Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society of Mechanical Engineers  

DOI： 10.1299/mej.22-00223

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

DOI： 10.1021/jacs.2c04096

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.snb.2021.131281

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.snb.2021.130587

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.chemphyslip.2021.105148

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：eLife Sciences Publications Ltd.  

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

DOI： 10.7554/eLife.66954

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

The conservation throughout evolution of membrane-bound structures that encapsulate genomic material indicates the existence of a simple, physical mechanism that facilitates the enclosing of long-stranded DNA by lipid bilayers. This study aimed to elucidate such a mechanism by investigating how molecular crowding promotes the spontaneous enveloping of model DNA into lipid bilayer membranes. Using fluorescence microscopy and giant unilamellar vesicles (GUVs) we showed that a 166 kb DNA molecule coencapsulated with a model crowder attaches to the inner membrane of the GUVs as they osmotically deflate and after the DNA–membrane complex buds out. The set of results is consistent with the hypothesis that the depletion volume effect is responsible for the spontaneous encapsulation of DNA in the GUVs. This phenomenon may offer novel insights into the basic mechanisms governing membrane encapsulation of long-stranded nucleic acids found in celluar sytems that are independent of genetic control.

DOI： 10.1021/acssynbio.0c00360

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

This paper presents a demonstration of the selective bonding of three different millimeter-scale components by using complementary patterning of hydrophilic and hydrophobic surfaces. The hydrophobic surface of millimeter-scale components fabricated from hydrophobic polydimethylsiloxane (PDMS) was made partially hydrophilic with a designed pattern by exposure to an excimer light through a stencil mask. The oil, as an adhesive, was only deposited on the hydrophobic surface. We prepared 30 components with 3 patterns, which were agitated in water by using a computer-controlled propeller stirrer. As a result, we obtained a significantly higher yield of correct bonds compared to erroneous bonds under an appropriate stirring condition. The result can be explained through the contrast in bonding strength between the correct and erroneous bonds. The design principle of using concentric patterns serves as a basic strategy for realizing programmed self-assembly with selective bonding patterns.

DOI： 10.1016/j.sna.2020.112143

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

Deformation of liposomes, or lipid vesicles, has been investigated extensively in terms of the thermodynamic equilibrium of the bending energy of the lipid bilayer membrane. However, the range of such deformation in previous literature has been limited within the moderate surface-to-volume ratio of the vesicles, in which axisymmetric shapes are dominant. Here, we show that neuron-like morphology, in which many lipid tubes extend radially from the mother vesicle, becomes dominant upon the slow osmotic shrinkage of giant unilamellar vesicles (GUVs) initially larger than several tens of micrometers. We show that, in the time-lapse confocal imaging, the emergence of lipid tubes is initiated from the instability that appeared along the annular rim of the flat stomatocyte shape. Since these deformation dynamics into the neuron-like morphology resemble that of the milk-crown formation in liquid splashing, we discuss that the Rayleigh–Plateau capillary instability drives this transformation into a nonaxisymmetric shape.

DOI： 10.1021/acs.langmuir.0c00872

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：RSC  

We investigated the capability of simple microfluidic devices with trenches having vertical sidewalls for live-cell fluorescence imaging of adherent cells. An epithelial cell line that forms a two-dimensional (2D) sheet was cultured to adhere to the vertical sidewall so that its vertical section can be imaged directly using ordinal inverted-type laser-scanning microscopy. The material and the structure of the device were characterized. We show that the detailed distribution of intracellular organelles, such as microtubules and mitochondria, and of intercellular apparatus, such as claudin and zonula occludens, can be imaged with high spatio-temporal resolution with a single scan.

DOI： 10.1039/C9AN02165E

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

Electroporation or electropermealization is a technique to open pores in the lipid bilayer membrane of cells and vesicles transiently to increase its permeability to otherwise impermeable molecules. However, the upper size limit of the materials permeable through this operation has not been studied in the past. Here, we investigate the size of the material that can be released (ejected) from giant unilamellar vesicles (GUVs) upon electrical pulsation. We confirm that the volume of GUV shrinks in a stepwise manner upon periodical pulsation, in accordance with previous studies. When the same operation is applied to GUVs that encapsulate microbeads, we find that beads as large as 20 μm can be ejected across the membrane without rupturing the whole GUV structure. We also demonstrate that functional bioactive particulate materials, such as gel balls, vesicles, and cells can be encapsulated in and ejected from GUVs. We foresee that this phenomenon can be applied to precisely regulate the time and location of release of these particulate materials in the microenvironment.

DOI： 10.1021/acs.langmuir.9b01617

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IEEE  

Three-dimensional (3D) self-assembly of mesoscale parts with tens to hundreds of micrometer scales, independent of the material properties, is demonstrated by simply adding an inert polymer, i.e., polyethylene glycol, with large molecular weight (4MDa) at a concentration of micrograms per milliliters. It is most likely that the depletion volume effect, a thermodynamical effect to maximize the entropy of the system, induces face-to-face bonds with microfabricated parts having flat faces. The optimum polymer concentration range yielding high assembling efficiency is found, where the bonding energy is large enough to stabilize the bonding and viscosity is small enough to allow frequent collisions. Using this strategy, a site-specific assembly of 60 μm cubic parts in cavities engraved on large receptor parts, distributed on either 2D or 3D faces, is successfully demonstrated.

DOI： 10.1109/JMEMS.2019.2919230

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

In this paper, we demonstrate that 20 μm microbeads can be preferentially assembled into substrate trenches of similar width by employing a polymer (depletant) that induces the depletion volume effect (depletion attraction). In previous works, we proved that this strategy is useful to assemble mesoscale parts in a site-specific manner. Here, we show that it is also applicable to assemble functional parts, such as fluorescent particles, into trenches engraved on the surface of two- and three-dimensional template components. A convenient advantage of this strategy is that it is independent of material properties for assembling mesoscale functional components into desired patterns.

