IKEBUKURO Kazunori

Activity information

Name and contact details

• Name

  イケブクロ　カズノリ, 池袋　一典, IKEBUKURO Kazunori

• E-mail

  ikebucc.tuat.ac.jp

Affiliation / Position

• Institute of Engineering　Division of Biotechnology and Life Science, Professor

Other affiliation

• Faculty of Engineering　Department of Biotechnology and Life Science
• Graduate School of Engineering　Department of Biotechnology and Life Sciences
• Institute of Global Innovation Research
• Graduate School of Bio-Applications and Systems Engineering

Research History

• -
  東京農工大学大学院工学研究院
  From 01 Apr. 2009
• -
  東京農工大学工学部　助教授
  From Apr. 2001
• -
  東京大学先端科学技術研究センター　講師
  From Apr. 1996, To Mar. 2001
• -
  東京大学先端科学技術研究センター　助手
  From Apr. 1993, To Mar. 1996

Education

• The University of Tokyo
  Faculty of Engineering
  工業化学科
  To 1989, graduated
• The University of Tokyo
  Graduate School, Division of Engineering
  合成化学専攻
  To 1992, completed, master course
• The University of Tokyo
  Graduate School, Division of Engineering
  合成化学専攻
  To 1993, withdrawn before completion, doctor course

Current state of research and teaching activities

• 私たちの身体の中では、遺伝情報を担うDNAと生理機能を担う蛋白質がお互いに作用して、生命現象を維持しています。私たちはDNAと蛋白質の相互作用を応用して、様々な病気の診断技術の開発を目的に研究を行っています。蛋白質を認識する核酸分子“アプタマー” を使って、癌やアルツハイマー病、感染症を診断する新規バイオセンサーの開発を進めています。また、核酸を認識する蛋白質“ジンクフィンガー” を使って、病原性微生物を検出するシステムの開発を進めています。

Research Areas

• Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering), Biofunction and bioprocess engineering

Research Interests

• Biosensor, aptamer, Evolutionary molecular engineering, DNA/SNP sensor

Subject of research

• Development of novel DNA/SNP sensor
  From 1996
• Development of novel DNA recognition device by the method mimicing evolution
  From 1997
• Development of novel DNA aptamer by the method mimicing evolution.
  From 1998
• Peptide ligand screening by in silico panning
  From 200104
• Development of novel molecular sensing method using the aptamers
  From 2002

Proposed theme of joint or funded research

• Development of novel DNA aptamer by the method uninchigecolution
  wish to undertake joint research with industry and other organizations including private sector.

Grants-in-Aid for Scientific Research

• 挑戦的研究（萌芽）
  DNA中の疎水場を利用したナノプラスチック検出センサの開発
  From 2024, To 2024
• 挑戦的研究（萌芽）
  DNAの構造安定性の変化を利用したCpGメチル化迅速検出法の開発
  From 2024, To 2025
• 基盤研究（C）
  疾患関連抗体に対する抗イディオタイプDNAアプタマーのファスト＆ファインデザイン
  From 2023, To 2023
• 挑戦的研究（萌芽）
  DNA中の疎水場を利用したナノプラスチック検出センサの開発
  From 2023, To 2023
• 基盤研究(B)
  酵素・抗体・アプタマーの近接を利用した単一工程電気化学センサーの開発
  From 2023, To 2025
• 基盤研究(C)
  疾患関連抗体に対する抗イディオタイプDNAアプタマーのファスト＆ファインデザイン
  From 2022, To 2022
• 挑戦的研究（萌芽）
  細胞内の標的蛋白質を可視化するturn-on型蛍光RNAアプタマーの開発
  From 2021, To 2022
• 基盤研究(B)
  抗体特異的な核酸アプタマーの創出とバイオ・プロセス・クリニカルアナリシスへの活用
  From 2021, To 2021
• 基盤研究(B)
  抗体特異的な核酸アプタマーの創出とバイオ・プロセス・クリニカルアナリシスへの活用
  From 2020, To 2020
• 基盤研究(C)
  分子内/分子間Gカルテット構造形成による免疫活性化CpG ODNの機能制御
  From 2020, To 2020
• 基盤研究(B)
  光架橋性グアニン四重鎖リガンドを用いたメチローム構成蛋白質解析技術の開発
  From 2019, To 2021
• 挑戦的研究（萌芽）
  気相中で標的分子を特異的に認識するDNAアプタマーの開発
  From 2019, To 2020
• 基盤研究(B)
  抗体特異的な核酸アプタマーの創出とバイオ・プロセス・クリニカルアナリシスへの活用
  From 2019, To 2019
• 基盤研究(C)
  分子内/分子間Gカルテット構造形成による免疫活性化CpG ODNの機能制御
  From 2019, To 2019
• 基盤研究(C)
  分子内/分子間Gカルテット構造形成による免疫活性化CpG ODNの機能制御
  From 2018, To 2018
• 基盤研究(C)
  眼内VEGF濃度の即時測定系開発と臨床応用?
  From 2017, To 2017
• 基盤研究（C）
  眼内VEGF濃度の即時測定系開発と臨床応用
  From 2016, To 2016
• 基盤研究（A）
  ナノニードルを用いた核輸送による高効率ゲノム編集
  From 2016, To 2016
• 挑戦的萌芽研究
  細胞内での異常な核酸分子挙動の解析
  From 2015, To 2015
• 基盤研究（C）
  眼内VEGF濃度の即時測定系開発と臨床応用
  From 2015, To 2015
• 基盤研究（A）
  ナノニードルを用いた核輸送による高効率ゲノム編集
  From 2015, To 2015
• 挑戦的萌芽研究
  細胞内での異常な核酸分子挙動の解析
  From 2014, To 2014
• 基盤研究(A)
  ナノニードルを用いた核輸送による高効率ゲノム編集
  From 2014, To 2014
• 挑戦的萌芽研究
  DNA折り紙を応用した新しい抗ウイルス薬の開発
  From 2013, To 2013
• 基盤研究(B)
  アプタマーセットを用いた血液中極微量疾病マーカーの多数同時検出法の開発
  From 2012, To 2014
• 挑戦的萌芽研究
  電気化学的グルコースセンサーとアプタマーのナノ構造変化を利用した検出法の開発
  From 2012, To 2013
• 挑戦的萌芽研究
  Ｚｎフィンガー蛋白質を用いたメチル化頻度解析法の開発
  From 2010, To 2011
• 基盤研究(B)
  ピロロキノリンキノンによるアミロイド蛋白質の選択的構造形成制御
  From 2010, To 2010
• 基盤研究(B)
  ピロロキノリンキノンによるアミロイド蛋白質の選択的構造形成制御
  From 2009, To 2009
• 萌芽研究
  磁場による誘導加熱を利用した金ナノ粒子修飾アプタマーの構造・機能制御
  From 2008, To 2009
• 基盤研究(B)
  ピロロキノリンキノンによるアミロイド蛋白質の選択的構造形成制御
  From 2008, To 2008
• 基盤研究(C)一般
  複数の疾病マーカー分子を特異的に認識するアプタマーの同時探索法の開発　　　　　　
  From 2006, To 2007
• 基盤研究(C)一般
  複数の疾病マーカー分子を特異的に認識するアプタマーの同時探索法の開発　　　　　　
  From 2006, To 2007
• 若手研究(B)
  コンピュータ内進化による疾病原因酵素阻害ＤＮＡアプタマ－の検索
  From 2003, To 2004
• 若手研究(B)
  コンピューター内進化による疾病原因酵素阻害DNAアプタマーの探索
  From 2003, To 2004
• 基盤研究(C)一般
  有害微生物のDNA検出のための新規ジンクフィンガー蛋白質の創出
  From 2001, To 2002
• その他
  超機能バイオセンサーの開発
  From 1998, To 1999

