Photolithographic synthesis of high-density DNA probe arrays: Challenges and opportunities
Scaling of sequence density on commercial DNA microarrays from Affymetrix fabricated with contact lithography and photoremovable protecting group chemistry. Sequence density on a chip has historically doubled approximately every 15 months, and currently exceeds .
Process flow for microarray fabrication using a photoresist imaging layer. (a) Substrates are prepared with an appropriate film stack, patterned with wafer and chip alignment marks, cleaned, and silanated to achieve the appropriate initial site density. (b) Substrates are coupled with a molecular spacer protected at the terminal end with a DMTr group. (c) Photoresist film is coated onto the substrate and exposed with the first mask image to generate a photoacid pattern. (d) During the postexposure delay, photoacid in the resist film detritylates DMTr protecting groups rendering free hydroxyl sites. (e) Resist film is removed, and the substrate is process through cycles of phosphoramidite coupling, capping, and oxidation in a flow cell to add the first monomer in the oligonucleotide sequence . (f) Steps (c) through (e) are repeated to pattern the remaining sequence of the array (three additional cycles illustrated for , , and ). (g) After completion of the entire sequence, substrates are processed through final base deprotection, dicing, and packaging.
Contrast curve for the extent of detritylation by photoacid in a typical PAG system. Contrast, , for this resist process was measured as 10.6, with a photospeed of approximately .
HPLC chromatogram from a stepwise efficiency determination for the photoresist process using a cytidine 6-mer. The oligos 1–6 monomers in length elude the column at retention times of approximately 11.5, 13.8, 16, 18, 19.8, and 21.5 min, respectively. Quantification of relative oligo concentration by peak integration resulted in a relative yield for 6-mers of and a stepwise efficiency of .
Histograms demonstrating the theoretical probe composition for the synthesis of 25-mers with 90, 95, and stepwise efficiency.
SEM micrographs for nominally line and space patterns exposed at 200, 270, and through postexposure delay.
CD as a function of postexposure delay as determined by analysis of SEM images shown in Fig. 6.
Fluorescence analogy to a traditional Bossung curve for nominally dark features in pitch checkerboard patterns. Fluorescence intensity plotted as a function of stepper focus offset for variations in exposure dose. Intensity data were not corrected for background or resolution limitations of the scanner. Selection of fluorescence images is included.
Hypothetical detritylation response as a function of relative intensity for PAG films of varying contrast from 15 to 2.
Simulated relative intensity and extent of detritylation images for an isolated bright and an isolated dark feature. The extent of detritylation was calculated for two values of contrast, 10 and 3.
Cut lines through the center of the two-dimensional images plotted in Fig. 9. (A) relative intensity of the aerial image; (B) extent of detritylation for a contrast of 10; and (C) extent of detritylation for a contrast of 3. Cut lines from both the isolated bright (dashed line) and isolated dark feature (solid line) were overlaid. The system with a contrast of 10 eliminated detritylation within the spaces between adjacent features and in the position of the isolated dark feature. A contrast of 3 was not sufficient to completely stop detritylation within the isolated dark feature location, and a relative light intensity of less than partially illuminated this area.
Extent of detritylation as a function of PAG film contrast for two locations within the isolated dark feature, the center of the feature and the location of maximum extent of detritylation. The inset plot displays an enlarged image of the central feature from Fig. 11(C) for a system with a contrast of 3.
Fluorescence micrograph of the top left corner of an experimental GeneChipTM expression array fabricated with a pitch. The chip was hybridized with a complex RNA target, and processed under conditions similar to standard product applications.
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