Hitachi High-Tech (Japan)

Electrochemiluminescence (ECL) is a powerful transduction technique with a leading role in the biosensing field due to its high sensitivity and low background signal. Although the intrinsic analytical strength of ECL depends critically on the overall efficiency of the mechanisms of its generation, studies aimed at enhancing the ECL signal have mostly focused on the investigation of materials, either luminophores or coreactants, while fundamental mechanistic studies are relatively scarce. Here, we discover an unexpected but highly efficient mechanistic path for ECL generation close to the electrode surface (signal enhancement, 128%) using an innovative combination of ECL imaging techniques and electrochemical mapping of radical generation. Our findings, which are also supported by quantum chemical calculations and spin trapping methods, led to the identification of a family of alternative branched amine coreactants, which raises the analytical strength of ECL well beyond that of present state-of-the-art immunoassays, thus creating potential ECL applications in ultrasensitive bioanalysis.

Abstract Multimetallic alloys (MMAs) with various compositions enrich the materials library with increasing diversity and have received much attention in catalysis applications. However, precisely shaping MMAs in mesoporous nanostructures and mapping the distributions of multiple elements remain big challenge due to the different reduction kinetics of various metal precursors and the complexity of crystal growth. Here we design a one-pot wet-chemical reduction approach to synthesize core–shell motif PtPdRhRuCu mesoporous nanospheres (PtPdRhRuCu MMNs) using a diblock copolymer as the soft template. The PtPdRhRuCu MMNs feature adjustable compositions and exposed porous structures rich in highly entropic alloy sites. The formation processes of the mesoporous structures and the reduction and growth kinetics of different metal precursors of PtPdRhRuCu MMNs are revealed. The PtPdRhRuCu MMNs exhibit robust electrocatalytic hydrogen evolution reaction (HER) activities and low overpotentials of 10, 13, and 28 mV at a current density of 10 mA cm −2 in alkaline (1.0 M KOH), acidic (0.5 M H 2 SO 4 ), and neutral (1.0 M phosphate buffer solution (PBS)) electrolytes, respectively. The accelerated kinetics of the HER in PtPdRhRuCu MMNs are derived from multiple compositions with synergistic interactions among various metal sites and mesoporous structures with excellent mass/electron transportation characteristics.

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.

Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

Mechanochromic organic molecules (MOMs) that exhibit a large difference of fluorescence wavelength between two states have important potential applications, but few such compounds are known. Here, we report a new MOM, cis-ABPX01(0), which shows switchable near-IR and blue fluorescence responses. Detailed spectrophotometric and single-crystal X-ray analyses revealed that the near-IR fluorescence is attributable to fluorescence from slip-stacked dimeric structures in crystals, while the blue fluorescence is attributable to fluorescence from the monomer. Switching between the two is achieved by dynamic structural interconversion between the two molecular packing arrangements in response to mechanical grinding and solvent vapor-fuming.

Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current method expands upon this by quantifying eight UGT1A isoforms by nanobore high-performance liquid chromatography (HPLC) coupled with a linear ion trap time-of-flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney, and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8, and 1A10 were below detection limits (<1 pmol/mg protein) in human liver microsome (HLMs). In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9, and 1A10 protein were higher relative to that of liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.

Abstract On the basis of observations using Cs‐corrected STEM , we identified three types of surface modification probably formed by space weathering on the surfaces of Itokawa particles. They are (1) redeposition rims (2–3 nm), (2) composite rims (30–60 nm), and (3) composite vesicular rims (60–80 nm). These rims are characterized by a combination of three zones. Zone I occupies the outermost part of the surface modification, which contains elements that are not included in the unchanged substrate minerals, suggesting that this zone is composed of sputter deposits and/or impact vapor deposits originating from the surrounding minerals. Redeposition rims are composed only of Zone I and directly attaches to the unchanged minerals (Zone III). Zone I of composite and composite vesicular rims often contains nanophase (Fe,Mg)S. The composite rims and the composite vesicular rims have a two‐layered structure: a combination of Zone I and Zone II, below which Zone III exists. Zone II is the partially amorphized zone. Zone II of ferromagnesian silicates contains abundant nanophase Fe. Radiation‐induced segregation and in situ reduction are the most plausible mechanisms to form nanophase Fe in Zone II. Their lattice fringes indicate that they contain metallic iron, which probably causes the reddening of the reflectance spectra of Itokawa. Zone II of the composite vesicular rims contains vesicles. The vesicles in Zone II were probably formed by segregation of solar wind He implanted in this zone. The textures strongly suggest that solar wind irradiation damage and implantation are the major causes of surface modification and space weathering on Itokawa.

