川野　竜司KAWANO Ryujiカワノ　リユウジ

業績情報

氏名・連絡先

• 氏名

  カワノ　リユウジ, 川野　竜司, KAWANO Ryuji

• 生年

  1976

• eメールアドレス

  rjkawanocc.tuat.ac.jp

• 個人ホームページ

  http://www.tuat.ac.jp/~rjkawano/

主たる所属・職名

• 工学研究院　生命機能科学部門, 教授

その他の所属

• 工学研究院
• 生物システム応用科学府

経歴

• -
  東京農工大学　工学研究院 生命機能科学部門　准教授
  自 2014年
• -
  公益財団法人神奈川科学技術アカデミー・非常勤研究員
  自 2014年
• -
  公益財団神奈川科学技術アカデミー　竹内プロジェクト 常勤研究員
  自 2009年, 至 2013年
• -
  東京大学生産技術研究所・協力研究員
  自 2009年, 至 2015年
• -
  ユタ大学化学科　博士研究員
  自 2008年, 至 2009年
• -
  ユタ大学化学科　日本学術振興会海外特別研究員
  自 2006年, 至 2008年
• -
  横浜国立大学　ベンチャービジネスラボラトリー　講師（中核的研究機関）
  自 2005年, 至 2006年

学歴

• 横浜国立大学
  工学研究科
  物質工学専攻
  至 2005年, 修了, 博士

教育・研究活動状況

• 当研究室ではマイクロ微細加工・マイクロ流体技術を利用して人工的に細胞を創り、また細胞機能の工学的応用を目指して研究を行っています。特にリン脂質や膜タンパク質で構成された細胞「膜」部分に注目し、生体機能模倣型バイオセンサの構築を進めています。

研究テーマ

• マイクロ加工技術を用いた細胞膜、膜タンパク質に関する研究
  自 9999

担当授業科目

• 国際専修科目　生体機能工学フロンティア特論
  2023年, 専門科目等
• 生命工学研究概論
  2023年, 専門科目等
• 生命工学実験III＆IV
  2023年, 専門科目等
• 生命工学の最先端
  2023年, 専門科目等
• 生命工学入門・医工学入門
  2023年, 教養科目等
• 計算機実験II
  2023年, 教養科目等

科学研究費助成事業

• 挑戦的研究（萌芽）
  猫マルチオーガンオルガノイドオンチップシステムの確立
  自 2023年, 至 2023年
• 基盤研究（B）
  クライオ電子顕微鏡によるタンパク質膜透過の動的構造解析
  自 2023年, 至 2023年
• 基盤研究（B）
  口腔癌の統合的リアルタイムモニタリングの社会実装の為の検証研究および治療への応用
  自 2023年, 至 2023年
• 基盤研究（A）
  嗅覚スーパーセンシングの分子機構解明と人工構築を実現する、細胞融合デバイスの構築
  自 2023年, 至 2023年
• 挑戦的研究（萌芽）
  猫マルチオーガンオルガノイドオンチップシステムの確立
  自 2022年, 至 2022年
• 基盤研究(B)
  細胞外物質による化学感覚機能機構の解明と、高機能性センサー開発への展開
  自 2022年, 至 2022年
• 基盤研究(B)
  クライオ電子顕微鏡によるタンパク質膜透過の動的構造解析
  自 2022年, 至 2022年
• 基盤研究(B)
  口腔癌の統合的リアルタイムモニタリングの社会実装の為の検証研究および治療への応用
  自 2022年, 至 2022年
• 新学術領域研究（研究領域提案型）
  酵素反応によるde novoペプチドナノポアの開閉制御
  自 2021年, 至 2021年
• 学術変革領域研究(A)（計画研究）
  De novo細胞膜分子システムのボトムアップ構築
  自 2021年, 至 2025年
• 挑戦的研究（萌芽）
  海水中、大気中で作動するリポソーム型分子ロボットの開発
  自 2021年, 至 2022年
• 基盤研究(B)
  クライオ電子顕微鏡によるタンパク質膜透過の動的構造解析
  自 2021年, 至 2021年
• 基盤研究(B)
  細胞外物質による化学感覚機能機構の解明と、高機能性センサー開発への展開
  自 2021年, 至 2021年
• 基盤研究(B)
  口腔癌の統合的リアルタイムモニタリングの社会実装の為の検証研究および治療への応用
  自 2021年, 至 2021年
• 基盤研究(B)
  金属錯体多面体を用いた外場応答型人工イオンチャネルの創製
  自 2020年, 至 2020年
• 基盤研究(B)
  細胞外物質による化学感覚機能機構の解明と、高機能性センサー開発への展開
  自 2020年, 至 2020年
• 基盤研究(A)
  分子演算システムによる腫瘍由来核酸の高速パターン診断
  自 2019年, 至 2022年
• 新学術領域研究（研究領域提案型）
  De novo設計による合成ナノポアの構築
  自 2019年, 至 2020年
• 基盤研究(B)
  金属錯体多面体を用いた外場応答型人工イオンチャネルの創製
  自 2019年, 至 2019年
• 基盤研究(B)
  自己複製する非平衡動的なDNA人工細胞の創成
  自 2019年, 至 2019年
• 基盤研究(B)
  細胞外物質による化学感覚機能機構の解明と、高機能性センサー開発への展開
  自 2019年, 至 2019年
• 基盤研究(B)
  嗅覚器の細胞外構成成分を利用した、高感度生物模倣型匂いセンサーの基盤技術創出
  自 2018年, 至 2018年
• 基盤研究(B)
  金属錯体多面体を用いた外場応答型人工イオンチャネルの創製
  自 2018年, 至 2018年
• 基盤研究(B)
  自己複製する非平衡動的なDNA人工細胞の創成
  自 2018年, 至 2018年
• 挑戦的研究（萌芽）
  ナノポアプローブ：チャネル膜タンパク質のナノ空間を利用したナノ環境計測
  自 2017年, 至 2018年
• 基盤研究(B)
  自己複製する非平衡動的なDNA人工細胞の創成
  自 2017年, 至 2017年
• 基盤研究(B)
  嗅覚器の細胞外構成成分を利用した、高感度生物模倣型匂いセンサーの基盤技術創出
  自 2017年, 至 2017年
• 若手研究(A)
  DNA演算技術による癌腫瘍マーカーmiRNAの自律的検出と治療
  自 2016年, 至 2019年
• 基盤研究（B）
  嗅覚器の細胞外構成成分を利用した、高感度生物模倣型匂いセンサーの基盤技術創出
  自 2016年, 至 2016年
• 新学術領域研究（公募）
  ナノポアセンシングによる分子演算システムの構築
  自 2015年, 至 2016年
• 挑戦的萌芽研究
  人工細胞回路を用いたDNAコンピュータの創生
  自 2014年, 至 2015年
• 基盤研究（C）
  猫マルチオーガンオルガノイドオンチップシステムの確立
  自 2024年, 至 2024年
• 基盤研究（A）
  尿サンプル由来膀胱オルガノイドを活用したがん進展・治療抵抗性獲得機序の解明
  自 2024年, 至 2024年
• 基盤研究（A）
  嗅覚スーパーセンシングの分子機構解明と人工構築を実現する、細胞融合デバイスの構築
  自 2024年, 至 2024年

