Integrated Microfluidic System for Rapid 89‐Plex Y‐SNP Profiling: Development and Forensic Validation

Current Y‐chromosomal single‐nucleotide polymorphism (Y‐SNP) detection technologies in forensic genetics often rely on bulky equipment, complex procedures, and lack adaptability to field conditions. To address these limitations, we developed an 89‐plex microfluidic Y‐SNP system comprising a disposable DNA extraction/amplification chip and a capillary electrophoresis chip. Using in situ lyophilization, the reagents are stabilized for long‐term storage. Integrated with a portable device, the system enables a fully automated “sample‐in‐answer‐out” workflow and delivers complete 89‐locus Y‐SNP genotyping within 139 min. The system includes two panels—AIYSNP42 for global high‐frequency haplogroups and AIYSNP47 for East Asian O‐haplogroup subclades—designed on the basis of the International Society of Genetic Genealogy (ISOGG) phylogenetic tree for multi‐level resolution. Validation showed a detection sensitivity of 2.5 ng of DNA, with a 93.6% genotyping success rate across 94 forensic samples. It maintained performance under environmental inhibitors (humic acid ≤ 100 ng/µL, hemin ≤ 300 µM, indigo ≤ 15 mM) and moderate UV‐induced DNA degradation. The system demonstrated excellent reproducibility (coefficient of variation, CV < 0.5%) and reliably detected male DNA in mixtures (≥2% in male–female, ≥33% in male–male). This microfluidic system reduces the reliance on the need for conventional laboratory workflows and supports rapid, on‐site Y‐SNP analysis for pedigree tracing, ancestry inference, and mixture interpretation.