Asparagine-linked oligosaccharides (N-glycans) usually show structural heterogeneity, especially in proteins with sialylated N-glycans and, therefore, their structural analysis is still very difficult. A zwitterionic type of hydrophilic interaction chromatography column with sulfobetaine functional groups (called a ZIC-HILIC column) was applied to the separation of tryptic peptides of alpha-1-acid glycoprotein. It was demonstrated that the ZIC-HILIC separation column has a selectivity for sialylated N-glycopeptides and a high capability for separation based on the structural recognition of sialylated N-glycan isomers as well as for the previously reported neutral N-glycans and N-glycopeptides. The retention characteristics of neutral and sialylated N-glycans derivatized with 2-aminopyridine (PA N-glycans) demonstrate that the retentions of the N-glycans are based primarily on hydrophilic interaction with the water-rich liquid layer generated on the surface of the ZIC-HILIC column. In addition, the electrostatic repulsion interaction shielded with counter ions effectively tunes the separation and recognition of sialylated N-glycan isomers.

Abstract In some high‐strength steels, a fatigue crack tends to occur at the interior inclusion after a long‐term sequence of the cyclic loadings at low stress levels, although the crack takes place at the surface in the usual life region at high stress levels. Thus, we have the duplex S–N curves consisting of the respective S–N curves for usual life region and very high‐cycle regime. It is well known that a significant fracture surface having the fine granular morphology is formed around the interior inclusion at the crack initiation site. This surface area is sometimes called as ‘fine granular area’. In this work, metallurgical structures around the interior inclusion at the fatigue crack initiation site were carefully observed by combining several special techniques such as focused ion beam technique and high‐resolution scanning electronic microscopes. Based on the current observation results, it was found that the microstructure around the interior inclusion was changed into the penny‐shape fine granular layer from the usual martensitic structure during long‐term cyclic loadings. Then, debondings along with the boundaries of the matrix and the fine granular layer have produced the small cracks inside the metallic material, and these interior cracks caused the final fatigue fracture after definite loading cycles of the crack propagation.

Nanomanipulation inside a scanning electron microscope (SEM) has been employed to maneuver and characterize nanomaterials. Despite recent efforts toward automated nanomanipulation, it is still largely conducted manually. In this paper, we demonstrate automated nanomanipulation inside an SEM for a well-structured nanomanipulation task via visual servo control and a vision-based contact-detection method using SEM as a vision sensor. Four-point probe measurement of individual nanowires is achieved automatically by controlling four nanomanipulators with SEM visual feedback. A feedforward controller is incorporated into the control system to improve response time. This technique represents an advance in nanomanipulation inside SEM and can be extended to other nanomanipulation tasks.

In recent years major advancements have been made in the directed self-assembly (DSA) of block copolymers (BCP). Insertion of DSA for IC fabrication is seriously considered for the 7nm node. At this node the DSA technology could alleviate costs for double patterning and limit the number of masks that would be required per layer. At imec multiple approaches for inserting DSA into the 7nm node are considered. One of the most straightforward approaches for implementation would be for via patterning through templated DSA (grapho-epitaxy), since hole patterns are readily accessible through templated hole patterning of cylindrical phase BCP materials. Here, the pre-pattern template is first patterned into a spin-on hardmask stack. After optimizing the surface properties of the template the desired hole patterns can be obtained by the BCP DSA process. For implementation of this approach to be implemented for 7nm node via patterning, not only the appropriate process flow needs to be available, but also appropriate metrology (including for pattern placement accuracy) and DSA-aware mask decomposition are required. In this paper the imec approach for 7nm node via patterning will be discussed.

