10-Plex Digital Polymerase Chain Reaction with Four-Color Melting Curve Analysis for Simultaneous KRAS and BRAF Genotyping.

Digital PCR (dPCR) is a promising method for performing liquid biopsies that quantifies nucleic acids more sensitively than real-time PCR. However, dPCR shows large fluctuations in the fluorescence intensity of droplets or wells due to insufficient PCR amplification in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. A sample was digitized into a silicon chip with up to 2 × 104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that were complementary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and the melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, the dye color of the probe, and the melting temperature (Tm). Because the Tm of the PCR products is not highly dependent on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF, and eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. To the best of our knowledge, this is the first demonstration of the genotyping of 10 DNA groups including single mutations of cancer-related genes by combining dPCR with four-color melting curve analysis.

Selection of Diacrylate Monomers for Sub-15 nm Ultraviolet Nanoimprinting by Resonance Shear Measurement.

In UV nanoimprinting, the selection of monomers suitable for sub-15 nm patterning is difficult because the filling behavior of resin at this scale still remains scientifically unclear. We demonstrate sub-15 nm patterning by UV nanoimprinting using silica molds with 20, 15, and 7 nm diameter holes; however, the 7 nm diameter pillar patterns were not fabricated using hydroxy-containing monomers. The filling behavior into silica holes of around 10 nm depended on the chemical structure of the monomers. Resonance shear measurements revealed the following: (1) The viscosities of hydroxy-containing monomers confined between chlorodimethyl(3,3,3-trifluoropropyl)silane (FAS3-Cl)-modified surfaces began to increase at distances shorter than those of the monomers between unmodified surfaces. (2) The monomers confined between tridecafluoro-1,1,2,2-tetrahydrooctyltrimethoxysilane-modified surfaces were squeezed out when the surface-surface distance decreased at less than 7 nm. The measured viscosities between the FAS3-Cl-modified silica surfaces were correlated with the insufficient filling behavior into the silica holes of around 10 nm in UV nanoimprinting. Contact angle measurements provided an additional insight that a higher wettability of the monomers onto the antisticking chemisorbed monolayers resulted in imprinted patterns with higher aspect ratios. Considering the increase in the monomer viscosity in the nanospace and the wettability of monomers onto chemisorbed monolayers, we concluded that the monomer showing low viscosity under confinement and high wettability onto the mold surface was suitable for single-digit nanometer UV nanoimprinting.

Parallel Evaluation of Melting Temperatures of DNAs in the Arrayed Droplets through the Fluorescence from DNA Intercalators.

Parallel evaluation of melting temperatures (Tm's) of DNA molecules in multiple floating droplets (20 µm in diameter) was demonstrated. The Tm values were evaluated from the melting curves which were observed through the fluorescence from the DNA intercalators. The Tm values measured in the droplets corresponded well to those measured in the bulk, indicating the validity of the measurement. The parallel evaluation of Tm's was realized by observing melting curves of DNAs in the different droplets at the same time using the "droplet guide", which guided and fixed the floating droplets to the designated points in the observing plane. This demonstration would pave the way for the improvement of the precision of droplet digital PCR (ddPCR), whose state-of-the-art ascribes color and intensity of fluorescence to the base sequence of DNA in the droplet.

KRAS genotyping by digital PCR combined with melting curve analysis.

Digital PCR (dPCR) has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR. However, dPCR exhibits large fluctuations in the fluorescence intensity of the compartment, resulting in low accuracy. The main cause is most likely due to insufficient PCR. In this study, we proposed a new method that combines dPCR with melting curve analysis and applied that method to KRAS genotyping. Since the melting temperature (Tm) of the PCR product hardly depends on the amplification efficiency, genotyping accuracy is improved by using the Tm value. The results showed that the peaks of the distribution of the Tm values of DNA in the wells were 68.7, 66.3, and 62.6 °C for wild-type KRAS, the G12R mutant, and the G12D mutant, respectively, and the standard deviation of the Tm values was 0.2 °C for each genotype. This result indicates that the proposed method is capable of discriminating between the wild-type sequence and the two mutants. To the best of our knowledge, this is the first demonstration of the genotyping of single mutations by combining melting curve analysis and dPCR. The application of this approach could be useful for the quantification and genotyping of cancer-related genes in low-abundance samples.

