Integrated microfluidic platform for programmable multi-window DNA fractionation and <i>in situ</i> recovery

Nucleic acid size selection underpins applications from sequencing to genome engineering, yet current methods impose trade-offs among separation breadth, recovery fidelity, and operational throughput. To address these trade-offs, we engineered a compact on-chip microfluidic field-inversion gel electrophoresis (MFIGE) platform that integrates programmable deoxyribonucleic acid (DNA) fractionation with <i>in situ</i> dual-membrane DNA recovery, which avoids manual gel excision in a closed, low-shear, and automation-ready format. MFIGE delivers multi-window fractionation beyond 140 kbp, with total recovery rate up to 57.9% and operates robustly across different sample types. In nanopore sequencing validation, MFIGE reshaped the read-length distribution and substantially increased long-read output. It generated more than 2900 reads exceeding 100 kbp, 976-fold higher than long-fragment accumulator (LA) reagent-based fractionation and 47.2-fold higher than the unfractionated control. It also raised the N50 to 33.58 kbp, 1.5-fold higher than LA and 3.4-fold higher than unfractionated. By programming the field strength, we concentrated over 94% of >50 kbp fragments in target wells, enabling precise, high-fidelity capture. Together, these results position MFIGE as a practical front end for long-read sequencing library preparation and other applications demanding precise DNA sizing.