Papers

• Proximity-Unlocked Luminescence by Sequential Enzymatic Reactions from Antibody and Antibody/Aptamer (PULSERAA): A Platform for Detection and Visualization of Virus-Containing Spots
  Miura, Daimei; Hayashi, Wakana; Hirano, Kensuke; Sasaki, Ikkei; Tsukakoshi, Kaori; Kakizoe, Hidehumi; Asai, Satomi; Vavricka, Christopher J.; Takemae, Hitoshi; Mizutani, Tetsuya; Tsugawa, Wakako; Sode, Koji; Ikebukuro, Kazunori; Asano, Ryutaro
  ADVANCED SCIENCE
  WILEY
  The SARS-CoV-2 pandemic has challenged more scientists to detect viruses and to visualize virus-containing spots for diagnosis and infection control; however, detection principles of commercially available technologies are not optimal for visualization. Here, a convenient and universal homogeneous detection platform named proximity-unlocked luminescence by sequential enzymatic reactions from antibody and antibody/aptamer (PULSERAA) is developed. This is designed so that the signal appears only when the donor and acceptor are in proximity on the viral surface. PULSERAA specifically detected in the range of 25-500 digital copies/mL of inactivated SARS-CoV-2 after simply mixing reagents; it is elucidated that the accumulation of chemical species in a limited space of the viral surface contributed to such high sensitivity. PULSERAA was quickly adapated to detect another virus variant, inactivated influenza A virus, and infectious SARS-CoV-2 in a clinical sample. Furthermore, on-site (direct, rapid, and portable) visualization of the inactivated SARS-CoV-2-containing spots by a conventional smartphone camera was achieved, demonstrating that PULSERAA can be a practical tool for preventing the next pandemic in the future. A homogeneous immunoassay named proximity-unlocked luminescence by sequential enzymatic reactions from antibody and antibody/aptamer (PULSERAA) is developed, which enabled not only rapid virus detection in 15 min with high sensitivity and specificity but also the on-site visualization of the virus-containing spots on a surface through a smartphone camera after spraying reagents. image
  Nov. 2024, Research paper (scientific journal), joint, 11, 43, DOI(公開)(r-map)
• A Versatile Method to Create Antibody/Split-Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System
  Tobita, Yuka; Hirano, Kensuke; Miura, Daimei; Hatano, Yuma; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji; Asano, Ryutaro
  ADVANCED SENSOR RESEARCH
  WILEY
  07 Oct. 2024, Research paper (scientific journal), joint, 4, 1, 2751-1219, DOI(公開)(r-map)
• Development of DNA aptamers universally bound to single-chain fragment variables and their applications in bioprocess monitoring
  Hamasaki, Mai; Takamatsu, Shouhei; Nagata, Madoka; Wilson, Ellie; Suzuki, Hirobumi; Tanaka, Ayumi; Ikebukuro, Kazunori; Sode, Koji; Asano, Ryutaro
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with K D = 2.1 mu M or K D = 1.6 mu M toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and antihemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10 -500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real -time and versatile monitoring of large-scale scFv production.
  01 Oct. 2024, Research paper (scientific journal), joint, 261, 0956-5663, DOI(公開)(r-map)
• Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology
  Yamada, Tomohiro; Tsukakoshi, Kaori; Furusho, Aogu; Sugiyama, Eiji; Mizuno, Hajime; Hayashi, Hideki; Yamano, Takeshi; Kumobayashi, Hideki; Hasebe, Takashi; Ikebukuro, Kazunori; Toyo'oka, Toshimasa; Todoroki, Kenichiro
  TALANTA
  ELSEVIER
  We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentrationdependent, falling within the range of 50-2000 mu g/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within +/- 24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.
  01 Sep. 2024, Research paper (scientific journal), joint, 277, 0039-9140, DOI(公開)(r-map)
• Potential of Enzymatically Synthesized Hemozoin Analog as Th1 Cell Adjuvant
  Hoshi, Kazuaki; Tu, Anh Thi Tram; Shobo, Miwako; Kettisen, Karin; Ye, Lei; Buelow, Leif; Hakamata, Yoji; Furuya, Tetsuya; Asano, Ryutaro; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji; Yamazaki, Tomohiko
  NANOMATERIALS
  MDPI
  Hemozoin (Hz) is a heme crystal produced during malaria infection that stimulates immune cells, leading to the production of cytokines and chemokines. The immunostimulatory action of Hz has previously been applied in the development of alternative adjuvants. Crystallization of hemin is a chemical approach for producing Hz. Here, we focused on an enzymatic production method for Hz using the heme detoxification protein (HDP), which catalyzes heme dimer formation from hemin in Plasmodium. We examined the immunostimulatory effects of an enzymatically synthesized analog of Hz (esHz) produced by recombinant Plasmodium falciparum HDP. Enzymatically synthesized Hz stimulates a macrophage cell line and human peripheral mononuclear cells, leading to the production of interleukin (IL)-6 and IL-12p40. In mice, subcutaneous administration of esHz together with an antigen, ovalbumin (OVA), increased the OVA-specific immunoglobulin (Ig) G2c isotype level in the serum, whereas OVA-specific IgG1 was not induced. Our findings suggest that esHz is a useful Th-1 cell adjuvant.
  Sep. 2024, Research paper (scientific journal), joint, 14, 17, DOI(公開)(r-map)
• The 2.5th generation enzymatic sensors based on the construction of quasi-direct electron transfer type NAD(P)-Dependent dehydrogenases