DOI： 10.3390/mi10070428

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

The steady streaming (SS) phenomenon is gaining increased attention in the microfluidics community, because it can generate net mass flow from zero-mean vibration. We developed numerical simulation and experimental measurement tools to analyze this vibration-induced flow, which has been challenging due to its unsteady nature. The validity of these analysis methods is confirmed by comparing the three-dimensional (3D) flow field and the resulting particle trajectories induced around a cylindrical micro-pillar under circular vibration. In the numerical modeling, we directly solved the flow in the Lagrangian frame so that the substrate with a micro-pillar becomes stationary, and the results were converted to a stationary Eulerian frame to compare with the experimental results. The present approach enables us to avoid the introduction of a moving boundary or infinitesimal perturbation approximation. The flow field obtained by the micron-resolution particle image velocimetry (micro-PIV) measurement supported the three-dimensionality observed in the numerical results, which could be important for controlling the mass transport and manipulating particulate objects in microfluidic systems.

DOI： 10.3390/mi9120668

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

Microelectromechanical systems (MEMS)-based high-performance accelerometers are ubiquitously used in various electronic devices. However, there is an existing need to detect physical impacts using low-cost devices with no electronic circuits or a battery. We designed and fabricated an impact sensor prototype using a commercial stereolithography apparatus that only consists of a plastic housing and working fluids. The sensor device responds to the instantaneous acceleration (impact) by deformation and pinch off of a water droplet that is suspended in oil in a sensor cavity. We tested the various geometrical and physical parameters of the impact sensor to identify their relations to threshold acceleration values. We show that the state diagram that is plotted against the dimensionless Archimedes and Bond numbers adequately describes the response of the proposed sensor.

DOI： 10.1371/journal.pone.0195741

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

The excluded volume effect, or depletion attraction, is the phenomenon describing induced aggregation of colloidal particles suspended in a densely crowded macromolecule solution. In this work, we attempted to utilize this effect for the self-assembly of artificially manufactured microcomponents on a 10-100 mu m scale. The bonding energy does not originate from the nature of the surfaces, but is generated by an increase of the translational entropy of macromolecules in solution. Thus, simple immersion of the microcomponents in the macromolecule solution allowed us to observe their assembly, based on shape complementarity, to minimize the free energy of the system. However, it became apparent that elaborate design is required for specific bonding between complementary shapes, in addition to merely increasing the contact surface area. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.sna.2016.11.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

Herein we examined the extent of replicability of the PDMS microchamber device transferred from the master mold with complex 3D structures fabricated via micro stereolithography. Due to the elastomeric properties of PDMS, the reversely tapered micromold, with the diameter ratio of ∼5 from the largest to the narrowest part, was precisely transferred without breaking. We obtained the mathematical model to estimate the stress exerted on the mold during the demolding process. Finally, we tested the applicability of this unusual microchamber for single-cell trapping and an enzyme assay.

DOI： 10.1039/c6lc01313a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Lipid vesicles, in particular Giant Unilamellar Vesicles (GUVs), have been increasingly important as compartments of artificial cells to reconstruct living cell-like systems in a bottom-up fashion. Here, we report shape transformations of lipid vesicles induced by polyethylene glycol-lipid conjugate (PEG lipids). Statistical analysis of deformed vesicle shapes revealed that shapes vesicles tend to deform into depended on the concentration of the PEG lipids. When compared with theoretically simulated vesicle shapes, those shapes were found to be more energetically favorable, with lower membrane bending energies than other shapes. This result suggests that the vesicle shape transformations can be controlled by externally added membrane molecules, which can serve as a potential method to control the replications of artificial cells.

DOI： 10.1371/journal.pone.0132963

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells over-expressing MDR1, an ATP-binding cassette transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level.

DOI： 10.3389/fbioe.2015.00032

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

This paper describes the utilization of giant unilamellar vesicles (GUVs) as a platform for handling chemical and biochemical reagents. GUVs with diameters of 5 to 10 mm and containing chemical/biochemical reagents together with inert polymers were fused with electric pulses (electrofusion). After reagent mixing, the fused GUVs spontaneously deformed to a budding shape, separating the mixed solution into sub-volumes. We utilized a microfluidic channel and optical tweezers to select GUVs of interest, bring them into contact, and fuse them together to mix and aliquot the reaction product. We also show that, by lowering the ambient temperature close to the phase transition temperature T-m of the lipid used, daughter GUVs completely detached (fission). This process performs all the liquid-handing features used in bench-top biochemistry using the GUV, which could be advantageous for the membrane-related biochemical assays.

DOI： 10.1371/journal.pone.0101820

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We here present a method to reconstruct effective free energy landscapes (FELs) of lipid vesicles from the statistical analysis of a large number of microscope images. This method, not only allows us to define possible energy landscapes, but also highlights minority vesicle shapes that were otherwise hidden in the majority. When compared with temporal evolution of deforming lipid vesicles, it was found that the trajectory of deforming vesicles was in accordance with the reconstructed landscape, in which the minority shapes play a key role. When compared with theoretical models, it revealed that the vesicle shapes characterised in the reconstructed FELs were consistent with the theoretically predicted shapes. These results suggest that the FEL analysis can be a useful tool to investigate the morphological dynamics of lipid vesicles, in conjunction with other analytical methods.

DOI： 10.1039/c4sm00992d

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

The permeability of giant unilamellar vesicle (GUV) membranes to various solutes was investigated at the single-vesicle level. Membrane permeability has primarily been studied by obtaining the average of vesicle ensembles using small or large unilamellar vesicles (SUVs and LUVs, respectively) <1 mu m in diameter. The average properties observed for biological molecules or systems may not necessarily represent those of individual vesicles. In addition, although the GUV (>1 mu m) is considered to be a primitive cell model, its membrane permeability has rarely been investigated. We investigated the permeation of various molecules, including amino acids and mononucleotides, through more than 20 000 GUV membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using flow cytometry. We observed a general trend of lower membrane permeability for polar or charged molecules than for nonpolar molecules, which is consistent with previous studies. However, we found that the lower permeation of charged molecules resulted from the presence of at least two distinct GUV populations; the larger population consisted of impermeable GUVs, and the smaller population consisted of those with a high permeability. The presence of phospholipid vesicles highly permeable to charged and small molecules (<3 nm) was found through the single vesicular measurement. The observed permeability of the GUVs may have played an essential role in the self-reproduction and evolution of primitive cells.

