In situ synthesis and direct immobilization of ssDNA on electron beam patterned hydrogen silsesquioxane
Scanning electron microscopy of electron beam patterned HSQ lines on silicon. lines and spaces, scale (left). lines and spaces, scale (middle). line and spaces, scale (right). All HSQ lines were patterned to be in length.
Optical microscope image of an array of HSQ lines spaced , scale (top left). A cartoon drawing showing the overlay of DNA synthesis by four different exposures (dose 1–dose 4) perpendicular to the long direction of HSQ lines is shown on the top right (not to scale). ssDNA synthesis should occur in the intersecting areas of HSQ and light-directed synthesis. Hybridization with fluorescent labeled oligonucleotides reveals the relative amount of DNA synthesized for a given dose. The Cy-3 fluorescence image for the same region in the top left is shown on the bottom left, scale . Over the total area of the HSQ array, DNA was synthesized in the spatially defined areas by the MAS. Individual mirrors can be identified in the fluorescence image, and at higher magnification we see that the fluorescence comes directly from the individual HSQ structures. A box outlines the higher resolution image (bottom right) of hybridization on patterned HSQ, scale . Letters indicate the different doses used in synthesis: , , , , line scans for these different dosed regions are given in Fig. 3.
Linescans taken through the central region of the synthesis area for different light exposure doses in ssDNA synthesis [corresponding to Figs. 2(a)–2(d)]. Fluorescence intensity (arbitrary units) vs position plots across HSQ (peaks) and silicon (minimum) surfaces. Graph A is for DNA synthesized with (highest dose) and shows the lowest amount of modulation in hybridization signal. Graph B, shows the highest amount of modulation with peaks higher than 3072. The second highest amount of modulation is shown in graph C for synthesis . For graph D, and shows relatively low synthesis. Peaks correspond to the edges of the mirrors were the light intensity tapers off as oppose to the central region of the mirrors where the delivered light intensity is highest is observable in A and D.
Fluorescent microscopy of Cy-3 labeled ssDNA hybridized to different sized HSQ line patterns (top row). Linescans as indicated by the white line in the microscope images are plotted below. For each area DNA was synthesized using a dose. lines and spaces showing clear modulation (a). lines and spaces with the line scan taken at the edge of the HSQ array (b). lines and spaces also measured at the edge of the HSQ array (c). For each image the scale . Peak intensities coincide with HSQ lines showing the detection of hybridized DNA.
Hybridization with Cy-3 labeled compliment to immobilized ssDNA imaged by fluorescence microscopy (top), scale bar is . Linescan measurement of the fluorescence intensity profile (bottom): lines with spaces (a). lines with spaces (b). Both A and B show clear evidence of localized hybridization (peak fluorescence) of complimentary oligonucleotide to DNA on HSQ patterns. A slight slope is noticeable in both line scans which is from nonuniform illumination in the optical microscope.
Passivation tests: Hybridization measured by fluorescence is plotted vs the light dose used for the synthesis of a 30 nucleotide sequence. Dose range: , for increments. For each trend, the background fluorescence is subtracted and the average intensity is plotted on a logarithmic scale. Glass (hash mark) performs as expected with high yield of DNA synthesis. Glassy carbon untreated (diamond) and hydrogen plasma treated glassy carbon (triangle) show relatively low synthesis yield for the range of doses tests. Piranha cleaned silicon and vapor deposited HMDS on silicon were both below the level of detection (BLD) and thus are not plotted on this figure. Piranha cleaned silicon was derivitized with a silane linker and its synthesis response is plotted (dots) showing moderate synthesis.
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