論文

• Real-time monitoring of the amyloid β1-42 monomer-to-oligomer channel transition using a lipid bilayer system
  Numaguchi, Yuri; Tsukakoshi, Kaori; Takeuchi, Nanami; Suzuki, Yuki; Ikebukuro, Kazunori; Kawano, Ryuji; Wand, Josh
  PNAS NEXUS
  OXFORD UNIV PRESS
  This study describes the observation of the transformation of monomeric amyloid β1–42 (Aβ42) into oligomers in a lipid membrane utilizing a lipid bilayer system for electrophysiological measurement. The relevance of oligomers and protofibrils in Alzheimer's disease (AD) is underscored given their significant neurotoxicity. By closely monitoring the shift of Aβ42 from its monomeric state to forming oligomeric channels in phospholipid membranes, we noted that this transformation transpired within a 2-h frame. We manipulated the lipid membrane's constitution with components such as glycerophospholipid, porcine brain total lipid extract, sphingomyelin (SM), and cholesterol (Chol.) to effectively imitate nerve cell membranes. Interesting findings showcased Chol.'s ability to foster stable oligomeric channel formation in the lipid membrane, with SM and GM1 lipids potentially enhancing channel formation as well. Additionally, the study identified the potential of a catechin derivative, epigallocatechin gallate (EGCG), in obstructing oligomerization. With EGCG present in the outer solution of the Aβ42-infused membrane, a noteworthy reduction in channel current was observed, suggesting the successful inhibition of oligomerization. This conclusion held true in both, prior and subsequent, stages of oligomerization. Our findings shed light on the toxicity of oligomers, promising invaluable information for future advancements in AD treatment strategies.
  2023年12月14日, 研究論文（学術雑誌）, 共同, 3, 1, DOI(公開)(r-map), 907, 911
• Enhancement of cell membrane permeability by using charged nanoparticles and a weak external electric field
  Nakamura, Hideya; Okamura, Takumi; Tajima, Masaya; Kawano, Ryuji; Yamaji, Misa; Ohsaki, Shuji; Watano, Satoru
  PHYSICAL CHEMISTRY CHEMICAL PHYSICS
  ROYAL SOC CHEMISTRY
  Because the cell membrane is the main barrier of intracellular delivery, it is important to facilitate and control the translocation of extracellular compounds across it. Our earlier molecular dynamics simulations suggested that charged nanoparticles under a weak external electric field can enhance the permeability of the cell membrane without disrupting it. However, this membrane permeabilization approach has not been tested experimentally. This study investigated the membrane crossing of a model compound (dextran with a Mw of 3000-5000) using charged nanoparticles and a weak external electric field. A model bilayer lipid membrane was prepared by using a droplet contact method. The permeability of the membrane was evaluated using the electrophysiological technique. Even when the applied electric field was below the critical strength for membrane breakdown, dextran was able to cross the membrane without causing membrane breakdown. These results indicate that adding nanomaterials under a weak electric field may enhance the translocation of delivery compounds across the cell membrane with less damage, suggesting a new strategy for intracellular delivery systems. Because the cell membrane is the main barrier of intracellular delivery, it is important to facilitate and control the translocation of extracellular compounds across it.
  2023年12月06日, 研究論文（学術雑誌）, 共同, 25, 47, 1463-9076, DOI(公開)(r-map), 32356, 32363
• Membrane-spanning DNA system: designing strand displacement across lipid bilayers
  Sotaro Takiguchi and Ryuji Kawano
  2023年11月, 研究論文（国際会議プロシーディングス）
• Effects of excess DNA probes on DNA detection at femtomolar levels using a biological nanopore
  Nanami Takeuchi and Ryuji Kawano
  Proceedings of DNA, 2023
  2023年11月, 研究論文（国際会議プロシーディングス）
• Construction of a DNA Switching Circuit with Consciousness based on Integrated Information Theory
  Fumika Kambara, Sotaro Takiguchi, Hiroki Watanabe, Masahiro Takinoue and Ryuji Kawano
  Proceedings of DNA, 2023
  2023年11月, 研究論文（国際会議プロシーディングス）
• Bottom-up creation of cell-free molecular systems: Basic research toward social implementation
  Kawamura, Izuru; Kawano, Ryuji; Matsuura, Tomoaki
  BIOPHYSICS AND PHYSICOBIOLOGY
  #N/A
  2023年11月, 研究論文（学術雑誌）, 共同, 20, 4, DOI(公開)(r-map)
• Nanopore sensing for nucleic acid detection at femtomolar levels without amplification
  Nanami Takeuchi and Ryuji Kawano
  MicroTAS, 2023
  2023年10月, 研究論文（国際会議プロシーディングス）
• Liposome Budding: Microfluidic generation of monodisperse liposomes
  Jiajiu Ji and Ryuji Kawano
  MicroTAS, 2023
  2023年10月, 研究論文（国際会議プロシーディングス）
• Theranostics molecular robot: detect a miRNA from tumor cells and generate the DNA drug in a liposome
  Harune Suzuki, Ken Komiya, and Ryuji Kawano
  MicroTAS, 2023
  2023年10月, 研究論文（国際会議プロシーディングス）
• Microfluidic Generation of Micro-soap Bubbles For Airborne Molecular Robot
  Rina Takagi and Ryuji Kawano
  MicroTAS, 2023
  2023年10月, 研究論文（国際会議プロシーディングス）
• Simultaneous Recognition of Over- and Under-Expressed MicroRNAs Using Nanopore Decoding
  Takiguchi, Sotaro; Kambara, Fumika; Tani, Mika; Sugiura, Tsuyoshi; Kawano, Ryuji
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  This paper describes a strategy for simultaneous recognition of over- and under-expressed microRNAs (miRNAs) using the method of signal classification-based nanopore decoding. MiRNA has attracted attention as a promising biomarker for cancer diagnosis owing to its cancer-type-specific expression patterns. While nanopore technology has emerged as a simple and label-free method to detect miRNAs and their expression patterns, recognizing patterns involving simultaneous over/under-expression is still challenging due to the inherent working principles. Here, inspired by the sequence design for DNA computation with nanopore decoding, we designed diagnostic DNA probes targeting two individual over/under-expressed miRNAs in the serum of oral squamous cell carcinoma. Through nanopore measurements, our designed probes exhibited characteristic current signals depending on the hybridized miRNA species, which were plotted on the scatter plot of duration versus current blocking ratio. The classified signals reflected the relative abundance of target miRNAs, thereby enabling successful pattern recognition of over/under-expressed miRNAs, even when using clinical samples. We believe that our method paves the way for miRNA-targeting simple diagnosis as a liquid biopsy.
  2023年09月07日, 研究論文（学術雑誌）, 共同, 95, 39, 0003-2700, DOI(公開)(r-map), 14675, 14685
• DNAコンピューティング技術とナノポア計測を組み合わせた体液診断技術
  竹内七海、滝口創太郎、神原史佳、川野竜司
  生物工学会誌
  生物工学会
  2023年09月, （MISC）総説・解説（学術雑誌）, DOI(公開)(r-map)
• マイクロ流体デバイスを用いた細胞サイズリポソームの作製手法
  鈴木春音、髙木里菜、吉家爵、川野竜司
  公益社団法人 日本化学会 バイオテクノロジー部会 NEWS LETTER
  2023年08月, （MISC）総説・解説（学術雑誌）
• Structure-Activity Relationship Study of Helix-Stabilized Antimicrobial Peptides Containing Nonproteinogenic Amino Acids
  Ito, Takahito; Hashimoto, Wakana; Ohoka, Nobumichi; Misawa, Takashi; Inoue, Takao; Kawano, Ryuji; Demizu, Yosuke
  ACS BIOMATERIALS SCIENCE & ENGINEERING
  AMER CHEMICAL SOC
  Helical amphipathic peptides containing cationic andhydrophobicamino acid residues can possess potent antimicrobial activity againstboth Gram-positive and Gram-negative bacteria. In this study, severalamphipathic peptides with enhanced helical structures containing nonproteinogenicamino acids were designed, and the relationships between the antimicrobialactivity, hemolytic activity, and cytotoxicity were evaluated. Inparticular, the effect on the antimicrobial activity and cytotoxicityof the number and position of stapling structures introduced intothe sequence was investigated. Peptide stp1 containing & alpha;,& alpha;-disubstituted amino acids showed potent antimicrobialactivity against multidrug-resistant bacteria (MDRP, SP45, and Staphylococcus aureus) without causing appreciablehemolytic activity or cytotoxicity. The cytotoxicity was found tobe somewhat correlated to the hydrophobicity of the peptides.
  2023年07月24日, 研究論文（学術雑誌）, 共同, 9, 8, 2373-9878, DOI(公開)(r-map), 4654, 4661
• Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore
  Tada, Asuka; Takeuchi, Nanami; Shoji, Kan; Kawano, Ryuji
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  Thispaper describes a method for the real-time counting and extractionof DNA molecules at the single-molecule level by nanopore technology.As a powerful tool for electrochemical single-molecule detection,nanopore technology eliminates the need for labeling or partitioningsample solutions at the femtoliter level. Here, we attempt to developa DNA filtering system utilizing an alpha-hemolysin (alpha HL)nanopore. This system comprises two droplets, one filling with andone emptying DNA molecules, separated by a planar lipid bilayer containing alpha HL nanopores. The translocation of DNA through the nanoporesis observed by measuring the channel current, and the number of translocatedmolecules can also be verified by quantitative polymerase chain reaction(qPCR). However, we found that the issue of contamination seems tobe an almost insolvable problem in single-molecule counting. To tacklethis problem, we tried to optimize the experimental environment, reducethe volume of solution containing the target molecule, and use thePCR clamp method. Although further efforts are still needed to achievea single-molecule filter with electrical counting, our proposed methodshows a linear relationship between the electrical counting and qPCRestimation of the number of DNA molecules.
  2023年06月06日, 研究論文（学術雑誌）, 共同, 95, 26, 0003-2700, DOI(公開)(r-map), 9805, 9812
• Analysis of direct transmembrane permeability of mono-amino acids by the planar membrane system
  Zheng, Kaiyi; Izumi, Kayano; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（国際会議プロシーディングス）, 共同, 122, 3, 0006-3495, 367A, 367A
• Construction of nanopores using β-hairpin peptides synthesized by a cell-free system
  Fujita, Shoko; Fukuda, Miyu; Mizoguchi, Ikuro; Kawamura, Izuru; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（国際会議プロシーディングス）, 共同, 122, 3, 0006-3495, 367A, 367A
• Discrimination of cationic peptides using malaria translocon, EXP2, nanopores
  Miyagi, Mitsuki; Kurokawa, Nina; Yohda, Masafumi; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（国際会議プロシーディングス）, 共同, 122, 3, 0006-3495, 438A, 438A
• Facilitating the nanopore detection of protein fragment with a neutral charge
  Yamaji, Misa; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（国際会議プロシーディングス）, 共同, 122, 3, 0006-3495, 435A, 435A
• Long-term survivable liposomes in seawater
  Izumi, Kayano; Koiwai, Keiichiro; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（学術雑誌）, 共同, 122, 3, 0006-3495, 82A, 82A
• Ultra-low concentration detection of DNA without its amplification using a biological nanopore
  Takeuchi, Nanami; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（学術雑誌）, 共同, 122, 3, 0006-3495, 287A, 287A
• Unfolding of β-hairpin and α-helical peptides through a biological nanopore
  Fukuda, Miyu; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2023年02月10日, 研究論文（学術雑誌）, 共同, 122, 3, 0006-3495, 439A, 439A
• Cell-Free Expression of De Novo Designed Peptides That Form β-Barrel Nanopores
  Shoko Fujita, Izuru Kawamura, Ryuji Kawano
  ACS Nano
  AMER CHEMICAL SOC
  Nanopore sensing has attracted much attention as a rapid, simple, and label-free single-molecule detection technology. To apply nanopore sensing to extensive targets including polypeptides, nanopores are required to have a size and structure suitable for the target. We recently designed a de novo fl-barrel peptide nanopore (SVG28) that constructs a stable and monodispersely sized nanopore. To develop the sizes and functionality of peptide nanopores, systematic exploration is required. Here we attempt to use a cell-free synthesis system that can readily express peptides using transcription and translation. Hydrophilic variants of SVG28 were designed and expressed by the PURE system. The peptides form a monodispersely sized nanopore, with a diameter 1.1 or 1.5 nm smaller than that of SVG28. Such cell-free synthesizable peptide nanopores have the potential to enable the systematic custom design of nanopores and comprehensive sequence screening of nanopore-forming peptides.
  2023年02月02日, 研究論文（学術雑誌）, 共同, 1936-0851, DOI(公開)(r-map)
• Liposome Deformation Induced by Membrane-Binding Peptides
  Izumi, Kayano; Saito, Chihiro; Kawano, Ryuji
  MICROMACHINES
  MDPI
  This paper presents an investigation of liposome deformation and shape distortion using four membrane-binding peptides: TAT and C105Y as cell-penetrating peptides (CPPs), and melittin and ovispirin as antimicrobial peptides (AMPs). Liposome deformation was monitored utilizing fluorescent microscopy, while the binding of peptides to the DOPC membrane was estimated through capacitance measurements. The degree of liposome deformation and shape distortion was found to be higher for the CPPs compared to the AMPs. Additionally, it was observed that C105Y did not induce liposome rupture, unlike the other three peptides. We propose that these variations in liposome distortion may be attributed to differences in secondary structure, specifically the presence of an alpha-helix or random coil. Our studies offer insight into the use of peptides to elicit control of liposome architecture and may offer a promising approach for regulating the bodies of liposomal molecular robots.
  2023年02月, 研究論文（学術雑誌）, 共同, 14, 2, DOI(公開)(r-map)
• 生体ナノポアを用いた核酸検出とDNA演算技術との融合による診断応用
  滝口創太郎、竹内七海、川野竜司
  応用物理学会M&BE分科会誌
  2022年12月13日, （MISC）総説・解説（学術雑誌）, 共同
• De novoペプチドナノポアの設計と精密1分子検出
  清水啓佑、宇佐美将誉、溝口郁朗、藤田祥子、川野竜司
  生物物理学会誌
  2022年11月01日, （MISC）総説・解説（学術雑誌）, 共同
• ナノポア計測によるDNA演算情報の復号化
  滝口創太郎、川野竜司
  高分子学会誌
  2022年10月10日, （MISC）総説・解説（学術雑誌）, 共同
• Novel uptake and degradation pathway of proteins by lysosomes required for neuromuscular homeostasis
  Fujiwara, Yuuki; Contu, Viorica Raluca; Kabuta, Chihana; Ogawa, Megumu; Miyagi, Mitsuki; Fujita, Hiromi; Kikuchi, Hisae; Sakai, Ryohei; Hase, Katsunori; Suzuki, Mari; Koyama-Honda, Ikuko; Inoue, Michio; Oya, Yasushi; Inoue, Yukiko U.; Kawano, Ryuji; Inoue, Takayoshi; Takahashi, Ryosuke; Nishino, Ichizo; Wada, Keiji; Noguchi, Satoru; Kabuta, Tomohiro
  JOURNAL OF NEUROCHEMISTRY
  WILEY
  2022年08月, 研究論文（国際会議プロシーディングス）, 共同, 162, 0022-3042, DOI(公開)(r-map), 103, 104
• Pattern Recognition of microRNA Expression in Body Fluids using Nanopore Decoding at Sub-femtomolar Concentrations
  Nanami Takeuchi, Moe Hiratani, Ryuji Kawano
  JACS Au
  2022年06月26日, 研究論文（学術雑誌）, 共同, DOI(公開)(r-map)
• Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic alpha-helical peptides
  Takechi-Haraya, Yuki; Ohgita, Takashi; Kotani, Mana; Kono, Hiroki; Saito, Chihiro; Tamagaki-Asahina, Hiroko; Nishitsuji, Kazuchika; Uchimura, Kenji; Sato, Takeshi; Kawano, Ryuji; Sakai-Kato, Kumiko; Izutsu, Ken-ichi; Saito, Hiroyuki
  SCIENTIFIC REPORTS
  NATURE PORTFOLIO
  We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.
  2022年03月23日, 研究論文（学術雑誌）, 共同, 12, 1, 2045-2322, DOI(公開)(r-map)
• Single polypeptide detection using a translocon EXP2 nanopore
  Miyagi, Mitsuki; Takiguchi, Sotaro; Hakamada, Kazuaki; Yohda, Masafumi; Kawano, Ryuji
  PROTEOMICS
  WILEY
  DNA sequencing using nanopores has already been achieved and commercialized; the next step in advancing nanopore technology is towards protein sequencing. Although trials have been reported for discriminating the 20 amino acids using biological nanopores and short peptide carriers, it remains challenging. The size compatibility between nanopores and peptides is one of the issues to be addressed. Therefore, exploring biological nanopores that are suitable for peptide sensing is key in achieving amino acid sequence determination. Here, we focus on EXP2, the transmembrane protein of a translocon from malaria parasites, and describe its pore-forming properties in the lipid bilayer. EXP2 mainly formed a nanopore with a diameter of 2.5 nm assembled from 7 monomers. Using the EXP2 nanopore allowed us to detect poly-L-lysine (PLL) at a single-molecule level. Furthermore, the EXP2 nanopore has sufficient resolution to distinguish the difference in molecular weight between two individual PLL, long PLL (Mw: 30,000-70,000) and short PLL (Mw: 10,000). Our results contribute to the accumulation of information for peptide-detectable nanopores.
  2022年03月, 研究論文（学術雑誌）, 共同, 22, 5-6, 1615-9853, DOI(公開)(r-map)
• Analysis of membrane transportation of cell penetrating peptides using lipid bilayer system
  Saito, Chihiro; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2022年02月11日, 研究論文（国際会議プロシーディングス）, 共同, 121, 3, 0006-3495, DOI(公開)(r-map), 223A, 223A