DOI： 10.1039/c4ra05332j

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society  

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：RSC  

We report a facile, versatile and highly efficient self-patterning method for depositing functional coatings for poly(dimethylsiloxane) (PDMS) microfluidics, inspired by the wetting phenomena of the desert beetle and plant leaves. Driven by the solvent evaporation, fluoropolymer coatings have been demonstrated to deposit only on the roughened PDMS surfaces with exact coverage, conformal thickness, no clogging and good bonding strength, which significantly suppressed the swelling of PDMS by the organic solvent and the nonspecific adsorption of the fluorescent dye. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3sm27625b

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition to modern cell membranes composed of phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108: 5249-5254]. Thus, a growth-division mechanism of membranes that does not depend on the chemical nature of amphiphilic molecules must have existed. Here, we show that giant unilamellar vesicles composed of phospholipids can undergo the coupled process of fusion and budding transformation, which mimics cell growth and division. After gaining excess membrane by electrofusion, giant vesicles spontaneously transform into the budded shape only when they contain macromolecules (polymers) inside their aqueous core. This process is a result of the vesicle maximizing the translational entropy of the encapsulated polymers (depletion volume effect). Because the cell is a lipid membrane bag containing highly concentrated biopolymers, this coupling process that is induced by physical and nonspecific interactions may have a general importance in the self-reproduction of the early cellular compartments.

DOI： 10.1073/pnas.1120327109

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：RSC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

To achieve a cell-mimetic reaction environment, we fabricated and tested quartz microchambers for conducting protein synthesis using an in vitro transcription and translation system, the PURE system. By introducing a glass microchamber and blocking the surface of the chamber with amino acids, the concentration of the synthesized marker protein (green fluorescent protein, GFP) was significantly improved compared to that in the poly(dimethylsiloxane) (PDMS) microchamber. The concentration was below the detection limit in the PDMS microchambers, whereas the glass microchambers yielded 700 nM GFP, representing 41% of the bulk reaction. There was no detectable difference when the GFP synthesis was performed in microchambers with sizes ranging from 40 fL to 7 pL, indicating that the present microchamber system can serve as a cell-sized test tube with a variable reaction volume. Finally, we demonstrated that two different proteins, GFP and beta-galactosidase, can be expressed from single genes in our experimental setup. Quantized and distinctive signals from proteins synthesized from 0, 1, or 2 copies of genes were obtained. The microchamber presented here can be utilized not only to study the effects of compartment volume on protein synthesis but also for the comprehensive analysis of complex biochemical reactions in cell-mimetic environments.

DOI： 10.1039/c2lc40098g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Microfluidic trapping technology has been widely applied for single-cell observation in order to reveal characteristic cell behaviors. However, this strategy has yet to be tested for monitoring highly motile cells, which are often biologically important. In this paper, we seek the conditions that enable effective and long-term trapping of a prominent model ciliate Tetrahymena thermophila within a hydrodynamic microfluidic device. Although motility and flexibility of T. thermophila make it difficult to avoid escaping from the trap, we show that tuning some key parameters in the hydrodynamic circuit was effective to achieve approximately 40 h cell retention, which is long enough to monitor cell behaviors over several generations. Here, we demonstrate the real-time observation of cell division and phagocytic digestion, revealing interesting phenomena such as a wide distribution in doubling time in a poor synthetic medium and heterogeneous time courses in digestion processes. Our results present a strategy for trapping highly motile ciliate cells in order to study the dynamic behaviors of single cells.

DOI： 10.1039/c2lc40367f

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EPL ASSOCIATION, EUROPEAN PHYSICAL SOCIETY  

Living cells replicate by passing through cycles of growth and division, and they act as containers for chemical reactions. To determine basic properties of such recursively growing and dividing micro-compartments, we constructed a model system in a micro-fluidic device. In this system, droplets in a water-in-oil emulsion underwent cycles of fusion and subsequent division with nutrient-mimicking droplets. We found that repeating the combinations of growth (by fusion) and division of the droplets brought about their steady size distribution that had a skewed shape, similar to that which had been observed for proliferating cells. Our numerical analysis suggested that the skewness was originated from stochasticity in the addition of volumes in the growth process. Copyright (C) EPLA, 2011

DOI： 10.1209/0295-5075/96/48006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EPL  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We present a simple method to form free-standing lipid membranes on arrayed microchambers (> 100). The formed membranes are perpendicular to an imaging plane with control of solute concentration on each side of the membranes. This platform let us quantitatively detect membrane transport of non-charged fluorescent molecules, induced by membrane proteins.

DOI： 10.1039/c1lc20334g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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DOI： 10.1007/978-1-60327-331-2_20

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane a-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the a-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications.

DOI： 10.1021/ac901732z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

DOI： 10.1021/la902237y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We present a micro fluidic chip for parallel ion channel recording in a large array of artificial planar lipid bilayer membranes. To realize a composite structure that features an array of recording wells with free-standing microapertures for lipid bilayer reconstitution, the device was fabricated by the hybrid stereolithography technology, in which a Parylene film with pre-formed microapertures was inserted during the rapid stereolithography process. We designed and tested a hybrid chip that has 96 (12x8) addressable recording wells to demonstrate recording of ion channel current in high-throughput manner. Measurement was done by sequentially moving the recording electrode, and, as a result, the channel current of model membrane protein was detected in 44 wells out of 96. We also showed that this hybrid fabrication process was capable of integrating micropatterned electrodes suitable for automated recording. These results support the efficiency of our present architecture of the parallel ion channel recording chip toward realization of the high-throughput screening of ion channel proteins in the artificial lipid bilayer system.