  Ikegai, Kurea; Okuda-Shimazaki, Junko; Tran, Truc Thanh; Hatada, Mika; Asano, Ryutaro; Ikebukuro, Kazunori; Tsugawa, Wakako; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  We introduce a versatile method to convert NAD+ or NADP+ -dependent dehydrogenases into quasi-direct electron transfer (quasi-DET)-type dehydrogenases, by modifying with a mediator on the enzyme surface toward the development of 2.5th generation enzymatic sensors. In this study, we use beta-hydroxybutyrate (BHB) dehydrogenase (BHBDh) from Alcaligenes faecalis (AfBHBDh) as a representative NAD+ or NADP+ -dependent dehydrogenase. BHBDhs are important in ketone monitoring, especially for the diagnosis of diabetic ketoacidosis. We modified AfBHBDh with a thiol-reactive phenazine ethosulfate (trPES). We designed, constructed, and modified mutant BHBDhs harboring cysteine residues within 20 & Aring; from the C4 nicotinamide in NAD+/NADH. Mutants Ser65Cys, Thr96Cys, and Lys106Cys showed indistinguishable catalytic activities from the wild-type enzyme, even after trPES modification. These trPES-modified mutants were immobilized on gold disk electrodes via amine coupling with succinimide-groups of dithiobis (succinimidyl hexanoate) self-assembled monolayers for electrochemical measurements. Considering there is a wide range of BHB concentrations, we exploited the linear regression in log scales. The linear range for the sensors with trPES-modified BHBDh mutants Ser65Cys, Thr96Cys, and Lys106Cys were 0.1-4.0 mM in both buffer solution and artificial interstitial fluid (ISF). They have limits of detection of 0.047 mM for Ser65Cys, 0.15 mM for Thr96Cys, and 0.060 mM for Lys106Cys in buffer solution, and 0.12 mM, 0.089 mM, and 0.044 mM in artificial ISF, respectively. These results indicate that redox mediator modification of NAD(P)-dependent dehydrogenases converts them into quasi-DET-type dehydrogenases, thereby enabling their utilization in 2.5th generation enzymatic sensors, which will facilitate the construction of enzymatic sensors suitable for continuous monitoring systems.
  01 Jul. 2024, Research paper (scientific journal), joint, 255, 0956-5663, DOI(公開)(r-map)
• Structure of cytotoxic amyloid oligomers generated during disaggregation
  Kaku, Toshisuke; Ikebukuro, Kazunori; Tsukakoshi, Kaori
  JOURNAL OF BIOCHEMISTRY
  OXFORD UNIV PRESS
  Amyloidosis is characterized by the abnormal accumulation of amyloid proteins. The causative proteins aggregate from monomers to oligomers and fibrils, among which some intermediate oligomers are considered as major toxins. Cytotoxic oligomers are generated not only by aggregation but also via fibril disaggregation. However, little is known about the structural characteristics and generation conditions of cytotoxic oligomers produced during disaggregation. Herein, we summarized the structural commonalities of cytotoxic oligomers formed under various disaggregation conditions, including the addition of heat shock proteins or small compounds. In vitro experimental data demonstrated the presence of high-molecular-weight oligomers (protofibrils or protofilaments) that exhibited a fibrous morphology and beta-sheet structure. Molecular dynamics simulations indicated that the distorted beta-sheet structure contributed to their metastability. The tendency of these cytotoxic oligomers to appear under mild disaggregation conditions, implied formation during the early stages of disaggregation. This review will aid researchers in exploring the characteristics of highly cytotoxic oligomers and developing drugs that target amyloid aggregates.
  02 Mar. 2024, Research paper (scientific journal), joint, 175, 6, 0021-924X, DOI(公開)(r-map), 575, 585
• Rapid and Convenient Single-Chain Variable Fragment-Employed Electrochemical C-Reactive Protein Detection System
  Miura, Daimei; Motohashi, Saki; Goto, Ayaka; Kimura, Hayato; Tsugawa, Wakako; Sode, Koji; Ikebukuro, Kazunori; Asano, Ryutaro
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Although IgG-free immunosensors are in high demand owing to ethical concerns, the development of convenient immunosensors that alternatively integrate recombinantly produced antibody fragments, such as single-chain variable fragments (scFvs), remains challenging. The low affinity of antibody fragments, unlike IgG, caused by monovalent binding to targets often leads to decreased sensitivity. We improved the affinity owing to the bivalent effect by fabricating a bivalent antibody-enzyme complex (AEC) composed of two scFvs and a single glucose dehydrogenase, and developed a rapid and convenient scFv-employed electrochemical detection system for the C-reactive protein (CRP), which is a homopentameric protein biomarker of systemic inflammation. The development of a point-of-care testing (POCT) system is highly desirable; however, no scFv-based CRP-POCT immunosensors have been developed. As expected, the bivalent AEC showed higher affinity than the single scFv and contributed to the high sensitivity of CRP detection. The electrochemical CRP detection using scFv-immobilized magnetic beads and the bivalent AEC as capture and detection antibodies, respectively, was achieved in 20 min without washing steps in human serum and the linear range was 1-10 nM with the limit of detection of 2.9 nM, which has potential to meet the criteria required for POCT application in rapidity, convenience, and hand-held detection devices without employing IgGs.
  Mar. 2024, Research paper (scientific journal), joint, 25, 5, 1661-6596, DOI(公開)(r-map)
• Aptamer-enhanced particle aggregation inhibition assay for simple homogeneous protein detection using DNA aptamer and thermo-responsive magnetic nanoparticles
  Rokutani, Shunsuke; Hiraka, Kentaro; Saitoh, Hiroshi; Saito, Taiki; Nonaka, Yoshihiko; Ueno, Kinuko; Tsukakoshi, Kaori; Ohnishi, Noriyuki; Ikebukuro, Kazunori