Organometal halide perovskites have attracted widespread attention as the most favorable prospective material for photovoltaic technology because of their high photoinduced charge separation and carrier transport performance. However, the microstructural aspects within the organometal halide perovskite are still unknown, even though it belongs to a crystal system. Here direct observation of the microstructure of the thin film organometal halide perovskite using transmission electron microscopy is reported. Unlike previous reports claiming each phase of the organometal halide perovskite solely exists at a given temperature range, it is identified that the tetragonal and cubic phases coexist at room temperature, and it is confirmed that superlattices composed of a mixture of tetragonal and cubic phases are self-organized without a compositional change. The organometal halide perovskite self-adjusts the configuration of phases and automatically organizes a buffer layer at boundaries by introducing a superlattice. This report shows the fundamental crystallographic information for the organometal halide perovskite and demonstrates new possibilities as promising materials for various applications.

Abstract Solid‐state synthesis from powder precursors is the primary processing route to advanced multicomponent ceramic materials. Designing reaction conditions and precursors for ceramic synthesis can be a laborious, trial‐and‐error process, as heterogeneous mixtures of precursors often evolve through a complicated series of reaction intermediates. Here, ab initio thermodynamics is used to model which pair of precursors has the most reactive interface, enabling the understanding and anticipation of which non‐equilibrium intermediates form in the early stages of a solid‐state reaction. In situ X‐ray diffraction and in situ electron microscopy are then used to observe how these initial intermediates influence phase evolution in the synthesis of the classic high‐temperature superconductor YBa 2 Cu 3 O 6+ x (YBCO). The model developed herein rationalizes how the replacement of the traditional BaCO 3 precursor with BaO 2 redirects phase evolution through a low‐temperature eutectic melt, facilitating the formation of YBCO in 30 min instead of 12+ h. Precursor selection plays an important role in tuning the thermodynamics of interfacial reactions and emerges as an important design parameter in planning kinetically favorable synthesis pathways to complex ceramic materials.

The fibroblast growth factor receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was >or=11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of 15 non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC.

High-entropy alloys (HEAs) are a class of metallic materials composed of solid solutions of five or more elements in equi- or near-equiatomic proportions. The fascinating properties of HEAs have recently attracted considerable attention for water-splitting applications. Mechanical alloying (MA) is a method for preparing HEAs that results in crystalline, homogeneous materials at room temperature. In this work, several CoCuFeNi-based HEAs were prepared through MA and evaluated as electrocatalysts for the oxygen evolution reaction (OER), hydrogen evolution reaction (HER), and overall water splitting in 1 M KOH. The results showed that CoCuFeNiMnMo1.5 with the highest amount of molybdenum exhibited the best OER performance (375 ± 15 mV at the current density of 10 mA cm–2), and CoCuFeNiMnMo0.5 with the lowest amount of molybdenum exhibited the best HER activity with lower overpotentials (275 ± 12 mV at the current density of 10 mA cm–2) and over 72 h of stability. The assembled CoCuFeNiMnMo1.5 (anode)∥CoCuFeNiMnMo0.5 (cathode) couple required 1.76 V to produce 10 mA cm–2, and the Faradaic efficiency for generated H2 was determined to be more than 80%.

Phosphorus is an essential element for life on earth and is also important for modern agriculture, which is dependent on inorganic fertilizers from phosphate rock. Polyphosphate is a biological polymer of phosphate residues, which is accumulated in organisms during the biological wastewater treatment process to enhance biological phosphorus removal. Here, we investigated the relationship between polyphosphate accumulation and electron-dense bodies in the green alga Parachlorella kessleri. Under sulfur-depleted conditions, in which some symporter genes were upregulated, while others were downregulated, total phosphate accumulation increased in the early stage of culture compared to that under sulfur-replete conditions. The P signal was detected only in dense bodies by energy dispersive X-ray analysis. Transmission electron microscopy revealed marked ultrastructural variations in dense bodies with and without polyphosphate. Our findings suggest that the dense body is a site of polyphosphate accumulation, and P. kessleri has potential as a phosphate-accumulating organism.

A new class of rhodamine luminophores, 3',3''-bis(oxospiroisobenzofuran)-3,7-bis(dialkylamino)benzopyrano-xanthene derivatives (ABPX), have been successfully developed. The emission behavior of ABPX series is directly opposite to the concentration quenching of conventional rhodamine dyes. ABPX series exhibit aggregation-induced emission enhancement (AIEE).