DOI： 10.1007/s10544-008-9205-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometersized apertures (phi = 15-50 mu m, t = 20 mu m) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5 x 5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

DOI： 10.1021/ac7016635

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We present a highly parallel and reproducible method for reconstituting an array of lipid bilayers to analyze membrane transport. We infuse buffer/lipid/buffer solutions sequentially into a microchannel with numerous microchambers in its walls and seal each chamber by a lipid bilayer containing membrane proteins. Due to the small volume of the chamber (2 pL), membrane transport of confined fluorescent molecules across the bilayer through the proteins is readily observed as changes in fluorescent intensity. We successfully perform quantitative measurement of the transport flux of fluorescent molecules (Calcein) through a-Hemolysin antibiotic pores.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER ADVANCED TECHNOLOGY  

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented. (c) 2006 Published by Elsevier B.V.

DOI： 10.1016/j.bios.2006.04.013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACS  

DOI： 10.1021/la052534p

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Language：English   Publishing type：Research paper (scientific journal)  

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC  

An efficient magnetic force driven mixer with simple configuration is designed, fabricated, and tested. It is designed to facilitate the mixing of magnetic beads and biomolecules in a microchannel, where mixing is unavoidably inefficient due to its low Reynolds number. With appropriate temporal variation of the force field, chaotic mixing is achieved, hence the mixing becomes effective. The mixing device consists of embedded microconductors as a magnetic field source and a microchannel that guides the streams of working fluid. It is demonstrated that a pair of integrated micro conductors provides a local magnetic field strong enough to attract nearby magnetic beads. Mixing of magnetic beads is accomplished by applying a time-dependent control signal to a row of conductors, at the Reynolds number of as low as 10(-2). Two-dimensional numerical simulation has been performed to design the configuration of the channel and electrodes, which creates chaotic motion of beads. It is found that a simple two-dimensional serpentine channel geometry with the transverse electrodes is able to create the stretching and folding of material lines, which is a manifestation of chaos. The mixing pattern predicted by the simulation has been confirmed by both flow visualization and PTV (Particle Tracking Velocimetry) in the chaotic mixer fabricated, which should greatly increase the attachment of beads onto the target biomolecules. The optimum frequency of applied control signal is searched by evaluating the Lyapunov exponent in both numerical, and experimental particle tracking. It is found that the range of optimum Strouthal number is 5 < St < 12, which is defined by the channel width and the mean velocity.

DOI： 10.1109/JMEMS.2004.835775

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 mm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

DOI： 10.1039/b405967k

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本機械学会  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.43.S118_2

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Publishing type：Research paper (scientific journal)   Publisher：AIP Publishing  

Enhanced mixing in microfluidic systems is necessary in many applications such as chemical processing, biological assays, and diagnosis. We are developing a microfluidic system to efficiently mix minute reagents (down to several microliters) using vibration-induced flow (VIF), in which a net flow is generated around a micropillar by applying periodic vibration. In this study, we numerically investigate the enhancement in chaotic mixing using the VIF technique and periodic switching of vibrations. By extending our previous numerical simulation model, we investigate the flow field and trajectories of fluid particles in three-dimensional space. We demonstrate that chaotic advection characteristics can be observed by periodically switching the vibrational direction of a substrate using simple cylindrical pillars. In addition, using an appropriate interval for switching the vibration axes yields better mixing performance. The extent of chaotic advection is evaluated quantitatively using the Lyapunov exponent considering various vibration parameters, such as the vibration amplitude, separation distance between each pillar and pillar shape. The flow field induced by a large-amplitude and sharp-edged wall pillar provides excellent mixing results. Thus, VIF is successfully applied to obtain an efficient mixing strategy with the aid of the chaotic theory.

DOI： 10.1063/5.0192387

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

The addition of surface acoustic wave (SAW) technologies to microfluidics has greatly advanced lab-on-a-chip applications due to their unique and powerful attributes, including high-precision manipulation, versatility, integrability, biocompatibility, contactless nature, and rapid actuation. However, the development of SAW microfluidic devices is limited by complex and time-consuming micro/nanofabrication techniques and access to cleanroom facilities for multistep photolithography and vacuum-based processing. To simplify the fabrication of SAW microfluidic devices with customizable dimensions and functions, we utilized the additive manufacturing technique of aerosol jet printing. We successfully fabricated customized SAW microfluidic devices of varying materials, including silver nanowires, graphene, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). To characterize and compare the acoustic actuation performance of these aerosol jet printed SAW microfluidic devices with their cleanroom-fabricated counterparts, the wave displacements and resonant frequencies of the different fabricated devices were directly measured through scanning laser Doppler vibrometry. Finally, to exhibit the capability of the aerosol jet printed devices for lab-on-a-chip applications, we successfully conducted acoustic streaming and particle concentration experiments. Overall, we demonstrated a novel solution-based, direct-write, single-step, cleanroom-free additive manufacturing technique to rapidly develop SAW microfluidic devices that shows viability for applications in the fields of biology, chemistry, engineering, and medicine.

DOI： 10.1038/s41378-023-00606-z

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Other Link： https://www.nature.com/articles/s41378-023-00606-z

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Vibration-induced flow (VIF), in which a mean flow is induced around a microstructure by applying periodic vibrations, is increasingly used as an active flow-control technique at the microscale. In this study, we have developed a microdevice that actively controls the VIF patterns using elastic membrane protrusions (microballoons) actuated by pneumatic pressure. This device enables on-demand spatial and temporal fluid manipulation using a single device that cannot be achieved using a conventional fixed-structure arrangement. We successfully demonstrated that the device achieved displacements of up to 38 µm using the device within a pressure range of 0 to 30 kPa, indicating the suitability of the device for microfluidic applications. Using this active microballoon array, we demonstrated that the device can actively manipulate the flow field and induce swirling flows. Furthermore, we achieved selective actuation of the microballoon using this system. By applying air pressure from a multi-input channel system through a connection tube, the microballoons corresponding to each air channel can be selectively actuated. This enabled precise control of the flow field and periodic switching of the flow patterns using a single chip. In summary, the proposed microdevice provides active control of VIF patterns and has potential applications in advanced microfluidics, such as fluid mixing and particle manipulation.

