multilayer, as described by QCM-D and SPR, is achieved using x-ray photoelectron spectroscopy (XPS). These results show that the multilayer surface is robustly attached to the substrate and consists of a large amount of water whilst being able to immobilise mixtures of four proteins. A custom protocol for fabricating these two layer devices was devised and is presented. Scale limitations have been overcome to provide mixing capabilities for large extracellular matrix molecules to be immobilised on the previously described, microfluidically generated surface immobilisation strategy. The optimisation and characterisation of the mixing within this microfluidic device, affected by the incorporated staggered herring bone mixer is also shown. Using dynamic force spectroscopy (DFS) along with a custom designed force curve data processing and analysis package, the spatial localisation of a mixture of four immobilised biomolecules was determined. The aim of this study was to compare the spatial localization of a mixture of four biomolecules created by; standard cell culture protocols (adsorbed from bulk onto tissue culture polystyrene) and a surface created via microfluidic deposition on top of a previously described surface immobilisation strategy. The design and robust application of this custom analysis package allows the definition of a “Barricade of Specificity” such that interactions between an antibody functionalised AFM tip and a surface composed of a mixture of proteins, to be categorised as either a “true” specific interaction, or a non-specific interaction. The application of this Barricade of Specificity thus allows the spatial localisation of four immobilized biomolecules to be determined with a large degree of accuracy as a result of the large rage of non-specific interactions surveyed and the strict definition of a valid rupture force. The final chapter details the application of the microfluidic platform to enable high throughput screening of the effects of extracellular matrix (ECM) molecules, singly and in combination, with regards to the effect on the expression of cell surface markers on umbilical cord blood (UCB) derived CD34+ cells. Careful selection of candidate ECM molecules, cytokine and oxygen concentration has resulted in little difference in the effect on UCB derived CD34+ cells differentiation state after seven days in culture. The major effect has been the maturation towards lymphocyte and leukocyte precursors. However, of the four ECM molecules tested individually, in binary and in quaternary combinations, osteopontin (Opn) and laminin (Ln) demonstrated differences compared to other surfaces tested. In order to further assess the effect of these protein surfaces on the cell surface marker expression of UCB derived CD34+ cells, further tests are warranted for increased periods of time to enable greater discrimination in marker expression and thus increase our understanding of the fundamental biology of this rare and clinically useful cell source.
| Identifer | oai:union.ndltd.org:ADTP/291226 |
| Creators | Michael Hines |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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