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We investigated the etching characteristics of hydrogen iodide (HI) neutral beam etching (NBE) of GaN and InGaN and compared with Cl2 NBE. We showed the advantages of HI NBE vs Cl2 NBE, namely: higher InGaN etch rate, better surface smoothness, and significantly reduced etching residues. Moreover, HI NBE was suppressed of yellow luminescence compared with Cl2 plasma. InClx is a product of Cl2 NBE. It does not evaporate and remains on the surface as a residue, resulting in a low InGaN etching rate. We found that HI NBE has a higher reactivity with In resulting in InGaN etch rates up to 6.3 nm/min, and low activation energy for InGaN of approximately 0.015 eV, and a thinner reaction layer than Cl2 NBE due to high volatility of In- I compounds. HI NBE resulted in smoother etching surface with a root mean square average (rms) of 2.9 nm of HI NBE than Cl2 NBE (rms: 4.3 nm) with controlled etching residue. Moreover, the defect generation was suppressed in HI NBE vs. Cl2 NBE, as indicated by lower yellow luminescence intensity increase after etching. Therefore, HI NBE is potentially useful for high throughput fabrication of μLEDs.

We have developed a new particle counting immunoassay system integrated with a resistive-pulse sensor with a low-aspect-ratio pore. The particle counting immunoassay is based on the principle of latex aggregation. The target single-molecule concentration is measured by counting aggregated latex particles that form multimers over the reaction time. To enable highly accurate identification of the number of aggregated particles, we designed and fabricated a low-aspect-ratio pore device to perform quantitative structural analysis of particles. The aggregation numbers of the conjugated particles were classified based on blockade current profiles when the particles translocated through the pore. Time-dependent agglutination reaction monitoring demonstrated that the reaction speed increased for a solution containing a higher concentration of antigens. The results prove the feasibility of using the proposed immunoassay system to count particles while determining the aggregation number based on their current profiles.

Visualization of periodically aligned pores in organic frameworks is a key to the understanding of their structural control. Comparing to monolayer-thick self-assembled molecular networks, real-space nanoscale characterization of thicker films, especially obtaining information on the stacking manner of molecules is challenging. Here, we report an atomic force microscopy study of hydrogen-bonded thin films fabricated at the air/liquid interface. The presence of one-dimensional channels is evidenced by resolving honeycomb structures over the films with the thickness variation of more than several nanometers. We also demonstrate that the film thickness can be controlled by the ratio of mixed solvent rather than the surface pressure during the fabrication at the air/liquid interface.

In this paper, we propose a combination of digital polymerase chain reaction (dPCR) and melting curve analysis for the highly multiplexed genotyping of cell-free DNA (cfDNA) with high sensitivity. A silicon chip with 7.7x104 high-aspect-ratio wells with controlled surface hydrophobicity/hydrophilicity was developed. A sample containing cfDNA was divided into this large number of wells and amplified by PCR to enable sensitive detection. In addition, high multiplexing was achieved by capturing fluorescence images while controlling the temperature of the chip, measuring the melting curve for each well, and genotyping using fluorescence intensity, probe dye color, and melting temperature (Tm). The simultaneous dPCR genotyping of 10 types of DNA including single-nucleotide mutations of cancer-related genes was demonstrated for the first time with up to 7.7x104 wells.

Nanopore-based biosensors have attracted attention as highly sensitive microscopes for detecting single molecules in aqueous solutions. However, the ionic current noise through a nanopore degrades the measurement accuracy. In this study, the magnitude of the low-frequency noise in the ionic current through a silicon nitride nanopore was found to change depending on the metal ion species in the aqueous solution. The order of the low-frequency noise magnitudes of the alkali metal ionic current was consistent with the order of the adsorption affinities of the metal ions for the silanol surface of the nanopore (Li i.e., metal ions having a very high or low adsorption affinity causing a reduction in low-frequency noise, suggests that the low-frequency noise was induced by the exchange reactions between protons and metal ions occurring on the silanol surface. In addition, the low-frequency noise in the ionic current remained low even after replacing the CaCl2 aqueous solution with a CsCl aqueous solution, indicating that Ca ions continued being adsorbed onto silanol groups even after removing the aqueous solution.

