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. The absolute copy number of a target molecule can be calculated from the fraction of FL(+) droplets relative to the total droplet number analyzed using Poisson statistics. It is crucial that the population of FL(+) droplets can be distinctly separated from that of the FL(-) droplets for accurately estimating the FL(+) droplet fraction and the absolute copy number. However, the distinct separation of the two populations is often impaired in actual ddPCR tests. Although many factors have been suggested to affect population separation, no study has addressed whether the droplet size influences the degree of separation. In this study, we compared the degrees of separation for ddPCR runs with three different droplet sizes. The experimental results showed an increasing degree of separation with decreasing droplet size. This discovery will potentially guide researchers to use smaller droplets in ddPCR to achieve higher accuracy and precision.

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. However, in microfluidic droplet PCR, ensuring successful amplification from single molecules within droplets has limited studies on accelerating assays through fast thermocycling. Our developed microfluidic cartridge, distinguished for its convenience in executing single-molecule droplet PCR with common laboratory equipment, features droplets positioned on a thin glass slide. We hypothesized that applying fast thermocycling to this cartridge would achieve single-molecule droplet PCR amplification. Indeed, the application of this fast protocol demonstrated successful amplification in just 22 min for 30 cycles (40 s/cycle). This breakthrough is noteworthy for its potential to expedite microfluidic droplet PCR assays, ensuring efficient single-molecule amplification within a remarkably short timeframe.

CO2-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. This modification was based on our idea that the bottom PDMS slab does not contribute to the automatic fluid manipulation and that replacing the bottom PDMS slab with a more affordable and accessible, ready-to-use polymer slab, such as a PMMA, would further facilitate the rapid and low-cost fabrication of the connection-free microchips. Using a new PMMA/PDMS microchip, we produced water-in-oil droplets with high degree of size-uniformity (a coefficient of variation for droplet diameters of <5%) without a decrease in the droplet production rate (~270 droplets/s) as compared with that achieved via the previous PDMS/PDMS microchip (~220 droplets/s).

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.e., (1) droplet generation; (2) PCR amplification; and (3) determination of the fluorescence intensity of the thermocycled droplets. In the current study, we developed a new microfluidic cartridge consisting of four layers, with a thin glass slide as the bottom layer. In this cartridge, droplets generated in the uppermost polydimethylsiloxane microfluidic layer are delivered to the glass slide in an online fashion. After the accumulation of many droplets on the glass slide, the cartridge is placed on the flatbed heat block of a thermocycler for PCR amplification. Direct fluorescence imaging of the thermocycled droplets on the glass slide is then carried out using a conventional fluorescence microscope. Efficient heat transfer from the heat block to the settled droplets through the thin glass slide was confirmed by successful PCR amplification inside the droplets, even from single template molecules. The new cartridge eliminates the need for manual droplet transfer between the major steps of droplet PCR analysis, allowing more convenient single-molecule droplet PCR than in our previous studies.

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. However, the incorporation of the additional PDMS layer (which was separate from the microfluidic PDMS layer itself) into the device required reversible bonding of five different layers. In the current study, we aimed to simplify the device architecture by reducing the number of constituent layers for enhancing usability of this microfluidic droplet generator while retaining its rapid production rate. The new device consisted of three layers. This comprised a degassed PDMS slab with microfluidic channels on one surface and tens of thousands of vacuum-generating micropillars on the other surface, which was simply sandwiched by PMMA layers. Despite its simplified configuration, this new device created monodisperse droplets at an even faster rate (>1000 droplets per second).

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. For the first 10 patients (120/100 kV group), we used a conventional tube voltage combined CTA protocol (an ECG-gated helical scan; 120 kV, non-gated helical scan; 100 kV). For the subsequent 30 patients (70-kV group), we adopted a low tube voltage CTA protocol. We evaluated vascular attenuation, image noise, contrast-to-noise ratio (CNR), and renal function. The mean contrast media (CM) volume was 77.7 ± 17.7 mL in the 120/100-kV group and 30.9 ± 6.3 mL in the 70-kV group (P < 0.001). In the images of the aortic valve complex, the mean attenuation was not significant difference for both groups. In the images of the aorto-femoral arteries, mean attenuation was > 250 Hounsfield Units and CNR was > 10 in all vascular segments for both groups. There was no significant difference in the change of renal function in the 70-kV group, but renal function in the 120/100-kV group decreased within 1-3 months after CTA. Low tube voltage CTA using third-generation dual-source CT is suitable to assess procedural planning for TAVI. This approach maintains image quality and reduces the required CM volume.

