Fabrication and Surface Characterization of DNA Microarrays Using Amine- and Thiol-Terminated Oligonucleotide Probes

Abstract

A versatile chemistry utilizing the homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDC) to attach both amine- and thiol-terminated oligonucleotides to aminosilane-coated slides was examined in a microarray format. Three common aminosilanes, 3-aminopropyltriethoxysilane (APS), N -(2-aminoethyl)-3-aminopropyltrimethoxysilane, and m, p -(aminoethyl-aminomethyl) phenethyltrimethoxysilane, were coated onto glass slides and silicon wafers and characterized using contact angle goniometry, ellipsometry, and X-ray photoelectron spectroscopy. Evaluation of the aminosilane-modified surfaces using contact angle measurements, UV−vis spectroscopy, and covalent attachment of a Cy5-conjugated N -hydroxysuccinimide ester reporter molecule suggested that derivatization of the surface with APS + PDC resulted in the best overall coverage. Microarrays printed using APS + PDC chemistry to immobilize both amine- and thiol-terminated oligonucleotides resulted in rapid attachment, uniform spot morphology, and minimal background fluorescence. Both amine- and thiol-terminated oligonucleotides showed comparable attachment, although greater attachment and hybridization efficiencies were observed with amine-functionalized molecules at saturating printing densities. The data highlight the influence of surface chemistry on both immobilization and hybridization and, by extrapolation, on microarray data analysis.