Pyrrole is a privileged five-membered aromatic nitrogen heterocycle, which is ubiquitous in natural products, drug molecules, and functional materials. Therefore, numerous synthetic routes to substituted pyrroles have been extensively developed. Nevertheless, the efficient and short-step synthesis of highly substituted and/or fused pyrroles has remained a significant challenge in organic chemistry. Here we report a ruthenium-catalyzed nitrogen-transfer [2 + 2 + 1] cycloaddition of α,ω-diynes involving cyclic biscarbenoid intermediates. To achieve the key nitrogen transfer to carbenoid carbons, sulfoximines are employed as nitrene surrogates. Consequently, diverse fused pyrroles are successfully synthesized in good yields with wide functional group compatibility. Moreover, this method allows the synthesis of N-alkyl, N-aryl, and even N-H pyrroles, which are difficult to obtain using previous [2 + 2 + 1]-type reactions. Nitrogen transfer from sulfoximines to cyclic biscarbenoid intermediates is supported by density functional theory calculations and control experiments. Cycloadditions involving nitrene transfer provide a useful route to valuable substituted pyrroles. Here the authors report a ruthenium-catalysed [2+2+1] cycloaddition of diynes using sulfoximines as unusual nitrogen transfer reagents.

Transfer hydrogenation of unsaturated nitrogen heterocycles using a rhodium catalyst immobilized on bipyridine-periodic mesoporous organosilica (BPy-PMO) is described. The immobilized catalyst was prepared by mixing [Cp*RhCl2]2 (Cp* = η5-C5Me5) with BPy-PMO powder in DMF at 60 °C and characterized by nitrogen adsorption measurements, solid-state NMR spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. In the presence of the catalyst, a wide variety of unsaturated nitrogen heterocycles underwent transfer hydrogenation to afford the corresponding products in good yields. The immobilized catalyst could be readily recovered by centrifugation and reused several times in the transfer hydrogenation process., ファイル公開：2019/01/21

Based on the results of rapid-flow electron spin resonance (RF-ESR) and spin-trapping (ST-ESR) measurements, carbonyl-conjugated radicals derived from the vicinal diol moiety (2, i.e., the head-to-head structure) of poly(vinyl alcohol) (PVA) have been proposed to be the most probable intermediate species involved in the initial stages of the graft reaction of PVA with methyl methacrylate (MMA). A modified poly(vinyl alcohol) (PPVA) bearing a 1,2-propanediol pendant moiety (3) with a molar ratio of 8% (based on the monomer unit) was prepared to clarify the role of the carbonyl-conjugated radicals of PPVA in the graft reaction with MMA. The RF-ESR spectra observed for the mixtures composed of PPVA, hydrogen peroxide (HPO) or ammonium persulfate (APS), and Ti2(SO4)3 (pH ca. 2) revealed the formation of four radical species (A, B1, B2, and C) derived from PPVA. By comparison of the RF-ESR spectra observed for PPVA and PVA, species A (g = 2.0041) was confirmed to be the pendant derived radical of PPVA, and spe...

Nanopore DNA sequencing with a solid-state nanopore requires deceleration of the ultrafast translocation speed of single-stranded DNA (ssDNA). We report an unexpected phenomenon: controlled dielectric breakdown (CBD) with a divalent metal cation, especially Ca2+, provides a silicon nitride nanopore with the ability to decelerate ssDNA speed to 100 μs per base even after solution replacement. This speed is two orders of magnitude slower than that for CBD with a conventional monovalent metal cation. Temperature dependence experiments revealed that the enthalpic barrier for a nanopore created via CBD with Ca2+ is 25–30kBT, comparable to that of a biological nanopore. The slowing effect originates from the strong interaction between ssDNA and divalent cations, which were coated on the sidewall of the nanopore during the CBD process. In addition, we found that the nanopore created via CBD with Ca2+ can decelerate the speed of even single-nucleotide monomers, dNMPs, to 0.1–10 ms per base. The four single nucleotides could be statistically identified according to their blockade currents. Our approach is simple and practical because it simultaneously allows nanopore fabrication, ssDNA deceleration and the identification of nucleotide monomers.

Continuous rapid-flow electron spin resonance (ESR) was used to detect poly(vinyl alcohol) (PVA)-derived carbon-centered radicals, in order to characterize the short-lived intermediate radicals in the initial stage of the graft polymerization of PVA. ESR spectra were recorded by mixing PVA, hydrogen peroxide, and Ti2(SO4)3 at pH 2.0. The observed spectrum indicated three paramagnetic species: a minor species A, and two major species B1, and B2. Based on the observed proton hyperfine coupling constant (hfcc) values, species A was assigned to the carbon-centered radical of PVA produced by hydrogen atom abstraction from the methine carbon bearing a hydroxyl group. Species B1 and B2 were ascribed to two structural isomers of the PVA-derived, carbonyl-conjugated radical produced by acid-catalyzed radical reaction at the vicinal diol moiety (i.e., head-to-head structure) of PVA. Spin-trapping ESR measurements were performed at 80 °C using a water-soluble spin-trapping reagent 3,5-dibromo-4-nitroso-benzenesulfon...

