An Affinity-Free, Centrifugal Microfluidic System for Rapid, Automated Differential Extraction

Some of the most common case types received in a forensic DNA laboratory are those involving sexual assault/battery, rape, or attempted rape. Often, the biological evidence collected for these cases is incorporated into a sexual assault evidence collection kit (SAECK). Samples in SAECKs vary but often include swabs from areas of reported penetration/ejaculation (e.g., skin, vaginal, and anal swabs); these swabs are likely to include sperm cells from a male suspect as well as cells from the victim. Differential extraction (DE) procedures exist that permit the separation of perpetrator sperm cells from the victims cells, greatly reducing the likelihood of complicated DNA mixtures. Existing DE methods are difficult to automate and most often require time-intensive, hands-on processes that are not 100% efficient in separating the perpetrator and victim DNA contributions. Microdevices may circumvent these issues by offering an alternative to traditional manual SAECK sample preparation. Most researchers interested in microdevice development are focused on complete integration of laboratory workflow in a single microdevice (i.e., lab-on-a-chip or micro total analysis systems [μTAS]), which is currently many years away from implementation for SAECK sample processing. Thus, our research group has shifted towards making more immediate use of microdevice advancements for replacing only a portion of the total SAECK workflow. Our approach would improve processing time, minimize variability associated with manual procedures, and shift manual labor to downstream processes that require more extensive training and examiner time (e.g., CE analysis, DNA profile interpretation, and reporting). The approach described in this proposal compromises a simple, inexpensive instrument and disc that does not rely on affinity-based capture of cells. It integrates two existing, recently developed on-chip microfluidic modules for DNA extraction and laser-based closable microvalving, as well as an existing rotational platform for microfluidic control. Furthermore, this approach exploits emerging enzyme-based, temperature-controlled methods for DE (TCDE) while avoiding the need for costly immunocapture (i.e., no antibodies). Prototype devices will produce purified, PCR-ready DNA that is compatible with existing chemistries for quantitation, PCR amplification, and amplicon separation/detection via capillary electrophoresis. Collectively, the microfluidic and TCDE strategies will replace some of the most laborious steps in sexual assault evidence processing. This proposed microdevice could be available for testing, commercialization, and technology transfer within a two-year window. Additionally, it could be easily validated/implemented by operational forensic DNA laboratories, i.e., this system would replace only upstream sample preparation steps and would not interfere with existing downstream PCR-based methods. 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