Forensic Lab-on-a-Disc for enhanced sperm cell purification in differential extraction workflows

Efficient separation of sperm cells from mixed biological samples is critical in forensic DNA analysis, particularly in sexual assault investigations. Sperm separation in conventional differential extraction (cDE) methods is labor-intensive, time-consuming, and prone to sample loss, often compromising DNA recovery and quality. Here, we present the Forensic Lab-on-a-Disc, a centrifugal microfluidic device that enables rapid, semi-automated sperm cell and non-sperm DNA separation. Leveraging centrifugal force and tangential flow filtration with dual membranes (10 µm and 1 µm pore sizes), the device effectively separates sperm cells from epithelial cell lysates and debris. Optimization experiments identified a 10 µm pore size for the first filter as optimal for maximizing sperm cell recovery while depleting non-sperm DNA. A single washing cycle was sufficient to preserve male DNA ratio (defined as Y-DNA divided by total gDNA) while minimizing sperm loss. Comparative analyses showed that the Forensic Lab-on-a-Disc, which specifically replaces the sperm-cell separation step within the cDE workflow, outperforms the conventional method by delivering significantly higher male DNA ratio, faster processing times (∼6 min), and improved reproducibility. STR profiling confirmed male DNA recovery with minimal non-sperm DNA carryover, even in samples with epithelial-to-sperm DNA equivalent ratios as high as 1000:1. Featuring a compact, semi-automated, and multi-sample processing capability, the Forensic Lab-on-a-Disc reduces operator variability and minimizes non-sperm-derived lysate carryover risks that compromise mixture interpretation, offering a transformative solution to accelerate forensic DNA analysis in sexual assault cases.