Completion of the SONIC-DE 2.0 System for Implementation in Forensic Laboratories

American forensic laboratories are overwhelmed with sexual assault DNA evidence for processing, emphasizing the need for a streamlined process that delivers high quality results with reduced cost and technician-required time. Microfluidics, in conjunction with acoustic trapping, offers an exciting paradigm shift in forensic science towards a fully integrated closed system capable of matching the selectivity of advanced laser microdissection, while enabling fiscally feasible high throughput. Here, we propose to utilize the difference in size of sperm cells and epithelial cells to separate the male and female DNA for successful differential extraction from evidentiary samples. We propose to further explore our hands-free cell sorting technique of acoustic differential extraction (ADE), bringing together the benefits of microfluidics, while achieving increased specificity of separation of male and female cells through the application of an ultrasonic acoustic wave. Here, the acoustic wave will trap sperm cells within the microfluidic device, separating them from the female cell lysate. The ADE method has shown successful trapping of sperm cells from a highly dilute sample (~ 1 cell/L), generating a full male STR profile. This demonstrates the first instance of ADE operating within forensically relevant concentrations while functioning at an elevated flow rate. Evaluation of our prototype ADE technology at the Palm Beach County Sheriffs Office showed that we can successfully isolate sperm cells from real sexual assault samples, and purify them in an automated fashion to produce male STR profiles. However, we also observed that samples with a high number of lysed epithelial cells demonstrate a shift in the optimal trapping frequency, moving 0.02-0.05 MHz higher than trapping in water. This small change is enough to impede trapping ability, and results in loss of sperm cells. Our continued development of this technology will incorporate an adaptive feedback system wherein periodic electronic monitoring will identify the optimal trapping frequency during a run, and continuously update the actual frequency being used. In this manner we can confidently trap sperm cells across any sample bandwidth, and eliminate the issues we observed during external evaluation. We will continue our partnerships with Jeff Hickey (Microlab Inc.), Mesa Police Department (Mesa, Arizona), and the Palm Beach County Sheriffs Office (Palm Beach, Florida) in order to make necessary hardware and chemical adjustments, and to conduct a second external evaluation of the modified technology. The new prototype ADE instrument will be field-ready, robust, and offer many advantages over current differential extraction techniques. Note: This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14). CA/NCF