DOI： 10.3390/mi14112010

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Fuji Technology Press Ltd.  

In this study, we developed a system for selective self-assembly of millimeter-scale components differentiated by adhesive patterns. This was achieved by designing concentric circular patterns having different radii but the same total length of peripheries. Small polymer sheets having solder adhesive patterns in these designs were simply attached to the millimeter-scale components to be assembled in a stirring container. This strategy was effective in avoiding an overlap between different patterns and enforcing the selective bonds between identical patterns among three types of components. Finally, the selective assembly of a functional structure (i.e., poly(N-isopropylacrylamide) gel actuator) was demonstrated.

DOI： 10.20965/jrm.2023.p1219

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Harnessing liquid crystals to control topological defects. Our innovative method empowers precise manipulation of liquid crystal topological defects, offering a platform for homogeneous isolated topological and light-driven control.

DOI： 10.1039/d3sm00838j

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

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Authorship：Corresponding author   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

DOI： 10.1109/nems54180.2022.9791090

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DOI： 10.1080/02678292.2021.1991016

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Institute of Electrical and Electronics Engineers Inc.  

We used microfluidics to separate immune cells from non-model organisms based on the cell size using the Deterministic Lateral Displacement (DLD) method. In this study, we targeted shrimp (Marsupenaeus japonicus) as a non-model organism that is widely cultured all over the world. We evaluated the efficiency of the size separation of target cells by flow cytometer (FCM) and image analysis. Besides, to predict the functions of the separated cells, we conducted gene expression analysis on the separated cells by qRT-PCR. The results showed that different sizes of cells have different immune functions.

DOI： 10.1109/Transducers50396.2021.9495666

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier  

DOI： 10.1016/j.jconrel.2021.06.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MyJove Corporation  

DOI： 10.3791/62129

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Institute of Electrical and Electronics Engineers Inc.  

On-chip mixing is important technology. In this paper, we propose micromixier based on vibration-induced flow with low dead volume. We show the proof of concept and quantitatively evaluated mixing performance of proposed mixing method.

DOI： 10.1109/MHS48134.2019.9249335

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Copyright© (2018) by Chemical and Biological Microsystems Society.All rights reserved. A mean flow is induced around a micro-pillar by applying a small circulating vibration (typically < 10 μm amplitude and 100-1000 Hz frequency) in parallel to the whole substrate (Figure 1). This technique has been applied for cell manipulation and mixing biochemical samples with no external pump. We constructed a numerical model to analyze a three-dimensional flow field induced by the oscillating micro-pillar. A flow profile obtained by simulation agreed well with that of PIV measurement. The present numerical model can be easily extended to three-dimensional flow fields with various pillar designs and arrangements.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Institute of Physics Publishing  

This study investigated the fracture properties of wrinkled metallic films on a polydimethylsiloxane (PDMS) soft substrate. In particular, the crack density of the wrinkled film during tensile deformation was examined. In order to achieve better deformability of metallic thin films, a method to fabricate a wrinkled thin film on a PDMS soft substrate was first established. The copper (Cu) nano-film fabricated in this study possessed a wrinkled geometry, which plays a critical role in determining the extent of large elastic deformation. To create the wrinkled structure, wet-etching with a polymeric sacrificial layer was used. A sacrificial layer was first deposited onto a silicone rubber sheet. During the curing process of the layer, a compressive strain was applied such that the hardened surface layer buckled, and a wrinkled form was obtained. Subsequently, a PDMS solution was used to cover the layer in order to form a wrinkled PDMS substrate. Finally, the Cu film was deposited onto the wrinkled PDMS, such that the wrinkled Cu film on a soft PDMS substrate was fabricated. The use of uni-axial tensile tests resulted in film crack generation at the stress concentration zone in the wrinkled structure of the films. When the tensile loading was increased, the number of cracks increased. It was found that the increase in crack density was strongly related to the inhomogeneous nature of the wrinkled structure. Such a trend in crack density was investigated using FEM (finite element method) computations, such that this study established a simple mechanical model that may be used to predict the increase in crack density during tensile deformation. This model was verified through several experiments using various wrinkle patterns. The proposed mechanical model may be useful to predict the crack density of a wrinkled metallic film subject to tensile loading.

DOI： 10.1088/1361-6463/aa95cb

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Language：English   Publishing type：Research paper (international conference proceedings)  

© 17CBMS-0001. Mixing of minute reagents is one of the common operational elements in integrated microfluidic devices. Although a variety of mixer units have been developed, most of them are designed to use external pumps. In this study, we designed a pumpless micro-mixer for mixing reagents in μL volume. We employed a vibration-induced flow, in which a mean circulating flow around a micro-pillar is induced by giving small circulating vibration to the whole substrate. By employing non-axisymmetric micro-pillar design and switching of circulating direction, we show that stretching and folding of the fluid element, a feature of chaotic advection, can be induced.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We have been testing the application of the entropic effect (the depletion volume effect) for the self-assembly of microcomponents. Microcomponents with the arbitrary shapes fabricated by the negative photoresist were immersed and gently agitated in the solution. When the solution contains nanometric macromolecule at relatively high concentration, they formed assembled structures, to increase the translational entropy of macromolecules in the solution. In the report, we attempted the assembly of the desired structure composed of multiple components, and found that it is important to design the accessible complementary shape with the greater interfacial area to render the specific bonding effectively.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We developed the microwell device for measurement of membrane transport of single adherent cells. As the cells in a population (e.g., tumor) is inevitably heterogeneous, a technique to measure the transport activities at a single-cell level is needed. When adherent cells were cultured on the microwells with similar to 10 mu m diameter, they spread over the opening to form the closed picoliter space. Thus, molecules exported from cells accumulate in such a space and be detected by fluorescence imaging. In this report, we show that, by employing horizontal microwell design, materials exported from the cell membrane can be visualized without overlapping with the cell, increasing the S/N ratio of the fluorescence signal. Efflux of the cancer drug transported by the multidrug resistance protein was detected.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We propose the use of the entropic effect (the depletion volume effect), which is at work in the assembly of biomolecules, for the self-assembly of artificially engineered microcomponents. When the solution contains macromolecule at relatively high concentration, microcomponents formed assembled structures. The bonding energy is not originated from the surface; it is generated by increasing the translational entropy of macromolecules in the solution. We expect that use of the depletion volume effect promotes the search of the global free-energy minimum in the system by avoiding being trapped to the local minima in the self-assembly process.