• De novo design of peptide nanopores with beta-barrel structure synthesized by cell-free expression
  Fujita, Shoko; Mizoguchi, Ikuro; Fukuda, Miyu; Usami, Masataka; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2022年02月11日, 研究論文（国際会議プロシーディングス）, 共同, 121, 3, 0006-3495, DOI(公開)(r-map), 222A, 222A
• De novo designed a-helix peptides which form barrel-stave nanopores
  Usami, Masataka; Sekiya, Yusuke; Mijiddorj, Batsaikhan; Kawamura, Izuru; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2022年02月11日, 研究論文（国際会議プロシーディングス）, 共同, 121, 3, 0006-3495, DOI(公開)(r-map), 222A, 222A
• Discrimination of polycationic peptides using a translocon EXP2 nanopore
  Miyagi, Mitsuki; Takiguchi, Sotaro; Hakamada, Kazuaki; Yohda, Masafumi; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2022年02月11日, 研究論文（国際会議プロシーディングス）, 共同, 121, 3, 0006-3495, DOI(公開)(r-map), 467A, 467A
• MicroRNA detection at femtomolar concentration using a probe-based nanopore technique
  Takeuchi, Nanami; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2022年02月11日, 研究論文（国際会議プロシーディングス）, 共同, 121, 3, 0006-3495, DOI(公開)(r-map), 539A, 539A
• Microfiber-Shaped Programmable Materials with Stimuli-Responsive Hydrogel
  Takeuchi, Nobuki; Nakajima, Shunsuke; Yoshida, Koki; Kawano, Ryuji; Hori, Yutaka; Onoe, Hiroaki
  SOFT ROBOTICS
  MARY ANN LIEBERT, INC
  Programmable materials have artificially designed physical shapes responding to external stimuli, as well as high design capability and high flexibility. Here, we propose a microfiber-shaped programmable material with an axial pattern of stimuli-responsive (SR) and nonresponsive hydrogels. The SR pre-gel solution was mixed to sodium alginate pre-gel solution for instantaneous gelation with ionic crosslinking and solidified on a nonresponsive hydrogel microfiber with a valve-controlled microfluidic system. A design of microfiber-shaped programmable material (patterned position of SR regions) could be flexibly altered by changing a coded sequence program. We confirmed that the three-dimensional (3D) coil-like structures were self-folded at the patterned SR regions responding to the thermal stimulus and that the chirality of the self-folded 3D coil-like structures depends on the condition of the stimulus to the microfiber. Finally, interaction with objects using the programmable microfiber as a soft actuator was demonstrated. Our microfiber-shaped programmable materials expand possibilities of fiber-based materials in biomimetics and soft robotics fields.
  2022年02月01日, 研究論文（学術雑誌）, 共同, 9, 1, 2169-5172, DOI(公開)(r-map), 89, 97
• De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide
  Keisuke Shimizu, Batsaikhan Mijiddorj, Masataka Usami, Ikuro Mizoguchi, Shuhei Yoshida, Shiori Akayama, Yoshio Hamada, Akifumi Ohyama, Kenji Usui, Izuru Kawamura, Ryuji Kawano
  Nature Nanotechnology
  2022年01月06日, 研究論文（学術雑誌）, 共同, DOI(公開)(r-map)
• Simple Fabrication of Solid-State Nanopores on a Carbon Film
  Takai, Natsumi; Shoji, Kan; Maki, Tei; Kawano, Ryuji
  MICROMACHINES
  MDPI
  Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe the simple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM) electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method, nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm were obtained. Since these nanopores were successfully fabricated in the commercially available carbon thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.
  2021年09月, 研究論文（学術雑誌）, 共同, 12, 9, DOI(公開)(r-map)
• Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process
  Koiwai, Keiichiro; Koyama, Takashi; Tsuda, Soichiro; Toyoda, Atsushi; Kikuchi, Kiyoshi; Suzuki, Hiroaki; Kawano, Ryuji
  ELIFE
  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.
  2021年06月16日, 研究論文（学術雑誌）, 共同, 10, 2050-084X, DOI(公開)(r-map)
• Nanopore decoding for a Hamiltonian path problem
  Takiguchi, Sotaro; Kawano, Ryuji
  NANOSCALE
  ROYAL SOC CHEMISTRY
  DNA computing has attracted attention as a tool for solving mathematical problems due to the potential for massive parallelism with low energy consumption. However, decoding the output information to a human-recognizable signal is generally time-consuming owing to the requirement for multiple steps of biological operations. Here, we describe simple and rapid decoding of the DNA-computed output for a directed Hamiltonian path problem (HPP) using nanopore technology. In this approach, the output DNA duplex undergoes unzipping whilst passing through an alpha-hemolysin nanopore, with information electrically decoded as the unzipping time of the hybridized strands. As a proof of concept, we demonstrate nanopore decoding of the HPP of a small graph encoded in DNA. Our results show the feasibility of nanopore measurement as a rapid and label-free decoding method for mathematical DNA computation using parallel self-assembly.
  2021年03月28日, 研究論文（学術雑誌）, 共同, 13, 12, 2040-3364, DOI(公開)(r-map), 6192, 6200
• 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年02月15日, 研究論文（学術雑誌）, DOI(公開)(r-map)
• Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence
  Hirano, Motoharu; Saito, Chihiro; Yokoo, Hidetomo; Goto, Chihiro; Kawano, Ryuji; Misawa, Takashi; Demizu, Yosuke
  MOLECULES
  MDPI
  Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing alpha,alpha-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.
  2021年01月, 研究論文（学術雑誌）, 共同, 26, 2, DOI(公開)(r-map)
• 細胞機能のボトムアップデザインとシステム化
  庄司観、川野竜司
  日本生物工学会誌
  日本生物工学会
  2020年12月01日, （MISC）総説・解説（学術雑誌）, 共同, 98, 12, 0919-3758, 651, 654
• Editorial on the Special Issue on Recent Advances of Molecular Machines and Molecular Robots
  Takinoue, Masahiro; Kawano, Ryuji
  MICROMACHINES
  MDPI
  2020年12月, 研究論文（学術雑誌）, 共同, 11, 12, DOI(公開)(r-map)
• Rational Design of Helix-Stabilized Antimicrobial Peptide Foldamers Containing alpha,alpha-Disubstituted Amino Acids or Side-Chain Stapling
  Hirano, Motoharu; Saito, Chihiro; Goto, Chihiro; Yokoo, Hidetomo; Kawano, Ryuji; Misawa, Takashi; Demizu, Yosuke
  CHEMPLUSCHEM
  WILEY-V C H VERLAG GMBH
  Antimicrobial peptides (AMPs) are expected to be good candidate molecules for novel antimicrobial therapies. Most AMPs exert their antimicrobial activity through disruption of microbial membranes due to their amphipathic properties. Recently, the helical peptide 'Stripe' was reported by our group, a rationally designed amphipathic AMP focused on distribution of natural cationic and hydrophobic amino acid residues. In this study, a set of Stripe-based AMP foldamers was designed, synthesized and investigated that contain alpha,alpha-disubstituted amino acids or side-chain stapling to stabilize their helical structures. Our results showed that a peptide containing 2-aminoisobutyric acid (Aib) residues exhibited potent antimicrobial activity against both Gram-positive S.aureus (MIC value: 3.125 mu M) and Gram-negative bacteria (including a multidrug-resistant strain, MDRP, MIC value: 1.56 mu M), without significant hemolytic activity (>100 mu M). Electrophysiological measurements revealed that this peptide formed stable pores in a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) bilayer but not in a dioleoylphosphocholine (DOPC) bilayer. The introduction of Aib residues into Stripe could be a promising way to increase the antimicrobial activity of AMP foldamers, and the peptide could represent a promising novel therapeutic candidate to treat multidrug-resistant bacterial infection.
  2020年12月, 研究論文（学術雑誌）, 共同, 85, 12, 2192-6506, DOI(公開)(r-map), 2731, 2736
• Editorial on the Special Issue on Recent Advances of Molecular Machines and Molecular Robots
  Masahiro Takinoue, Ryuji Kawano
  Micromachines
  MDPI
  2020年11月24日, （MISC）総説・解説（学術雑誌）, 共同, 11, 12, DOI(公開)(r-map), 1031, 1032
• Analytical Model for Particle Capture in Nanopores Elucidates Competition among Electrophoresis, Electroosmosis, and Dielectrophoresis
  Chinappi, Mauro; Yamaji, Misa; Kawano, Ryuji; Cecconi, Fabio
  ACS NANO
  AMER CHEMICAL SOC
  The interaction between nanoparticles dispersed in a fluid and nanopores is governed by the interplay of hydrodynamical, electrical, and chemical effects. We developed a theory for particle capture in nanopores and derived analytical expressions for the capture rate under the concurrent action of electrical forces, fluid advection, and Brownian motion. Our approach naturally splits the average capture time in two terms, an approaching time due to the migration of particles from the bulk to the pore mouth and an entrance time associated with a free-energy barrier at the pore entrance. Within this theoretical framework, we described the standard experimental condition where a particle concentration is driven into the pore by an applied voltage, with specific focus on different capture mechanisms: under pure electrophoretic force, in the presence of a competition between electrophoresis and electroosmosis, and finally under dielectrophoretic reorientation of dipolar particles. Our theory predicts that dielectrophoresis is able to induce capture for both positive and negative voltages. We performed a dedicated experiment involving a biological nanopore (alpha-hemolysin) and a rigid dipolar dumbbell (realized with a beta-hairpin peptide) that confirms the theoretically proposed capture mechanism.
  2020年11月24日, 研究論文（学術雑誌）, 共同, 14, 11, 1936-0851, DOI(公開)(r-map), 15816, 15828
• Recognition of Single-Point Mutation Using a Biological Nanopore
  Liu, Ping; Kawano, Ryuji
  SMALL METHODS
  WILEY-V C H VERLAG GMBH
  Here the recognition of a single-point mutation in oligonucleotides is described by using nanopore measurements. The translocation behavior of a series of mutated DNA strands, hybridized with a complementary DNA probe, is analyzed via blocking current and unzipping time. Discernment of the mutation position at the single nucleotide level is achieved by analysis of a 2D graph of the bootstrapped translocation data. The proposed approach provides a useful tool for the mutation detection of oligonucleotides secreted from tumor cells and is applicable in simple and label-free diagnoses as a nanopore liquid biopsy.
  2020年11月, 研究論文（学術雑誌）, 共同, 4, 11, 2366-9608, DOI(公開)(r-map)
• Enhancement of direct membrane penetration of arginine-rich peptides by polyproline II helix structure
  Ohgita, Takashi; Takechi-Haraya, Yuki; Okada, Keisuke; Matsui, Saki; Takeuchi, Misaki; Saito, Chihiro; Nishitsuji, Kazuchika; Uchimura, Kenji; Kawano, Ryuji; Hasegawa, Koki; Sakai-Kato, Kumiko; Akaji, Kenichi; Izutsu, Ken-ichi; Saito, Hiroyuki
  BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
  ELSEVIER
  The left-handed, extended polyproline II (PPII) helix is a unique secondary structure which potently modulates peptide/protein functions through its constraint conformation. To investigate the effect of PPII helix on the direct cell membrane penetration of arginine-rich peptides, we designed a polyproline-containing arginine-rich peptide P9R7W (PPPPPPPPPPRRRRRRRW) by introducing nine proline residues into a linear R7W (RRRRRRRW) peptide. Circular dichroism spectroscopy showed that P9R7W has the PPII helix structure in solution whereas R7W is predominantly in random coil structure. Tryptophan fluorescence measurements demonstrated that P9R7W binds to negatively charged lipid vesicles with similar affinity to R7W, in which there was no significant change in the PPII helix structure. Flow cytometry and confocal laser scanning microscopy analyses showed that P9R7W has an ability to penetrate into CHO-K1 cells more efficiently compared to R7W with no cytotoxicity. Consistently, a channel current analysis unveiled that P9R7W penetrates planar lipid bilayer membranes more efficiently than R7W without significant membrane perturbation. Our results indicate that the PPII helix structure can enhance the membrane penetration efficiency of arginine-rich peptides without lipid membrane perturbation.
  2020年10月01日, 研究論文（学術雑誌）, 共同, 1862, 10, 0005-2736, DOI(公開)(r-map)
• NANOPORE DECODING FOR DNA COMPUTATION WITH PARALLEL SELF-ASSEMBLY
  Sotaro Takiguchi and Ryuji Kawano
  Proceedings of MicroTAS
  MicroTAS
  2020年09月01日, 研究論文（国際会議プロシーディングス）, 共同, 1, 1, DOI(公開)(r-map), 486, 487
• Analysis of Membrane Protein Deinsertion-Associated Currents with Nanoneedle-Supported Bilayers to Discover Pore Formation Mechanisms
  Shoji, Kan; Kawano, Ryuji; White, Ryan J.
  LANGMUIR
  AMER CHEMICAL SOC
  Analysis of the pore formation mechanisms of biological nanopores can provide insight into pore-forming peptide-induced diseases and into the characterization of nanopores employed in sensing methods. Evaluation of pore formation mechanisms is typically performed using microscopy including atomic force microscopy, transmission electron microscopy, as well as electrically via channel current measurements using a patch-clamp amplifier. However, due to the relatively low temporal resolution of the above-mentioned microscopy techniques and the low analysis accuracy of the channel current measurements, new analytical methods are required. Here, we describe a new analytical strategy to measure and analyze both ionic currents associated with biological nanopore insertion and deinsertion into and out of lipid bilayers to determine pore formation mechanisms for several representative proteins. The current changes associated with protein deinsertion are monitored as the lipid membrane leaflets are pulled apart-a unique phenomenon enabled by our gold nanoneedle measurement probe. This deinsertion current analysis (DiCA) is performed using a gold nanoneedle-supported lipid bilayer at which a bilayer membrane is formed by bringing together two lipid monolayers on the surface of the nanoneedle and at the interface of an aqueous solution and a lipid/oil mixture. The lipid bilayer can be pulled apart by removing the nanoneedle from this interface. In this study, we demonstrate the determination of pore formation mechanisms for four different pore-forming proteins and peptides-alpha-hemolysin, streptolysin O, alamethicin, and amyloid beta 1-42 using DiCA. As a result, we successfully discern the pore formation mechanism, either addition or expansion, for each protein/peptide by analyzing the ratio and magnitude of insertion and deinsertion current events.
  2020年09月01日, 研究論文（学術雑誌）, 共同, 36, 34, 0743-7463, DOI(公開)(r-map), 10012, 10021
• Recent Advances in Liposome-Based Molecular Robots
  Shoji, Kan; Kawano, Ryuji
  MICROMACHINES
  MDPI
  A molecular robot is a microorganism-imitating micro robot that is designed from the molecular level and constructed by bottom-up approaches. As with conventional robots, molecular robots consist of three essential robotics elements: control of intelligent systems, sensors, and actuators, all integrated into a single micro compartment. Due to recent developments in microfluidic technologies, DNA nanotechnologies, synthetic biology, and molecular engineering, these individual parts have been developed, with the final picture beginning to come together. In this review, we describe recent developments of these sensors, actuators, and intelligence systems that can be applied to liposome-based molecular robots. First, we explain liposome generation for the compartments of molecular robots. Next, we discuss the emergence of robotics functions by using and functionalizing liposomal membranes. Then, we discuss actuators and intelligence via the encapsulation of chemicals into liposomes. Finally, the future vision and the challenges of molecular robots are described.
  2020年09月, 研究論文（学術雑誌）, 共同, 11, 9, DOI(公開)(r-map)
• Recessed Ag/AgCl Microelectrode-Supported Lipid Bilayer for Nanopore Sensing
  Shoji, Kan; Kawano, Ryuji; White, Ryan J.
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  Biological nanopores reconstituted into supported lipid bilayer membranes are widely used as a platform for stochastic nanopore sensing with the ability to detect single molecules including, for example, single-stranded DNA (ssDNA) and miRNA. A main thrust in this area of research has been to improve overall bilayer stability and ease of measurements. These improvements are achieved through a variety of clever strategies including droplet- based techniques; however, they typically require specific microfabrication techniques to prepare devices or special manipulation techniques for microdroplets. Here, we describe a new method to prepare lipid bilayers using a recessed-in-glass Ag/AgCl microelectrode as a support structure. The lipid bilayer is formed at the tip of the microelectrode by immersing the microelectrode into a layered bath solution consisting of an oil/lipid mixture and an aqueous electrolyte solution. In this paper, we demonstrate this stable, supported lipid bilayer structure for channel current measurements of pore-forming toxins and single-molecule detection of ssDNA. This Ag/AgCl-supported lipid bilayer can potentially be widely adopted as a lipid membrane platform for nanopore sensing because of its simple and easy procedure needed to prepare lipid bilayers.
