住友 弘二

We have completed a system that can achieve both deep x-ray lithography and submicron x-ray lithography with a single beamline by introducing the combination of x-ray plane and cylindrical mirrors. This x-ray lithography system can provide a large-scale microfabrication processing with 210 × 300 mm2 (A4 size). To exploit multiscale lithography, the beamline has a beam transport vacuum duct with a two-stage stacked structure and a 5-axis stage. This two-stage stacked structure allows us to fabricate both micron scale structures with high aspect ratios and submicron scale structures using the same beamline. In addition, x-ray imaging and computer tomography (CT) system are connected to the x-ray lithography system for nondestructive inspection and evaluation of the fabricated microstructures. The x-ray imaging system constructed this study has a relatively low energy range of x-ray energy in the beamline, which is in the range of 2–15 keV or less. Therefore, relatively good absorption contrast can be obtained for plastic materials, biomaterials, and the like. Since nondestructive imaging of the processed shape by x-ray lithography is possible, it is a very useful system in processing and evaluation can be performed simultaneously. This system also enables us to obtain the live images with keeping the creature alive in liquid using an indirect x-ray imaging system which converts x-ray images to visible light images through the fluorescent plate.

<jats:title>Abstract</jats:title> <jats:p>The reduction of graphene oxide (GO) through atomic hydrogen annealing (AHA) and soft X-ray irradiation is investigated using microwell substrates with μm-sized holes with and without Ni underlayers. The GO film is reduced through AHA at 170 °C and soft X-ray irradiation at 150 °C. In contrast, some GO films are not only reduced but also amorphized through soft X-ray irradiation. The effect of the Ni underlayer on GO reduction differs between AHA and soft X-ray irradiation. In AHA, the difference in GO reduction between SiO<jats:sub>2</jats:sub> and Ni underlayer was originated from the atomic hydrogen density on sample surface. On the other hand, in soft X-ray irradiation, the difference in GO reduction between SiO<jats:sub>2</jats:sub> and the Ni underlayer originates from the excited electrons generated by soft X-ray irradiation. Reduction without damage is more likely to occur in the suspended GO than in the supported GO.</jats:p>

The effect of atomic hydrogen exposure on hydrogenated amorphous carbon (a-C:H) films was investigated by X-ray photoelectron spectroscopy (XPS). From the dependence of the wide-scan XPS spectra of an a-C:H film on atomic hydrogen exposure, it was shown that the film was etched with an etching rate of 0.2 nm min-1. In addition, by analyzing the C 1s XPS spectra, the coordination of C atoms in the a-C:H film was investigated as a function of the atomic hydrogen exposure and photoelectron emission angle. This indicated that the coordination of C atoms at the surface of the a-C:H film was not influenced by atomic hydrogen exposure. Therefore, we propose that the depth profile of a-C:H films can be measured with no damage using atomic hydrogen etching.

An extreme ultraviolet (EUV) light with a wavelength of 13.5 nm has been introduced to 7 nm FinFET technology. Optical elements such as Mo/Si multilayer mirror in lithography equipment are contaminated with hydrocarbon during the EUV light irradiation. The reflectance of the mirrors is decreased by carbon contamination. Therefore, the removal method of the carbon contamination is required for reduction of maintenance cost. The surface treatment using atomic hydrogen generated by a heated tungsten mesh, called as atomic hydrogen annealing (AHA), have been investigated for cleaning of the optical elements used in the synchrotron facility. The Au/Cr/Si substrate, Ni mirror and Ni diffraction grating with carbon contamination were cleaned and the reflectance of the mirrors was recovered by AHA. In addition, the AHA conditions could be optimized for cleaning of Mo/Si multilayer mirrors from the relationship between the treatment conditions and degradation. Furthermore, to clarify the reaction of atomic hydrogen with not only C-C bond but also C-O bond, the graphene oxide (GO) film was also treated by AHA. The C-O-C bonds in the GO films were preferentially reduced by AHA. It is found that the surface contamination consisting of hydrocarbon and/or C-O bond on the optical elements is removed without damage. The ability of atomic hydrogen to clean the optical elements had been confirmed. The findings are useful for the advanced lithography technology using EUV light.

© 2020 The Institute of Electrical Engineers of Japan. We propose the system to analyze water permeability through the lipid bilayer. The lipid bilayer suspended over microwells changes its shape when the difference of osmotic pressure is formed by solution exchange. When the solution concentration outside the microwell is lower than the inside, water permeates through the lipid bilayer membrane and bulges outward. The interference fringes observed in the suspended lipid bilayer allowed us to accurately analyze its structural changes. Water permeability through the lipid bilayer could be accurately estimated based on analysis of the changes in the interference fringes observed in the suspended lipid bilayer caused by the solution exchange.

