Fast Thermocycling in Custom Microfluidic Cartridge for Rapid Single-Molecule Droplet PCR.

The microfluidic droplet polymerase chain reaction (PCR), which enables simultaneous DNA amplification in numerous droplets, has led to the discovery of various applications that were previously deemed unattainable. Decades ago, it was demonstrated that the temperature holding periods at the denaturation and annealing stages in thermal cycles for PCR amplification could be essentially eliminated if a rapid change of temperature for an entire PCR mixture was achieved. Microfluidic devices facilitating the application of such fast thermocycling protocols have significantly reduced the time required for PCR.

Droplet size affects the degree of separation between fluorescence-positive and fluorescence-negative droplet populations in droplet digital PCR.

In droplet digital polymerase chain reaction (ddPCR) tests, a single sample solution is divided into many water-in-oil droplets. At the endpoint of PCR amplification, individual droplets are classified as either fluorescence-positive (FL(+)) or fluorescence-negative (FL(-)) droplets based upon their fluorescence amplitudes. Populations of FL(+) and FL(-) droplets can be seen in the histogram of fluorescence amplitude.

CO-Laser-Micromachined, Polymer Microchannels with a Degassed PDMS slab for the Automatic Production of Monodispersed Water-in-Oil Droplets.

In our recent study, we fabricated a pump/tube-connection-free microchip comprising top and bottom polydimethylsiloxane (PDMS) slabs to produce monodispersed water-in-oil droplets in a fully automated, fluid-manipulation fashion. All microstructures required for droplet production were directly patterned on the surfaces of the two PDMS slabs through CO2-laser micromachining, facilitating the fast fabrication of the droplet-production microchips. In the current extension study, we replaced the bottom PDMS slab, which served as a microfluidic layer in the microchip, with a poly(methyl methacrylate) (PMMA) slab.

Convenient microfluidic cartridge for single-molecule droplet PCR using common laboratory equipment.

We have previously established a cost-efficient in-house system for single-molecule droplet polymerase chain reaction (PCR) using a polydimethylsiloxane microfluidic cartridge and common laboratory equipment. However, the microfluidic cartridge was only capable of generating monodisperse water-in-oil droplets. Therefore, careful and time-consuming manual droplet handling using a micropipette was required to transfer droplets between the three discrete steps involved in the workflow of droplet PCR-i.

A simplified PDMS microfluidic device with a built-in suction actuator for rapid production of monodisperse water-in-oil droplets.

We previously established an automatic droplet-creation technique that only required air evacuation of a PDMS microfluidic device prior to use. Although the rate of droplet production with this technique was originally slow (∼10 droplets per second), this was greatly improved (∼470 droplets per second) in our recent study by remodeling the original device configuration. This improvement was realized by the addition of a degassed PDMS layer with a large surface area-to-volume ratio that served as a powerful vacuum generator.

Clinical Advantages of Using Low Tube Voltage in Third-Generation 192-Slice Dual-Source Computed Tomographic Angiography Before Transcatheter Aortic Valve Implantation.

Low-voltage computed tomographic angiography (CTA) is a highly effective technique to reduce contrast media volume. We sought to examine the suitability of low tube voltage CTA with a reduced contrast media volume protocol using third-generation 192-slice dual-source CT in patients undergoing transcatheter aortic valve implantation (TAVI). CTA was performed to aid TAVI planning for 40 consecutive patients with severe aortic stenosis.

Vacuum-driven fluid manipulation by a piezoelectric diaphragm micropump for microfluidic droplet generation with a rapid system response time.

Recently, we developed a convenient microfluidic droplet generation device based on vacuum-driven fluid manipulation with a piezoelectric diaphragm micropump. In the present study built on our previous work, we investigate the influence of settings applied to the piezoelectric pump, such as peak-to-peak drive voltage (V ) and wave frequency, on droplet generation characteristics. Stepwise adjustments to the drive voltage in ±10-V increments over the range of 200-250 V during droplet creation revealed that the droplet generation rate could be reproducibly controlled at a specific drive voltage.