  2020年08月04日, 研究論文（学術雑誌）, 共同, 92, 15, 0003-2700, DOI(公開)(r-map), 10856, 10862
• Competing Roles of Two Kinds of Ligand during Nonclassical Crystallization of Pillared-Layer Metal-Organic Frameworks Elucidated Using Microfluidic Systems
  Tanaka, Yoko; Kitamura, Yu; Kawano, Ryuji; Shoji, Kan; Hiratani, Moe; Honma, Tetsuo; Takaya, Hikaru; Yoshikawa, Hirofumi; Tsuruoka, Takaaki; Tanaka, Daisuke
  CHEMISTRY-A EUROPEAN JOURNAL
  WILEY-V C H VERLAG GMBH
  To diversify metal-organic frameworks (MOFs), multi-component MOFs constructed from more than two kinds of bridging ligand have been actively investigated due to the high degree of design freedom afforded by the combination of multiple ligands. Predicting the synthesis conditions for such MOFs requires an understanding of the crystallization mechanism, which has so far remained elusive. In this context, microflow systems are efficient tools for capturing non-equilibrium states as they facilitate precise and efficient mixing with reaction times that correspond to the distance from the mixing point, thus enabling reliable control of non-equilibrium crystallization processes. Herein, we prepared coordination polymers with pillared-layer structures and observed the intermediates in the syntheses with an in-situ measurement system that combines microflow reaction with UV/Vis and X-ray absorption fine-structure spectroscopies, thereby enabling their rapid nucleation to be monitored. Based on the results, a three-step nonclassical nucleation mechanism involving two kinds of intermediate is proposed.
  2020年07月22日, 研究論文（学術雑誌）, 共同, 26, 41, 0947-6539, DOI(公開)(r-map), 8889, 8896
• 平面脂質膜システム評価系を用いたポア形成ペプチドの de novo 設計
  川野竜司
  ペプチドニュースレター
  日本ペプチド学会
  2020年07月01日, （MISC）総説・解説（学術雑誌）, 単独, 117, 1, DOI(公開)(r-map), 6, 8
• Microfluidic Formation of Honeycomb-Patterned Droplets Bounded by Interface Bilayers via Bimodal Molecular Adsorption
  Fujiwara, Shougo; Shoji, Kan; Watanabe, Chiho; Kawano, Ryuji; Yanagisawa, Miho
  MICROMACHINES
  MDPI
  Assembled water-in-oil droplets bounded by lipid bilayers are used in synthetic biology as minimal models of cell tissue. Microfluidic devices successfully generate monodispersed droplets and assemble them via droplet interface bilayesr (DIB) formation. However, a honeycomb pattern of DIB-bounded droplets, similar to epithelial tissues, remains unrealized because the rapid DIB formation between the droplets hinders their ability to form the honeycomb pattern. In this paper, we demonstrate the microfluidic formation of a honeycomb pattern of DIB-bounded droplets using two surfactants with different adsorption rates on the droplet surface. A non-DIB forming surfactant (sorbitan monooleate, Span 80) was mixed with a lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, PC), whose adsorption rate on the droplet surface and saturated interfacial tension were lower than those of Span 80. By changing the surfactant composition, we established the conditions under which the droplets initially form a honeycomb pattern and subsequently adhere to each other via DIB formation to minimize the interfacial energy. In addition, the reconstituted membrane protein nanopores at the DIBs were able to transport molecules. This new method, using the difference in the adsorption rates of two surfactants, allows the formation of a honeycomb pattern of DIB-bounded droplets in a single step, and thus facilitates research using DIB-bounded droplet assemblies.
  2020年07月, 研究論文（学術雑誌）, 共同, 11, 7, DOI(公開)(r-map)
• Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy
  Ueno, Takafumi; Niwase, Kento; Tsubokawa, Daisho; Kikuchi, Kosuke; Takai, Natsumi; Furuta, Tadaomi; Kawano, Ryuji; Uchihashi, Takayuki
  NANOSCALE
  ROYAL SOC CHEMISTRY
  Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.
  2020年04月21日, 研究論文（学術雑誌）, 共同, 12, 15, 2040-3364, DOI(公開)(r-map), 8166, 8173
• Nanopore Decoding for Solution of Hamiltonian Path Problem in DNA Computing
  S. Takiguchi, N. Takeuchi and R. Kawano
  Proceedings of FNANO
  FNANO
  2020年04月10日, 研究論文（国際会議プロシーディングス）, 共同, 1, 1, DOI(公開)(r-map), 185, 186
• Biological Nanopore Probe: Probing of Viscous Solutions in a Confined Nanospace
  Matsushita, Masaki; Shoji, Kan; Takai, Natsumi; Kawano, Ryuji
  JOURNAL OF PHYSICAL CHEMISTRY B
  AMER CHEMICAL SOC
  This paper describes a nanospace probing system constructed with a pore-forming toxin and a hairpin DNA (hpDNA) molecule. The single hpDNA molecule can be inserted and can move in the confined nanospace of the alpha-hemolysin (aHL) pore. The molecular motion of the hpDNA can be determined based on the fluctuation of the blocking current via channel current measurements. Using this system, we investigated the effect of viscosity of the aqueous solution in the macrospace (bulk) and in the confined nanospace with a small molecule (glycerol) and a polymer (PEG600). The molecular motion of the hpDNA in the nanospace differed in glycerol and PEG600 solutions, while the viscosity remained the same in the bulk solution. The fundamental factors for the viscosity in glycerol and PEG600 solutions are hydrogen bonding and the entanglement of polymer chains, respectively. This difference in factors becomes significant in confined nanospaces, and our system allows us to observe its effect. Additionally, we constructed a spatially resolved nanopore probe integrated into a gold nanoneedle. The alpha HL-hpDNA nanoprobe system was constructed with the nanoneedle and can be used to monitor the nanospace with nanometer spatial resolution.
  2020年03月26日, 研究論文（学術雑誌）, 共同, 124, 12, 1520-6106, DOI(公開)(r-map), 2410, 2416
• Lipid Membrane Deformation Induced by Transmembrane Peptides
  Izumi, Kayano; Shimizu, Keisuke; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2020年02月07日, 研究論文（国際会議プロシーディングス）, 共同, 118, 3, 0006-3495, DOI(公開)(r-map), 231A, 231A
• Development of Simple and Rapid Fabrications for Solid-State Nanopores
  Takai, Natsumi; Matsushita, Masaki; Shoji, Kan; Maki, Tei; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2020年02月07日, 研究論文（国際会議プロシーディングス）, 共同, 118, 3, 0006-3495, DOI(公開)(r-map), 349A, 349A
• Construction of Programmable Nanopore using de novo Designed beta-Sheet Peptide
  Shimizu, Keisuke; Sakashita, Shungo; Hamada, Yoshio; Usui, Kenji; Mijiddorj, Batsaikhan; Kawamura, Izuru; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2020年02月07日, 研究論文（国際会議プロシーディングス）, 共同, 118, 3, 0006-3495, DOI(公開)(r-map), 474A, 474A
• Analyzing Single-Molecule Behavior of a Small Protein in Confined Nanospace of a Biological Nanopore
  Yamaji, Misa; Takai, Natsumi; Chinappi, Mauro; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2020年02月07日, 研究論文（国際会議プロシーディングス）, 共同, 118, 3, 0006-3495, DOI(公開)(r-map), 474A, 474A
• Osmotic-engine-driven liposomes in microfluidic channels
  Shoji, Kan; Kawano, Ryuji
  LAB ON A CHIP
  ROYAL SOC CHEMISTRY
  Self-propelled underwater microrobots that locomote without external sources of energy have potential application as drug carriers and probes in narrow spaces. In this study, we focused on an osmotic engine model, which is a migration mechanism, and applied it as a negative chemotaxis mechanism to induce liposome displacement. First, we confirmed the osmotic flow across the lipid bilayer and calculated the osmotic flow velocity to be 8.5 fL min(-1) mu m(-2) when a salt concentration difference was applied to the lipid bilayer. Next, we designed and fabricated a microchannel that can trap a giant liposome and apply a salt concentration difference to the front and rear of the liposome. Then, we demonstrated the movement of the liposome by flowing it to the microchannel. The liposome successfully moved in the direction of the lower ion concentration at a speed of 0.6 mu m min(-1) owing to the osmotic pressure difference. Finally, we visualized the inner flow in the liposome by encapsulating microbeads in the liposome and observed the movement of the microbeads to verify that an osmotic flow was generated on the liposome. As a result, we observed the circulation of the microbeads in the liposome when the concentration difference was applied to the front and rear of the liposome, suggesting that the movement of the liposome was driven by the osmotic flow generated by the osmotic pressure difference. These results indicate that the osmotic-pressure-based migration mechanism has the potential to be utilized as the actuator of molecular robots.
  2019年10月21日, 研究論文（学術雑誌）, 共同, 19, 20, 1473-0197, DOI(公開)(r-map), 3472, 3480
• DNA Origami Nanoplate-Based Emulsion with Nanopore Function
  Ishikawa, Daisuke; Suzuki, Yuki; Kurokawa, Chikako; Ohara, Masayuki; Tsuchiya, Misato; Morita, Masamune; Yanagisawa, Miho; Endo, Masayuki; Kawano, Ryuji; Takinoue, Masahiro
  ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
  WILEY-V C H VERLAG GMBH
  Bio-inspired functional microcapsules have attracted increasing attention in many fields from physical/chemical science to artificial-cell engineering. Although particle-stabilised microcapsules are advantageous for their stability and functionalisation potential, versatile methods for their functionalisation are desired to expand their possibilities. This study reports a water-in-oil microdroplet stabilised with amphiphilic DNA origami nanoplates. By utilising DNA nanotechnology, DNA nanoplates were designed as a nanopore device for ion transportation and to stabilise the oil-water interface. Microscopic examination revealed the microcapsule formed by the accumulation of amphiphilic DNA nanoplates at the oil-water interface. Ion current measurements revealed the nanoplate pores functioned as channel to transport ions. These findings provide a general strategy for the programmable design of microcapsules to engineer artificial cells and molecular robots.
  2019年10月21日, 研究論文（学術雑誌）, 共同, 58, 43, 1433-7851, DOI(公開)(r-map), 15299, 15303
• Electrophysiological Analysis of Membrane Disruption by Bombinin and its Isomer using Lipid Bilayer System
  Yusuke Sekiya, Keisuke Shimizu, Yuki Kitahashi, Akifumi Ohyama, Izuru Kawamura, and Ryuji Kawano
  ACS Appl. Bio. Mater.,
  ACS
  2019年08月06日, 研究論文（学術雑誌）, 共同, 2, 4, DOI(公開)(r-map), 13124, 13130
• Electrophysiological Analysis of Antimicrobial Peptides in Diverse Species
  Saigo, Naoki; Izumi, Kayano; Kawano, Ryuji
  ACS OMEGA
  AMER CHEMICAL SOC
  This study describes a technical platform that allows us to measure the pore-forming activity of antimicrobial peptides (AMPs) in the lipid bilayer and estimate antimicrobial activity. We selected six different AMPs of diverse species from urochordata to vertebrata and measured the channel current signals using a microfabricated lipid bilayer system. As a result of the electrophysiological measurements, we were able to estimate the pore-forming activity and roughly predict the antimicrobial activity although there was not a strong correlation between the pore-forming activity and the variety of species. Our method will be a unique tool for analyzing a wide variety of diverse AMPs.
  2019年08月, 研究論文（学術雑誌）, 共同, 4, 8, 2470-1343, DOI(公開)(r-map), 13124, 13130
• Spatially Resolved Chemical Detection with a Nanoneedle-Probe-Supported Biological Nanopore
  Shoji, Kan; Kawano, Ryuji; White, Ryan J.
  ACS NANO
  AMER CHEMICAL SOC
  In this article, we describe the quantitative characterization of a gold nanoneedle ion channel probe and demonstrate the utility of this probe for spatially resolved detection of a small molecule using ion channel activity. Our report builds on recent reports of Ide and co-workers, who reported the use of an etched gold wire modified with a poly(ethylene) glycol monolayer as a support for a lipid bilayer and subsequent single ion channel recordings. Although this nanoneedle electrode approach was reported previously, in our report, we investigate the effects of several operational parameters on the performance of the ion channel measurement and electrochemical phenomenon that occur in the nanoconfined space between the supported bilayer and the gold electrode. More specifically, we address the effects of length of the supporting monolayer and the composition of the electrolyte baths on channel current measurements and provide a quantitative description of what carries current at the working electrode (double-layer charging). In addition, we demonstrate the ability to control the direction of protein insertion (tip side vs bath side) with freely diffusing protein, which has not been previously reported, with the former method (tip side) enabling single-molecule detection of beta-icyclodextrin (beta CD) using a reconstituted a-hemolysin channel. Finally, anticipating future use of a nanoneedle-based biological nanopore probe in a scanned-probe microscopy, we demonstrate the ability to quantify and spatially resolve the concentration of beta CD molecules in a microfluidic channel. We believe, in the long term, the described nanoneedle-based biological nanopore probe can be employed in, for example, scanning ion conductance microscopy using ion channels.
  2019年02月, 研究論文（学術雑誌）, 共同, 13, 2, 1936-0851, DOI(公開)(r-map), 2606, 2614
• Nanopore Decoding of Oligonucleotides in DNA Computing
  Kawano, Ryuji
  BIOTECHNOLOGY JOURNAL
  WILEY-V C H VERLAG GMBH
  In conventional DNA-computation methods involving logic gate operations, the output molecules are detected and decoded mainly by gel electrophoresis or fluorescence measurements. To employ rapid and label-free decoding, nanopore technology, an emerging methodology for single-molecule detection or DNA sequencing, is proposed as a candidate for electrical and simple decoding of DNA computations. This review describes recent approaches to decoding DNA computation using label-free and electrical nanopore measurements. Several attempts have been successful in DNA decoding with the nanopore either through enzymatic reactions or in water-in-oil droplets. Additionally, DNA computing combined with nanopore decoding has clinical applications, including microRNA detection for early diagnosis of cancers. Because this decoding methodology is still in development and not yet widely accepted, this review aims to inform the scientific community regarding usefulness.
  2018年12月, 研究論文（学術雑誌）, 単独, 13, 12, 1860-6768, DOI(公開)(r-map)
• Electrophysiological measurement of ion channels on plasma/organelle membranes using an on-chip lipid bilayer system
  Kamiya, Koki; Osaki, Toshihisa; Nakao, Kenji; Kawano, Ryuji; Fujii, Satoshi; Misawa, Nobuo; Hayakawa, Masatoshi; Takeuchi, Shoji
  SCIENTIFIC REPORTS
  NATURE PUBLISHING GROUP
  Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.
  2018年11月30日, 研究論文（学術雑誌）, 共同, 8, 2045-2322, DOI(公開)(r-map)
• Spring-shaped stimuli-responsive hydrogel actuator with large deformation
  Yoshida, Koki; Nakajima, Shunsuke; Kawano, Ryuji; Onoe, Hiroaki
  SENSORS AND ACTUATORS B-CHEMICAL
  ELSEVIER SCIENCE SA
  This study describes a novel microfluidics-based method for compressive/expanding actuation of stimuli-responsive hydrogel microsprings with large deformations. A continuous flow of mixed alginate and poly(N-isopropylacrylamide- co-acrylic acid) pre-gel solution can spontaneously form a hydrogel microspring with a wide range of gradient pitches via buoyancy force. This technique enables fabrication of hydrogel microsprings using only simple capillaries and syringe pumps. The resulting microsprings can be patterned via laminar flow inside the capillary, which can contribute to large deformation. Single-layered hydrogel microsprings shrunk isotropically while maintaining the shape of the spring. Compressing stimuli-responsive microsprings can be done by patterning the shrinking part of the spring. Here, the degree of compression in the double-layered spring depends on the initial pitch. Furthermore, large axial expansion of microsprings can be achieved by shrinking part of a microspring. Our large compression/expansion stimuli-responsive hydrogel microsprings have immense potential to be applied in various microengineering products including soft actuators, chemical sensors, and medical applications.
  2018年11月01日, 研究論文（学術雑誌）, 共同, 272, 0925-4005, DOI(公開)(r-map), 361, 368
• Chemical sensing using a nanoneedle-based nanopore probe
  Shoji, Kan; Kawano, Ryuji; White, Ryan
  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
  AMER CHEMICAL SOC
