| Description |
This dissertation describes the integration of a microfluidic flow cell array (MFCA) with surface plasmon resonance microscopy (SPRM) and the application of MFCASPRM system for microarray analysis of biomolecule interactions. The design and construction of a SPR microscope with a sensing area that is compatible with the fluidics footprint of the MFCA is described first. Antibody-antibody interactions are used as the model system to demonstrate the capability of the integrated MFCA-SPRM for in situ microarray fabrication and analysis. Impacts of physicochemical parameters, such as reactant concentrations, reaction constants and the flow rate, on the performance of the MFCA-SPRM are investigated by experiments and modeling. Optimized experimental conditions will support the future application of the MFCA-SPRM. Statistical analysis of microarray data (24 micro spots) shows that the spot-to-spot coefficient of variation is within 15%. Major sources of signal variance are from the deviation of light incident angle, heterogeneous sensing surface and the mass transport. Next, a proof-of-principle experiment demonstrates the potential of the MFCA-SPRM system for immunogenicity assays of Daclizumab to analyze anti-drug antibodies (ADA) from serum samples. Daclizumab is a monoclonal antibody drug for treatment of multiple sclerosis patients. Biotinylated-Daclizumab immobilized on a streptavidin monolayer is used to assess the presence of ADA in serum samples of three patients with multiple sclerosis. The result shows that the sample from a patient without the treatment of Daclizumab generates the highest SPR signal. In the future, more samples are required to generate statistically significant data to evaluate the immunogenicity of Daclizumab. Matrix-assisted laser desorption/ionization mass spectrometer (MALDI MS) is an ideal tool to be combined with SPR for protein analysis. An antibody microarray created by MFCA and coupled to MS is utilized to demonstrate the potential of microarray analysis with SPR-MS. Finally, we apply MFCA-SPRM system for characterization of in situ immobilized vesicles on solid surfaces. Hydrophilicity of surface, vesicle size and composition are investigated as factors that affect the structure of vesicles adsorbed on surfaces. A model for the calculation of the surface area of the bilayer is proposed to correlate the SPR response with vesicle structures at the surface. |