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  A simple and sensitive homogeneous protein detection system is required for the early detection of biomarkers. Thermo-responsive magnetic particles (TM) have already been developed to achieve easy bound/free separation at the homogeneous protein detection system, but they are still limited owing to the requirement of secondary antibodies and negatively charged polymers, and it is challenging to control the TM aggregation behavior because of the size of the TM. Therefore, at new method to control TM aggregation behavior that is simple, easy, and highly sensitive is required. In this study, we developed a DNA aptamer-based TM assay as a simple protein detection system without additional secondary molecular recognition elements or negatively charged polymer. In the first attempt, a DNA aptamer was modified on the TM surface, and its aggregation behavior was monitored depending on the target molecule concentration. The TM aggregation rate during the heating process decreased depending on the amount of the DNA aptamer and increased depending on the target protein level. This suggests that the DNA aptamer prevented TM aggregation owing to its negative charge and achieved target protein detection owing to the cancellation of repulsion. Capturable aptamers were used in the TM assay to improve the sensitivity and limit of detection. The designed Capture DNA was modified on the TM surface, and the aptamer was captured in the presence of the target protein through a conformational change. Eventually, Capturable aptamer-based TM assay achieved a sub-nanomolar limit of detection and higher sensitivity than that of our initial investigation. Through this study and the ease of the DNA aptamer design, it was shown that the DNA aptamer-modified TM assay enabled the development of a simple and sensitive homogeneous protein detection system.
  01 Feb. 2024, Research paper (scientific journal), joint, 245, 0956-5663, DOI(公開)(r-map)
• Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element
  Komiya, Erika; Takamatsu, Shouhei; Miura, Daimei; Tsukakoshi, Kaori; Tsugawa, Wakako; Sode, Koji; Ikebukuro, Kazunori; Asano, Ryutaro
  JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
  AMER CHEMICAL SOC
  DNA-protein complexes are attractive components with broad applications in various research fields, such as DNA aptamer-enzyme complexes as biosensing elements. However, noncovalent DNA-protein complexes often decrease detection sensitivity because they are highly susceptible to environmental conditions. In this study, we developed a versatile DNA-protein covalent-linking patch (D-Pclip) for fabricating covalent and stoichiometric DNA-protein complexes. We comprehensively explored the database to determine the DNA-binding ability of the candidates and selected UdgX as the only uracil-DNA glycosylase known to form covalent bonds with DNA via uracil, with a binding efficiency >90%. We integrated a SpyTag/SpyCatcher protein-coupling system into UdgX to create a universal and convenient D-Pclip. The usability of D-Pclip was shown by preparing a stoichiometric model complex of a hemoglobin (Hb)-binding aptamer and glucose oxidase (GOx) by mixing at 4 C-degrees. The prepared aptamer-GOx complexes detected Hb in a dose-dependent manner within the clinically required detection range in buffer and human serum without any washing procedures. D-Pclip covalently connects any uracil-inserted DNA sequence and any SpyCatcher-fused protein stoichiometrically; therefore, it has a high potential for various applications.
  31 Jan. 2024, Research paper (scientific journal), joint, 146, 6, 0002-7863, DOI(公開)(r-map), 4087, 4097
• 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.
  14 Dec. 2023, Research paper (scientific journal), joint, 3, 1, DOI(公開)(r-map), 13066, 13069
• Development of a liquid chromatography-based versatile bioanalysis for bevacizumab based on pretreatment combining aptamer affinity purification and centrifugal ultrafiltration concentration
  Todoroki, Kenichiro; Hamada, Daichi; Yamada, Tomohiro; Saito, Taro; Shimizu, Yutaka; Sugiyama, Eiji; Mizuno, Hajime; Hayashi, Hideki; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  ANALYTICAL SCIENCES
  SPRINGERNATURE
  We report on the development of a versatile and accurate bioanalytical method for bevacizumab using a pretreatment method combining affinity purification with anti-idiotypic DNA aptamers and centrifugal ultrafiltration concentration, followed by liquid chromatography (LC)-fluorescence analysis. An affinity purification method using Sepharose beads as an affinity support removed immunoglobulin G and a large amount of coexisting substances in the serum sample. Purified bevacizumab was separated as a single peak by conventional LC and detected fluorometrically, showing good linearity (R2 = 0.999) in the range of 5-200 & mu;g/mL, sufficient to analyze bevacizumab concentrations in the blood of bevacizumab-treated patients. By combining this purification method with a concentration method using a centrifugal filtration device that inhibits non-specific adsorption of bevacizumab, the quantitative range was reduced by a factor of 10 while showing good linearity (R2 = 0.999) in the 0.5-20 & mu;g/mL range. The developed analytical method is expected to be used not only for general bioanalysis of therapeutic mAbs in clinical settings, but also for next-generation antibody drugs that show drug efficacy at low concentrations and for analysis of trace samples.
  Nov. 2023, Research paper (scientific journal), joint, 39, 11, 0910-6340, DOI(公開)(r-map), 1805, 1811
• Protein engineering of antibody fragments for pharmaceutical production
  Kuwahara, Atsushi; Ikebukuro, Kazunori; Asano, Ryutaro
  APPLIED PHYSICS REVIEWS
  AIP Publishing
