| Protein microarrays, or protein chips, is a new and promising technology for the emerging Proteomic studies. However, the current design of most protein chips is based on protein microarrays printed on protein-impermeable substrates, and the hybridization is performed by incubation with shaking. The consequent diffusion-limited kinetics compromises the overall performance of the protein microarray. In this thesis work, I have developed a novel filtration-based protein microarray technique, and studied its hybridization kinetics using both experimental and theoretical methods. The protein microarray was constructed on nitrocellulose filter membranes, and the sample was filtrated through the filter to facilitate the hybridization. Compared to the current protein microarray technique, the filtration-based protein technique was found to have significantly accelerated kinetics, extended dynamic ranges, reduced backgrounds, and improved specificity. Multiple chips could be stacked and hybridized simultaneously to further improve the assay throughput. Moreover, the advantages of some new-generation dyes could be fully exploited by this technique. The potential clinical application of this technique was also demonstrated by a filtration-based microarray sandwich assay for detecting CEA in pancreatic caner patient's plasma. To gain a fundamental understanding of filtration-hybridization, the hybridization kinetics of the filtration-based microarray was measured and analyzed by a two-compartment model. The predicted intrinsic rate constants of the reaction were compared with those measured in solution. Finally, the kinetics of microarray assays was studied using a computational fluid dynamics model, which further revealed the parameters that influence the performance of the filtration-based microarray technique. The filtration-based protein chips developed in this work promise a new platform for faster and more sensitive high-throughput protein detection and analysis, and have a wide range of applications in biomedical and biological studies of human health and disease. |
