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Micro and nano electroporation for direct delivery of molecules to cell nucleus

Research Project

Project/Area Number 26289035
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypePartial Multi-year Fund
Section一般
Research Field Fluid engineering
Research InstitutionInstitute of Physical and Chemical Research (2018)
Kyoto University (2014-2017)

Principal Investigator

Shintaku Hirofumi  国立研究開発法人理化学研究所, 開拓研究本部, 理研白眉研究チームリーダー (80448050)

Co-Investigator(Kenkyū-buntansha) 小寺 秀俊  京都大学, 工学研究科, 教授 (20252471)
Project Period (FY) 2014-04-01 – 2019-03-31
Project Status Completed (Fiscal Year 2018)
Budget Amount *help
¥16,380,000 (Direct Cost: ¥12,600,000、Indirect Cost: ¥3,780,000)
Fiscal Year 2018: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2017: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2016: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2015: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2014: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
KeywordsRNA / DNA / 電気泳動 / マイクロ流体システム / シーケンシング / 1細胞 / 電気穿孔 / マイクロ流路 / 動電現象 / 生体高分子 / 細胞 / 一細胞 / マイクロ流体 / 物質輸送
Outline of Final Research Achievements

We developed a microfluidic system that offered a highly reproducible single-cell isolation and electroporation (97%, n>120). We showed that the hydrodynamic trap effectively captured a single cell and focused the electric field for electroporation to reduce the applied voltage by ten-fold in comparison to a uniform electric field. We also developed an equation that estimated the magnitude of the electric field exerted on the nuclear membrane as a function of the distance between the hydrodynamic trap and the nuclear membrane. Furthermore, we applied our microfluidic system for integrated nuclear and cytoplasmic RNA-sequencing of single cells and coined the method SINC-seq.

Academic Significance and Societal Importance of the Research Achievements

本研究で開発したマイクロ流体システムは集中電場を活用して選択的に細胞膜を穿孔し,細胞内外の物質輸送を制御することを実現した.これを活用して,再現性の高い電気穿孔法を確立した.さらに,世界で初めて1細胞の細胞質RNAと核RNAを独立かつ並列でシーケンス解析するSINC-seq法(1細胞RNA分画解読法)を確立した.

Report

(6 results)
  • 2018 Annual Research Report   Final Research Report ( PDF )
  • 2017 Annual Research Report
  • 2016 Annual Research Report
  • 2015 Annual Research Report
  • 2014 Annual Research Report

Research Products

(47 results)

All 2019 2018 2017 2016 2015 2014 Other

All Int'l Joint Research (5 results) Journal Article (11 results) (of which Int'l Joint Research: 5 results,  Peer Reviewed: 9 results,  Open Access: 6 results,  Acknowledgement Compliant: 3 results) Presentation (27 results) (of which Int'l Joint Research: 13 results,  Invited: 15 results) Book (1 results) Remarks (2 results) Patent(Industrial Property Rights) (1 results)

  • [Int'l Joint Research] Stanford 大学/NIST/San Jose State University(米国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] TriLink/University of Minnesota(米国)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] The University of Jordan(ヨルダン)

    • Related Report
      2018 Annual Research Report
  • [Int'l Joint Research] Stanford University(米国)

    • Related Report
      2017 Annual Research Report
  • [Int'l Joint Research] Stanford University(米国)

    • Related Report
      2015 Annual Research Report
  • [Journal Article] SINC-seq: Dissecting Nuclear and Cytoplasmic RNA of Single Cells2019

    • Author(s)
      OGUCHI Yusuke、ABDELMOEZ Mahmoud Nady、SHINTAKU Hirofumi
    • Journal Title

      Biophysics

      Volume: 59 Issue: 2 Pages: 088-090

    • DOI

      10.2142/biophys.59.088

    • NAID

      130007620379

    • ISSN
      0582-4052, 1347-4219
    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] SINC-seq: correlation of transient gene expressions between nucleus and cytoplasm reflects single-cell physiology2018

    • Author(s)
      Abdelmoez Mahmoud N.、Iida Kei、Oguchi Yusuke、Nishikii Hidekazu、Yokokawa Ryuji、Kotera Hidetoshi、Uemura Sotaro、Santiago Juan G.、Shintaku Hirofumi
    • Journal Title

      Genome Biology

      Volume: 19

    • DOI

      10.1186/s13059-018-1446-9

    • NAID

      120007133998

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters2018

    • Author(s)
      Khnouf Ruba、Shore Sabrina、Han Crystal M.、Henderson Jordana M.、Munro Sarah A.、McCaffrey Anton P.、Shintaku Hirofumi、Santiago Juan G.
    • Journal Title

