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.

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).

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.

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.

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).

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.

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.

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.

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.

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. The microbeads were accumulated in a confined space within the capillary using a bar magnet. The packed bead sample was then scanned by a fluorescence scanning imager to detect the presence of any mutations. With the developed methodology, we were able to successfully detect one cancer mutation in a mixture of 400 wild-type templates (t test at 95% confidence level). Furthermore, the post-LDR processing, typically the most laborious and time-consuming step in LDR-based mutation detection assays, could be carried out much more rapidly (approximately 20 min). This was enabled by the simple bead and fluid manipulations involved in the present assay.

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). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system.

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.

Development of a ligase detection reaction/CGE method using a LIF dual-channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild-type DNA.

We developed an inexpensive LIF dual-channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low-abundance point mutations in a large excess of wild-type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K-ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K-ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild-type sequence-dominated population, and to disclose their allelic composition, was thus demonstrated successfully.

Michrochip chromatography using an open-tubular microchannel and a ternary water-ACN-ethyl acetate mixture carrier solution.

A capillary chromatography system has been developed using a ternary mixed-solvents solution, i.e. water-hydrophilic/hydrophobic organic solvent mixture as a carrier solution. Here, we tried to carry out the chromatographic system on a microchip incorporating the open-tubular microchannels. A model analyte solution of isoluminol isothiocyanate (ILITC) and ILITC-labeled biomolecule was injected to the double T-junction part on the microchip. The analyte solution was delivered in the separation microchannel (40 µm deep, 100 µm wide, and 22 cm long) with the ternary water-ACN-ethyl acetate mixture carrier solution (3:8:4 volume ratio, the organic solvent rich or 15:3:2 volume ratio, the water-rich). The analyte, free-ILITC and labeled BSA mixture, was separated through the microchannel, where the carrier solvents were radially distributed in the separation channel generating inner and outer phases. The outer phase acts as a pseudo-stationary phase under laminar flow conditions in the system. The ILITC and the labeled BSA were eluted and detected with chemiluminescence reaction.

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).

Fluorescence observation supporting capillary chromatography based on tube radial distribution of carrier solvents under laminar flow conditions.

We developed a capillary chromatography system by using an open capillary tube made of fused-silica, polyethylene, or polytetrafluoroethylene, and a water-hydrophilic/hydrophobic organic mixture carrier solution, called tube radial distribution chromatography (TRDC) system. By comparing with chromatograms obtained via the TRDC system, fluorescence photographs and profiles of the fluorescent dyes dissolved in the carrier solvents in capillary tubes were observed under laminar flow conditions. The chromatograms were obtained for a model mixture analyte consisting of 1-naphthol and 2,6-naphthalenedisulfonic acid with the TRDC system, by using a fused-silica capillary tube and a water-acetonitrile-ethyl acetate carrier solution. By altering the carrier flow rates, we examined the fluorescence photographs and profiles of the dyes, perylene and Eosin Y, dissolved in the carrier solvents in the capillary tube by using a fluorescence microscope equipped with a CCD camera. As confirmed by fluorescence observations, the major inner and minor outer phases generated in the capillary tube were based on the tube's radial distribution of the carrier solvents. We designed and manufactured a microreactor incorporating microchannels in which three narrow channels combined to form one wide channel. When the carrier solvents containing the dyes were fed into the channels, the inner and outer phase generations were also observed in the narrow and wide channels, strongly supporting the conclusions concerning the tube radial distribution phenomenon of the solvents.

Use of tube radial distribution of ternary mixed carrier solvents for introduction of absorption reagent for metal ion separation and online detection into capillary.

When ternary mixed solvents consisting of water-hydrophilic/hydrophobic organic solvents are fed into a micro-space under laminar flow conditions, the solvent molecules are radially distributed in the micro-space. The specific fluidic behavior of the solvents is called the "tube radial distribution phenomenon (TRDP)". A novel capillary chromatography method was developed based on the TRDP that creates the inner major and outer minor phases in a tube, where the outer phase acts as a pseudo-stationary phase. This is called "tube radial distribution chromatography (TRDC)". In this study, Chrome Azurol S as an absorption reagent was introduced into the TRDC system for metal ion separation and online detection. The fused-silica capillary tube (75 µm id and 110 cm length) and water-acetonitrile-ethyl acetate mixture (3:8:4 volume ratio) including 20 mM Chrome Azurol S as a carrier solution were used. Metal ions, i.e. Co(II), Cu(II), Ni(II), Al(III), and Fe(III), as models were injected into the present TRDC system. Characteristic individual absorption characteristics and elution times were obtained as the result of complex formation between the metal ions and Chrome Azurol S in the water-acetonitrile-ethyl acetate mixture solution. The elution times of the metal ions were examined based on their absorption behavior; Co(II), Ni(II), Al(III), Fe(III), and Cu(II) were eluted in this order over the elution times of 4.7-6.8 min. The elution orders were determined from the molar ratios of metal ion to Chrome Azurol S and Irving-Williams series for bivalent metal ions.

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 case or resection of synchronous multiple liver metastases from rectal cancer after FOLFOX4 therapy].

A 7 0-year-old man with multiple liver metastases from rectal cancer was admitted to our hospital. From imaging inspection, a resection was determined to be difficult, so he was treated with FOLFOX4 therapy. After ten courses of FOLFOX4, the liver metastases showed 52% reduction in size and were judged to be PR. Therefore low anterior resection of the rectum, left lobectomy of the liver and partial resection of the liver(S7, S8)were performed. He was able to undergo curative surgery after FOLFOX4 therapy.