The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the β-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the β-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time.

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In an aqueous solution the phospholipids dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudogels) in which small temperature changes of 5-6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. Advantages of utilizing phospholipid pseudogels for sieving are the ease with which they are introduced into the separation channel and the potential to implement gradient separations. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 μm i.d. fused silica capillary. Assessment of the phospholipid pseudogel with a Ferguson plot yields an apparent pore size of ~31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Nearly single base resolution of short tandem repeats relevant to human identification is accomplished with 30 min separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudogel preparation described here provides a new innovation to sieving-based capillary separations.

We report an improved separation method for the isolation of sperm cells from dilute, "large volume" samples containing female DNA using beadassisted acoustic trapping. In an enclosed glass−PDMS−glass (GPG) resonator, we exploit a three-layer microfluidic architecture to generate "trapping nodes" in ultrasonic standing waves. We investigate the dependence of trapping efficiency on particle concentration for both sperm cells and polymeric beads. After determination of the critical concentration of polymeric beads required to seed the trapping event, sperm cells in dilute solution are trapped as a result of the enhanced secondary radiation force (SRF). Sperm-cell-containing samples with volumes up to 300 μL and cell concentrations as low as ∼10 cells/μL are amenable to effective trapping in the presence of an abundance of female DNA in solution. Complete processing of samples is accomplished with separation of the female and male fractions within 15 min. We demonstrate that the collected fractions are amenable to subsequent DNA extraction, short tandem repeat PCR, and the generation of STR profiles for the isolated sperm cells.

Biochemical techniques, such as the polymerase chain reaction (PCR), can take up to 3.5 h for completion using a commercial bench-top instrument, creating a bottleneck in sample preparation processes. PCR has been successfully adapted to microfluidic devices, reducing the time needed to as little as 7-10 min. Recently, a trend in the field is to use alternative substrates, such as poly(methyl methacyrlate) (PMMA), for the fabrication of microfluidic devices. PMMA has several advantages over more expensive substrates including rigidity without fragility, disposability, and it is easy to fabricate, using techniques such as hot embossing or CO 2 laser ablation. Here, we report the fabrication of PMMA microdevices to explore their effectiveness for PCR amplification. Several types of PMMA microdevices were fabricated using a CO 2 laser ablation system, with two or three PMMA layers of different thicknesses. Bonding of the microdevices was significantly improved through the use of a grid system etched into the device, allowing for trapped air to escape, eliminating leakage. Using infrared thermal cycling, the ramping rates were determined to be dependent on the thickness of the PMMA used in fabrication, allowing for customization of cycling conditions. Further reduction of the thermal mass by isolation of the chambers provided a significant increase in the heating and cooling rates (up to 6.19 ( ± 0.32 • C s −1 ) and −7.99 ( ± 0.06 • C s −1 ), respectively). Bubble formation, a chronic problem in microfluidic systems in general and problematic during the heating phase of PCR, was minimized through the use of a biocompatible adhesive/manifold combination to seal the reservoirs. Finally, successful PCR amplification was demonstrated with both a fragment from the β-globin gene and 15 tetra-nucleotide repeat regions with a sex-typing marker in a conventional STR kit with the latter facilitated by the dynamic coating of the fluidic architecture with poly(ethylene glycol) (PEG).

The majority of microfluidic devices used as a platform for low-cost, rapid DNA analysis are glass devices; however, microchip fabrication in glass is costly and laborious, enhancing the interest in polymeric substrates, such as poly (methyl methacrylate) (PMMA), as an inexpensive alternative. Here, we report amplification in PMMA polymerase chain reaction (PCR) microchips providing full short tandem repeat profiles (16 of 16 loci) in 30-40 min, with peak height ratios and stutter percentages that meet literature threshold requirements. In addition, partial profiles (15 of 16 loci) were generated using an ultrafast PCR method in 17.1 min, representing a ~10-fold reduction in reaction time as compared to current amplification methods. Finally, a multichamber device was demonstrated to simultaneously amplify one positive, one negative, and five individual samples in 39 min. Although there were instances of loci dropout, this device represents a first step toward a microfluidic system capable of amplifying more than one sample simultaneously.

Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

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