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., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5'-end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite-converted genomic DNA, and a DNA polymerase. A single base-extended primer (i.e., SBE product) that was 5'-Cy3- and 3'-Cy5-tagged was formed by incorporation of the Cy5-labeled terminator into the 3'-end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.

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 (Vp-p ) and wave frequency, on droplet generation characteristics. Stepwise adjustments to the drive voltage in ±10-Vp-p increments over the range of 200-250 Vp-p during droplet creation revealed that the droplet generation rate could be reproducibly controlled at a specific drive voltage. The droplet generation rate switched within <0.5 s after the input of a new voltage. Although the droplet generation rate depended on the drive voltage, this setting had almost no influence on droplet size. The frequency over the selected range (50-60 Hz) did not markedly influence the droplet generation rate or droplet size. We show that the current fluid manipulation system can be conveniently used for both droplet generation and for rapid droplet reading, which is required in many microfluidic-based applications.

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. METHODS: Sixteen patients with acute intracranial hemorrhage within 1 day after head trauma were enrolled in this study. All CT examinations were performed on a dual-source dual-energy CT scanner. BRCT images were generated using the Bone Removal Application. Simulated standard CT and BRCT images were visually reviewed in terms of detectability (presence or absence) of acute hemorrhagic lesions. RESULTS: DECT depicted 28 epidural/subdural hemorrhages, 17 contusional hemorrhages, and 7 subarachnoid hemorrhages. In detecting epidural/subdural hemorrhage, BRCT [28/28 (100%)] was significantly superior to simulated standard CT [17/28 (61%)] (p = .001). In detecting contusional hemorrhage, BRCT [17/17 (100%)] was also significantly superior to simulated standard CT [11/17 (65%)] (p = .0092). CONCLUSION: BRCT was superior to simulated standard CT in detecting acute intracranial hemorrhage. BRCT could improve the detection of small intracranial hemorrhages, particularly those adjacent to bone, by removing bone that can interfere with the visualization of small acute hemorrhage. In an emergency such as head trauma, BRCT can be used as support imaging in combination with simulated standard CT and bone scale CT, although BRCT cannot replace a simulated standard CT.

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. To achieve this aim, we developed a new device with a much larger PDMS surface area-to-volume ratio within the air-trapping void space (178 cm-1 ), than that of our earlier device (5.0 cm-1 ). This design approach was based on the idea that a larger PDMS surface area-to-volume ratio was likely to create a higher vacuum inside the void space, thereby contributing to faster liquid flow and an increased droplet generation rate. The new device consisting of five layers featuring a degassed PDMS slab as a detachable liquid-suction actuator, which was stacked on a lower microfluidic layer. In this device, the rate of droplet production increased during the time-course droplet formation and reached ca. 470 droplets per second immediately before completely consuming the loaded aqueous solution (20 µL).

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).

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. We confirmed that the onset of droplet formation occurred immediately after turning on the pump (<1 s). Over repeated runs, droplet formation was highly reproducible, with droplet size purity (polydispersity, <4%) comparable to that achieved using other microfluidic droplet preparation techniques. We also demonstrated single-molecule PCR amplification in the created droplets, suggesting that the device could be used for effective droplet digital PCR platforms in most laboratories without requiring great expense, space, or time for acquiring technical skills.