  Antibody fragments without the Fc region are attracting attention in the pharmaceutical industry due to their high ability to penetrate solid tissues, cost-effective expression using microbial expression systems, and distinctive modes of action compared to those of full-size antibodies. Based on these characteristics, several antibody fragment agents have been approved. However, developing platform engineering methodologies to accelerate their development is important. In this review, we summarize and discuss protein engineering strategies for preparing therapeutic antibody fragments composed of antibody variable domains. Three (introduction of high-solubility tag systems, complementarity-determining region grafting, and domain arrangements) and two (introduction of purification tag systems and mutagenesis studies for protein L- or protein A-binding) protein engineering strategies have been reported for the cultivation and purification processes, respectively. Fusion tags might negatively impact molecular folding, function, immunogenicity, and final yield. If the production behavior of antibody fragments is not improved through complementarity-determining region grafting, domain arrangements, or human sequence-based mutagenesis, using additional fusion tag systems should be considered, with careful attention to the points described above. This summarized knowledge regarding protein engineering strategies for effectively producing antibody fragments will further accelerate therapeutic antibody fragment development.
  Sep. 2023, Research paper (scientific journal), joint, 10, 3, 1931-9401, DOI(公開)(r-map)
• Regulation of thrombin activity by ligand-induced topological alteration in a thrombin-binding aptamer
  Sasaki, Shogo; Ma, Yue; Hirokawa, Takatsugu; Ikebukuro, Kazunori; Tera, Masayuki; Nagasawa, Kazuo
  CHEMICAL COMMUNICATIONS
  ROYAL SOC CHEMISTRY
  Thrombin-binding aptamer (TBA), which forms a G-quadruplex (G4) structure with anti-parallel topology, interacts with thrombin to inhibit its enzymatic activity. Here we show that the G4-topology-altering ligand L2H2-2M2EA-6LCO (6LCO) changes the anti-parallel topology of TBA G4 to the parallel topology, thereby abrogating the thrombin-inhibitory activity of TBA. This finding suggests that G4 ligands that alter topology may be promising drug candidates for diseases involving G4-binding proteins.
  13 Jul. 2023, Research paper (scientific journal), joint, 59, 57, 1359-7345, DOI(公開)(r-map), 8862, 8865
• Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis
  Clarke, O. E.; Pelling, H.; Bennett, V.; Matsumoto, T.; Gregory, G. E.; Nzakizwanayo, J.; Slate, A. J.; Preston, A.; Laabei, M.; Bock, L. J.; Wand, M. E.; Ikebukuro, K.; Gebhard, S.; Sutton, J. M.; Jones, B. V.
  FRONTIERS IN MICROBIOLOGY
  FRONTIERS MEDIA SA
  Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as resistant to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited similar to 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.
  05 Apr. 2023, Research paper (scientific journal), joint, 14, DOI(公開)(r-map)
• Identification of novel amyloidosis in dogs: α-S1-casein acquires amyloidogenicity in mammary tumor by overexpression and N-terminal truncation
  Murakami, Tomoaki; Kaku, Toshisuke; Tsukakoshi, Kaori; Iwaide, Susumu; Itoh, Yoshiyuki; Hisada, Miki; Nomura, Kohji; Kubo, Rikako; Ikebukuro, Kazunori; Sassa-O'Brien, Yukiko; Kametani, Fuyuki
  VETERINARY PATHOLOGY
  SAGE PUBLICATIONS INC
  Mammary tumor-associated amyloidosis (MTAA) in dogs is characterized by amyloid deposition in the stroma of mammary adenoma or carcinoma; however, the amyloid precursor protein remains unknown. We attempted to identify an amyloid precursor protein and elucidated its etiology by characterizing 5 cases of canine MTAA. Proteomic analyses of amyloid extracts from formalin-fixed paraffin-embedded specimens revealed alpha-S1-casein (CASA1) as a prime candidate and showed the N-terminal truncation of canine CASA1. Both immunohistochemistry and immunoelectron microscopy showed that amyloid deposits or fibrils in MTAA cases were positive for CASA1. Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction revealed the complete mRNA sequence encoding CASA1, whose expression was significantly higher in the amyloid-positive group. The recombinant protein of the N-terminal-truncated canine CASA1 and the synthetic peptides derived from canine and human CASA1 formed amyloid-like fibrils in vitro. Structural prediction suggested that the N-terminal region of CASA1 was disordered. Previously, full-length CASA1 was reported to inhibit the amyloidogenesis of other proteins; however, we demonstrated that CASA1 acquires amyloidogenicity via excessive synthesis followed by truncation of its disordered N-terminal region. By identifying a novel in vivo amyloidogenic protein in animals and revealing key mechanistic details of its associated pathology, this study provides valuable insights into the integrated understanding of related proteopathies.
  Mar. 2023, Research paper (scientific journal), joint, 60, 2, 0300-9858, DOI(公開)(r-map), 203, 213
• Development of an Anti-Idiotype Aptamer-Based Electrochemical Sensor for a Humanized Therapeutic Antibody Monitoring
  Nagata, Madoka; Lee, Jinhee; Saito, Taro; Ikebukuro, Kazunori; Sode, Koji
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Therapeutic monoclonal antibodies (mAbs) are currently the most effective medicines for a wide range of diseases. Therefore, it is expected that easy and rapid measurement of mAbs will be required to improve their efficacy. Here, we report an anti-idiotype aptamer-based electrochemical sensor for a humanized therapeutic antibody, bevacizumab, based on square wave voltammetry (SWV). With this measurement procedure, we were able to monitor the target mAb within 30 min by employing the anti-idiotype bivalent aptamer modified with a redox probe. A fabricated bevacizumab sensor achieved detection of bevacizumab from 1-100 nM while eliminating the need for free redox probes in the solution. The feasibility of monitoring biological samples was also demonstrated by detecting bevacizumab in the diluted artificial serum, and the fabricated sensor succeeded in detecting the target covering the physiologically relevant concentration range of bevacizumab. Our sensor contributes to ongoing efforts towards therapeutic mAbs monitoring by investigating their pharmacokinetics and improving their treatment efficacy.
