| High-throughput technology has become an essential tool for bioanalytical analysis at the genomic and proteomic level. Two different types of potentially high-throughput bioanalytical system have been studied specifically.; A diazo-coupling method for covalent immobilization of nucleic acid and protein molecules onto solid substrates was developed, characterized and shown to be capable for forming both microarrays and micro-particle based. DNA and protein microarrays formed using this method provided consistent, high-quality signals and low background intensities, and the immobilized biomolecules maintained stability and bioactivity. DNA microarray formed using this method could be reused for at least 5 times. The diazo-coupling method is also of use in the fabrication of high-reactivity microarrays with preferably oriented polyhistidine tagged proteins.; A small volume rapid mixing device (SVRMD) was designed and characterized for flow cytometric analysis. This instrument demonstrates robust and versatile sample handling for subsecond kinetic analysis of biological reaction systems using just muL volume of samples. The device was validated by measuring the kinetics of the fast dequenching of fluorescein biotin from streptavidin upon the addition of biotin. A simple and quantitative assay for biotin and biotin conjugates, as well as the detection limit, dynamic range, and response of the assay were also determined. This device was used to analyze subsecond G-protein activation over particulate surfaces. High affinity ligand-receptor-G-protein (LRG) ternary complexes were formed on beads, and the dissociation kinetics of seven different LRG systems was determined for the first time using the device. The small volume rapid mixing device was thus proven to be a powerful tool for mechanistic study associated with rapid kinetic events in biological reaction systems. |
