The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform

Shoji Takeuchi - One of the best experts on this subject based on the ideXlab platform.

  • Lipid Bilayer on a microdroplet integrated with a patterned ag agcl microelectrode for voltage clamp fluorometry of membrane transport
    Sensors and Actuators B-chemical, 2021
    Co-Authors: Toshihisa Osaki, Taishi Tonooka, Koji Sato, Ryuji Kawano, Shoji Takeuchi
    Abstract:

    Abstract Voltage-clamp fluorometry (VCF) has been combined with artificial Lipid Bilayer systems to optically measure the dynamics and contribution of membrane proteins on molecular transport at the clamped membrane potential. However, the previous methods for VCF require expensive apparatus, such as total internal reflection fluorescence microscope, or require extensive experience for voltage-clamping, or possibly lead to imprecise membrane potential. This study describes VCF performed using a pico-liter-sized Lipid Bilayer chamber with an embedded Ag/AgCl microelectrode. Owing to the size of the picoliter order, molecular transport through the Lipid Bilayer can be detected using a normal fluorescent microscope. Easy electrical access into the Lipid Bilayer chamber can be realized because the microelectrode is spontaneously embedded inside when forming the Lipid Bilayer chamber. The microelectrode is made of Ag/AgCl; therefore, it does not tend to polarize, which results in reliable regulation of the membrane potential. Using the developed system, we performed the VCF of molecular transport through the Lipid Bilayer by incorporating the pore-forming membrane protein, α-hemolysin. This proposed method will increase the number of researches that can perform the VCF of molecular transport through artificial Lipid Bilayers.

  • droplet based Lipid Bilayer system integrated with microfluidic channels for solution exchange
    Lab on a Chip, 2013
    Co-Authors: Yutaro Tsuji, Toshihisa Osaki, Ryuji Kawano, Koki Kamiya, Norihisa Miki, Shoji Takeuchi
    Abstract:

    This paper proposes a solution exchange of a droplet-based Lipid Bilayer system, in which the inner solution of a droplet is replaced for the purpose of efficient ion channel analyses. In our previous report, we successfully recorded the channel conductance of alpha-hemolysin in a Bilayer Lipid membrane using a droplet contact method that can create a spontaneous Lipid Bilayer at the interface of contacting droplets; this method is widely used as highly efficient method for preparing planar Lipid membranes. When only pipetting droplets of the solution, this method is highly efficient for preparing Lipid membranes. However, the drawback of droplet-based systems is their inability to exchange the solution within the droplets. To study the effect of inhibitors and promoters of ion channels in drug discovery, it would be beneficial to conduct a solution exchange of droplets to introduce membrane proteins and to apply or wash-out the chemicals. In this study, we propose a droplet contact method that allows for the solution exchange of droplets via microfluidic channels. We experimentally and numerically investigated the Bilayer stability with respect to exchanging flow rates, and then demonstrated a binding assay of an alpha-hemolysin using one of its blockers. The solution exchange in this system was conducted in less than 20 s without rupturing the membrane. We believe that the proposed system will enhance the efficiency of ion channel analyses.

  • Microfluidic formation of Lipid Bilayer array for membrane transport analysis
    2008 IEEE 21st International Conference on Micro Electro Mechanical Systems, 2008
    Co-Authors: Sadao Ota, Wei-heong Tan, Hiroaki Suzuki, Shoji Takeuchi
    Abstract:

    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 alpha-hemolysin antibiotic pores.

  • highly reproducible method of planar Lipid Bilayer reconstitution in polymethyl methacrylate microfluidic chip
    Langmuir, 2006
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Shoji Takeuchi
    Abstract:

    We developed a highly reproducible method for planar Lipid Bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar Lipid Bilayers are formed at apertures, 100 microm in diameter, by flowing Lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the Lipid solution at the aperture determines the state of the Lipid Bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of Lipid solution is distributed at the aperture. Then, the layer of Lipid solution was thinned by applying an external pressure and finally became a Bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for Bilayer formation was 90%. Using this technique, four Lipid Bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.

  • planar Lipid Bilayer reconstitution with a micro fluidic system
    Lab on a Chip, 2004
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Yasuyuki Katoyamada, Shoji Takeuchi
    Abstract:

    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 μm 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.

Rikiya Watanabe - One of the best experts on this subject based on the ideXlab platform.

  • High-throughput formation of Lipid Bilayer membrane arrays with an asymmetric Lipid composition
    Scientific Reports, 2014
    Co-Authors: Rikiya Watanabe, Naoki Soga, Tomoko Yamanaka, Hiroyuki Noji
    Abstract:

    We present a micro-device in which more than 10,000 asymmetric Lipid Bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric Lipid Bilayers, where Lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The Lipid compositional asymmetry is an intrinsic property of bio-membranes and therefore, this micro-device extends the versatility of artificial Lipid-Bilayer systems, which were previously limited to symmetric Bilayer formation and could contribute to the understanding of the role of Lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

  • arrayed Lipid Bilayer chambers allow single molecule analysis of membrane transporter activity
    Nature Communications, 2014
    Co-Authors: Naoki Soga, Rikiya Watanabe, Kazuhito V Tabata, Daishi Fujita, Lisa Yamauchi, Daisuke Asanuma, Mako Kamiya, Yasuteru Urano
    Abstract:

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed Lipid Bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter Lipid Bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function. The development of small volume chamber arrays has greatly facilitated high throughput biological assays of soluble proteins. Here, Watanabe et al.adapt this approach to develop an arrayed Lipid Bilayer chamber system for single molecule level measurements of membrane transporter activity.