Flavin-containing opine dehydrogenase from Bradyrhizobium japonicum forms a heterooligomeric α4β4γ4 enzyme complex. An electron paramagnetic resonance spectroscopy analysis using wild-type and site-directed mutants revealed that [4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the γ- and α-subunits, respectively. The latter was found to be important for structural folding and enzyme catalysis.

A graphene cantilever beam of arbitrary shape by patterning suspended graphene using a focused ion beam (FIB) is fabricated. Suspended graphene was formed by transferring graphene over a trench structure and patterned the suspended graphene into a cantilever beam structure. When the suspended graphene is patterned by an FIB, the high-ion irradiation of FIB deforms the shape of the cantilever beam; in contrast, low-ion irradiation cannot penetrate the suspended graphene. An evaluation of the ion irradiation dose of the FIB and the damages caused to the suspended graphene confirmed that in the case of few-layer graphene with a maximum of three layers, optimum processing can be performed by an ion irradiation of 6 × 1016 ions/cm2. Under this condition, a graphene cantilever beam with a width of 1.34 μm and a length of 2.13 μm could be fabricated. Furthermore, the Raman spectroscopy results indicate that the FIB process does not significantly change the properties of the graphene.

The first example of the Ru-catalyzed transfer oxygenative [2 + 2 + 1] cycloaddition of silyldiynes to produce bicyclic 2-silylfurans is described. This cyclization process was realized using nitrones as readily available and adjustable oxygen atom donors. The bicyclic silylfuran products could be used as platforms for a diverse range of functionalized furans.

Ruthenium-catalyzed cycloisomerization of 2-ethynylbiaryls was investigated to identify an optimal ruthenium catalyst system. A combination of [η(6) -(p-cymene)RuCl2 (PR3 )] and two equivalents of AgPF6 effectively converted 2-ethynylbiphenyls into phenanthrenes in chlorobenzene at 120 °C over 20 h. Moreover, 2-ethynylheterobiaryls were found to be favorable substrates for this ruthenium catalysis, thus achieving the cycloisomerization of previously unused heterocyclic substrates. Moreover, several control experiments and DFT calculations of model complexes were performed to propose a plausible reaction mechanism.

The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans , which is known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrates. However, studies have investigated only cellulosomal proteins; profile changes in noncellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans , including cellulosomal and noncellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid-swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica capillary column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 noncellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than noncellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrates. Drastic changes were observed mainly among the noncellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate and that noncellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of noncellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.

Candida albicans is an opportunistic pathogen that causes fatal disease if the host immunity is compromised. The mortality rate of systemic candidiasis is very high; hence, there is a ceaseless demand for novel pharmaceuticals. In this study, quantitative time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) is described. Survival in blood is essential for virulence of C. albicans, and a detailed analysis is required. We cultivated C. albicans in FBS for 0–180 min, and determined quantitative time-course variations of 1024 proteins in the cultured cells by using a LC–MS/MS system with a long monolithic silica capillary column. Clustering analysis identified FBS-induced proteins associated with detoxification of oxidative species, high-affinity glucose transport, citrate cycle, oxidative phosphorylation, and iron acquisition. Furthermore, we identified possible virulence factors such as orf19.4914.1 (named Blood-induced peptide 1, Blp1). Heterologous expression of BLP1 in Saccharomyces cerevisiae shortened the lag phase and resulted in a pleiotropic stress-tolerance phenotype, indicating a possible role for quick adaptation to a stressful environment. While further experiments are necessary to prove virulence of the identified factors, systematic identification of candidate virulence proteins in this study will lead to profound understanding of virulence of C. albicans. Biological significance This paper describes time-course proteomics of C. albicans during adaptation to serum, which is an essential process for fatal systemic candidiasis. Using a LC–MS/MS system with a monolithic silica capillary column, we have successfully characterized time-course variations of 1024 proteins. Among them, orf19.4914.1 (Blp1) was identified as a novel pleiotropic stress-tolerance peptide, which could have an important role for virulence of C. albicans.

The use of N-(p-chlorophenyl)methylbenzoxazole-2-thione as a sulfur-atom donor enables the catalytic [2+2+1] cycloaddition of diynes in wet DMF at 80 °C when open to air, thus affording diverse fused thiophenes with good yields and wide functional-group compatibility. A plausible mechanism, involving a cationic ruthenacycle intermediate, was also proposed on the basis of several control experiments.