      Analytical Chemistry

      Volume: 90 Pages: 12609-12615

    • DOI

      10.1021/acs.analchem.8b02773

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate)2018

    • Author(s)
      Subramanian Parimalam Sangamithirai、Oguchi Yusuke、Abdelmoez Mahmoud N.、Tsuchida Arata、Ozaki Yuka、Yokokawa Ryuji、Kotera Hidetoshi、Shintaku Hirofumi
    • Journal Title

      Analytical Chemistry

      Volume: 90 Pages: 12512-12518

    • DOI

      10.1021/acs.analchem.8b02338

    • Related Report
      2018 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Correlation of gene expressions between nucleus and cytoplasm reflects single-cell physiology2017

    • Author(s)
      Abdelmoez Mahmoud Nady、Iida Kei、Oguchi Yusuke、Nishikii Hidekazu、Yokokawa Ryuji、Kotera Hidetoshi、Uemura Sotaro、Santiago Juan Gabriel、Shintaku Hirofumi
    • Journal Title

      bioRxiv

      Volume: NA

    • DOI

      10.1101/206672

    • Related Report
      2017 Annual Research Report
    • Open Access / Int'l Joint Research
  • [Journal Article] Protocol for microfluidic system to automate the preparation and fractionation of the nucleic acids in the cytoplasm versus nuclei of single cells2016

    • Author(s)
      Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
    • Journal Title

      Bio-protocol

      Volume: 6(12)

    • DOI

      10.21769/bioprotoc.1844

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Protocol for microfluidic system to automate the preparation and fractionation of the nucleic acids in the cytoplasm versus nuclei of single cells2016

    • Author(s)
      Kentaro Kuriyama*, Hirofumi Shintaku*, and Juan G. Santiago [*: Equal contributors]
    • Journal Title

      Bio-protocol

      Volume: -

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
  • [Journal Article] Linearisation of λDNA Molecules by Instantaneous Variation of the Trapping Electrode Voltage Inside a Micro-Channel2015

    • Author(s)
      Itsuo HANASAKI, Naoya YUKIMOTO, Satoshi UEHARA, Hirofumi SHINTAKU, and Satoyuki KAWANO
    • Journal Title

      Journal of Physics D: Applied Physics

      Volume: 48 Pages: 135402-135402

    • DOI

      10.1088/0022-3727/48/13/135402

    • Related Report
      2015 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Isotachophoresis for Fractionation and Recovery of Cytoplasmic RNA and Nucleus from Single Cells2015

    • Author(s)
      Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
    • Journal Title

      Electrophoresis

      Volume: -

    • DOI

      10.1002/elps.201500040

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Extraction and Fractionation of RNA and DNA from Single Cells Using Selective Lysing and Isotachophoresis2015

    • Author(s)
      Hirofumi Shintaku and Juan G. Santiago
    • Journal Title

      Proc. of SPIE

      Volume: 9320

    • DOI

      10.1117/12.2084981

    • Related Report
      2014 Annual Research Report
  • [Journal Article] Increasing Hybridization Rate and Sensitivity of Bead Based Assays Using Isotachophoresis2014

    • Author(s)
      Hirofumi Shintaku, James W. Palko, Glenn M. Sanders, and Juan G. Santiago
    • Journal Title

      Angewandte Chemie International Edition

      Volume: 53 Pages: 13813-13816

    • DOI

      10.1002/anie.201408403

    • NAID

      120005647508

    • Related Report
      2014 Annual Research Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Presentation] RNA extraction from single cells via focused electric field at a hydrodynamic trap in a microfluidic channel2018

    • Author(s)
      Mahmoud N. Abdelmoez, Ryuji Yokokawa, Hidetoshi Kotera, Hirofumi Shintaku
    • Organizer
      the EMBS Micro and Nanotechnology in Medicine Conference
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Single-Cell Small RNA-Seq by Microfluidic Chip Isotachophoresis with sRNA Library preparation using Chemically Modified Adapters2018

    • Author(s)
      L. Dick Keys, Sabrina Shore, Ruba Khnouf, Crystal M. Han, Jordana M. Henderson, Richard I. Hogrefe, Anton P. McCaffrey, Sarah A. Munro, Hirofumi Shintaku, Juan G. Santiago
    • Organizer
      Next Generation Dx Summit
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Dissecting correlation between cytoplasmic and nuclear gene expressions of single cells by microfluidic fractionation2018

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      the EMBS Micro and Nanotechnology in Medicine Conference
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] RNA Extraction from Single Cells Using Electrical Lysis and Electrophoresis2018

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      International Conference on Complex Fluids and Soft Matter
    • Related Report
      2018 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] SINC-seq:電場を用いた一細胞分画RNA-sequencing法2018

    • Author(s)
      新宅博文
    • Organizer
      新化学技術推進協会 電子情報技術 マイクロナノシステムと材料・加工分科会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] マイクロ流路における電場誘起流れを用いた核酸操作と1細胞解析2018

