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

Paper DN+BI-MoA9
Quantification of Immobilized and Hybridized Oligonucleotide Surface Density on Commercial Amine-Reactive Microarray Slides using Radiometric Assay, Fluorescence Imaging and X-Ray Photoelectron Spectroscopy

Monday, October 31, 2005, 4:40 pm, Room 311

Session: DNA Detection and Sensing
Presenter: P. Gong, Colorado State University
Authors: P. Gong, Colorado State University
G.M. Harbers, Colorado State University
D.W. Grainger, Colorado State University
Correspondent: Click to Email
In an effort to establish a quantitative understanding of the correlation between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples, we have characterized amine-derivatized, single-stranded DNA probes attached to amine-reactive commercial microarray slides and its complementary DNA target hybridization using fluorescence imaging, X-ray photoelectron spectroscopy (XPS) and @super 32@P-radiometric assays. Importantly, we have reproduced immobilization efficiencies of DNA probes under microarray formats using high ionic strength and increased DNA concentrations in macroscopic spotted dimensions to permit XPS surface analysis with good reliability and reproducibility. Target hybridization efficiency with complementary DNA was studied on these capture surfaces and shown to exhibit an optimum at intermediate probe densities. The macroscopic model provides a new platform for study of DNA surface chemistry using highly sensitive, quantitative surface analytical techniques. (e.g., XPS, ToF-SIMS) Sensitive @super 32@P-DNA radiometric measurements are now calibrated with more routine XPS DNA signals, facilitating future routine DNA density determinations without the use of hazardous radioactive assay. The objective is to provide new insight into the surface chemistry influences on DNA probe environments that influences the efficiency of target capture form solution in order to improve microarray assay performance.