  2018年08月19日, 研究論文（学術雑誌）, 共同, 256, 0065-7727, DOI(公開)(r-map)
• Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides
  Sekiya, Yusuke; Sakashita, Shungo; Shimizu, Keisuke; Usui, Kenji; Kawano, Ryuji
  ANALYST
  ROYAL SOC CHEMISTRY
  We measured the current signal of the transmembrane model peptides using the barrel-stave, toroidal pore, and penetration models in order to establish a precise assignment of the channel signals. In addition, we analyzed the spike signals to estimate the membrane penetration of model cell-penetration peptides of different lengths.
  2018年08月07日, 研究論文（学術雑誌）, 共同, 143, 15, 0003-2654, DOI(公開)(r-map)
• DNA Logic Operation with Nanopore Decoding To Recognize MicroRNA Patterns in Small Cell Lung Cancer
  Hiratani, Moe; Kawano, Ryuji
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  Although DNA computation has traditionally been developed for parallel calculations in molecular analyses, this approach has recently been considered for use in diagnostic or medical applications in living systems. In this study, we propose that the DNA logic operation may be a powerful tool for the recognition of microRNA patterns, which may have applications for the early diagnosis of cancers. We developed a rapid, label-free decoding method for output diagnostic molecules using nanopore measurements. We designed diagnostic DNAs that autonomously recognized two microRNAs, miR-20a and miR-17-5p, and formed a four-way junction structure that was captured in the nanopore, showing long blocking currents. We analyzed the blocking duration based on the central limit theorem and found that four different operations, i.e., (0, 0), (0, 1), (1, 0), and (1, 1), could be discriminated. This pattern recognition method has been differentiated from simple detection methods based on DNA computing and nanopore technologies.
  2018年07月17日, 研究論文（学術雑誌）, 共同, 90, 14, 0003-2700, DOI(公開)(r-map), 8531, 8537
• Microfluidic Formation of Double-Stacked Planar Bilayer Lipid Membranes by Controlling the Water-Oil Interface
  Shoji, Kan; Kawano, Ryuji
  MICROMACHINES
  MDPI
  This study reports double-stacked planar bilayer lipid membranes (pBLMs) formed using a droplet contact method (DCM) for microfluidic formation with five-layered microchannels that have four micro guide pillars. pBLMs are valuable for analyzing membrane proteins and modeling cell membranes. Furthermore, multiple-pBLM systems have broadened the field of application such as electronic components, light-sensors, and batteries because of electrical characteristics of pBLMs and membrane proteins. Although multiple-stacked pBLMs have potential, the formation of multiple-pBLMs on a micrometer scale still faces challenges. In this study, we applied a DCM strategy to pBLM formation using microfluidic techniques and attempted to form double-stacked pBLMs in micro-meter scale. First, microchannels with micro pillars were designed via hydrodynamic simulations to form a five-layered flow with aqueous and lipid/oil solutions. Then, pBLMs were successfully formed by controlling the pumping pressure of the solutions and allowing contact between the two lipid monolayers. Finally, pore-forming proteins were reconstituted in the pBLMs, and ion current signals of nanopores were obtained as confirmed by electrical measurements, indicating that double-stacked pBLMs were successfully formed. The strategy for the double-stacked pBLM formation can be applied to highly integrated nanopore-based systems.
  2018年05月, 研究論文（学術雑誌）, 共同, 9, 5, 2072-666X, DOI(公開)(r-map)
• Well-Controlled Cell-Trapping Systems for Investigating Heterogeneous Cell-Cell Interactions
  Kamiya, Koki; Abe, Yuta; Inoue, Kosuke; Osaki, Toshihisa; Kawano, Ryuji; Miki, Norihisa; Takeuchi, Shoji
  ADVANCED HEALTHCARE MATERIALS
  WILEY
  Microfluidic systems have been developed for patterning single cells to study cell-cell interactions. However, patterning multiple types of cells to understand heterogeneous cell-cell interactions remains difficult. Here, it is aimed to develop a cell-trapping device to assemble multiple types of cells in the well-controlled order and morphology. This device mainly comprises a parylene sheet for assembling cells and a microcomb for controlling the cell-trapping area. The cell-trapping area is controlled by moving the parylene sheet on an SU-8 microcomb using tweezers. Gentle downward flow is used as a driving force for the cell-trapping. The assembly of cells on a parylene sheet with round and line-shaped apertures is demonstrated. The cell-cell contacts of the trapped cells are then investigated by direct cell-cell transfer of calcein via connexin nanopores. Finally, using the device with a system for controlling the cell-trapping area, three different types of cells in the well-controlled order are assembled. The correct cell order rate obtained using the device is 27.9%, which is higher than that obtained without the sliding parylene system (0.74%). Furthermore, the occurrence of cell-cell contact between the three cell types assembled is verified. This cell-patterning device will be a useful tool for investigating heterogeneous cell-cell interactions.
  2018年03月21日, 研究論文（学術雑誌）, 共同, 7, 6, 2192-2640, DOI(公開)(r-map)
• Design of protein-responsive micro-sized hydrogels for self-regulating microfluidic systems
  Hirayama, Mayu; Tsuruta, Kazuhiro; Kawamura, Akifumi; Ohara, Masayuki; Shoji, Kan; Kawano, Ryuji; Miyata, Takashi
  JOURNAL OF MICROMECHANICS AND MICROENGINEERING
  IOP PUBLISHING LTD
  Diagnosis sensors using micro-total analysis systems (mu-TAS) have been developed for detecting target biomolecules such as proteins and saccharides because they are signal biomolecules for monitoring body conditions and diseases. In this study, biomolecularly stimuli-responsive micro-sized hydrogels that exhibited quick shrinkage in response to lectin concanavalinA (ConA) were prepared in a microchannel by photopolymerization using a fluorescence microscope. In preparing the micro-size hydrogels, glycosyloxyethyl methacrylate (GEMA) as a ligand monomer was copolymerized with a crosslinker in the presence of template ConA in molecular imprinting. The ConA-imprinted micro-hydrogel showed greater shrinkage in response to target ConA than nonimprinted micro-hydrogel. When a buffer solution was switched to an aqueous ConA solution in the Y-shaped microchannel, the flow rates changed quickly because of the responsive shrinkage of the micro-hydrogel prepared in the microchannel. These results suggest that the ConA-imprinted micro-hydrogel acted as a self-regulated microvalve in microfluidic systems.
  2018年03月, 研究論文（学術雑誌）, 共同, 28, 3, 0960-1317, DOI(公開)(r-map)
• Synthetic Ion Channels and DNA Logic Gates as Components of Molecular Robots
  Kawano, Ryuji
  CHEMPHYSCHEM
  WILEY-V C H VERLAG GMBH
  A molecular robot is a next-generation biochemical machine that imitates the actions of microorganisms. It is made of biomaterials such as DNA, proteins, and lipids. Three prerequisites have been proposed for the construction of such a robot: sensors, intelligence, and actuators. This Minireview focuses on recent research on synthetic ion channels and DNA computing technologies, which are viewed as potential candidate components of molecular robots. Synthetic ion channels, which are embedded in artificial cell membranes (lipid bilayers), sense ambient ions or chemicals and import them. These artificial sensors are useful components for molecular robots with bodies consisting of a lipid bilayer because they enable the interface between the inside and outside of the molecular robot to function as gates. After the signal molecules arrive inside the molecular robot, they can operate DNA logic gates, which perform computations. These functions will be integrated into the intelligence and sensor sections of molecular robots. Soon, these molecular machines will be able to be assembled to operate as a mass microrobot and play an active role in environmental monitoring and invivo diagnosis or therapy.
  2018年02月19日, 研究論文（学術雑誌）, 単独, 19, 4, 1439-4235, DOI(公開)(r-map), 359, 366
• MicroRNA detection at femtomolar concentrations with isothermal amplification and a biological nanopore
  Zhang, Haolin; Hiratani, Moe; Nagaoka, Kentaro; Kawano, Ryuji
  NANOSCALE
  ROYAL SOC CHEMISTRY
  One of the greatest challenges faced by chemists and biologists is the detection of molecules at extremely low concentrations. This paper describes a method to detect ultra-low concentrations (1 femtomole) of nucleotides using isothermal amplification and a biological nanopore.
  2017年11月, 研究論文（学術雑誌）, 共同, 9, 42, 2040-3364, DOI(公開)(r-map), 16124, 16127
• pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis
  Kunthic, Thittaya; Watanabe, Hirokazu; Kawano, Ryuji; Tanaka, Yoshikazu; Promdonkoy, Boonhiang; Yao, Min; Boonserm, Panadda
  BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
  ELSEVIER SCIENCE BV
  Vip3Aa insecticidal protein is produced from Bacillus thuringiensis and exerts a broad spectrum of toxicity against lepidopteran insect species. Although Vip3Aa has been effectively used as part of integrated pest management strategies, the mechanism of the toxin remains unclear. Here, we investigated the effect of pH in a range from 5.0 to 10.0 on the pore-forming activity of the trypsin activated Vip3Aa (actVip3Aa) by in vitro pore-forming assays. Based on calcein release assay, actVip3Aa could permeabilize the artificial neutral liposomes under all the pH tested, except pH 10.0. The maximum membrane permeability of actVip3Aa was detected at pH 8.0 and the permeability decreased and abolished when exposing to acidic and alkaline conditions, respectively. The planar lipid bilayer experiment revealed that actVip3Aa formed ion channels at pH 5.0-8.0 but no current signals were detected at pH 10.0, consistent with the observation from calcein release assay. The toxin formed ion channels with a diameter of 1.4 nm at pH 8.0 and pore size was gradually decreased when reducing the pH. This study provided a view of the molecular mechanism of Vip3Aa by which the pore-forming activity is regulated by pH.
  2017年11月, 研究論文（学術雑誌）, 共同, 1859, 11, 0005-2736, DOI(公開)(r-map), 2234, 2241
• Analysis of Pore Formation and Protein Translocation Using Large Biological Nanopores
  Watanabe, Hirokazu; Gubbiotti, Alberto; Chinappi, Mauro; Takai, Natsumi; Tanaka, Koji; Tsumoto, Kouhei; Kawano, Ryuji
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  This paper describes the analysis of pore formation and detection of a single protein molecule using a large nanopore among five different pore-forming proteins. We demonstrate that the identification of appropriate pores for nanopore sensing can be achieved by classifying the channel current signals and performing noise analysis. Through these analyses, we selected a perforin nanopore from the membrane attack complex/perforin superfamily and attempted to use it to detect the granzyme B protein, a serine protease. As a result, we found that granzyme B might pass through the perforin nanopore if it adopts an unfolded structure. Our proposed analytical approach should be useful for exploring several types of nanopore as large biological nanopores other than alpha-hemolysin.
  2017年11月, 研究論文（学術雑誌）, 共同, 89, 21, 0003-2700, DOI(公開)(r-map), 11269, 11277
• Nanopore Logic Operation with DNA to RNA Transcription in a Droplet System
  Ohara, Masayuki; Takinoue, Masahiro; Kawano, Ryuji
  ACS SYNTHETIC BIOLOGY
  AMER CHEMICAL SOC
  This paper describes an AND logic operation with amplification and transcription from DNA to RNA, using T7 RNA polymerase. All four operations, (0 0) to (1 1), with an enzyme reaction can be performed simultaneously, using four-droplet devices that are directly connected to a patch-clamp amplifier. The output RNA molecule is detected using a biological nanopore with single-molecule translocation. Channel current recordings can be obtained using the enzyme solution. The integration of DNA logic gates into electrochemical devices is necessary to obtain output information in a human-recognizable form. Our method will be useful for rapid and confined DNA computing applications, including the development of programmable diagnostic devices.
  2017年07月, 研究論文（学術雑誌）, 共同, 6, 7, 2161-5063, DOI(公開)(r-map), 1427, 1432
• Serial DNA relay in DNA logic gates by electrical fusion and mechanical splitting of droplets
  Yasuga, Hiroki; Inoue, Kosuke; Kawano, Ryuji; Takinoue, Masahiro; Osaki, Toshihisa; Kamiya, Koki; Miki, Norihisa; Takeuchi, Shoji
  PLOS ONE
  PUBLIC LIBRARY SCIENCE
  DNA logic circuits utilizing DNA hybridization and/or enzymatic reactions have drawn increasing attention for their potential applications in the diagnosis and treatment of cellular diseases. The compartmentalization of such a system into a microdroplet considerably helps to precisely regulate local interactions and reactions between molecules. In this study, we introduced a relay approach for enabling the transfer of DNA from one droplet to another to implement multi-step sequential logic operations. We proposed electrical fusion and mechanical splitting of droplets to facilitate the DNA flow at the inputs, logic operation, output, and serial connection between two logic gates. We developed Negative-OR operations integrated by a serial connection of the OR gate and NOT gate incorporated in a series of droplets. The four types of input defined by the presence/absence of DNA in the input droplet pair were correctly reflected in the readout at the Negative-OR gate. The proposed approach potentially allows for serial and parallel logic operations that could be used for complex diagnostic applications.
  2017年07月, 研究論文（学術雑誌）, 共同, 12, 7, 1932-6203, DOI(公開)(r-map)
• Stimuli-responsive hydrogel microfibers with controlled anisotropic shrinkage and cross-sectional geometries
  Nakajima, Shunsuke; Kawano, Ryuji; Onoe, Hiroaki
  SOFT MATTER
  ROYAL SOC CHEMISTRY
  sStimuli-responsive microfibers are fabricated by extruding mixed solutions of poly(N-isopropylacrylamideco-acrylic acid) (pNIPAM-AAc) and sodium alginate (Na-alginate) using a microfluidic spinning system. The fabricated microfibers shrink and swell with temperature and/or pH. By controlling the extruded laminar flow, microfibers capable of anisotropic shrinkage are fabricated. Cross-sectional microscale geometries of microfibers, including double layering and hollowness, are successfully controlled by patterning the laminar flow during microfiber formation, resulting in hydrogels capable of folding/unfolding motions and fluid pumping. In addition, macroscopic 3D-bundle structures are assembled with these microfibers. We believe that our microfibers can be applied to various applications such as soft actuators, soft robots, and micropumps.
  2017年05月, 研究論文（学術雑誌）, 共同, 13, 20, 1744-683X, DOI(公開)(r-map), 3710, 3719
• Metal-Organic Cuboctahedra for Synthetic Ion Channels with Multiple Conductance States
  Kawano, Ryuji; Horike, Nao; Hijikata, Yuh; Kondo, Mio; Carne-Sanchez, Arnau; Larpent, Patrick; Ikemura, Shuya; Osaki, Toshihisa; Kamiya, Koki; Kitagawa, Susumu; Takeuchi, Shoji; Furukawa, Shuhei
  CHEM
  CELL PRESS
  Emulation of biological ion channels by synthetic molecules is not only a challenge for chemists in designing highly complex (supra) molecules but also key to developing a new tool for exploring subcellular electrochemical activity. Despite efforts to create a single pore in a lipid bilayer by synthetic channels, a general synthetic strategy for realizing more complex two-pore channels has yet to be proposed. Here, we demonstrate two distinct ion conductance states by embedding a singlemetal-organic porous molecule with the geometry of an Archimedean cuboctahedron into an artificially reconstructed lipid bilayer membrane in which triangular and square apertures in the cuboctahedron work independently as ion-transporting pathways. By changing the aliphatic chain length introduced on the periphery of the cuboctahedron, we found that the rotational dynamics of the cuboctahedron regulate the open pore time of each conductance state through distinct apertures and the switching between them.
  2017年03月, 研究論文（学術雑誌）, 共同, 2, 3, 2451-9294, DOI(公開)(r-map), 393, 403
• Amplification and Quantification of an Antisense Oligonucleotide from Target microRNA Using Programmable DNA and a Biological Nanopore
  Hiratani, Moe; Ohara, Masayuki; Kawano, Ryuji
