MicroArrays

Figure 1: The process of patterning a surface. (a) We place a solid support that has been coated with gold into a 1mM ethanolic solution of 11-mercaptaoundecanoic acid (MUA) for at least 18 hours. The surface is subsequently covered with poly-L-lysine. The electrostatic interaction between the carboxylic acid groups of the MUA and the amine groups on the PL allows PL to bind to the MUA surface. (b) When the MUA-PL surface is exposed to an UV light source through a quartz mask, the gold-sulfur bond between the MUA and the gold surface is oxidized. (c) Those regions where the UV light was blocked through the mask are conserved. Rinsing the surface completely removes the alkanethiol in the exposed areas. (d) When the surface is immersed into a 1mM ethanolic solution of n-octyldecyl mercaptan (ODM), the ODM fills the bare gold regions and transforms the exposed areas into hydrophobic regions. (e) The surface is then reacted with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SSMCC) and finally with 5'-thiol modified DNA. The SSMCC only reacts with the MUA-PL regions of the surface, not to the ODM areas, and the 5'-thiol modified DNA will only react at the SSMCC sites. In addition, the ODM regions are hydrophobic while the MUA-PL-SSMCC all create hydrophilic regions. The DNA solutions are confined to specific areas on the surface through the hydrophobic/hydrophilic interactions of the patterned sample, and the DNA strands selectively attach to the surface only on the reactive MUA-PL-SSMCC sites.

Figure 2: A DNA array viewed in fluorescence. (a) Forty-nine squares with DNA strands attached to the surface at each array site are created with the process described in figure 1. We deposited a 0.5mM DNA solution on the surface and placed a clean coverslip on top of the surface. The DNA only attached at the array sites and we did not see any non-specific adsorption onto the background of the n-octadecyl mercaptan (ODM) film. Each array site is 500 μm x 500 μm and is separated by 1 mm center to center. We detect the attached DNA at the array site by flooding the surface with a solution of the complementary strands that are 5'-fluorescein-labeled oligonucleotides. When the surface is scanned with a fluorImager at 530nm, an image of the surface is obtained, and the areas where there are double-stranded DNA with the fluorescein-tagged complement are highlighted. (b) In arbitrary fluorescent units, we scale the intensity of the signal and represent the intensity in the color diagram.

Figure 3:  Demonstration of selectivity in hybridization and isolation to array elements. Two different DNA solutions are placed with an automated pin tool onto the patterned surface that is created as described in figure 1. Confined to the array site through the difference in surface tension between the hydrophobic n-octyldecyl mercaptan (ODM) background layer and the hydrophilic array elements consisting of 11-mercaptoundecanoic acid (MUA) and poly-L-lysine (PL), the DNA solutions react with the surface to form bonds and become tethered. When we place the DNA solution in a pattern described in (a), we can demonstrate the selective hybridization of the array elements. After the DNA solutions have reacted to the surface and have become bound to the specific array sites, we expose the entire surface to the 5'-fluorescein-labeled complements of both. When we scan the surface at 530nm with a fluorImager, we obtain an image of the surface and the areas where the DNA strands have formed duplexes with the fluorescein-tagged complements are highlighted, as seen in (b). We remove all the of complements though a wash with an 8.3M urea solution at 40ºC for 20 minutes. When we flood the surface with the fluorescein-labeled complement of A and image the surface, the highlighted areas of the image correspond to the array sites where we placed the DNA solution A, as seen in (c). We remove the complement of A though a wash with an 8.3M urea solution at 40ºC for 20 minutes. When we expose the surface with the fluorescein-labeled complement of B and image the surface, the highlighted areas of the image correspond to the array sites where we placed the DNA solution B, as seen in (d). (e) The DNA sequence legend for each position on the array. (f) In arbitrary fluorescent units, we scale the intensity of the signal and represent the intensity in the color diagram. The figure shows that each array element can be individually loaded with a specific DNA solution and that the solution stays confined in the array site. When we apply a fluorescent complement, only the array elements with the corresponding sequence become highlighted.