The practical use of solid-state nanopores for DNA sequencing requires easy fabrication of the nanopores, reduction of the DNA movement speed and reduction of the ionic current noise. Here, we report an integrated nanopore platform with a nanobead structure that decelerates DNA movement and an insulating polyimide layer that reduces noise. To enable rapid nanopore fabrication, we introduced a controlled dielectric breakdown (CDB) process into our system. DNA translocation experiments revealed that single nanopores were created by the CDB process without sacrificing performance in reducing DNA movement speed by up to 10 μs/base or reducing noise up to 600 pArms at 1 MHz. Our platform provides the essential components for proceeding to the next step in the process of DNA sequencing.

Reducing device capacitance is effective for decreasing current noise observed in a solid-state nanopore-based DNA sequencer. On the other hand, we have recently found that voltage stress causes pinhole-like defects in such low-capacitance devices. The origin of voltage stress, however, has not been determined. In this research, we identified that a dominant origin is static charge on the outer surface of a flow cell. Even though the outer surface was not in direct contact with electrolytes in the flow cell, the charge induces high voltage stress on a membrane according to the capacitance coupling ratio of the flow cell to the membrane.

This paper summarizes the latest developments regarding solid-state nanopores achieved in our laboratory. For direct DNA sequencing with solid-state nanopores, a simple method has been developed to precisely fabricate nanopores with diameters of 1 to 3 nm. By measuring DNA translocation events through the fabricated nanopores, it was confirmed that single-stranded DNA (ssDNA) can pass through nanopores with diameters as small as 1.2 nm. In addition, it was discovered that the translocation speed of ssDNA is higher than that for double-stranded DNA (dsDNA) by three or four orders of magnitude. This difference in speed was explained by a difference in the forces acting on the DNA. From molecular dynamics (MD) simulations, it was found that the force of solution-DNA interactions on dsDNA is approximately 10 times larger than that on ssDNA during DNA translocation through a nanopore.

The reaction conditions developed for the cycloisomerization of various 2-ethinylbiaryls to phenanthrenes are also applied in the cycloisomerization of heteroaryl analogues.

To achieve DNA sequencing using a solid-state nanopore, it is necessary to reduce the electric noise current. The noise current can be decreased by reducing the capacitance (C) of the nanopore device. However, we found that an electric-charge difference (ΔQ) between the electrolyte in one chamber and the electrolyte in another chamber occurred. For low capacitance devices, this electric-charge imbalance can lead to unexpectedly high voltage (ΔV = ΔQ/C) which disrupted the membrane when the two electrolytes were independently poured into the chambers. We elucidated the mechanism for the generation of initial defects and established new procedures for preventing the generation of defects by connecting an electric bypass between the chambers.

A new tandem process is developed comprising a transfer-hydrogenative cyclization followed by intramolecular Diels—Alder reaction using a cationic Ru-catalyst and Hantzsch ester as hydrogen source.

A tandem transfer-hydrogenative cyclization/intramolecular Diels-Alder reaction of enediyne substrates, containing 1,6-diyne, acrylate dienophile, and phenol tether moieties, was successfully accomplished using the combination of a cationic ruthenium complex, [CpRu(AN)3]PF6 (1b, Cp = η(5)-C5H5, AN = MeCN), as the catalyst and a Hantzsch ester as the H2 surrogate to afford interesting dihydrocoumarin-fused polycyclic products as single diastereomers.

We report the resonant frequency measurement of few-layer (1-, 2-, and 3-layer) graphene (FLG) cantilevers by optical heterodyne interferometry. The micro-sized FLG cantilevers with and without the diamond-like carbon weights were fabricated using focused ion beam. The Young's modulus was able to be calculated from the measured resonant frequency. The calculated Young's modulus was larger than the literature data [1]. This result suggests that the overlapped structure of the FLG cantilever makes the structure rigid.

We performed a focused proteome analysis of cellulosomal proteins predicted by a genome analysis of Clostridium cellulovorans [Tamaru, Y., et al.. 2010. J. Bacteriol. 192:901–902]. Our system employed a long monolithic column (300 cm), which provides better performance and higher resolution than conventional systems. Twenty-three cellulosomal proteins were, without purification, identified by direct analysis of the culture medium. Proteome analysis of the C. cellulovorans cellulosome after culture in various carbon sources demonstrated the production of carbon source-adapted cellulosome components.