Electronically controlled deprotection chemistry for multiplex enzymatic DNA synthesis on a chip with single-base...

Enzymatic deoxyribonucleic acid (DNA) synthesis (EDS) is an environmentally friendly approach capable of generating longer and more complex sequences than chemical synthesis, making it a promising next-generation technology for high-throughput single-stranded DNA production. However, precise sequence control at high throughput remains a key challenge. Here, we present a novel electronically controlled deprotection chemistry (ECDC) integrated with a hydrogel-primer modification system on-chip for efficient multiplexed EDS. Electrochemically generated HNO₂ at the working electrodes selectively converts the 3'-oxyamino group of DNA into a hydroxyl group, enabling precise spatiotemporal control of a multipixel synthesis array and facilitating future automation. This platform enables parallel EDS with single-base resolution on silicon chips. In four-nucleotide validation experiments, single-sequence synthesis could achieve 100% accuracy, while dual-sequence synthesis reached an average accuracy of approximately 96%. Our approach provides a highly accurate solution for high-throughput ssDNA synthesis, laying the foundation for scalable and automated enzymatic DNA manufacturing.