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  This paper describes a strategy for autonomous diagnoses of cancers using microRNA (miRNA) and therapy for tumor cells by DNA computing techniques and nanopore measurement. Theranostics, which involves the combination of diagnosis and therapy, has emerged as an approach for personalized medicine or point-of-care cancer diagnostics. DNA computing will become a potent tool for theranostics because it functions completely autonomously without the need for external regulations. However, conventional theranostics using DNA computing involves a one-to-one reaction in which a single input molecule generates a single output molecule; the concentration of the antisense drug is insufficient for the therapy in this type of reaction. Herein we developed an amplification system involving an isothermal reaction in which a large amount of the antisense DNA drug was autonomously generated after detecting miRNA from small cell lung cancer. In addition, we successfully quantified the amount of the generated drug molecule by nanopore measurement with high accuracy, which was more accurate than conventional gel electrophoresis. This autonomous amplification strategy is a potent candidate for a broad range of theranostics using DNA computing.
  2017年02月, 研究論文（学術雑誌）, 共同, 89, 4, 0003-2700, DOI(公開)(r-map), 2312, 2317
• Expression and characterization of the Plasmodium translocon of the exported proteins component EXP2
  Hakamada, Kazuaki; Watanabe, Hirokazu; Kawano, Ryuji; Noguchi, Keiichi; Yohda, Masafumi
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
  ACADEMIC PRESS INC ELSEVIER SCIENCE
  The malaria parasite Plasmodium falciparum requires the Plasmodium translocon of exported proteins (PTEX) to proliferate in human red blood cells. During the blood stages of malaria, several hundred parasite-encoded proteins are exported from the parasite into the cytosol of red blood cells. PTEX is the translocon for protein export and comprises 5 proteins: EXP2, PTEX150, PTEX88, Hsp101 and TRX2. Among them, EXP2 is thought to constitute the transmembrane pore, whereas the other components seem to play a role in unfolding the luggage proteins or providing a driving force. However, detailed functional and structural characterizations of PTEX proteins have not been performed. In this study, we expressed and characterized the membrane-associated component EXP2. Because expression of EXP2 is lethal to E. coli, EXP2 was expressed as a fusion protein with GST, and the recombinant EXP2 was obtained by protease digestion. The recombinant EXP2 formed pores in bilayer lipid membranes. The inner diameter of the pore was estimated to be approximately 3.5 nm based on electron microscopy images and channel currents. From this size and the molecular mass as determined by size exclusion chromatography and blue native polyacrylamide gel electrophoresis, we determined that the pore comprises approximately 10-12 EXP2 subunits. However, there is a possibility that the pore structure is different in the PTEX complex. These results provide important insights in the protein transport mechanism of PTEX, which will aid in developing new drugs targeting PTEX. (C) 2016 Elsevier Inc. All rights reserved.
  2017年01月, 研究論文（学術雑誌）, 共同, 482, 4, 0006-291X, DOI(公開)(r-map), 700, 705
• Rhodium–Organic Cuboctahedra as Porous Solids with Strong Binding Sites
  S. Furukawa, N. Horike, M. Kondo, Y. Hijikata, A. Carné-Sánchez, P. Larpent, N. Louvain, S. Diring, H. Sato, R. Matsuda, R. Kawano, S. Kitagawa
  Inorganic Chemistry
  2016年01月, 研究論文（学術雑誌）, 共同, DOI(公開)(r-map), ASAP
• Scalable fabrication of microneedle arrays via spatially controlled UV exposure
  H. Takahashi, Y.J. Heo, N. Arakawa, T. Kan, K. Matsumoto, R. Kawano, I. Shimoyama
  Microsystems & Nanoengineering
  2016年01月, 研究論文（学術雑誌）, 共同, 2, DOI(公開)(r-map), 16049
• Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes
  K. Kamiya, R. Kawano, T. Osaki, K. Akiyoshi, S. Takeuchi
  Nature Chemistry
  2016年01月, 研究論文（学術雑誌）, 共同, 8, DOI(公開)(r-map), 881–889
• Hairpin DNA Unzipping Analysis Using a Biological Nanopore Array
  M. Ohara, Y. Sekiya, R. Kawano
  Electrochemistry
  2016年01月, 研究論文（学術雑誌）, 共同, 84, 5, DOI(公開)(r-map), 338, 341
• Logic Gate Operation by DNA Translocation through Biological Nanopores
  H. Yasuga, R. Kawano, M. Takinoue, T. Tsuji, T. Osaki, K. Kamiya, N. Miki, S. Takeuchi
  PLoS One
  2016年01月, 研究論文（学術雑誌）, 共同, 11, 2, DOI(公開)(r-map), e0149667
• Channel Current Analysis for Pore-forming Properties of an Antimicrobial Peptide, Magainin 1, using the Droplet Contact Method
  H. Watanabe and R. Kawano
  Analytical Sciences
  2016年01月, 研究論文（学術雑誌）, 共同, 32, 1, DOI(公開)(r-map), 57, 60
• Nonlinear concentration gradients regulated by the width of channels for observation of half maximal inhibitory concentration (IC50) of transporter proteins
  Y. Abe, K. Kamiya, T. Osaki, H. Sasaki, R. Kawano, N. Miki and S. Takeuchi
  Analyst
  2015年01月, 研究論文（学術雑誌）, 共同, 140, DOI(公開)(r-map), 5557, 5562
• Towards combinatorial mixing devices without any pumps by open-capillary channels: fundamentals and applications
  M. Tani, R. Kawano, K. Kamiya & K. Okumura
  Scientific Reports
  2015年01月, 研究論文（学術雑誌）, 共同, 5, DOI(公開)(r-map), 10263
• Preparation of Molecule-Responsive Microsized Hydrogels via Photopolymerization for Smart Microchannel Microvalves
  Y. Shiraki, K. Tsuruta, J. Morimoto, C. Ohba, A. Kawamura, R. Yoshida, R. Kawano, T. Uragami and T. Miyata
  Macromol. Rapid Commun
  2015年01月, 研究論文（学術雑誌）, 共同, 36, DOI(公開)(r-map), 515–519
• Preparation of Structurally Colored, Monodisperse Spherical Assemblies Composed of Black and White ColloidalParticles using a Micro Flow-Focusing Device
  M. Teshima, T. Seki, R. Kawano, S. Takeuchi, S. Yoshioka and Y. Takeoka
  Journal of Materials Chemistry C
  2015年01月, 研究論文（学術雑誌）, 共同, 3, 769, DOI(公開)(r-map)
• A Portable Lipid Bilayer System for Environmental Sensing with a Transmembrane Protein
  R. Kawano, Y. Tsuji, K. Kamiya, T. Kodama, T. Osaki, N. Miki, S. Takeuchi
  PLoS ONE
  2014年07月, 研究論文（学術雑誌）, 共同, 9, 7, DOI(公開)(r-map), e102427
• Lipid bilayers on a picoliter microdroplet array for rapid fluorescence detection of membrane transport
  T. Tonooka, K. Sato, T. Osaki, R. Kawano, S. Takeuchi
  Small
  2014年03月, 研究論文（学術雑誌）, 共同, 10, 6, 3275, 3282
• Round-Tip Dielectrophoresis-Based Tweezers for Single Micro-Object Manipulation
  T. Kodama, T. Osaki, R. Kawano, K. Kamiya, N. Miki, S. Takeuchi
  Biosensors and Bioelectronics
  2013年11月, 研究論文（学術雑誌）, 共同, 47, 206, 212
• Droplet based lipid bilayer system integrated with microfluidic channels for solution exchange
  †Y. Tsuji,†R. Kawano, T. Osaki, K. Kamiya, N. Miki, S. Takeuchi
  Lab on a Chip
  2013年11月, 研究論文（学術雑誌）, 共同, 13, 1476, 1481
• Automated Parallel Recordings of Topologically Identified Single Ion Channels
  R. Kawano, Y. Tsuji, K. Sato, T. Osaki, K. Kamiya, M. Hirano, T. Ide, N. Miki, S. Takeuchi
  Scientific Reports
  2013年11月, 研究論文（学術雑誌）, 共同, 3, DOI(公開)(r-map), 1995
• Droplet Split-and-Contact Method for High-Throughput Transmembrane Electrical Recording
  †Y. Tsuji,†R. Kawano, T. Osaki, K. Kamiya, N. Miki, S. Takeuchi
  Anal. Chem.
  2013年11月, 研究論文（学術雑誌）, 共同, 85, 10913, 10919
• Single-vesicle estimation of ATP-binding cassette transporters in microfluidic channels
  H. Sasaki, R. Kawano, T. Osaki, K. Kamiya, S. Takeuchi
  Lab on a Chip
  2012年11月, 研究論文（学術雑誌）, 共同, 12, 702, 704
• Microfluidic Control of the Internal Morphology in Nanofiber-based Macroscopic Cables
  D. Kiriya, R. Kawano, H. Onoe, S. Takeuchi
  Angewandte Chemie Int. Ed
  2012年11月, 研究論文（学術雑誌）, 共同, 51, 7942, 7947
• A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing
  Y. Oyama, T. Osaki, K. Kamiya, R. Kawano, T. Honjoh, H. Shibata, T. Ide, S. Takeuchi
  Lab on a Chip
  2012年11月, 研究論文（学術雑誌）, 共同, 12, 5155, 5159
• Electrical Access to Lipid Bilayer Membrane Microchambers for Transmembrane Analysis
  T. Osaki, Y. Watanabe, R. Kawano, H. Sasaki, S. Takeuchi
  Journal of Microelectromechanical Systems
  2011年11月, 研究論文（学術雑誌）, 共同, 20, 797, 799
• Rapid Detection of a Cocaine-Binding Aptamer Using Biological Nanopores on a Chip
  R. Kawano, T. Osaki, H. Sasaki, M. Takinoue, S. Yoshizawa, S. Takeuchi
  J. Am. Chem. Soc
  2011年11月, 研究論文（学術雑誌）, 共同, 133, 8474, 8477
• Simple and Stable Lipid Bilayer Formation: A Droplets Contacting Method using Parylene Micropores for Multiple Ion Channel Recordings
  Y. Tsuji, R. Kawano, T. Osaki, H. Sasaki, N. Miki, S. Takeuchi
  IEEJ Trans.E
  2011年11月, 研究論文（学術雑誌）, 共同, 131, 419, 424
• Solid-State Dye-Sensitized Solar Cells using Polymerized Ionic Liquid Electrolyte and Platinum-Free Counter Electrode
  R. Kawano*, T. Katakabe, H. Shimosawa, M. K. Nazeeruddinn, M. Gratzel, M. Watanabe
  Phys. Chem. Chem. Phys.
  2010年11月, 研究論文（学術雑誌）, 共同, 12, 1916, 1921
• Monitoring the Escape of DNA from a Nanopore Using an Alternating Current Signal
  D. K. Lathrop, E. N. Ervin, G. A. Barrall, M. G. Keehan, R. Kawano, M. A. Krupka, H. S. White, and A. H. Hibbs
  J. Am. Chem. Soc.
  2010年11月, 研究論文（学術雑誌）, 共同, 132, 1878, 1885
• Quartz Nanopore Membranes for Suspended Bilayer Ion Channel Recordings
  A. Schibel, T. Edwards, R. Kawano, W. Lan, and H. White
  Anal. Chem.
  2010年11月, 研究論文（学術雑誌）, 共同, 82, 7259–7266
• A Polymer-based Nanopore-integrated Microfluidic Device for Generating Stable Bilayer Lipid Membranes
  R. Kawano, T. Osaki, H. Sasaki, S. Takeuchi
  Small
  2010年11月, 研究論文（学術雑誌）, 共同, 6, 2100, 2104
• A rupture detection algorithm for the DNA translocation detection through biological nanopore
  T. Osaki, J. P. Barbot, R. Kawano, H. Sasaki, O. Francais, B. Le Pioufle, S. Takeuchi
  Procedia Engineering
  2010年11月, 研究論文（学術雑誌）, 共同, 5, 796, 799
• Parylene-coating in PDMS channels to prevent the absorption of fluorescent dyes
  H. Sasaki, H. Onoe, T. Osaki, R. Kawano, S. Takeuchi
  Sensors & Actuators: B. Chemical
  2010年11月, 研究論文（学術雑誌）, 共同, 150, 478, 482
• Simultaneous Alternating and Direct Current Readout of Protein Ion Channel Blocking Events using Glass Nanopore Membranes
  E. N. Ervin, R. Kawano, R. J. White, H. S. White
  Anal. Chem.
  2009年11月, 研究論文（学術雑誌）, 共同, 80, 2069, 2076
• Controlling the Translocation of Single-Stranded DNA through α-Hemolysin Ion Channels Using Viscosity
  R. Kawano*, A. Schibel, C. Cauley, H. S. White
  Langmuir
  2009年11月, 研究論文（学術雑誌）, 共同, 25, 2, 1233–1237
• Fabrication of nanopores composed of beta-sheet peptides
  Kayamori, Fumihiro; Usui, Kenji; Kariya, Takuto; Hamada, Yoshio; Sato, Daisuke; Peng, Zugui; Kawamura, Izuru; Kawano, Ryuji
  JOURNAL OF PEPTIDE SCIENCE
  WILEY
  2024年08月, 研究論文（国際会議プロシーディングス）, 共同, 30, 1075-2617
• Membrane permeation of antimicrobial peptides through asymmetric lipopolysaccharide lipid bilayer
  Hashimoto, Wakana; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2024年02月08日, 研究論文（国際会議プロシーディングス）, 共同, 123, 3, 0006-3495, 508A, 508A
• Rapid synthesis of de novo β-barrel peptide nanopores using cell-free expression
  Fujita, Shoko; Kawamura, Izuru; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2024年02月08日, 研究論文（国際会議プロシーディングス）, 共同, 123, 3, 0006-3495, 163A, 163A
• Time series analysis for detecting peptide fragments using EOF-enhanced nanopore
  Yamaji, Misa; Kawano, Ryuji
  BIOPHYSICAL JOURNAL
  CELL PRESS
  2024年02月08日, 研究論文（国際会議プロシーディングス）, 共同, 123, 3, 0006-3495, 292A, 292A
• Controlled Self-Assembly of Vesicles by Electrospray Deposition
  Osaki, Toshihisa; Kamiya, Koki; Kawano, Ryuji; Kuribayashi-Shigetomi, Kaori; Takeuchi, Shoji
  SMALL STRUCTURES
  WILEY
  The self-assembly of amphiphilic molecules produces structures of diverse dimensions, encompassing micelles, tubules, lamellae, and vesicles. This study focuses on elucidating the controllability of amphiphilic lipid molecule self-assembly in size and uniformity to facilitate our understanding of the molecular characteristics that correlate with the functions and attributes of the assembled structures. Electrospray deposition allows micropatterning of lipid molecules in conjunction with a conductive-nonconductive patterned substrate. The solvent in the sprayed mist undergoes evaporation during flight, leading to the deposition of dry lipids exclusively on the conductive regions of the substrate. This process enables homogeneous lipid micropatterning, effectively circumventing the coffee-ring effect. Subsequent hydration of the lipid pattern triggers the spontaneous formation of a size-controlled, unilamellar vesicle array on the substrate, spanning an area of a few square millimeters. The vesicles exhibits monodispersity, with a coefficient of variation below 8% for sizes ranging from 5 to 20 mu m. The size-controlled self-assembly process is adaptable to various lipid compositions, thereby demonstrating that the molecular characteristics manifest in the morphological features appear as phase separation, budding, and curvature of vesicle membranes. The approach further validates its suitability for conducting time-resolved analyses of molecular transport and ligand binding on the monodispersed vesicle array. This study focuses on controlling the self-assembly of lipid molecules. Using electrospray deposition, lipids are patterned on a conductive-nonconductive substrate, enabling homogeneous micropatterning. Subsequent hydration generates size-controlled, monodispersed vesicles with various lipid mixtures, demonstrating the correlation between molecular characteristics and morphological features. The approach is suitable for time-resolved analyses of molecular transport and ligand binding on the vesicle array.image (c) 2024 WILEY-VCH GmbH
  2024年06月, 研究論文（学術雑誌）, 共同, 5, 6, DOI(公開)(r-map)
• Long-Term Stable Liposome Modified by PEG-Lipid in Natural Seawater
  Izumi, Kayano; Ji, Jiajue; Koiwai, Keiichiro; Kawano, Ryuji
  ACS OMEGA
  AMER CHEMICAL SOC
  This paper describes the stabilization of liposomes using a PEGylated lipid, N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (DSPE-PEGs), and the evaluation of the survival rate in natural seawater for future environmental applications. Liposomes in natural seawater were first monitored by confocal microscopy, and the stability was compared among different lengths and the introduction ratio of DSPE-PEGs. The survival rate increased with an increase in the PEG ratio. In addition, the survival rate in different cationic solutions (Na+, K+, Mg2(+), and Ca2(+) solutions) was studied to estimate the effects of the DSPE-PEG introduction. We propose that these variations in liposome stability are due to the cations, specifically the interaction between the poly(ethylene glycol) (PEG) chains and divalent ions, which contribute to making it difficult for cations to access the lipid membrane. Our studies provide insights into the use of PEG lipids and may offer a promising approach to the fabrication of liposomal molecular robots using different natural environments.
  2024年02月22日, 研究論文（学術雑誌）, 共同, 9, 9, 2470-1343, DOI(公開)(r-map), 10958, 10966
• Lipid vesicle-based molecular robots
  Peng, Zugui; Iwabuchi, Shoji; Izumi, Kayano; Takiguchi, Sotaro; Yamaji, Misa; Fujita, Shoko; Suzuki, Harune; Kambara, Fumika; Fukasawa, Genki; Cooney, Aileen; Di Michele, Lorenzo; Elani, Yuval; Matsuura, Tomoaki; Kawano, Ryuji
  LAB ON A CHIP
  ROYAL SOC CHEMISTRY
  A molecular robot, which is a system comprised of one or more molecular machines and computers, can execute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology. The core parts of molecular robots are fairly consistent from system to system and always include (i) a body to encapsulate molecular machines, (ii) sensors to capture signals, (iii) computers to make decisions, and (iv) actuators to perform tasks. This review aims to provide an overview of approaches and considerations to develop molecular robots. We first introduce the basic technologies required for constructing the core parts of molecular robots, describe the recent progress towards achieving higher functionality, and subsequently discuss the current challenges and outlook. We also highlight the applications of molecular robots in sensing biomarkers, signal communications with living cells, and conversion of energy. Although molecular robots are still in their infancy, they will unquestionably initiate massive change in biomedical and environmental technology in the not too distant future. A molecular robot, which is a system comprised of one or more molecular machines and computers, can execute sophisticated tasks in many fields that span from nanomedicine to green nanotechnology.
  2024年02月27日, 研究論文（学術雑誌）, 共同, 24, 5, 1473-0197, DOI(公開)(r-map), 996, 1029
• The potential of nanopore technologies toward empowering biophysical research: Brief history, basic principle and applications
  Yamazaki, Hirohito; Peng, Zugui; Kawano, Ryuji; Shoji, Kan
  BIOPHYSICS AND PHYSICOBIOLOGY
  BIOPHYSICAL SOC JAPAN
  2024年, 研究論文（学術雑誌）, 共同, 21, 1, DOI(公開)(r-map)

