DNA (deoxyribonucleic acid) microarrays are used as a tool in high-throughput methods to obtain genetic information. Using atomic force microscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry, and confocal scanning fluorescence microscopy, we investigated how the surface properties and topography of (3-mereaptopropyl)trimethoxysilane (MPTS)-coated mica influence the immobilization of 5'-thiolated oligonucleotides. Mica surfaces were grafted with concentrations of MPTS varied over 3 orders of magnitude (5.38 mM to 5.38 M). At low concentration (5.38 mM), condensed MPTS appeared to form "island-like" structures with an average surface area of 100 nm(2). The MPTS islands grew in size to 2400 nm2 as the concentration of MPTS was increased, suggesting the formation of an MPTS monolayer. XPS analysis indicated a corresponding increase in the elemental concentration of sulfur. At high MPTS concentrations (107.6 mM and 5.38 M), the island structures disappeared, suggesting the formation of a multilayered film. Fluorescence-labeled oligonucleotides were attached onto the MPTS-mica by covalent bonding via disulfide formation. Confocal scanning fluorescence microscopy was used to quantify the coverage of oligonucleotides. The data demonstrated a bell-shaped curve, in which the coverage of oligonucleotides on diluted MPTS incubated mica was similar to5 times higher than the coverage on mica incubated with pure MPTS. The maximum mean oligonucleotide density was 8.95 x 10(13) probes/cm(2). The highest immobilization density was achieved when the MPTS formed a near monolayer. Increased MPTS concentration led to the formation of a multilayer. The reduced number of thiol groups and the increased presence of silanol groups on the surface led to a significant reduction of oligonucleotide surface density.