Preparation and characterization of long-lived anode catalyst for direct methanol fuel cells.

Entry of direct methanol fuel cells into the market requires anode catalyst with stable activity. This paper presents a novel method for stabilizing the activity by immobilizing silica on the catalytic PtRu nanoparticles. Characterization was performed by STEM-EDX, XRD, and ICP. The silica-immobilized PtRu nanoparticles showed high and stable activity toward methanol oxidation. The activity was maintained for 1000 h in sulfuric acidic solution, while the activity of the catalyst with "bare" PtRu nanoparticles decayed after 100 h, showing high durability of the silica-immobilized PtRu nanoparticles catalyst in quasi-anodic acidic environment.

Preparation and characterization of aqueous colloids of Pt-Ru nanoparticles.

A synthetic method for platinum-ruthenium (PtRu) nanoparticles in aqueous media is proposed. This method employs citric acid as a capping agent and NaBH(4) as a reducing agent with the aid of pH control. The number-averaged size of the PtRu nanoparticles was ca. 2 nm. The crystal phase and chemical composition of the nanoparticles was investigated by X-ray diffraction measurement and scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy analysis, which indicated that the nanoparticles mainly consisted of an alloy of Pt and Ru. Electrochemical measurement showed that the PtRu nanoparticles had catalytic activity for methanol oxidation.

Reduction in viscosity of quasi-2D-confined nanoimprint resin through the addition of fluorine-containing monomers: shear resonance study.

The fluidity of the resin for the nanoimprint lithography was investigated in a quasi-2D space by the shear resonance measurement, which enables us to measure the viscous property of the resin confined in a nanometer gap between mica surfaces. With this technique, we could observe that the resin fluidity was enhanced by the addition of the fluorine-containing monomer. The fluidity of the resin with the fluorine-containing monomer was maintained until the distance between mica surfaces became smaller than 15 nm, wheras the resin without the fluorine-containing monomer started losing its fluidity when the distance became 30 nm. Two reasons possibly explain the result; (i) the interaction between the resin and mica was reduced by the existence of the fluorine-containing segregated layer at the interface, and (ii) the reduction in viscosity took place by the addition of the fluorine-containing monomer that reduced the degree of chain entanglement in the resin because of its small intermolecular interaction force. The effect of the length of the perfluoroalkyl chain in the fluorine-containing monomer is also discussed.

A durable PtRu/C catalyst with a thin protective layer for direct methanol fuel cells.

A methanol oxidation catalyst with improved durability in acidic environments is reported. The catalyst consists of PtRu alloy nanoparticles on a carbon support that were stabilized with a silane-coupling agent. The catalyst was prepared by reducing ions of Pt and Ru in the presence of a carbon support and the silane-coupling agent. The careful choice of preparatory conditions such as the concentration of the silane-coupling agent and solution pH resulted in the preparation of catalyst in which the PtRu nanoparticles were dispersively adsorbed onto the carbon support. The catalytic activity was similar to that of a commercial catalyst and was unchanged after immersion in sulfuric acid solution for 1000 h, suggesting the high durability of the PtRu catalyst for the anode of direct methanol fuel cells.

Highly thermoconductive polymer nanocomposite with a nanoporous alpha-alumina sheet.

A highly thermoconductive insulative polymer nanocomposite with a nanoporous alpha-alumina sheet was reported. The thermal conductivity of the nanocomposite along the surface normal was 12 W m(-1) K(-1) (41 vol % alumina), a value as high as that predicted theoretically for a nanocomposite with thermoconductive fillers that form a perfectly connected thermoconductive network. The high thermal conductivity is probably due to the continuous alpha-alumina phase that functions as an efficient phonon path in the nanocomposite. The results suggest that the structure of the filler is important for the design of highly thermoconductive materials.