Towards an affinity-free, centrifugal microfluidic system for rapid, automated forensic differential extraction

Biological evidence originating from victims of sexual assault is often comprised of unbalanced cellular mixtures with significantly higher contributions from the victim's genetic material. Enrichment of the forensically-critical sperm fraction (SF) with single-source male DNA relies on differential extraction (DE), a manually-intensive process that is prone to contamination. Due to DNA losses from sequential washing steps, some existing DE methods often fail to generate sufficient sperm cell DNA recovery for perpetrator(s) identification. Here, we propose an enzymatic, 'swab-in' rotationally-driven microfluidic device to achieve complete, self-contained, on-disc automation of the forensic DE workflow. This 'swab-in' approach retains the sample within the microdevice, enabling lysis of sperm cells directly from the evidence cutting to improve sperm cell DNA yield. We demonstrate clear proof-of-concept of a centrifugal platform that provides for timed reagent release, temperature control for sequential enzymatic reactions, and enclosed fluidic fractionation that allows for objective evaluation of the DE process chain with a total processing time of ≤15 min. On-disc extraction of buccal or sperm swabs establishes compatibility of the prototype disc with: 1) an entirely enzymatic extraction method, and 2) distinct downstream analysis modalities, such as the PicoGreen® DNA assay for nucleic acid detection and the polymerase chain reaction (PCR).