© 2020 SPST. The surface treatment using atomic hydrogen genareted by a heated tungsten mesh was investigated for the cleaning of the optical elements used in the synchrotron facility. We call the surface-tretament by the atomic hydrogen annealing (AHA). The Au and Ni mirors and Ni and Mo diffraction gratings with carbon contamination were cleaned by the chemical reaction and thermal effect due to the recombination of the atomic hydrogens during AHA. The carbon contamination was removed and the reflactance of the Au and Ni mirrors was recovered by AHA. The AHA conditions could be optimaized for cleaning of Mo/Si multilayer mirrors from the relationship between the treatment condition and degradation. In addition, to clarify the removal reaction of the carbon contamination, the two-types of amorphous carbon (a-C) films were used. The etching rate of the a-C film by AHA was strongly related to the hydrogen content, atomic density and sp2/sp3 component of a-C film.

The localisation of liquid-ordered (L-o) and liquid-crystalline (La) phase domains on a silicon substrate with a microwell array is investigated. Although the phase separation of the Lo and La phases on both a giant unilamellar vesicle (GUV) and a supported membrane remains stable for a long time, the lateral diffusion of lipids across each domain boundary occurs quickly. Since the phase separation and domain arrangement are governed by the stiffness and lateral tension of the lipid membrane, the phase separation is rearranged on a rnicropatterned substrate. Similar phase separation of the Lo and La phases is observed at a lipid membrane suspended over a microwell. However, the La phase is preferred at a suspended membrane, and saturated lipids and cholesterol are excluded toward the supporting membrane on the periphery. Since the Lo domain area is reduced by anisotropic diffusion through the boundary between the suspended and supported membranes, a very slow reduction rate with a La phase domain is observed at a membrane suspended over a microwell, which is membrane. linear functional relation is observed. Finally, a localized surrounded by an Lo phase supported membrane.

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.

Microfabricated mobile electrodes, nanopallets, designed to provide adherent cells with electrical stimulation are reported. Nanopallets composed of a cross-linked silk fibroin hydrogel matrix incorporated with poly(3,4-ethyle nedioxythiophene): polystyrene sulfonate (PEDOT:PSS) are used. The silk fibroin composite is characterized not only by mechanical and electrical conductive properties, but also by its optical transparency in both the visible and ultraviolet regions, and by its biocompatibility with adherent cells. It is demonstrated that the adherent cells, including normal cell-lined cells and primary neuronal cells, loaded on the nanopallets can be manipulated while faithfully retaining their adhesive properties. By applying voltages via the nanopallets, the voltage-dependent calcium channels expressed in the cells are selectively stimulated, and this is confirmed by using confocal fluorescent microscopy during manipulation and performing multiangle observations. These features are attributed to both the mobile operation of the transparent nanopallets, and the ability to simultaneously measure electrical signals and perform fluorescent observations.

Membrane proteins play vital roles in a wide variety of functions in living organisms. The development of nanobiodevices that fuse semiconductor nanotechnology and membrane proteins makes it possible to understand various biological processes as well as engineering applications. In this article, we introduce the fabrication of nanobiodevices that are functionalized by membrane proteins and designed to mimic the synaptic signal transmission mechanism in the nervous system.

Neuronal patterning is useful for understanding signal propagation between neurons as well as for biosensors and cell-based assays. The patterning of living cells has been made possible by employing surface physicochemical and topographic features. This study investigated neuronal growth on patterned nanopillars. Rat cortical neurons were cultivated on quartz substrates with amorphous silicon (a-Si) and Au pillars 100 and 500 nm in diameter. The neurites grew better with the larger diameter pillars, and the partly-selective neurite growth was observed for a-Si pillars but not for Au pillars. These results reveal the possibility of controlling neuronal growth by using a-Si nanopillars. (C) The Electrochemical Society of Japan, All rights reserved.

Interfaces between single neurons and conductive substrates were investigated using focused ion beam (FIB) milling and subsequent scanning electron microscopy (SEM) observation. The interfaces play an important role in controlling neuronal growth when we fabricate neuron-nanostructure integrated devices. Cross sectional images of cultivated neurons obtained with an FIB/SEM dual system show the clear affinity of the neurons for the substrates. Very few neurons attached themselves to indium tin oxide (ITO) and this repulsion yielded a wide interspace at the neuron-ITO interface. A neuron-gold interface exhibited partial adhesion. On the other hand, a neuron-titanium interface showed good adhesion and small interspaces were observed. These results are consistent with an assessment made using fluorescence microscopy. We expect the much higher spatial resolution of SEM images to provide us with more detailed information. Our study shows that the interface between a single neuron and a substrate offers useful information as regards improving surface properties and establishing neuron-nanostructure integrated devices.

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.

