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).

Characterization of a Centrifugal Microfluidic Orthogonal Flow Platform.

To bring to bear the power of centrifugal microfluidics on vertical flow immunoassays, control of flow orthogonally through nanoporous membranes is essential. The on-disc approach described here leverages the rapid print-cut-laminate (PCL) disc fabrication and prototyping method to create a permanent seal between disc materials and embedded nanoporous membranes. Rotational forces drive fluid flow, replacing capillary action, and complex pneumatic pumping systems. Adjacent microfluidic features form a flow path that directs fluid orthogonally (vertically) through these embedded membranes during assay execution. This method for membrane incorporation circumvents the need for solvents (e.g., acetone) to create the membrane-disc bond and sidesteps issues related to undesirable bypass flow. In other recently published work, we described an orthogonal flow (OF) platform that exploited embedded membranes for automation of enzyme-linked immunosorbent assays (ELISAs). Here, we more fully characterize flow patterns and cellulosic membrane behavior within the centrifugal orthogonal flow (cOF) format. Specifically, high-speed videography studies demonstrate that sample volume, membrane pore size, and ionic composition of the sample matrix significantly impact membrane behavior, and consequently fluid drainage profiles, especially when cellulosic membranes are used. Finally, prototype discs are used to demonstrate proof-of-principle for sandwich-type antigen capture and immunodetection within the cOF system.