著書

• マイクロ流体デバイスを用いた細胞サイズリポソームの作製手法
  鈴木春音、髙木里菜、吉家爵、川野竜司
  公益社団法人 日本化学会 バイオテクノロジー部会
  2023年08月01日
• Cell-Penetrating Peptides: Design, Development and Applications
  Makoto Oba, Yosuke Demizu
  WILEY-VCH
  2022年12月21日
• Molecular Robotics: An Introduction
  2022年08月17日, 978-981-19-3986-0
• 疾患バイオマーカーとしてのマイクロRNAと診断応用
  2022年06月20日, 978-4-7813-1667-3
• Reference Module in Biomedical Sciences
  Elsevier
  2021年04月30日
• 分子でつくるロボットーリポソーム型分子ロボットの開発
  小祝敬一郎、和泉佳弥乃、川野竜司
  化学同人
  2021年03月01日
• DNA- and RNA-Based Computing Systems
  John Wiley & Sons
  2021年01月15日
• 人工細胞膜システムの構築：情報科学から医療応用まで
  竹内七海、和泉佳弥乃、庄司観、川野竜司
  日本膜学会
  2019年08月24日
• 分子ロボティクス概論～分子のデザインでシステムをつくる
  村田智（編）高井なつみ、多田あすか、竹内七海、川野竜司
  2. 8 ナノポアによる一分子計測と膜ゲートとしての可能性
  分子ロボティクス研究会
  2019年05月02日