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.53.S202_4

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Language：English   Publisher：AMER CHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER INST PHYSICS  

We report a facile, low-cost, and high-yielding microfluidic technology for in situ generating and arraying water-in-oil droplets by shrinking them to the order of femtolitres (fLs) as scalable batch micro-reactors. Instead of generating ultra-small droplets by the direct atomization, which requires dedicate control and high energy input, we shrink droplets to stable smaller ones by utilizing the controlled water diffusion in oil. This "shrunk to fL" method is combined with a three-dimensional microwell design to create high-density addressable droplet arrays. As the result, scalable, high-throughput, and well-aligned W/O arrays with excellent long-term stability and predicable droplet sizes have been achieved. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4746754]

DOI： 10.1063/1.4746754

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Increasingly complex reactions are being constructed by bottom-up approaches with the aim of developing an artificial cell. We have been engaged in the construction of a translation-coupled replication system of genetic information from RNA and a reconstituted translation system. Here a mathematical model was established to gain a quantitative understanding of the complex reaction network. The sensitivity analysis predicted that the limiting factor for the present replication reaction was the appearance of parasitic replicators. We then confirmed experimentally that repression of such parasitic replicators by compartmentalization of the reaction in water-in-oil emulsions improved the duration of self-replication. We also found that the main source of the parasite was genomic RNA, probably by nonhomologous recombination. This result provided experimental evidence for the importance of parasite repression for the development of long-lasting genome replication systems.

DOI： 10.1016/j.chembiol.2012.01.019

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.52.S139_5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSICAL SOC  

Lognormal statistical distributions are observed in a variety of scientific fields. The widths of these distributions in the log scale are often similar, but the underlying mechanism that maintains these widths within a small range has not been well explained. We show that a stochastic process of halving followed by addition can yield a stationary distribution that resembles the universal lognormal distribution with a certain width. The mechanism that we propose here would provide insight into the essence of why lognormal-like distributions in many systems have a common width.

DOI： 10.1103/PhysRevE.83.031118

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Netherlands  

To realize the minimal cell by a bottom-up approach, increasingly -complex biochemical reactions are being encapsulated in lipid vesicles (liposomes). Here, we describe the encapsulation of one of the general properties of living organisms into liposomes, i.e., replication of genetic information with self-encoded replicase, and evolvability. We also discuss the possibility of realizing a minimal cell that can -proliferate autonomously. © 2011 Springer Science+Business Media B.V.

DOI： 10.1007/978-90-481-9944-0_15

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We report a novel, simple and effective 3D self-patterning method for functional coatings on poly(dimethylsiloxane) (PDMS) microfluidics before bonding, inspired by the wettability-based fast evaporation of water film on superhydrophilic plant leaves. Induced by evaporation, liquid-phase functional coatings are selectively deposited on rough PDMS surfaces, enabling the conventional PDMS-glass bonding. Several control factors are investigated to improve the coating quality including roughness, coverage, uniformity and clogging issues. Teflon-like CYTOP coatings have been demonstrated on PDMS channels with good coverage, uniformity and bonding strength, and significantly reduced the swelling, deformation and collapse of PDMS channels in toluene. © 2011 IEEE.

DOI： 10.1109/TRANSDUCERS.2011.5969541

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

A novel, facile and effective microfluidic approach to achieve monodisperse femtoliter (fL, 1015L) water-in-oil (W/O) droplet array is proposed for constructing artificial cell-sized micro reactors. By combining the controlled diffusion of water into oil and capillary-based 2D micro trap array (capillary traps), in-situ generation and 1000-fold volume shrinkage down to ∼4 fL have been achieved for monodisperse W/O array. With improved 3D trap array, high-density and well-aligned fL W/O droplet array with good long-time stability and coefficient of variation (CV) smaller than 5% has also been demonstrated. This approach also enables dynamically tuning of droplet sizes and solution concentration useful for many applications. © 2011 IEEE.

DOI： 10.1109/TRANSDUCERS.2011.5969434

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.51.S84_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

Understanding the origin of life requires knowledge not only of the origin of biological molecules such as amino acids, nucleotides and their polymers, but also the manner in which those molecules are integrated into the organized systems that characterize cellular life. In this article, we introduce a constructive approach to understand how biological molecules can be arranged to achieve a higher-order biological function: replication of genetic information.

DOI： 10.1101/cshperspect.a004945

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S143_5

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.50.S125_2

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DOI： 10.2142/biophys.50.S126_6

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DOI： 10.2142/biophys.50.S31_4

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We present a method to control the size of giant unilamellar vesicles (GUVs), in which biochemical reaction can be encapsulated and conducted, with the aid of microfluidics. GUVs were formed by passing the water-in-oil (W/O) emulsion droplets through the second oil/water interface by centrifugation. Last year we showed that, by controlling the size of the initial W/O emulsion with a flow-focusing device, size of the lipid vesicles can also be controlled. Here we examined the physical condition that determines the vesicle size, and showed that our method is compatible with biochemical reactions for artificial cell-model studies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cell Press  

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Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

In living cells, biochemical reaction systems are enclosed in small lipidic compartments. To experimentally simulate various biochemical reactions occurring in extant cells, giant liposomes are used to reconstruct an artificial model cell. We present methods for conducting a protein synthesis reaction, followed by the reaction catalyzed by the synthesized proteins inside liposomes, and for measurement of the in liposome reaction using a fluorescence-activated cell sorter (FACS). These techniques enable us to perform detailed analysis of the biochemical reactions occurring in the microcompartments, and have the potential to reveal the role of compartmentalization in cellular systems.