  Mar. 2023, Research paper (scientific journal), joint, 24, 6, DOI(公開)(r-map)
• Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System
  Yanase, Takumi; Okuda-Shimazaki, Junko; Asano, Ryutaro; Ikebukuro, Kazunori; Sode, Koji; Tsugawa, Wakako
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method.
  Feb. 2023, Research paper (scientific journal), joint, 24, 3, DOI(公開)(r-map)
• Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic A beta Oligomer Detection
  Tsukakoshi, Kaori; Kubo, Rikako; Ikebukuro, Kazunori
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Amyloid beta (A beta) oligomers play a key role in the progression of Alzheimer's disease (AD). Multiple forms of A beta assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic A beta oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic A beta oligomers. Since the N-terminus domain of PrPc (residue 23-111) derived from mice is known to bind to toxic A beta oligomers in vitro, we genetically fused PrPc23-111 to ALP. The developed fusion protein, PrP-ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP-ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP-ALP neutralized the toxic effect of A beta oligomers indicated an interaction between PrP-ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting A beta oligomers. PrP-ALP may serve as a powerful tool for detecting toxic A beta oligomers that may be related to AD progression.
  Dec. 2022, Research paper (scientific journal), joint, 23, 23, DOI(公開)(r-map)
• The state of water molecules induces changes in the topologies and interactions of G-quadruplex DNA aptamers in hydrated ionic liquid
  Fujita, Kyoko; Honda, Takuya; Tsukakoshi, Kaori; Ohno, Hiroyuki; Ikebukuro, Kazunori
  JOURNAL OF MOLECULAR LIQUIDS
  ELSEVIER
  Herein, the effects of the bound state of water molecules were investigated on the topologies and inter-actions of G-quadruplex DNA aptamers by using hydrated ionic liquids (ILs). Since intracellular water molecules are generally expected to exist in the bound state and not as free molecules that exist in the typical in vitro media, we proposed hydrated ILs as potential candidates for controlling the state of water molecules. Hydrated ILs have been reported to dissolve proteins without compromising their higher-order structures. In this study, the structures and interactions of three G-quadruplex DNA apta-mers (one thrombin-binding aptamer and two vascular endothelial growth factor 165-binding aptamers), with their target molecules were analyzed with circular dichroism (CD) spectroscopy and enzyme-linked oligonucleotide assay by changing the water content of hydrated cholinium dihydrogen phosphate (Hy [ch][dhp]). Hy[ch][dhp] allowed the effective dissolution of G-quadruplex DNA aptamers while maintain-ing their structure and binding affinity to the target molecule. The water content of Hy[ch][dhp] induced changes in the CD spectra, suggesting changes in the topology of G-quadruplex structure. Increased struc-tural stability and binding properties indicated molecular recognition and smooth dehydration progress in Hy[ch][dhp] via regulation of the state of water molecules. (c) 2022 Elsevier B.V. All rights reserved.
  15 Nov. 2022, Research paper (scientific journal), joint, 366, 0167-7322, DOI(公開)(r-map)
• Structure of lactate oxidase from Enterococcus hirae revealed new aspects of active site loop function: Product-inhibition mechanism and oxygen gatekeeper
  Hiraka, Kentaro; Yoshida, Hiromi; Tsugawa, Wakako; Asano, Ryutaro; La Belle, Jeffrey T.; Ikebukuro, Kazunori; Sode, Koji
  PROTEIN SCIENCE
  WILEY
  l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with d-lactate form ligand, which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.
  Oct. 2022, Research paper (scientific journal), joint, 31, 10, 0961-8368, DOI(公開)(r-map)
• Effects of G-Quadruplex Ligands on the Topology, Stability, and Immunostimulatory Properties of G-Quadruplex-Based CpG Oligodeoxynucleotides
  Tu, Anh Thi Tram; Hoshi, Kazuaki; Ma, Yue; Oyama, Taiji; Suzuki, Satoko; Tsukakoshi, Kaori; Nagasawa, Kazuo; Ikebukuro, Kazunori; Yamazaki, Tomohiko
  ACS CHEMICAL BIOLOGY
  AMER CHEMICAL SOC
  We previously reported that the formation of guanine-quadruplex (G4) structures provides phosphodiester oligodeoxynucleotides containing unmethylated cytosine-phosphate-guanine (CpG ODNs) with higher nuclease resistance and cellular uptake, thereby increasing their immunostimulation efficiency through TLR9 activation. CpG ODNs forming G4 structures (G4 CpG ODNs) are thus potential vaccine adjuvants against infectious diseases. However, the G4 structure changes topology depending on the surrounding environment. Recently, G4 ligands, which are small molecules that bind to G4 ODNs with high affinity, were reported to improve the stability of G4. In this study, we propose to increase the stability and function of G4 CpG ODNs using G4 ligands. We show the effects of two G4 ligands, named L2H2-6OTD (L2H2) and L2G2-2M2EG-6OTD (L2G2), on the topology, stability, and immunostimulatory properties of a monomeric hybrid-type G4 CpG ODN containing CpG motifs in the central loop, named GD3. We found that L2H2 helps maintain the hybrid G4 topology of GD3, whereas L2G2 induces parallel G4 formation. Both G4 ligands increase the thermodynamic and nuclease stability of GD3. However, only GD3 associated with L2H2 binds efficiently to TLR9 and evokes a higher immune response from mouse macrophage-like RAW264 cells. GD3 associated with L2G2 does not bind efficiently to TLR9 and elicits lower cytokine production. Our results demonstrate that the potential to enhance immunostimulatory properties depends on the ability of G4 ligands to maintain and stabilize the hybrid G4 of GD3. We anticipate that our findings will facilitate the development of more effective G4 CpG ODN-based vaccine adjuvants against infectious diseases.
