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.