DOI： 10.1016/S0076-6879(09)64002-7

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.49.S102_2

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DOI： 10.2142/biophys.49.S91_2

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DOI： 10.1007/s10544-008-9218-z

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We report the first imaging of a dye-doped artificial bilayer using electro-optical microscopy. The applied voltage dependence confirms the origin of the effect. This result is an important step towards a contactless optical patch-clamp.

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DOI： 10.2142/biophys.48.S13_6

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DOI： 10.2142/biophys.48.S170_4

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：社団法人生産技術振興協会  

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.48.S159_6

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DOI： 10.2142/biophys.48.S170_2

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Chemical and Biological Microsystems Society  

This paper describes a multichannel nanopore sensory system for single molecule detection. We use α-hemolysin, a bacterial membrane ionchannel, as the nanopores. The fabricated microfluidic chip is able to form planar bilayer lipid membranes in an array, and integrate the nanopores into the membranes. The system achieves simultaneous monitoring of ionic currents through the individual pores. In conclusion we succeeded in discriminating different concentrations of nucleic acid polymers, which promises its high potentiality for high-throughput screening applications. © 2008 CBMS.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：IOP Science  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We describe our effort to realize an assay system to study clathrin-mediated endocytosis (CME) in vitro. Since CME related proteins are known to bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2), we propose an accessible method to create an array of supported lipid bilayers (SLBs) with different lipid compositions, using a PDMS stamp and a microchannel. Formation of a uniform SLB of a complex lipid mixture containing PI(4,5)P-2 was confirmed by fluorescent imaging and fluorescence recovery after photobleaching (FRAP). Our technique should allow us to vary the concentration of PI(4,5)P-2 or other membrane constituents to study their requirements. In preliminary experiments we showed that dynamin binds to the planar bilayer containing PI(4,5)P-2, but without causing membrane tubulation as it does when dynamin binds liposomes.

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.47.S173_3

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This paper describes two novelties which will contribute to the precise examination of the functions of membrane proteins. First, we describe a method to reconstitute purified membrane proteins in giant liposomes (giant proteoliposomes). Second, we established a system to observe the chemical/physical responses of liposomes by confining them in microchamber arrays during the perfusion of surrounding solution through a semi-permeable membrane. Since the position of proteoliposomes does not change during perfusion, we can continuously observe the response of single liposomes. Using this technique, we successfully detected the function of water channel, humanaquaporin1 (hAQP1), by observing rapid shrinkage of its proteoliposomes due to the change in osmotic gradient. © 2006 Society for Chemistry and Micro-Nano Systems.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We report a method for forming ultra giant lipid vesicles. A chip device with a micro-jet nozzle was fabricated. The vesicles were produced by applying pulsed micro jets to a planar lipid bilayer, analogous to blowing soap bubbles from a liquid film. Using this method, lipid vesicles with diameters of about 500 mu m were formed one-by-one. This method enables the encapsulation of target molecules or nano particles into the vesicles rapidly and efficiently.

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We have been developing a measurement system for membrane transport across a reconstituted planar lipid bilayer to examine the functions of transporter membrane proteins using a microchamber array (Membrane Microchamber System). In this system, an artificial planar lipid bilayer is pressed on the Parylene microchamber array (typically 0.1 similar to 1 pL in volume) fabricated on a coverglass to enclose the volume. After membrane proteins are reconstituted in the bilayer, fluorescently labeled molecules transported across the bilayer are accumulated in microchambers, and detected by fluorescent imaging. The microchamber array is compatible with high-sensitive biological imaging techniques, such as a confocal microscopy and a total internal reflection fluorescence microscopy (TIRFM). Due to the tiny volume of microchambers, concentration of the transported molecules rapidly increases. In this study, the detection system was characterized in detail. With the aid of microchambers, the concentration reached to the detectable level with as small as 10(2)similar to 10(3) fluorescent molecules.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We present a novel method for the optical detection of membrane transport across an artificial planar lipid bilayer through membrane proteins (e.g., ion pumps and transporters) using a microchamber array. Parylene microchambers are fabricated on a coverglass, using photolithography, and a planar lipid bilayer is pressed on the microchambers to cover. After membrane proteins are reconstituted in the bilayer, fluorescently labeled molecules transported through them are accumulated in microchambers and detected by fluorescent imaging. The microchamber array is shown to be compatible with high-sensitive microscopic imaging techniques, such as a confocal microscopy and a total internal reflection fluorescence microscopy (TIRFM). Using this system, transport of fluorescent dye through peptide nanopore (alpha-hemolysin) is examined.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Japan Academic Association Inc  

We describe a simple method for the batch fabrication of solid-state pores with controllable size ranging from few micrometers down to few hundred nanometers. Based on our method, a 'Coulter counter' device was developed and we could demonstrate the straightforward detection of particles translocation through the pore, opening possibility for the detection of biomolecules such as DNA. © 2006 Society for Chemistry and Micro-Nano Systems.

Scopus

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S284_1

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

In this paper, the formation process of planar lipid bilayer in PMMA micro-fluidic device is investigated in detail by observing distribution of fluorescent lipid molecules with the aid of confocal microscopy. In our bilayer formation process, initially thick layer (similar to 50Rm) of lipid solution, which is stuck at the vertical section of the aperture edge, gradually thins down by applying static pressure in the upper compartment. It finally becomes a bilayer with the pressure of 200-400Pa. It is confirmed by recording single channel current through gramicidin. The yield of bilayer formation reached 90% in maximum.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

Biomolecular linear motor proteins --kinesin and microtubules -- are finely patterned on a conventional, flat glass substrate using parylene lift-off process; these patterns are useful for single molecule analysis and biohybrid transportation. The patterning was performed from 5 mu m - 40 mu m in width with minimal non-specific binding of the proteins. A conventional gliding assay was realized using this glass substrate and the behaviors of the single proteins were analyzed.

