A simple approach to fabricating hydrogel-based DNA microarrays is reported by physically entrapping the rolling circle amplification (RCA) product inside printable in situ gelling hydrazone cross-linked poly(oligoethylene glycol methacrylate) hydrogels. The hydrogel-printed RCA microarray facilitates improved RCA immobilization (>65% even after vigorous washing) and resistance to denaturation relative to RCA-only printed microarrays in addition to size-discriminative sensing of DNA probes (herein, 27 or fewer nucleotides) depending on the internal porosity of the hydrogel. Furthermore, the high number of sequence repeats in the concatemeric RCA product enables high-sensitivity detection of complementary DNA probes without the need for signal amplification, with signal/noise ratios of 10 or more achieved over a short 30 min assay time followed by minimal washing. The inherent antifouling properties of the hydrogel enable discriminative hybridization in complex biological samples, particularly for short (∼10 nt) oligonucleotides whose hybridization in other assays tends to be transient and of low affinity. The scalable manufacturability and efficient performance of these hydrogel-printed RCA microarrays thus offer potential for rapid, parallel, and inexpensive sensing of short DNA/RNA biomarkers and ligands, a critical current challenge in diagnostic and affinity screening assays.
Keywords: DNA microarrays; DNA sensing; antifouling; printable hydrogels; rolling circle amplification product; size selectivity.