    • Author(s)
      新宅博文
    • Organizer
      バイオナノミクス基盤技術研究会
    • Related Report
      2018 Annual Research Report
    • Invited
  • [Presentation] Isotachophoresis-based RNA extraction from fixed single cells2018

    • Author(s)
      Sangamithirai Subramanian Parimalam, Yusuke Oguchi, Mahmoud N. Abdelmoez, Arata Tsuchida, Yuka Ozaki, Ryuji Yokokawa, Hidetoshi Kotera, Hirofumi Shintaku,
    • Organizer
      日本機械学会第9回マイクロ・ナノ工学シンポジウム
    • Related Report
      2018 Annual Research Report
  • [Presentation] Numerical analyses on single-cell electroporation and RNA extraction under focused electric field2018

    • Author(s)
      Mahmoud N. Abdelmoez, Ryuji Yokokawa, Hidetoshi Kotera, and Hirofumi Shintaku
    • Organizer
      日本機械学会 2018年度年次大会
    • Related Report
      2018 Annual Research Report
  • [Presentation] Integrated nuclear and cytoplasmic RNA sequencing of single cells2017

    • Author(s)
      Mahmoud Nady Abdel-Moez Atta, Kei Iida, Yusuke Oguchi, Sotaro Uemura, Juan G. Santiago, and Hirofumi Shintaku
    • Organizer
      Advances in Genome Biology and Technology (AGBT) General Meeting 2017
    • Place of Presentation
      Hollywood, FL
    • Year and Date
      2017-02-13
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research
  • [Presentation] On-chip Electric Fractionation of Cellular Components for Sequencing at Subcellular Resolution2017

    • Author(s)
      Mahmoud Nady Abdel-Moez Atta, Kei Iida, Yusuke Oguchi, Sotaro Uemura, Juan G. Santiago, and Hirofumi Shintaku
    • Organizer
      2017 Microfluidics, Physics and Chemistry of GRC
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Electrokinetic microfluidic chip and chemically modified adapters streamline single cell next-generation sequencing of small non-coding RNA. Poster presented at: RNA-Seq2017

    • Author(s)
      Shore S, Khnouf R, Han CM, Henderson JM, Hogrefe RI, McCaffrey AP, Munro SA, Shintaku H, Santiago JG
    • Organizer
      Single Cell Analysis & Single Molecule Analysis 2017
    • Related Report
      2017 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Electrical fractionation of cytoplasmic and nuclear nucleic acids of single cells2017

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      Fundamentals and Applications of Microfluidic Compartmentalization
    • Related Report
      2017 Annual Research Report 2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 一細胞核内核酸と細胞質核酸の並列シークエンス解析2017

    • Author(s)
      新宅博文
    • Organizer
      生命科学系学会合同年次大会
    • Related Report
      2017 Annual Research Report
    • Invited
  • [Presentation] A microfluidic system for integrated analyses on nuclear and cytoplasmic nucleic acids of single cells2017

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      The 12th Annual IEEE International Conference on Nano/Micro Engineered and Molecular Sytems, IEEE-NEMS 2017
    • Place of Presentation
      Los Angeles, CA
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] Massively parallel quantification of miRNA in single cells via duplex-specific nuclease reaction in pico-liter wells2016

    • Author(s)
      Sangamithirai Subramanian Parimalam, Ruji Yokokawa, Hidetoshi Kotera and Hirofumi Shintaku
    • Organizer
      2016 International conference of Microfluidics, Nanofluidics, and Lab-on-a-chip
    • Place of Presentation
      Dalian, China
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Electrical Fractionation of Cytoplasmic RNA And Nucleus From Single Cells For Parallel RNA and DNA Analyses2016

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      2nd Annual Next Generation Sequencing & Single Cell Analysis USA Congress
    • Place of Presentation
      Boston, MA
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] On-Chip Electrical Lysis and Extraction of Cytoplasmic RNA and Genomic DNA from Single Cells2015

    • Author(s)
      Hirofumi Shintaku, Kentaro Kuriyama, and Juan G. Santiago
    • Organizer
      28th International Microprocesses and Nanotechnology Conference
    • Place of Presentation
      富山
    • Year and Date
      2015-11-10
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 電気穿孔および等速電気泳動を用いた一細胞前処理技術におけるRNA純度計測2015

    • Author(s)
      畑 翔太,横川 隆司,小寺 秀俊,新宅 博文
    • Organizer
      日本機械学会2015年度年次大会
    • Place of Presentation
      札幌
    • Year and Date
      2015-09-13
    • Related Report
      2015 Annual Research Report
  • [Presentation] On-Chip Preparation System for Simultaneous Cytoplasmic RNA and Genomic DNA Analyses of Single Cells2015