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S88_3

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ROYAL SOC CHEMISTRY  

Planar bilayer membranes have been widely used for electrophysiological analysis of membrane proteins in laboratories. We previously reported that planar lipid bilayer can be reconstituted at 100 similar to 150 mu m tapered hole fabricated in a silicon-based micro-fluidic channel system. However, for the measurement of ion channel current, which requires pico-ampere sensitivity, the substrate material should be non-conductive material to eliminate the electric noise. Thus, we fabricated PMMA plastic based micro-fluidic system made by precise machining. The activity of channel proteins incorporated into the planar membrane is successfully recorded.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ROYAL SOC CHEMISTRY  

We propose a fine protein patterning technique using a patterned Parylene film on a substrate. This method prevents non-specific binding, and can be performed in the wet condition to avoid drying of proteins. FITC and IC3-PE-maleimid labeled bovine albumin are successfully patterned in 20 x 20 mu m(2) spots with 2 mu m intervals. Patterning of different materials on different spots becomes possible by introducing sample solution through PDMS micro fluidic channel place on top. This technique will easily be applied to the analysis of protein in an array. We have demonstrated a selective patterning of beads with different fluorescence in 3 x 3 array (80 x 80 mu m(2) spots with 50 mu m intervals).

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

This paper describes a method for patterning proteins selectively using a combination of microfluidics and a lift-off process. Microchannels are used to introduce different protein solutions, allowing us to immobilize spots of proteins on a substrate. We fabricated microfluidic channels in polymethylmethacrylate (PMMA) substrate and deposited a thin Parylene film as a mask containing small holes patterned on the PMMA layer. Solutions of proteins were introduced in p channels and immobilized on the PMMA surface at positions defined by the parylene mask. The parylene mask was lifted-off, leaving different proteins and/or nanoparticles patterned on a PMMA substrate. Using microfluidic channel, different kinds of samples were able to be delivered and patterned on a same substrate in the wet condition; this method avoids denaturing of proteins without non-specific binding.

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Language：English  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

We report the novel methods to reconstitute artificial lipid membranes in an array on a chip, using the micro-fluidic system, for the batch analysis of membrane proteins. The micro-fabricated device should render the ease of injection/recirculation of the reagents as well as multi-channel and high-sensitive detection of protein activities incorporated in the bilayer membrane. It is shown that the lipid bilayer membrane can be formed on 100 similar to 200 mum square apertures fabricated in a silicon substrate in two methods. In the first method, the bilayer is formed by flowing the lipid solution and the buffer alternately. In the second method, it is done by placing a buffer droplet on a thin layer of the lipid solution. Parylene coating is found to be suitable for both bilayer formation and noise reduction. The future application includes the high-sensitive ion sensor chip and high-throughput drug screening chip.

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Mixing of magnetic beads and bio-molecules becomes critically important in a micro-scale cell sorting system. Particle tracking velocimetry (PTV) has been employed to examine the motion of magnetic beads in a model chaotic micro-mixer. The measured trajectories of beads show good agreement with the numerical simulation previously reported [5], which has predicted the onset of chaos. The bead trajectory is reconstructed from the phase-averaged velocity field of beads to estimate the Lyapunov exponent. The optimum frequency of applied control signal exists in the range of the Strouhal number of 5<St<12,which is also in accordance with the numerical simulation. The present method of estimating the Lypaunov can be used to determine the performance of the mixing device for micro particles and large molecules.

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

An integrated magnetic micro-mixer, which consists of embedded micro-conductors and a micro-channel, is developed by using MEMS technology for the mixing of magnetic beads in bio-fluids. In the previous report, it was shown that the simple 2-D micro-conductors provided a magnetic field strong enough to attract the nearby magnetic beads. Numerical simulation is used to search for the channel and electrode design that can generate chaotic trajectories of beads. It is found that the serpentine channel geometry with the perpendicular electrode arrangement is able to create the stretching and folding of material lines, which is a sign of chaos. Similar patterns are observed experimentally in the chaotic mixing in the fabricated device.

DOI： 10.1299/jsmemecjo.2002.6.0_103

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本機械学会  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

A novel axisymmetric jet nozzle equipped with a row of miniature electromagnetic flap actuators on its circular lip is developed for active flow control. Each of the flaps fabricated by photolithography is independently driven according to a control signal supplied by a PC. The spatio-temporal flow structures of the controlled jet are studied through flow visualization and laser Doppler velocimetry (LDV) measurement. It is found that artificial disturbances generated by the flaps can modify the formation and evolution of large scale vortical structures significantly. When each half cluster of these flaps are driven out of phase, the jet clearly bifurcates into two branches and its mixing is markedly enhanced in the plane of bifurcation.

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Language：English   Book type：Scholarly book

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Language：Japanese   Book type：Scholarly book

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Language：Japanese  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本生物物理学会  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本生物物理学会  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：SPRINGER HEIDELBERG  

Despite the rapid and enormous progress in biotechnologies, the biochemical analysis of membrane proteins is still a difficult task. The presence of the large hydrophobic region buried in the lipid bilayer membrane (transmembrane domain) makes it difficult to analyze membrane proteins in standard assays developed for water-soluble proteins. To handle membrane proteins, the lipid bilayer membrane may be used as a platform to sustain their functionalities. Relatively slow progress in developing micro total analysis systems (mu TAS) for membrane protein analysis directly reflects the difficulty of handling lipid membranes, which is a common problem in bulk measurement technologies. Nonetheless, researchers are continuing to develop efficient and sensitive analytical microsystems for the study of membrane proteins. Here, we review the latest developments, which enable detection of events caused by membrane proteins, such as ion channel current, membrane transport, and receptor/ligand interaction, by utilizing microfabricated structures. High-throughput and highly sensitive detection systems for membrane proteins are now becoming a realistic goal. Although most of these systems are still in the early stages of development, we believe this field will become one of the most important applications of mu TAS for pharmaceutical and clinical screenings as well as for basic biochemical research.

DOI： 10.1007/s00216-008-1916-0

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：社団法人日本機械学会  

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese  

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Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Oral presentation (general)  

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Application no：特願2014-014450  Date applied：2014.1.29

Announcement no：特開2015-141120  Date announced：2015.8.3

Applicant (Organization)：学校法人中央大学