Fabrication of microcolumn arrays for high-throughput oligonucleotide synthesis using 3D printing

Advances in high-density oligonucleotide array synthesis have enabled the creation of increasingly large DNA libraries. However, current planar array platforms are designed to synthesize only femtomole-scale amounts of oligonucleotides per feature, due to their inherently small feature sizes and high spatial density. Conversely, column-based synthesis supports higher per-feature output but accommodates fewer sequences. In this study, we introduce a 3D-printed DNA synthesis substrate that combines the strengths of both approaches. Using an LCD-based 3D printer and photopolymer resins, we fabricated micrometer-scale columns filled with controlled pore glass (CPG), enabling high-density oligonucleotide synthesis with sub-nanomole-scale output per feature. We screened commercial resins for chemical resistance and optimized printing parameters to fabricate rectangular microholes with gap widths smaller than CPG beads, capable of securely retaining CPG beads. A 15-mer poly(dT) sequence was synthesized across 1000 microcolumns on the array using an inkjet-based phosphoramidite system. The substrate was fabricated in ~ 2.5 h at an estimated material cost of just $0.12 per unit.