Based on abstract submitted by the proposer: Forensic laboratories are often faced with sexual assault DNA evidence that requires labor-intensive, time-consuming sample processing, and there is a need for streamlined workflows that deliver high quality results with reduced cost and analyst time. A microfluidic solution that exploits acoustic trapping offers an exciting paradigm shift from conventional differential extraction, one that can match the selectivity of advanced technologies, such as laser microdissection and increases the sensitivity while reducing the analysis time. Here, we propose to exploit the basic chemistry of current differential extraction (DE), but trap sperm cells specifically and directly using acoustics as a means of physically separating the male fraction (cells) from the female fraction (lysed epithelial cells) in evidentiary sexual assault samples. The currently utilized DE methodology, while efficient, is labor-intensive, time-consuming, presents a higher risk for contamination, and contributes to a significant backlog of forensic evidence to be processed in many laboratories. Consequently, a need exists for a multiplex system that addresses current method limitations and provides the following advantages: is amendable to low cell sperm count samples; has comparable selectivity to conventional DE methods; provides enhanced cell sperm recovery and fraction purity per separation; doesn't use risky procedures; is automated; provides a rapid turn-around time; leaves the sperm cells intact for future methods that separate sperm from multiple donors; permits decreased instrument and reagent costs; is easily interfaced with rapid microfluidic DNA technologies. Acoustic-based, microfluidic adaptation is an attractive option for this application as it combines high-throughput potential with low reagent and sample volume needs, while in a closed system that can be entirely automated (no intermediate sample transfer), and can ultimately be interfaced with a fully-integrated microfluidic STR profiling system. Prior NIJ funding facilitated the evolution of Acoustic Differential Extraction (ADE) to evolve from concept to successful demonstration of sperm cell trapping. Two challenges remain to be addressed with the existing ADE system for inclusion in standard laboratory work flows and these will be addressed in the proposed work. First, the current methodology (optimized with mock samples) needs to be shown effective with more relevant forensic samples. We need to ascertain that potential interferents likely encountered in vaginal swabs (bacteria, blood and yeast) do not affect the trapping of sperm cells. The ADE system needs to be challenged with bona fide sexual assault samples; samples containing high and low sperm cell numbers, and samples in small and large volumes. This will be facilitated by ADE of casework vaginal swabs provided by the Mesa PD Crime Laboratory. Second, two ADE instruments will be built for deployment and testing in bona fide forensic environments.