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. This droplet preparation technique only required evacuation of air dissolved in the PDMS before loading of oil and aqueous phases into separate inlet reservoirs. Degassing of the PDMS chip at approximately 300 Pa for 1.5 h in a vacuum desiccator gave 40 000 droplets in 80 min, which corresponded to a generation frequency of up to nine droplets per second. Over multiple runs the droplet creation was very reproducible, and the size reproducibility of generated droplets (polydispersity of up to 4.1%) was comparable to that acquired using other microfluidic droplet preparation techniques. Because the PDMS chip can be peeled off the glass plate, blocked channels can easily be fixed when they arise, and this extends the lifetime of the chip. Single DNA molecules partitioned into the droplets were successfully amplified by PCR. In addition, the droplet digital PCR platform allowed absolute quantification of low copy numbers of target DNA, and was robust against instrumental variance.

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. When the difference in the viscosity between the two phases was large (approximately >0.73 mPa·s), the phase with the higher viscosity formed as an inner phase regardless of the volume ratio. The distribution pattern of the solvents was supported by the viscous dissipation principle. Contrarily, when the difference was small (approximately <0.49 mPa·s), the phase with the larger volume formed as the inner phase. The distribution pattern of the solvents did not always correspond to the viscous dissipation principle. The current findings are expected to be useful in analytical science including microflow analysis research.

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). Here, we attempted to apply the TRDC system to a microchip incorporating microchannels with a double T-junction for injection of analyte solution and a three-to-one, narrow-to-wide channel confluence point for tube radial distribution phenomenon (TRDP) at room temperature. A ternary mixed solvent of water, acetonitrile and ethyl acetate was used as a carrier solution. TRDP in the wide microchannel was examined using various flow rates, temperatures, and component solvent ratios. Successful observation was carried out using a fluorescence microscope-CCD camera. Model analytes perylene (hydrophobic) and Eosin Y (hydrophilic) were separated by flowing through the microchannel, without any treatment such as packed columns or coating, at room temperature (25°C).

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.) with a ternary solvent flow rate of 0.8 µL min(-1), the compounds were eluted in the following order: [Ni(II)-(8-quinolinol)3] complex, [Ni(II)-(8-quinolinol)]-nitrate ion interaction complex, [Ni(II)-(8-quinolinol)]-chloride ion interaction complex, and [Ni(II)-(8-quinolinol)]-sulfate ion interaction complex. The elution of mixtures of the Al(III) and Fe(III) compounds showed similar trends.

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. Long-lasting fluid transport in the current system enabled us to create ca. 51,000 monodispersed droplets (with a coefficient of variation of <3% for the droplet diameter) in 80 min with a maximum droplet generation rate of ca. 12 Hz using a PDMS chip that had been degassed overnight. With multiple time-course measurements, the reproducibility in the current method of droplet preparation was confirmed, with tunable droplet sizes achieved simply by changing the cross-sectional dimensions of the microchannel. Furthermore, it was verified that the resultant droplets could serve as microreactors for digital polymerase chain reactions. This hands-free technique for preparing monodispersed droplets in a very facile and inexpensive fashion is intended for, but not limited to, bioanalytical applications and is also applicable to material syntheses.

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. We examined phase diagrams, viscosities of the two phases (upper and lower phases in a batch vessel), volume ratios of the phases, and bright-light or fluorescence photographs of the TRDP. When the difference in viscosities between the two phases was large (> approximately 0.73 mPa·s), the phase with the larger viscosity formed an inner phase regardless of the volume ratios, whereas when the difference was small (< approximately 0.42 mPa·s), the phase with the larger volume formed an inner phase. The TRDP using a water-surfactant mixed solution was also investigated in capillary chromatography based on TRDP.

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., 71:29 volume ratio) was delivered in a capillary tube that was controlled at 10°C, the tube radial distribution phenomenon (TRDP) of the solvents was clearly observed through fluorescence images of the dye, perylene, dissolved in the mixed solution. The homogeneous mixed solution (single phase) changed to a heterogeneous solution (two phases) with inner tetradecafluorohexane and outer hexane phases in the tube under laminar flow conditions, generating the dynamic liquid-liquid interface. We also tried to apply TRDP to a separation technique for metal compounds. A model analyte mixture, copper(II) and hematin, was separated through the capillary tube, and detected with a chemiluminescence detector in this order within 4 min.