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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.

Microwave energy has been used to rapidly heat food and drinks for decades, in addition to assisting other chemical reactions. However, only recently has microwave energy been applied in microfluidic systems to heat solution in reaction chambers, in particular, the polymerase chain reaction (PCR). One of the difficulties in developing microwave-mediated heating on a microchip is the construction of the appropriate architecture for delivery of the energy to specific micro-areas on the microchip. This work employs commercially-available microwave components commonly used in the wireless communications industry to generate a microwave signal, and a microstrip transmission line to deliver the energy to a 1 μL reaction chamber fabricated in plastic microdevices. A model was developed to create transmission lines that would optimally transmit energy to the reaction chamber at a given frequency, minimizing energy usage while focusing microwave delivery to the target chamber. Two different temperature control methods were demonstrated, varying microwave power or frequency. This system was used to amplify a fragment of the lambda-phage genome, thereby demonstrating its potential for integration into a portable PCR system.

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).

BACKGROUND Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. METHODS We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. RESULTS Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). CONCLUSIONS This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

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Further development of 'lab-on-a-chip' or 'micro total analysis system' technologies ultimately aims to streamline and miniaturize the entire genetic analysis process, enabling rapid, point-of-care analysis for molecular diagnostics.