Microspotting Streptavidin and Double-Stranded DNA Arrays on Gold for High-Throughput Studies of Protein−DNA Interactions by Surface Plasmon Resonance Microscopy

We present two strategies for microspotting 10 × 12 arrays of double-stranded DNAs (dsDNAs) onto a gold-coated glass slide for high-throughput studies of protein−DNA interactions by surface plasmon resonance (SPR) microscopy. Both methods use streptavidin (SA) as a linker layer between a biotin-containing mixed self-assembled monolayer (SAM) and biotinylated dsDNAs to produce arrays with high packing density. The primary mixed SAM is produced from biotin- and oligo(ethylene glycol)-terminated thiols bonded as thiolates onto the gold surface. In the first method, a robotic microspotter is used to deliver nanoliter droplets of dsDNA solution onto a uniform layer of this SA (∼2 × 1012 SA/cm2). SPR microscopy shows a density of (5−6) × 1011 dsDNA/cm2 (0.2−0.3 dsDNA/SA) in the array elements. The second method uses instead a microspotted array of this SA linker layer, onto which the microspots of dsDNA are added with spatial registry. SPR microscopy before addition of the dsDNA shows a SA coverage of 2 × 1012 SA/cm2 within the spots and a dsDNA density of 8.5 ± 3.5 × 1011 dsDNA/cm2 (0.3−0.7 dsDNA/SA, depending on the length of dsDNA) after dsDNA spotting. We demonstrate the ability to simultaneously monitor protein binding with the SPR microscope in many 200-μm spots with 1-s time resolution and sensitivity to