期刊
ACS APPLIED MATERIALS & INTERFACES
卷 11, 期 34, 页码 30534-30541出版社
AMER CHEMICAL SOC
DOI: 10.1021/acsami.9b07755
关键词
microarray; hydrogel; phosphoramidite; DNA synthesis; silanation; photoresist
资金
- National Human Genome Research Institute of the National Institutes of Health [R43HG008582]
Current techniques for making high-resolution, photolithographic DNA microarrays suffer from the limitation that the 3' end of each sequence is anchored to a hard substrate and hence is unavailable for many potential enzymatic reactions. Here, we demonstrate a technique that inverts the entire microarray into a hydrogel. This method preserves the spatial fidelity of the original pattern while simultaneously removing incorrectly synthesized oligomers that are inherent to all other microarray fabrication strategies. First, a standard 5'-up microarray on a donor wafer is synthesized, in which each oligo is anchored with a cleavable linker at the 3' end and an Acrydite phosphoramidite at the 5' end. Following the synthesis of the array, an acrylamide monomer solution is applied to the donor wafer, and an acrylamide-silanized acceptor wafer is placed on top. As the polyacrylamide hydrogel forms between the two wafers, it covalently incorporates the acrydite-terminated sequences into the matrix. Finally, the oligos are released from the donor wafer upon immersing in an ammonia solution that cleaves the 3'-linkers, thus freeing the oligos at the 3' end. The array is now presented 3'-up on the surface of the gel-coated acceptor wafer. Various types of on-gel enzymatic reactions demonstrate a versatile and robust platform that can easily be constructed with far more molecular complexity than traditional photolithographic arrays by endowing the system with multiple enzymatic substrates. We produce a new generation of microarrays where highly ordered, purified oligos are inverted 3'-up, in a biocompatible soft hydrogel, and functional with respect to a wide variety of programable enzymatic reactions.
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