    • Author(s)
      Hirofumi Shintaku and Juan G. Santiago
    • Organizer
      the 3rd Annual Single Cell Analysis Investigators Meeting
    • Place of Presentation
      Bethesda, MD, USA
    • Year and Date
      2015-04-20
    • Related Report
      2015 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Microfluidic System for Simultaneous Analyses of RNA and DNA in Single Cells2015

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      Berkeley Nanosciences and Nanoengineering Institute Seminar
    • Place of Presentation
      Berkeley, CA, USA
    • Year and Date
      2015-02-27
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Extraction and Fractionation of RNA and DNA from Single Cells Using Selective Lysing and Isotachophoresis2015

    • Author(s)
      Hirofumi Shintaku and Juan G. Santiago
    • Organizer
      SPIE Photonics West BiOS
    • Place of Presentation
      San Francisco, CA, USA
    • Year and Date
      2015-02-07 – 2015-02-12
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Microfluidic System for Correlation Analyses of RNA and DNA in Single Cells2014

    • Author(s)
      Hirofumi Shintaku, Kentaro Kuriyama, and Juan G. Santiago
    • Organizer
      Cold Spring Harbor Asia Conferences, Single Cell
    • Place of Presentation
      Suzhou, China
    • Year and Date
      2014-12-08 – 2014-12-12
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Coupling Isotachophoresis with Bead-Based Assay for Rapid and Multiplexed Nucleic Acids Detection2014

    • Author(s)
      Hirofumi Shintaku, James W. Palko, Glenn M. Sanders, and Juan G. Santiago,
    • Organizer
      Lab-on-a-Chip Asia - Microfluidics and Point Of Care Diagnostics
    • Place of Presentation
      Singapore
    • Year and Date
      2014-11-20 – 2014-11-21
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] 基板上に固定した液滴アレイの電気的結合と分裂2014

    • Author(s)
      岡 洸佑,横川 隆司,小寺 秀俊,新宅 博文
    • Organizer
      日本機械学会第6回マイク ロ・ナノ工学シンポジウム
    • Place of Presentation
      松江
    • Year and Date
      2014-10-20
    • Related Report
      2014 Annual Research Report
  • [Presentation] Sample Preparation for Simultaneous Analysis of RNA and DNA from Single Cells Using Electrophoretic Techniques2014

    • Author(s)
      Hirofumi Shintaku
    • Organizer
      2nd Annual Single Cell Genomics & Transcriptomics Asia Congress 2014
    • Place of Presentation
      Singapore
    • Year and Date
      2014-10-07 – 2014-10-08
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] Development of Microfluidic System for Isolation and Analyses of RNA and DNA from Single Cells2014

    • Author(s)
      Kentaro Kuriyama, Hirofumi Shintaku, and Juan G. Santiago
    • Organizer
      2nd Annual Single Cell Genomics & Transcriptomics Asia Congress 2014
    • Place of Presentation
      Singapore
    • Year and Date
      2014-10-07 – 2014-10-08
    • Related Report
      2014 Annual Research Report
  • [Presentation] Correlating DNA and RNA Amounts in Single Cells Using Selective Lysing and Isotachophoresis2014

    • Author(s)
      Hirofumi Shintaku, Kentaro Kuriyama, Hidekazu Nishikii, Lewis A. Marshall, Hidetoshi Kotera, and Juan G. Santiago
    • Organizer
      SCIX 2014
    • Place of Presentation
      Reno, Nevada, USA
    • Year and Date
      2014-10-01
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Book] 化学とマイクロ・ナノシステム学会 会誌 1細胞解析とマイクロフルイディックスの今後,Vol. 18, No.1 (2019)2018

    • Author(s)
      新宅博文
    • Total Pages
      2
    • Publisher
      化学とマイクロ・ナノシステム学会
    • Related Report
      2018 Annual Research Report
  • [Remarks] RIKEN Microfluidics Laboratory

    • URL

      https://www.hshintaku.com/

    • Related Report
      2018 Annual Research Report
  • [Remarks] Kyoto University Microfluidics Group

    • URL

      https://sites.google.com/site/kyotouniversitymicrofluidics/

    • Related Report
      2015 Annual Research Report
  • [Patent(Industrial Property Rights)] 核酸分離用チップ、それを用いた核酸分離方法、および核酸分析方法2015

    • Inventor(s)
      新宅博文,藁谷卓哉,上村想太郎,小口祐伴
    • Industrial Property Rights Holder
      京都大学
    • Industrial Property Rights Type
      特許
    • Filing Date
      2015
    • Acquisition Date
      2018
    • Related Report
      2018 Annual Research Report

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Published: 2014-04-04   Modified: 2022-02-21  

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