Microspotting streptavidin and double-stranded DNA arrays on gold for high-throughput studies of protein-DNA interactions by surface plasmon resonance microscopy

Publisher:
ACS Publications
Publication Type:
Journal Article
Citation:
Analytical Chemistry, 2004, 76 (4), pp. 918 - 929
Issue Date:
2004-01
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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-m spots with 1-s time resolution and sensitivity to <1 pg of protein.
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