Patterned functionalization layer for sub-μL DNA solid-phase immobilization and hybridization

The present work focuses on the patterning of a silane layer resulting from the reaction between a SiO2 surface and alkoxysilane (3-aminopropyl) triethoxysilane, APTES. The chemical composition and topography of the APTES layer are characterized using X-ray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopy (AFM), respectively. The effect of organic versus aqueous silanization media at different pH values, are compared. Micropatterning of the silanized layer is demonstrated and used to electrostatically immobilize single-stranded DNA probes, and to covalently immobilize thiolated single-stranded DNA probes after a cross-linking reaction with sulfo-EMCS. The hybridization efficiency of complementary DNA targets for both immobilization strategies is presented. The solid-phase DNA reactions were successfully demonstrated on surface squares of patterned functionalization layer, with dimensions in the micron scale, integrated on a surface microfluidic structure for sub-μL reaction volumes.