Separation-free single-base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites.

A separation-free single-base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele-specific oligonucleotide primer (i.e.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

Dual-energy bone removal computed tomography (BRCT): preliminary report of efficacy of acute intracranial hemorrhage detection.

Purpose: One of the major applications of dual-energy computed tomography (DECT) is automated bone removal (BR). We hypothesized that the visualization of acute intracranial hemorrhage could be improved on BRCT by removing bone as it has the highest density tissue in the head. This preliminary study evaluated the efficacy of a DE BR algorithm for the head CT of trauma patients.

Rapid automatic creation of monodisperse emulsion droplets by microfluidic device with degassed PDMS slab as a detachable suction actuator.

We previously developed a technique that enabled automatic creation of monodisperse water-in-oil droplets with the use of an air-evacuated PDMS microfluidic device. Although the device generated droplets over a long-time period, the production rate was slow (∼10 droplets per second). In the current study, we aimed to improve this rate, using the same fluid pumping principle described in our previous work, by remodeling our device configuration.

A compact and facile microfluidic droplet creation device using a piezoelectric diaphragm micropump for droplet digital PCR platforms.

We have exploited a compact and facile microfluidic droplet creation device consisting of a poly(dimethylsiloxane) microfluidic chip possessing T-junction channel geometry, two inlet reservoirs, and one outlet reservoir, and a piezoelectric (PZT) diaphragm micropump with controller. Air was evacuated from the outlet reservoir using the PZT pump, reducing the pressure inside. The reduced pressure within the outlet reservoir pulled oil and aqueous solution preloaded in the inlet reservoirs into the microchannels, which then merged at the T-junction, successfully forming water-in-oil emulsion droplets at a rate of ∼1000 per second with minimal sample loss.

A poly(dimethylsiloxane) microfluidic sheet reversibly adhered on a glass plate for creation of emulsion droplets for droplet digital PCR.

A PDMS microfluidic chip with T-junction channel geometry, two inlet reservoirs, and one outlet reservoir was reversibly adhered on a glass plate through the viscoelastic properties of PDMS. This formed a detachable microfluidic device for creation of water-in-oil emulsion droplets that were used as discrete reaction compartments for the droplet digital PCR. The PDMS/glass device could continuously produce monodisperse droplets without leakage of fluids using a vacuum-driven autonomous micropumping method.

Consideration of Inner and Outer Phase Configuration in Tube Radial Distribution Phenomenon Based on Viscous Dissipation in a Microfluidic Flow Using Various Types of Mixed Solvent Solutions.

When mixed solvent solutions, such as ternary water-hydrophilic/hydrophobic organic solvents, water-surfactant, and water-ionic liquid, are delivered into a microspace under laminar flow conditions, the solvent molecules radially distribute in the microspace, generating inner and outer phases. This specific fluidic behavior is termed "tube radial distribution phenomenon", and has been used in separation technologies such as chromatography and extraction. The factors influencing the configuration of the inner and outer phases in "tube radial distribution phenomenon" using the above-mentioned mixed solvent solutions were considered from the viewpoint of viscous dissipation in fluidic flows.

Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent.

Tube Radial Distribution Chromatography on a Microchip Incorporating Microchannels with a Three-to-One Channel Confluence Point.

We developed a capillary chromatography system using a phase-separated solvent mixture as a carrier solution--i.e., a water-hydrophilic/hydrophobic organic solvent mixture--which we call "tube radial distribution chromatography" (TRDC).

Separation of Metal Complexes with Counter Ions by Tube Radial Distribution Chromatography Using a Ternary Solvent Containing 8-quinolinol.

An open-tubular capillary chromatography system (tube radial distribution chromatography, TRDC) was developed using a ternary solvent (water-acetonitrile-ethyl acetate; volume ratio, 3:8:4) containing 10 mmol L(-1) 8-quinolinol for the separation of nitrate, chloride, and sulfate compounds of Ni(II), Al(III), and Fe(III). When a mixed solution of the Ni(II) compounds was injected into an untreated fused-silica capillary tube (90 cm × 75 μm i.d.