To provide a platform for biodevices designed to characterize membrane proteins, we fabricated a new type of microwell sealed with a lipid membrane on a SiO2 substrate. The microwell is surrounded by a Au ring with a self-assembled monolayer, on which a lipid membrane is formed to create a seal. Fluorescence and electrophysiological studies reveal that the structure prevents unfavorable ion leakage from/ into microwells. By separating the microwells and outer regions, ion diffusion through the water layer between the membrane and the substrate is reduced. This study offers a promising approach for the functional analysis of membrane proteins. (C) 2015 The Japan Society of Applied Physics

A membrane protein is one of the targets for understanding cellular function and for biosensor and drug screening applications. An artificial lipid membrane suspended over microwells can act as a platform when we attempt to realize a nanobio device based on a membrane protein function. With the aim of preparing a lipid membrane array on a substrate, we investigated lipid membrane formation on a hydrogel-confined micropatterned substrate because a hydrogel can support a lipid membrane both mechanically and functionally. The interaction between the positive charge of the hydrogel and the negative charge of the substrate surface allows the hydrogel to be confined in the microwells. When small unilamellar vesicles with a diameter smaller than the aperture of the microwells were added to the solution over the substrate, hydrogel-supported lipid membranes were formed. The vesicle fusion was facilitated by the electrostatic interaction between the vesicle charge and the opposite charge of the hydrogel. The continuous and fluid properties of the hydrogel-supported lipid membrane were confirmed by the fluorescent recovery after photobleaching method. Our results suggest that support provided by hydrogels in the microwells enables us to arrange membrane proteins on a nanobio device by the direct formation of a planar lipid membrane from proteoliposomes with a small diameter. (C) 2015 Elsevier B.V. All rights reserved.

Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes. (C) 2015 Elsevier Inc. All rights reserved.

We propose a hydrophilic-raised-ring structure at the aperture of a hole for the efficient suspension of a lipid bilayer. This ring structure can be fabricated by using a combination consisting of a focused ion beam (FIB) and a hydrogen silsesquioxane (HSQ) negative electron beam (EB) resist layer on a substrate. Negative EB resist at the aperture of a hole can be selectively reacted with a scattered gallium ion beam during the FIB milling process, and a ring structure can be obtained by employing a development process. Assessments of the cross section of the ring revealed a two-step structure. The suspension of lipid bilayers on the ring structures was assessed by observing the green fluorescence of calcein dye inside the holes. Our observation of fluorescent images showed that the lipid bilayers on the ring structures sealed the calcein solutions. This result indicates that our fabrication processes can be used to produce nanobio devices that need lipid bilayer suspensions. (C) 2014 The Japan Society of Applied Physics

The electrostatic control of lipid bilayer self-spreading was investigated using a device equipped with a nanogap gate. A series of mixtures containing negatively charged and uncharged lipids were employed to tune the charge of a membrane. We found that when a voltage is applied on a lipid bilayer passing through a nanogap, the effect of a voltage application on the dynamics depended largely on the charge of the membrane. For rich charged lipid compositions (&gt;10 mol%), the self-spreading was electrostatically controlled applying an electric field to the nanogap. The origin of the behaviour is the electrostatic trapping of charged lipids. The trapped lipids close the nanogap gate, thus preventing any lipid molecules from passing through it. For poor charged lipid compositions (similar to 1 mol%), no electrostatic trapping occurred even when a lipid bilayer reached the nanogap. Instead, we observed the cessation of self-spreading after a sufficient post-passage time interval, indicating that the translational flow force of self-spreading overcomes the trapping force. For uncharged lipid compositions, there was no electrostatic trapping throughout the measurement. The results suggest that the lipid charge plays a vital role in the electrostatic control mechanism and allow us to control lipid bilayer formation both spatially and temporally.

We observed neurons cultivated on an indium tin oxide (ITO) substrate using scanning electron microscopy (SEM) whose high spatial resolution allows us to observe neuronal morphological details. First, we optimized the fixation condition of cultivated neurons for SEM observation. The first fixation with paraformaldehyde and glutaraldehyde, and the second fixation with OsO4 were both necessary to avoid cell removal during the preparation before SEM observation. After optimization, we examined the morphological changes of neurons under an apoptotic condition, induced by staurosporine, by using both SEM and an immunochemical technique. Interestingly, the addition of staurosporine induced both apoptosis and a necrotic phenomenon. Immunostaining analysis revealed late-stage apoptosis after early-stage apoptosis, which was observed induced in cortical neurons by staurosporine for the first time. We confirmed that the SEM imaging of neurons is very useful as regards observing the apoptotic process. It is also a promising tool for understanding such neuronal activities as synaptic formation and axonal growth. Further examination of the interaction between neurons and substrates will contribute to the implementation of the artificial neurological devices. © 2014 The Surface Science Society of Japan.

