Surface and analyte capture analysis of DNA microarrays on model gold surfaces and commercial microarray slides.

摘要

Nucleic acid microarrays represent a powerful assay tool for large-scale parallel analysis of genome sequences and gene expression in biological and biomedical research. This would imply that DNA microarray assay results are quantitative and reproducible. However, numerous challenges preclude microarray reliability, sensitivity, specificity, direct assay from complex milieu, and direct abundance quantitation required for accurate clinical performance.;The objective of this dissertation research is to provide new insight into the surface chemistry influences on DNA probe environments that affect the efficiency of target capture from solution in order to improve microarray assay performance. To this end, this study is focused on DNA surface and analyte capture analysis of (1) model surfaces with mixed thiol-DNA/mercaptoundecanol (MCU) adlayers on gold, and (2) DNA microarrays on commercial amine-reactive microarraying polymer slides (CodeLink(TM) and OptArray(TM)). Assay surface reliability, DNA density, hybridization efficiency, and influence of complex milieu (serum dilutions, cell lysate) on hybridization were assessed using X-ray photoelectron spectroscopy (XPS), surface plasmon resonance (SPR), as well as more traditional biological and microarray analysis techniques such as radiometric assay and fluorescence intensity measurements.;Immobilization efficiencies of DNA probes on commercial slides under microarray formats were reproduced using high ionic strength and increased DNA concentrations in macroscopic dimensions to permit analysis with highly sensitive, quantitative surface analytical techniques (e.g., XPS) that are currently incompatible with microarray dimensions. Surface densities of immobilized DNA probes (2x1011 ∼ 4.4x103 probes cm 2) and hybridized DNA targets (and 2x1011 ∼ 8.9x10 12 targets/cm2) on gold and commercial slides were quantified using sensitive 32P-DNA radiometric measurements. Optimum target hybridization occurred at intermediate probe densities with more upright probe orientation. The more sensitive radiometric results were calibrated with more routine XPS and fluorescence intensity measurements to facilitate future routine DNA density determinations without the use of hazardous radioactive assay. Furthermore, influences of complex milieu and fluorescence dye labels on microarray DNA hybridization were investigated. Serum protein adsorption onto SPR sensor surfaces were found to significantly affect the SPR curve shape, impeding hybridization detection beyond 30% serum, while only minimal effect from complex milieu was observed on the commercial microarray slides.