Multi-technique comparison of immobilized and hybridized oligonucleotide surface density on commercial amine-reactive...

To establish a quantitative, corroborative understanding of observed correlations between immobilized probe DNA density on microarray surfaces and target hybridization efficiency in biological samples, we have characterized amine-terminated, single-stranded DNA probes attached to amine-reactive commercial microarray slides and complementary DNA target hybridization using fluorescence imaging, X-ray photoelectron spectroscopy (XPS) and 32P-radiometric assays. Importantly, we have reproduced DNA probe microarray immobilization densities in macroscopic spotted dimensions using high ionic strength, high-concentration DNA probe solutions to permit direct XPS surface analysis of DNA surface chemistry with good reliability and reproducibility. Target capture hybridization efficiency with complementary DNA exhibited an optimum value at intermediate DNA probe immobilization densities. The macroscopic array model provides a new platform for the study of DNA surface chemistry using highly sensitive, quantitative surface analytical techniques (e.g., XPS, ToF-SIMS). Sensitive 32P-DNA radiometric density measurements were calibrated with more routine XPS DNA signals, facilitating future routine DNA density determinations without the use of a hazardous radioactive assay. The objective is to provide new insight into different surface chemistry influences on immobilized DNA probe environments that affect target capture efficiency from solution to improve microarray assay performance.