研究発表、招待講演等

• 脂質膜にナノポアを形成するペプチドの設計とタンパクシーケンスへの応用
  第42回ケムステVシンポ
  2023年11月23日, 口頭発表（招待・特別）
• De nove design of peptide nanopore
  Black Forest Nanopore Meeting 2023
  2023年11月06日, 口頭発表（招待・特別）
• ナノポアを形成する人工膜輸送体の構築
  CBI学会2023年大会
  2023年10月25日, 口頭発表（招待・特別）
• ナノポアによるDNA演算の電気的デコーディング
  分子モーター討論会
  2023年09月27日, 口頭発表（招待・特別）
• Artificial cell-membranes: from construction to nanopore applications
  IPR & iCeMS Joint Seminar
  2023年08月28日, 口頭発表（招待・特別）
• ナノポアを⽤いたペプチド断⽚計測とプロテオームへの応⽤
  日本プロテオーム学会2023
  2023年07月26日, 口頭発表（招待・特別）
• ナノポアで一分子を「観る」
  第9回細胞生物若手の会シンポジウム
  2023年06月28日, 口頭発表（招待・特別）
• 細胞膜を人工的に造る：バイオセンシングから分子ロボットまで
  関東高分子若手研究会
  2023年06月24日, 口頭発表（招待・特別）
• 微細加工技術を基盤とした人工細胞膜システムの構築
  第89回酵素工学研究会
  2023年04月21日, 口頭発表（招待・特別）
• バイオナノポアを用いた核酸・ペプチド断片の単分子計測
  ATIバイオ単分子研究会
  2023年02月06日, 口頭発表（招待・特別）
• ナノポアリキッドバイオプシー：血中由来短鎖核酸の一分子計測
  第7回 Liquid Biopsy 研究会
  2023年01月27日, 口頭発表（招待・特別）
• De novo design of nanopores reconstituted in bilayer lipid membrane
  CEMS Topical Meeting Online
  2022年12月13日, 口頭発表（招待・特別）
• 人工細胞膜システムによる電気的膜透過評価
  第5回ナノバイオ交流会
  2022年12月09日, 口頭発表（招待・特別）
• ヘモリシントーク：ヘモリシンマニア2人が語る　ナノポア形成タンパク質「ヘモリシン」
  第6回分子ロボティクス年次大会
  2022年11月12日, 口頭発表（招待・特別）
• 分子演算システムによる複数miRNAのパターン認識
  第42回キャピラリー電気泳動シンポジウム
  2022年10月27日, 口頭発表（招待・特別）
• 人工細胞膜システムを創る
  細胞を創る研究会15.0
  2022年10月17日, 口頭発表（招待・特別）
• ⾼次機能性分⼦システム〜創る⽅法の解明に向けて〜
  第60回日本生物物理学会年会
  2022年09月28日, 口頭発表（招待・特別）
• De novo細胞膜分子システムのボトムアップ構築
  超越分子システム第二回領域会議
  2022年09月25日, 口頭発表（招待・特別）
• Artificial cell-membrane: Construction of peptide nanopores with de novo design
  IMRC 2022
  2022年08月17日, 口頭発表（招待・特別）
• Bottom-up construction of de novo designed nanopore using peptides
  Nano Korea2022
  2022年07月07日, 口頭発表（招待・特別）
• 人工細胞膜システムを用いた一分子計測
  CRESTチーム会議
  2022年06月10日, 口頭発表（招待・特別）
• De novo 設計ナノポアによる一分子計測と分子ロボット工学への応用
  第22回日本蛋白質科学会年会
  2022年06月07日, 口頭発表（招待・特別）
• 人工細胞膜と膜ペプチド
  第4回ナノバイオ交流会・サイエンスライブチケット
  2021年12月14日, 口頭発表（招待・特別）
• De novo細胞膜分子システムのボトムアップ構築
  学術変革領域A「超越分子システム」キックオフシンポジウム
  2021年12月04日, 口頭発表（招待・特別）
• De novo設計ペプチドによるナノポア構築と分子ロボットへの展開
  学術変革領域A「マルチファセット・プロテインズ」講演会
  2021年12月03日, 口頭発表（招待・特別）
• De novo design of nanopore using transmembrane peptides
  第59回 日本生物物理学会年会
  2021年11月25日, 口頭発表（招待・特別）
• 分析化学へアプローチする人工細胞膜研究
  生物工学フォーラム「最先端の分析化学アプローチからみる生物工学研究」
  2021年08月27日, 口頭発表（招待・特別）
• リポソーム型分子ロボットの開発
  日本生物物理学会サブグループ「人工細胞モデル＆分子ロボティクス」のキックオフミーティング
  2021年08月18日, 口頭発表（基調）
• ナノポアを用いた一分子計測法の開発（奨励賞受賞講演）
  化学とマイクロ・ナノシステム学会
  2021年05月17日, 口頭発表（基調）
• 平面膜システムによる膜ペプチド解析
  大阪大学蛋白質研究所セミナー
  2021年03月04日, 口頭発表（招待・特別）
• Nanopore decoding for DNA computing
  Nanopore Electrochemistry Meeting
  2020年10月11日, 口頭発表（基調）
• Analysis of transmembrane peptides using a lipid bilayer system
  第58回 生物物理学会年会
  2020年09月16日, 口頭発表（招待・特別）
• ボトムアップ配列設計ペプチドによるナノポアの構築
  第72回 日本生物工学会大会
  2020年09月02日, 口頭発表（招待・特別）
• Artificial Cell-membrane System Constructed by Microfluidic Technologies
  Microfluidics & Organ-on-a Chip Asia 2019
  2019年11月14日, 口頭発表（招待・特別）
• Artificial Cell Membrane System Integrated by Microfluidic Technologies
  The SPIRITS International Symposium -Shaping Self-Assembled Mesoscale (Bio)Materials with Microengineering-
  2019年03月28日, 口頭発表（招待・特別）

委員歴

• 日本学術振興会
  科学研究費委員会専門委員
  自 202308, 至 202403
• 日本学術振興会
  特別研究員等審査会専門委員
  自 20210719, 至 20230331
• 日本生物物理学会
  分野別専門委員
  自 20200401, 至 20220331
• 社団法人電気化学会
  代議員
  自 20200401, 至 202403
• 人工知能学会
  第2種研究会　計測インフォマティクス研究会　発起人
  自 20170401, 至 202403
• 日本分析化学会
  ナノ・マイクロ化学分析研究懇談会運営委員
  自 20170401, 至 202403
• 文部科学省科学技術政策研究所科学技術動向研究センター
  専門調査員
  自 20170401, 至 202403
• サイボウニクス研究会
  運営委員
  自 20140401, 至 202403

メディア報道

• アルツハイマー病の原因物質が毒性を示す過程の実時間観察に成功
  農工大らの共同研究チームは、アルツハイマー病の原因物質であるアミロイドβが人工細胞膜中で毒性を持つ構造に変化する様子をリアルタイムに観察することに成功したと、紹介される。
  MIT Technology Review
  自 2024年01月18日, 至 2024年01月18日
• ナノポアで迅速がん診断マイクロRNA増減 同時検出東京農工大など
  滝口さん・川野先生らの共同研究チームは、「ナノポア」を用いて口腔がんマーカーとなるマイクロRNAの発現上昇と減少を同時検出することに成功したと、紹介される。
  日刊工業新聞
  自 2023年09月18日, 至 2023年09月18日
• DNAを1分子レベルで抽出東京農工大などナノポア利用
  農工大・川野教授らが、生体の分子膜中の微細な孔「ナノポア」を使い、デオキシリボ核酸（DNA)を1分子レベルでカウント・抽出することに成功したと紹介される。
  日刊工業新聞
  自 2023年07月11日, 至 2023年07月11日
• 東京農工大、ペプチドで人工細胞変形　分子ロボットに
  東京農工大学大学院の川野竜司氏らの研究グループは、人工細胞（リポソーム）の変形を、細胞膜に結合するペプチドによって誘起することに成功したと紹介される。
  NIKKEI Tech Foresight／日経XTECH
  自 2023年03月02日, 至 2023年03月09日
• ペプチド1分子、検出容易に
  東京農工大学や横浜国立大学の研究チームは、人工合成したナノサイズの穴で1分子のペプチドを検出する技術を開発したと紹介され、川野竜司教授のコメントが掲載される。
  日経産業新聞
  自 2023年02月24日, 至 2023年02月24日
• 東京農工大、細胞膜に結合するペプチドによって人工細胞の変形を誘起することに成功
  国立大学法人東京農工大学大学院工学研究院生命機能科学部門の川野竜司教授と同大学卓越大学院生 和泉佳弥乃、齋藤千尋は、人工細胞（リポソーム、（注1））の変形を膜に結合するペプチドの二次構造によって誘起し、人工細胞を形状制御できる可能性を示唆したと紹介される。
  日本経済新聞
  自 2023年02月13日, 至 2023年02月13日
• ペプチド1分子、ナノサイズの穴で検出　東京農工大など
  東京農工大学や横浜国立大学の研究チームは、人工合成したナノサイズの穴で1分子のペプチドを検出する技術を開発したと紹介される。
  日本経済新聞
  自 2023年02月09日, 至 2023年02月09日
• Nanopore Technology Detects Cancer Biomarkers from Patients’ Blood
  “DNA computing uses the biochemical reactions of the information-encoding DNA molecules to solve problems based on formal logic, in the same way that normal computers do,” said Ryuji Kawano, PhD, professor, Tokyo University of Agriculture and Technology (TUAT). “In this case, a diagnostic DNA molecule was designed to be able to bind five different kinds of miRNA associated with bile duct cancer.
  Genetic Engineering & Biotechnology News／D１SoftballNews.com
  自 2022年06月28日, 至 2022年06月29日
• Detection of cancer biomarkers from blood samples using nanopore-based DNA computing technology
  Researchers at the Tokyo University of Agriculture and Technology have developed a new method for the detection of cancer miRNA patterns based on DNA computing technology.
  EurekAlert!
  自 2022年06月27日, 至 2022年06月27日
• 東京農工大、ナノポアを用いた血中がんマーカーのパターン識別に成功
  東京農工大学大学院工学研究院生命機能科学部門の川野竜司教授と同大学大学院工学府大学院生 竹内七海（卓越大学院生）、平谷萌恵（当時）は、DNA分子を用いて情報処理を行う「DNAコンピューティング技術」と「ナノポア」により血液中の複数のmicroRNA(血中がんマーカー)を同時検出し、胆管がんを識別することに成功したと紹介される。
  日本経済新聞／マイナビニュース
  自 2022年06月27日, 至 2022年06月28日
• De Novo-Designed Nanopore Used for the First Time to Detect DNA, Proteins
  東京農工大学の川野竜司教授らの研究グループが、人工設計したペプチドにより「ナノポア」を作製し、分子（DNA、タンパク質）の検出に成功したことが紹介される。
  AZONANO
  自 2021年11月25日, 至 2021年11月25日
• For the first time, DNA and proteins sensed by de novo-designed nanopore
  東京農工大学の川野竜司教授らの研究グループが、人工設計したペプチドにより「ナノポア」を作製し、分子（DNA、タンパク質）の検出に成功したことが紹介される。
  PHYS ORG
  自 2021年11月25日, 至 2021年11月25日
• 東京農工大、ナノポアを形成する新規ベータシートペプチドを設計しDNAおよびタンパク質1分子を検出することに成功
  東京農工大学の川野竜司教授らの研究グループが、人工設計したペプチドにより「ナノポア」を作製し、分子（DNA、タンパク質）の検出に成功したことが紹介される。
  日経バイオテク
  自 2021年11月24日, 至 2021年11月24日
• ナノポアを用いたDNAの一塩基変異位置の検出に成功
  EurekAlert!／PHYS ORG
  自 2020年08月18日, 至 2020年08月18日
• マイクロ流路を利用
  関西学院大、農工大などの研究グループ　多孔性材料生成メカニズムを解明
  科学新聞
  自 2020年08月07日, 至 2020年08月07日
• 関西学院大など、ＭＯＦの生成機構解明　合成条件検討に一役
  関西学院大学の田中大輔准教授らが、多孔性材料の「金属有機構造体（ＭＯＦ）」の生成メカニズムをマイクロ流路を利用して解明したことを紹介する記事で、東京農工大学との共同研究であることが紹介される。
  日刊工業新聞／WEB
  自 2020年08月06日, 至 2020年08月06日
• 世界初、DNAオリガミによる人工細胞微小カプセルの開発に成功
  東京工業大学、東北大学、東京農工大学、東京大学、京都大学の研究グループが、DNAオリガミで作製したDNAナノプレートによって細胞膜を模倣した、人工細胞の開発に世界で初めて成功したことが紹介される。
  大学ジャーナル
  自 2019年09月26日, 至 2019年09月26日
• 東京農工大　人工DNAをコンピューターに NextTech2030
  東京農工大学の川野竜司准教授らがDNAコンピューターを使い、体内の物質の増減から病気を判定する技術の開発に取り組んでいることが紹介される。
  日本経済新聞
  自 2019年06月14日, 至 2019年06月14日
• 東京農工大　人工DNAをコンピューターに NextTech2030
  東京農工大学の川野竜司准教授らがDNAコンピューターを使い、体内の物質の増減から病気を判定する技術の開発に取り組んでいることが紹介される。
  日経産業新聞
  自 2019年06月14日, 至 2019年06月14日
• EzineOne for the toad: Electrophysiological analysis
  "D- and L-amino acids are mirror images of each other, and most amino acids in nature have the L structure," explains TUAT's Ryuji Kawano. "A few proteins are modified to have D-amino acids. The role of having D-amino acids is not fully understood in the case of [this species]."
  spectroscopy now
  自 2019年05月15日, 至 2019年05月15日
• カエルの免疫 仕組み解明
  東京農工大学の川野竜司准教授らが、カエルが病原体などから体を守る免疫の仕組みを解明したことが紹介される。
  日経産業新聞
  自 2019年04月24日, 至 2019年04月24日
• 極細針使い化学物質検出東京農工大 細胞解析に応用へ
  東京農工大学の川野竜司准教授らが、極細針を使って化学物質を検出する技術を開発したことが紹介される。
  日経産業新聞
  自 2019年03月13日, 至 2019年03月13日
• 東京農工大、早期がん診断法開発　微量ＲＮＡで肺がん検出
  東京農工大学大学院工学研究院の川野竜司テニュアトラック特任准教授らが、がんから分泌される短いＲＮＡ（リボ核酸）「マイクロＲＮＡ」を活用した早期がん診断法を開発したことが紹介される。
  日刊工業新聞/WEB
  自 2017年11月03日, 至 2017年11月03日
• スポットライトリサーチ複数のイオン電流を示す人工イオンチャネルの開発
  川野竜司テニュアトラック特任准教授が、二つの電流値を持つ人工イオンチャネルの開発に成功したことが紹介される。
  Chem Station
  自 2017年06月12日, 至 2017年06月12日
• 生体内で働く分子ロボ実現に一歩-人工細胞内でDNAをコンピュータとして利用
  東京工業大学と東京農工大学は、DNA分子を用いて計算を行うDNAコンピューティングの計算結果である出力分子をナノポアと呼ばれるチャネル型の膜タンパク質により、電気情報として検出することに成功したことが紹介される。
  マイナビニュース/Livedoor News/ニコニコニュース/gooニュース/exciteニュース/グノシー
  自 2017年05月17日, 至 2017年05月17日
• 東京農工大、流路デバイスを用いて抗がん剤の効果と副作用の同時評価システムを開発～副作用発症の予測や細胞間相互作用の解明への利用に期待～
  農工大 川野竜司教授、臼井達哉准教、大松勉准教授らは、がんと正常のオルガノイドを搭載したマイクロ流路デバイスを用いて抗がん剤の効果と副作用を同時に評価可能なシステムを確立することに成功した、と掲載される。
  日経バイオテク
  自 2025年01月14日, 至 2025年01月14日

受賞

• 化学とマイクロ・ナノシステム学会
  化学とマイクロ・ナノシステム学会奨励賞
  化学とマイクロ・ナノシステムに関連する研究において顕著な業績をあげた、選考年度の4月1日現在において満45才未満の本会正会員が対象です。
  2021年03月07日