  15 Jul. 2022, Research paper (scientific journal), joint, 17, 7, 1554-8929, DOI(公開)(r-map), 1703, 1713
• CpG Methylation Altered the Stability and Structure of the i-Motifs Located in the CpG Islands
  Oshikawa, Daiki; Inaba, Shintaro; Kitagawa, Yudai; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Cytosine methylation within the 5 '-C-phosphate-G-3 ' sequence of nucleotides (called CpG methylation) is a well-known epigenetic modification of genomic DNA that plays an important role in gene expression and development. CpG methylation is likely to be altered in the CpG islands. CpG islands are rich in cytosine, forming a structure called the i-motif via cytosine-cytosine hydrogen bonding. However, little is known about the effect of CpG methylation on the i-motif. In this study, The CpG methylation-induced structural changes on the i-motif was examined by thermal stability, circular dichroism (CD) spectroscopy, and native-polyacrylamide gel electrophoresis (Native-PAGE) evaluation of five i-motif-forming DNAs from four cancer-related genes (VEGF, C-KIT, BCL2, and HRAS). This research shows that CpG methylation increased the transitional pH of several i-motif-forming DNAs and their thermal stability. When examining the effect of CpG methylation on the i-motif in the presence of opposite G4-forming DNAs, CpG methylation influenced the proportion of G4 and i-motif formation. This study showed that CpG methylation altered the stability and structure of the i-motif in CpG islands.
  Jun. 2022, Research paper (scientific journal), joint, 23, 12, DOI(公開)(r-map)
• Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection
  Saito, Taro; Shimizu, Yutaka; Tsukakoshi, Kaori; Abe, Koichi; Lee, Jinhee; Ueno, Kinuko; Asano, Ryutaro; Jones, Brian, V; Yamada, Tomohiro; Nakano, Tatsuki; Tong, Jiaxing; Hishiki, Asami; Hara, Kodai; Hashimoto, Hiroshi; Sode, Koji; Toyo'oka, Toshimasa; Todoroki, Kenichiro; Ikebukuro, Kazunori
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity. As the CDR of bevacizumab is predicted to be highly positively charged under mildly acidic conditions and that DNA is negatively charged, the affinity of A14#1 to bevacizumab markedly increased at pH 4.7 (K-D = 44 pM) than at pH 7.4 (K-D = 12 nM). A14#1-based electrochemical detection method capable of detecting 31 pM of bevacizumab at pH 4.7 was thus developed. A14#1 could be potentially useful for therapeutic drug measurement as a novel ligand of bevacizumab.
  01 May 2022, Research paper (scientific journal), joint, 203, 0956-5663, DOI(公開)(r-map)
• Development of a POCT type insulin sensor employing anti-insulin single chain variable fragment based on faradaic electrochemical impedance spectroscopy under single frequency measurement
  Khanwalker, Mukund; Fujita, Rinko; Lee, Jinhee; Wilson, Ellie; Ito, Kohei; Asano, Ryutaro; Ikebukuro, Kazunori; LaBelle, Jeffrey; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  To improve glycemic control managed through insulin administration, recent studies have focused on developing hand-held point-of-care testing (POCT) electrochemical biosensors for insulin measurement. Amongst them, anti insulin IgG-based sensors show promise in detecting insulin with high specificity and sensitivity. However, fabrication of electrochemical sensors with IgG antibodies can prove challenging because of their larger molecular size. To overcome these limitations, this study focuses on utilizing the anti-insulin single chain variable fragment (scFv) as a biosensing molecule with single-frequency faradaic electrochemical impedance spectroscopy (EIS). By comparing two different immobilization methods, covalent conjugation via succinimidyl ester and non-covalent poly-histidine chelation, we demonstrated effective modification of the electrode surface with anti insulin scFv, while retaining its specific recognition toward insulin. Sensor performance was confirmed via the concentration-dependent faradaic electrochemical impedance change using potassium ferricyanide as a redox probe. The optimal frequency for measurement was determined to be the peak slope of the calculated impedance correlation with respect to frequency. Based on the identified optimized frequency, we performed single frequency measurement of insulin within a concentration range of 10 pM-100 nM. This study can aid in developing a future point-of-care sensor which rapidly and sensitively measures insulin across a dynamic range of physiological concentrations, with label-free detection.
  15 Mar. 2022, Research paper (scientific journal), joint, 200, 0956-5663, DOI(公開)(r-map)
• Transient potentiometry based D-serine sensor using engineered D-amino acid oxidase showing quasi-direct electron transfer property
  Takamatsu, Shouhei; Lee, Inyoung; Lee, Jinhee; Asano, Ryutaro; Tsugawa, Wakako; Ikebukuro, Kazunori; Dick, Jeffrey E.; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  D-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent n-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for o-serine, transient potentiometry based o-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydmgenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with o-serine concentration range between 0.5 mM and 5 mM, dOCP/d root t based monitoring is suitable for o-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 mu M-30 mM). The maximum dOCP/d root t was -39.2 +/- 2.0 mV/s(1/2), the K-m(app) was 1.9 mM, and the lower limit of detection was 20 mu M. In addition, D-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for o-serine monitoring.
  15 Mar. 2022, Research paper (scientific journal), joint, 200, 0956-5663, DOI(公開)(r-map)
• An Amine-Reactive Phenazine Ethosulfate (arPES)-A Novel Redox Probe for Electrochemical Aptamer-Based Sensor