We investigated structural changes in ligand-gated ion channel proteins reconstituted into supported lipid bilayers. The ion channels' extracellular parts were highly dynamic and exhibited agonist-induced changes, which were inhibited by antagonists. Our data demonstrate that, by using small exogenously applied compounds (i.e., ligands), ion channel proteins reconstituted into supported lipid bilayers on a substrate can be controlled. (C) 2014 The Japan Society of Applied Physics

To provide a platform for a nanobiodevice, we fabricated microcavities on a Si/SiO2 substrate covered by a thin SiO2 layer with nanohole arrays that we call a pepper shaker substrate. Fluorescence and atomic force microscopy images showed that the structure of the pepper shaker substrate improved both the probability of membrane sealing over the microcavities by rupturing giant unilameller vesicles and the lifetime of the lipid membrane suspended over the microcavities. The success of this study reveals the potential for fabricating an artificial cell array as a tool for the functional and high throughput analysis of membrane proteins. (C) 2014 The Japan Society of Applied Physics

We developed a conductive fiber by coating the surface of silk or synthetic fiber with a conductive polymer and fabricated wearable electrodes to achieve long-term biosignal monitoring. This new bioelectrode made of a complex conductive material is flexible, biocompatible, and hydrophilic and allows stable recording equivalent to that obtained with conventional medical electrodes but without the need for any electrolyte paste, which can irritate the patients&#039; skin. We can expect this wearable electrode to be utilized in various applications related to sports, health improvement, and early medical diagnosis by providing long-term biosignal monitoring without patient discomfort or irritation.

We successfully sealed a gel-confined microwell array on a Si substrate with a lipid membrane by rupturing giant unilamellar vesicles. Atomic force microscope measurements suggested that gel with a hillock structure supported lipid membranes gently because of its soft and elastic properties and improved the stability of the lipid membrane over the microwell array. We found that the lipid membrane sealed the gel-confined microwells and no Ca2+ leakage through the lipid membrane was observed within the detection range of our calcium indicator. We expect that gel with properties similar to cytoplasm consisting of a cytoskeletal network is a potential candidate for providing lipid membranes with mechanical support. This study proposes an artificial cell array system for fluorescence and atomic force microscope observations of functional membrane proteins on silicon-based nanobiodevices. (C) 2014 The Japan Society of Applied Physics

Gold nanoparticles are generally considered to be biologically inactive. However, in this study we show that the addition of 1.4 nm diameter gold nanoparticle induces the remodeling of the ring-shaped protein TRAP into a hollow, capsid-like configuration. This structural remodeling is dependent upon the presence of cysteine residues on the TRAP surface as well as the specific type of gold nanoparticle. The results reveal an apparent novel catalytic role of gold nanoparticles.

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca2+ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca2+ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm2, and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices. © 2011 Elsevier B.V.

We investigated techniques for regulating the orientation of ion channel-type membrane proteins reconstituted in lipid bilayers. Free ion channel proteins aligned their long axis parallel to the substrate. In contrast, immunochemical and atomic force microscopy images revealed that ion channels reconstituted in supported lipid bilayers oriented upward, with their long axis perpendicular to the substrate. Our data demonstrates that the reconstitution of ion channels into planar lipid bilayers by rupturing small unilamellar proteoliposomes is a promising way of aligning ion channels upward in a membrane and of obtaining ion channels with controlled functions. (C) 2011 The Japan Society of Applied Physics

We fabricated a heterogeneous supported lipid bilayer (SLB) by employing binary lipid mixtures comprising a saturated acyl chain DSPC and an unsaturated acyl chain nickel-chelating lipid. By using the specific adsorption properties of histidine-tagged proteins (His-tagged GFPs) in relation to nickel-chelating lipids, we demonstrated protein pattern formation on the SLB corresponding to the phase separation pattern of the SLB. In addition, by using a lipid mixture consisting of an unsaturated acyl chain DOPC and a nickel-chelating lipid, and His-tagged GFPs, we succeeded in transporting the proteins along a hydrophilic micropattern on a SiO(2) substrate. The protein transport is induced by the self-spreading behavior of a fluid SLB with a kinetic spreading coefficient beta = 10.4 mu m(2)s(-1). This method provides a guide for strategically carrying various biomolecules to specific positions by using a soft biointerface on a solid surface. In addition, the results demonstrate the importance of using techniques that allow the controlled manipulation of biomolecules based on the static or dynamic properties of the SLB platform.

We investigated the optimum architecture for confining fluorescent probes in microwells on a Si substrate by covering it with a lipid bilayer. We modified the structure of the wells to prevent the lipid membrane from falling into them, and the overhang shape at the aperture improved the probability of confinement. The fluorescence intensity from the calcein confined in the wells remained unchanged for one hour or more, indicating that the probes remain stably in the wells without flowing out. An artificial cell sealed with the suspended membrane is a promising tool for the functional analysis of membrane proteins. (C) 2010 The Japan Society of Applied Physics