Reversible static-site PCR via dual-phase confinement in digital microfluidics.

Digital microfluidics (DMF) enables programmable manipulation of discrete droplets for multi-step biochemical workflows; however, integrating polymerase chain reaction (PCR) into reconfigurable DMF systems remains challenging due to evaporation, surface fouling, and loss of droplet mobility during PCR. Here, we present a dual-phase pinning system (DPPS) for stable and reversible static-site PCR in DMF. In this approach, an aqueous PCR droplet is locally encapsulated within an immiscible lubricating phase and stabilized through combined mechanical confinement and continuous electrowetting actuation. During thermocycling, the surrounding phase is pinned by microfabricated posts while the aqueous droplet remains electrically anchored, enabling stable confinement without permanent surface modification. Following amplification, the droplet can be released and re-mobilized for downstream processing. Evaporation remained below 10% during PCR, and amplification performance remained comparable to conventional benchtop PCR. The approach was further integrated into next-generation sequencing (NGS) library preparation and target enrichment workflows, where amplified droplets remained compatible with downstream processing and sequencing. These results establish a practical strategy for reversible static-site PCR in digital microfluidics and support broader integration of PCR within reconfigurable droplet-based workflows.