Hands-off preparation of monodisperse emulsion droplets using a poly(dimethylsiloxane) microfluidic chip for droplet digital PCR.

A fully autonomous method of creating highly monodispersed emulsion droplets with a low sample dead volume was realized using a degassed poly(dimethylsiloxane) (PDMS) microfluidic chip possessing a simple T-junction channel geometry with two inlet reservoirs for oil and water to be loaded and one outlet reservoir for the collection of generated droplets. Autonomous transport of oil and water phases in the channel was executed by permeation of air confined inside the outlet reservoir into the degassed PDMS. The only operation required for droplet creation was simple pipetting of oil and aqueous solutions into the inlet reservoirs.

Investigation of inner and outer phase formation in tube radial distribution phenomenon using various types of mixed solvent solutions.

When mixed solvent solutions, such as ternary water-hydrophilic/hydrophobic organic solvents, water-surfactant, water-ionic liquid, and fluorous-organic solvents are delivered into a microspace under laminar flow conditions, the solvent molecules are radially distributed in the microspace, generating inner and outer phases. This specific fluidic behavior is termed "tube radial distribution phenomenon" (TRDP). In this study, the factors influencing the formation of inner and outer phases in the TRDP using the above-mentioned mixed solvent solutions were investigated.

Tube radial distribution phenomenon with a two-phase separation solution of a fluorocarbon and hydrocarbon organic solvent mixture in a capillary tube and metal compounds separation.

A fluorocarbon and hydrocarbon organic solvent mixture is known as a temperature-induced phase-separation solution. When a mixed solution of tetradecafluorohexane as a fluorocarbon organic solvent and hexane as a hydrocarbon organic solvent (e.g.

Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.

We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences).

Microbead-based ligase detection reaction assay using a molecular beacon probe for the detection of low-abundance point mutations.

A microbead-based ligase detection reaction (LDR) assay using a molecular beacon probe was developed for the facile and rapid detection of point mutations present in low copy numbers in a mixed population of wild-type DNA. Biotin-tagged ligation products generated in the LDR were captured on the surface of streptavidin-modified magnetic beads for purification and concentration. The resulting product-tethered microbeads were combined with a molecular beacon probe solution, and the suspension was directly flowed into a capillary.

Specific distribution behavior of a ternary mixture of solvents fed into bent and wound microchannels in microchips.

The tube radial distribution of ternary solvents (water-hydrophilic/hydrophobic organic mixture) fed into bent and wound microchannels in a microchip was examined by fluorescence observations of dyes dissolved in the solvents under laminar flow conditions. Four kinds of microchips incorporating bent microchannels were used, together with a microchip with a straight channel. The microchannels had different bending times (2, 4, or 12 times), bending radii (0.

Development of a PCR/LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and chemiluminescence detection.

A polymerase chain reaction (PCR)/ligase detection reaction (LDR)/flow-through hybridization assay using chemiluminescence (CL) detection was developed for analyzing point mutations in gene fragments with high diagnostic value for colorectal cancers. A flow-through hybridization format using a capillary tube, in which probe DNA-immobilized magnetic beads were packed, provided accelerated hybridization kinetics of target DNA (i.e.

Two-phase extraction of metal ions using a water-acetonitrile-ethyl acetate ternary mixed-solvent separation system.

A ternary mixed-solvent solution of water-acetonitrile-ethyl acetate changes from a homogeneous (single-phase) to a heterogeneous (two-phase) system with temperature and/or pressure changes. In this study, we used this system in a batch vessel to extract metal ions. Water-acetonitrile-ethyl acetate at a volume ratio of 3:8:4 containing 8-hydroxyquinoline was used as a ternary mixed-solvent solution, changing from homogeneous at 25°C to heterogeneous after 30 min at 0°C.