  • arrayed Lipid Bilayer chambers allow single molecule analysis of membrane transporter activity
    Nature Communications, 2014
    Co-Authors: Naoki Soga, Rikiya Watanabe, Kazuhito V Tabata, Daishi Fujita, Lisa Yamauchi, Daisuke Asanuma, Soo Hyeon Kim, Mako Kamiya
    Abstract:

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed Lipid Bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter Lipid Bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

Donald M. Engelman - One of the best experts on this subject based on the ideXlab platform.

  • energetics of peptide phlip binding to and folding across a Lipid Bilayer membrane
    Biophysical Journal, 2009
    Co-Authors: Yana K. Reshetnyak, Michael Segala, Vladislav S Markin, Oleg A. Andreev, Donald M. Engelman
    Abstract:

    The pHLIP peptide (pH Low Insertion Peptide) serves as a model system for peptide insertion and folding across a Lipid Bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a Lipid Bilayer as an unstructured monomer, and (III) inserted across the Bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a POPC Lipid Bilayer and to calculate the transition energies between states. We found that the Gibbs Free Energy of binding to a POPC surface at low pHLIP concentration (state I - state II transition) at 37°C is about -7 kcal/mol near neutral pH and that the free energy of insertion and folding across a Lipid Bilayer at low pH (state II - state III transition) is nearly -2 kcal/mol. We plan to discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

  • energetics of peptide phlip binding to and folding across a Lipid Bilayer membrane
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Yana K. Reshetnyak, Michael Segala, Vladislav S Markin, Oleg A. Andreev, Donald M. Engelman
    Abstract:

    The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a Lipid Bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a Lipid Bilayer as an unstructured monomer, and (III) inserted across the Bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) Lipid Bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I–state II transition) at 37°C is approximately −7 kcal/mol near neutral pH and that the free energy of insertion and folding across a Lipid Bilayer at low pH (state II–state III transition) is nearly −2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

Hiroyuki Noji - One of the best experts on this subject based on the ideXlab platform.

  • High-throughput formation of Lipid Bilayer membrane arrays with an asymmetric Lipid composition
    Scientific Reports, 2014
    Co-Authors: Rikiya Watanabe, Naoki Soga, Tomoko Yamanaka, Hiroyuki Noji
    Abstract:

    We present a micro-device in which more than 10,000 asymmetric Lipid Bilayer membranes are formed at a time on micro-chamber arrays. The arrayed asymmetric Lipid Bilayers, where Lipid compositions are different between the inner and outer leaflets, are formed with high efficiency of over 97% by injecting several types of liquids into a micro-device that has hydrophilic-in-hydrophobic surfaces. The Lipid compositional asymmetry is an intrinsic property of bio-membranes and therefore, this micro-device extends the versatility of artificial Lipid-Bilayer systems, which were previously limited to symmetric Bilayer formation and could contribute to the understanding of the role of Lipid compositional asymmetry in cell physiology and also to further analytical and pharmacological applications.

  • highly reproducible method of planar Lipid Bilayer reconstitution in polymethyl methacrylate microfluidic chip
    Langmuir, 2006
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Shoji Takeuchi
    Abstract:

    We developed a highly reproducible method for planar Lipid Bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar Lipid Bilayers are formed at apertures, 100 microm in diameter, by flowing Lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the Lipid solution at the aperture determines the state of the Lipid Bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of Lipid solution is distributed at the aperture. Then, the layer of Lipid solution was thinned by applying an external pressure and finally became a Bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for Bilayer formation was 90%. Using this technique, four Lipid Bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.

  • planar Lipid Bilayer reconstitution with a micro fluidic system
    Lab on a Chip, 2004
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Yasuyuki Katoyamada, Shoji Takeuchi
    Abstract:

    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 μm 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.

Kazuhito V Tabata - One of the best experts on this subject based on the ideXlab platform.

  • arrayed Lipid Bilayer chambers allow single molecule analysis of membrane transporter activity
    Nature Communications, 2014
    Co-Authors: Naoki Soga, Rikiya Watanabe, Kazuhito V Tabata, Daishi Fujita, Lisa Yamauchi, Daisuke Asanuma, Soo Hyeon Kim, Mako Kamiya
    Abstract:

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed Lipid Bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter Lipid Bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins' function.

  • arrayed Lipid Bilayer chambers allow single molecule analysis of membrane transporter activity
    Nature Communications, 2014
    Co-Authors: Naoki Soga, Rikiya Watanabe, Kazuhito V Tabata, Daishi Fujita, Lisa Yamauchi, Daisuke Asanuma, Mako Kamiya, Yasuteru Urano
    Abstract:

    Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed Lipid Bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter Lipid Bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function. The development of small volume chamber arrays has greatly facilitated high throughput biological assays of soluble proteins. Here, Watanabe et al.adapt this approach to develop an arrayed Lipid Bilayer chamber system for single molecule level measurements of membrane transporter activity.

  • highly reproducible method of planar Lipid Bilayer reconstitution in polymethyl methacrylate microfluidic chip
    Langmuir, 2006
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Shoji Takeuchi
    Abstract:

    We developed a highly reproducible method for planar Lipid Bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar Lipid Bilayers are formed at apertures, 100 microm in diameter, by flowing Lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the Lipid solution at the aperture determines the state of the Lipid Bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of Lipid solution is distributed at the aperture. Then, the layer of Lipid solution was thinned by applying an external pressure and finally became a Bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for Bilayer formation was 90%. Using this technique, four Lipid Bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.

  • planar Lipid Bilayer reconstitution with a micro fluidic system
    Lab on a Chip, 2004
    Co-Authors: Hiroaki Suzuki, Hiroyuki Noji, Kazuhito V Tabata, Yasuyuki Katoyamada, Shoji Takeuchi
    Abstract:

    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 μm 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.