Isolation of a Low Number of Sperm Cells from Female DNA in a Glass-PDMS-Glass Microchip via Bead-Assisted Acoustic Differential Extraction.

We report an improved separation method for the isolation of sperm cells from dilute, "large volume" samples containing female DNA using bead-assisted 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.

A disposable laser print-cut-laminate polyester microchip for multiplexed PCR via infra-red-mediated thermal control.

Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-µL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s(-1) with a cooling rate of roughly -12 ± 0.9 °C s(-1) assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human ß-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human ß-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.

Low-power microwave-mediated heating for microchip-based PCR.

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.

A thermally responsive phospholipid pseudogel: tunable DNA sieving with capillary electrophoresis.

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.

Epoxy insulated carbon fiber and carbon nanotube fiber microelectrodes.

Carbon-fiber microelectrodes (CFMEs) are typically constructed from glass capillaries pulled to a fine taper or from a polyimide-coated capillary that is 90 µm in outer diameter. Here, a new fabrication method is developed to insulate carbon-fiber microelectrodes with a thin epoxy coating. A polytetrafluoroethylene (Teflon) mold was laser etched with channels 30-40 µm deep and wide and each channel filled with Armstrong C7 epoxy. A carbon fiber was laid into each channel so that the fiber extended past the mold, and the epoxy cured in an oven. One end of the fiber was trimmed to about 100 µm to form a cylindrical carbon-fiber microelectrode, while the other end was attached to a pin and connected to a potentiostat. Epoxy-insulated electrodes were tested with fast-scan cyclic voltammetry. For dopamine, the sensitivity is similar to glass and polyimide-coated capillary electrodes with a linear range of 0.1 to 10 µM and a LOD of 24 nM. SU-8 epoxy was tested as an alternative insulator because it cures at a lower temperature using light, but it was more brittle. Carbon nanotube fibers were also successfully insulated with epoxy. Epoxy- insulated CFMEs were used to detect stimulated dopamine release in vivo. Epoxy-insulated electrodes are smaller in diameter than polyimide-coated capillary electrodes and amenable to mass production. They are advantageous for use in higher order mammals, where glass is not permitted, and with alternative electrode materials, such as carbon nanotube fibers, that cannot be fabricated in a capillary puller.

Warfarin genotyping in a single PCR reaction for microchip electrophoresis.

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.

Electrophoretic microfluidic devices for mutation detection in clinical diagnostics.

BACKGROUND: In an era of growing interest in personalized medicine - where ubiquitous patient genotyping holds unprecedented clinical utility - rapid, sensitive and low-cost methodologies will be required for the detection of genetic variants correlative with disease. Electrophoretic microfluidic devices have emerged as a promising platform for such analyses, inherently offering faster analysis, excellent reagent economy, a small laboratory footprint and potentially seamless integration of multiple analytical steps. OBJECTIVE: Although glass and polymeric microchips have recently been developed for a wide variety of medical applications, this review focuses on their application to the detection of clinically relevant genomic DNA mutations and polymorphisms. METHOD: Mutation analysis techniques, including direct gene sizing, enzyme-based assays, heteroduplex analysis, single-strand conformational polymorphism analysis, and multiplex, allele-specific and methylation-specific PCR are included. CONCLUSION: 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.