Immobilization of Biological Matter using Transparent Metal Electrodes and Silicon Microstructures




Sankaran, Bharat

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This thesis describes the development of two different methods to produce an optimal platform for immobilizing biological matter (cells and proteins). Firstly, transparent indium tin oxide (ITO) microelectrodes were fabricated and used to immobilize suspended NIH 3T3 fibroblast cells by positive dielectrophoresis (DEP). The ITO electrodes facilitated microscopic observation of immobilized cells as compared to metallized electrodes. DEP was used to capture arrays of individual cells and small cell clusters within a microfluidic network. The extent of cellular immobilization (no-cell, single-cell, or multiple-cell capture) directly correlated with the applied voltage and inversely with the flow velocity. Specific conditions yielding predominantly single-cell capture were identified. The viability of immobilized cells was confirmed using fluorescence microscopy. In the second method, silicon microtechnology was used to make silicon microarray sector slides for facilitating high accuracy protein interactions and identifications. Photolithography and anisotropic chemical etching was used for creating pyramid-like array structures in each sector, to increase the sector surface area and hence the concentration of the reactant. The silicon microarrays were coated with different dielectric films to investigate if they improve the presence and relative abundance of specific variants of key signaling molecules. The microarray structures were also modified with a chemical surface coating: 3-metcaptopropyltrimethoxysilane (MPTMS). Competitive binding assays were then used to test the protein binding accuracy and sensitivity of the silicon based microarrays. Native silicon and dielectric layer microarrays produced poor protein molecule capture during Reverse Phase Antibody process. The presence of MPTMS was found to improve the extent of protein immobilization, thereby improving characterization of immobilized proteins on microarray structures.



DEP, Proteomics, ITO, Cell, Immobilization, Silicon