Comparison of Methods for Generating Planar DNA-Modified Surfaces for Hybridization Studies

The surface conformation and accessibility of oligonucleotides within arrays are two key parameters that affect the utility of immobilized nucleic acids in sensor technologies. In this work, a novel combination of analytical techniques was used to compare two methods for DNA immobilization on glass. The aim of the study was to identify a method that generated a high surface density of hybridization-accessible oligonucleotides in a true planar monolayer. The first method based on direct coupling of silanized DNA to the glass surface showed a high immobilization density of 0.013 molecules/nm2 but low surface accessibility, as shown by the hybridization measurements (< or =15%). The second method, based on the biotin-streptavidin interaction, generated a high immobilization density (0.02 molecules/nm2) and high surface accessibility (90%). Atomic force microscopy and X-ray photoelectron spectroscopy indicated that both methods achieved uniform surfaces. Using the biotin-streptavidin system, the intermolecular distance between the hybridized molecules could be tightly controlled by titrating biotinylated complementary and noncomplementary oligonucleotides.