AVS 52nd International Symposium
    DNA Topical Conference Monday Sessions
       Session DN+BI-MoM

Paper DN+BI-MoM6
Surface Characterization of DNA Immobilization on Silane-modified SiO@sub x@

Monday, October 31, 2005, 10:00 am, Room 311

Session: DNA Structures and Surfaces
Presenter: G.M. Harbers, Colorado State University
Authors: G.M. Harbers, Colorado State University
L.J. Gamble, University of Washington
D.G. Castner, University of Washington
D.W. Grainger, Colorado State University
Correspondent: Click to Email
Fundamental studies must better understand and characterize DNA-immobilized surfaces to improve DNA microarray assay performance. DNA probe immobilization and target capture in microarray formats are quantified with standard fluorescence and radiometric assays. However, stable, efficient bulk immobilization methods that faithfully replicate microarray formats, but also permit high-sensitivity surface analysis with XPS and NEXAFS, are desired. While thiol-immobilized DNA probes on gold remain popular, silane coupling layers on oxides are more practical by closely duplicating commercial polymer microarray slides. Yet, silane coupling remains problematic, and common amine-terminated silane chemistry requires heterobifunctional crosslinking to immobilize thiol- or amine-modified oligo-DNA. Both commercial microarray substrates and silanated surfaces contain C, O, and N, confounding DNA surface analysis. Although phosphorus (at ~ 5%) is unique to DNA, at low DNA densities, P is difficult to detect and its XPS binding energies overlap with silicon plasmon peaks, making analysis confusing. Therefore, we compare silane coupling agents and substrates lacking nitrogen to follow unique DNA nitrogen and when possible phosphorus signals. Several amine- and thiol-reactive silanes on silicon oxide substrates were monitored with XPS at each surface coupling stage. In addition to a qualitative assessment of DNA density using XPS with expected N/P ratios of ~3.8, immobilized oligo-DNA densities and hybridization efficiencies were quantified using @super 32@P-DNA radiolabeling. DNA densities scaled with oligo-DNA feed concentrations (5nM-2µM) and target hybridization depended on oligo-DNA densities, ranging from ~100% at low densities (~10@super 11@ molecules/cm@super 2@) to <10% at high densities (~10@super 13@ molecules/cm@super 2@). NEXAFS, used to determine oligo-DNA probe and target duplex orientation, demonstrated little to no DNA surface orientation under these conditions.