The 193nm photoresist (ArF resist) degradation mechanism in dielectric etching was investigated by using an ultra-high-frequency electron-cyclotron-resonance plasma. This investigation focused on via-hole etching. It was found that the bottom-antireflection coating (BARC) etching condition is a critical factor in the reduction of striation and pitting after via-hole etching. X-ray photoelectron spectroscopy and scanning electron spectroscopy studies revealed that argon-less and low-incident-ion-energy conditions in BARC etching can keep the resist surface smooth and maintain a carbon-rich micromask-less state because decomposition of the C–H or OC–O bonds is suppressed. As a result, resist damage after via-hole etching is reduced remarkably. Furthermore, in the via-hole etching, it was also found that the characteristics of the fluorocarbon polymer, i.e., deposition rate and flourine-to-carbon ratio of the fluorocarbon polymer, stacked on the resist surface during etching strongly affect the ArF resist degradation. Low-sticking-coefficient radicals such as CF2 and a low amount of deposition thickness are suitable for damage-less etching. In regard to the formation of striations at the pattern corner, the sputtering effect was taken into consideration. As a result, in the case of via-hole etching, line-edge-roughness in the trench pattern was improved by about 50%, and a striation-less and pitting-less hole etched profile was obtained by using either an argon-and-xenon (20%) mixture as a dilution gas or a fluorocarbon gas at low flow rate under low gas pressure.

&lt;div class="htmlview paragraph"&gt;A diesel particulate filter (DPF) controls and maintains a constant pressure drop across the filter by repeating the regeneration process for PM (Particulate Matter). However, the regeneration results in ash accumulation on the DPF. Although this ash accumulation is very slow, it eventually causes increased pressure drop which affects the product life of the DPF.&lt;/div&gt; &lt;div class="htmlview paragraph"&gt;Metal elements in the lubricant additives in the engine oil are the source of the ash. Since ash is an oxidized substance, the amount of ash produced depends on such factors as the amount of oil consumed in the engine and the kinds of lubricant additives contained in engine oil. According to the reference literature [&lt;span class="xref"&gt;1&lt;/span&gt;-&lt;span class="xref"&gt;3&lt;/span&gt;], ash accumulates on a DPF differently depending on use of either a passive regeneration system or an active regeneration system. With the passive regeneration system, ash accumulates uniformly on the filter wall while ash accumulates near the outlet of the filter with the active regeneration system. Even though the amount of ash collected on the filter is same regardless of the regeneration method, the difference in the way ash accumulates on the filter between the two regeneration systems affects the characteristics of pressure drop across the filter. Therefore, the regeneration method is another parameter that affects filter performance, and the selection of a regeneration strategy is important to extend the useful life of the DPF.&lt;/div&gt; &lt;div class="htmlview paragraph"&gt;According to observations of ash accumulating on a DPF through both bench and engine tests of the regeneration process and accumulated ash removed from the DPFs of fleets, the authors found that ash is different in its particle size depending on temperature of PM combustion during regeneration. The particle size was smaller at lower combustion temperatures and larger at higher combustion temperatures as in the cases of the passive and active regeneration systems, respectively. As a result of these observations, a hypothesis was developed to explain the effect that the regeneration system had on how ash would accumulate on a DPF.&lt;/div&gt; &lt;div class="htmlview paragraph"&gt;The authors investigated the effect that ash accumulating on a DPF had on pressure drop across the filter and PM filtering performance. DPFs were sampled from a high-mileage fleet and evaluated for both pressure drop and PM filtering performance. Subsequently, the DPFs were cut to observe how ash had accumulated on the filter. Various patterns of ash collection on a DPF were artificially created using fly ash, and their effects on both pressure drop and PM filtering performance were evaluated. The results of these investigations revealed how ash collected on the filter would affect the pressure drop and PM filtering performance.&lt;/div&gt; &lt;div class="htmlview paragraph"&gt;According to the results of various tests and evaluations, the authors describe the mechanism of ash loading on the DPF and propose a DPF regeneration system that produces an ideal accumulation pattern of ash on the filter to keep PM filtering performance at the optimal level for useful life.&lt;/div&gt;