  Nagata, Madoka; Lee, Jinhee; Henley, Stephen; Ikebukuro, Kazunori; Sode, Koji
  SENSORS
  MDPI
  Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at -0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.
  Mar. 2022, Research paper (scientific journal), joint, 22, 5, DOI(公開)(r-map)
• Stabilization of VEGF i-motif structure by CpG methylation
  Kimura, Kosuke; Oshikawa, Daiki; Ikebukuro, Kazunori; Yoshida, Wataru
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
  ACADEMIC PRESS INC ELSEVIER SCIENCE
  The intercalated motif (i-motif) is a non-canonical nucleic acid structure formed by intercalated hemiprotonated cytosine base pairs (C-C+) under acidic conditions. The i-motif structure formation is involved in biological processes such as transcription regulation. Therefore, the identification of factors controlling i-motif formation is important in elucidating the cellular functions it controls. We previously reported that the VEGF G-quadruplex structure is stabilized by CpG methylation. In this study, the effect of CpG methylation on the stability of the VEGF i-motif structure was investigated. The VEGF i-motifforming oligonucleotide contains four cytosines on CpG sites, and three of the four cytosines (C4, C15, and C20) are involved in C-C+ formation in the i-motif structure. Circular dichroism (CD) spectra analysis demonstrated that full CpG methylation increased the pH of mid transition (pHT) of the i-motif structure by 0.1, and the melting temperature (Tm) by 5.1 degrees C in 25 mM sodium cacodylate buffer at pH 5.0. Moreover, single methylation at C4, C15, and C20 increased Tm by 0.5, 1.7, and 2.0 degrees C in the buffer, respectively. These results demonstrated that CpG methylation stabilized the VEGF i-motif structure. (c) 2022 Elsevier Inc. All rights reserved.
  26 Feb. 2022, Research paper (scientific journal), joint, 594, 0006-291X, DOI(公開)(r-map), 88, 92
• Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria
  Kobayashi, Shunichi; Atsumi, Shota; Ikebukuro, Kazunori; Sode, Koji; Asano, Ryutaro
  MICROBIAL CELL FACTORIES
  BMC
  Background: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). Results: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. Conclusion: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers.The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.
  06 Jan. 2022, Research paper (scientific journal), joint, 21, 1, DOI(公開)(r-map)
• A Thiol-reactive Phenazine Ethosulfate - A Novel Redox Mediator for Quasi-direct Electron-transfer-type Sensors
  Fitriana, Maya; Hiraka, Kentaro; Ikebukuro, Kazunori; Sode, Koji; Tsugawa, Wakako
  SENSORS AND MATERIALS
  MYU, SCIENTIFIC PUBLISHING DIVISION
  A novel redox mediator, thiol-reactive phenazine ethosulfate (trPES), was used to modify an enzyme for the first time for biosensor development. Aerococcus viridans-lactate oxidase (LOx), widely used to study lactate biosensors, was modified with a single trPES molecule. A cysteine mutation was introduced into the vicinity of the LOx cofactor to enable the modification by trPES. LOx cysteine mutants were then successfully modified using trPES, thus acquiring quasi-direct electron transfer ability. An electrode immobilized with the trPES-LOx A96L/S210C mutant showed the highest amperometric response currents among the modified LOx cysteine mutants, indicating efficient electron transfer. The position around residues S210 to N212 on the LOx surface (distance <14 angstrom from FMN N5) is important for the mediator to access the reduced flavin. Then, the performances of the lactate sensor were improved by utilizing LOx A96L/S210C modified with trPES and arPES, another redox mediator used to modify a lysine residue. The lactate sensor has a detection range of up to 1 mM, a sensitivity of 6.62 mu A/mM.cm(2), and a limit of detection of 9.9 mu M. Furthermore, Aspergillus flavus-derived FAD glucose dehydrogenase was successfully modified with trPES and the response currents were obtained, showing the versatility of trPES for modifying other oxidoreductases.
  2022, Research paper (scientific journal), joint, 34, 6, 0914-4935, DOI(公開)(r-map), 2105, 2124
• Cytotoxic A beta Protofilaments Are Generated in the Process of A beta Fibril Disaggregation
  Kaku, Toshisuke; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Significant research on Alzheimer's disease (AD) has demonstrated that amyloid beta (A beta) oligomers are toxic molecules against neural cells. Thus, determining the generation mechanism of toxic A beta oligomers is crucial for understanding AD pathogenesis. A beta fibrils were reported to be disaggregated by treatment with small compounds, such as epigallocatechin gallate (EGCG) and dopamine (DA), and a loss of fibril shape and decrease in cytotoxicity were observed. However, the characteristics of intermediate products during the fibril disaggregation process are poorly understood. In this study, we found that cytotoxic A beta aggregates are generated during a moderate disaggregation process of A beta fibrils. A cytotoxicity assay revealed that A beta fibrils incubated with a low concentration of EGCG and DA showed higher cytotoxicity than A beta fibrils alone. Atomic force microscopy imaging and circular dichroism spectrometry showed that short and narrow protofilaments, which were highly stable in the beta-sheet structure, were abundant in these moderately disaggregated samples. These results indicate that toxic A beta protofilaments are generated during disaggregation from amyloid fibrils, suggesting that disaggregation of A beta fibrils by small compounds may be one of the possible mechanisms for the generation of toxic A beta aggregates in the brain.
  Dec. 2021, Research paper (scientific journal), joint, 22, 23, DOI(公開)(r-map)
• Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands
  Hasegawa, Hijiri; Sasaki, Ikkei; Tsukakoshi, Kaori; Ma, Yue; Nagasawa, Kazuo; Numata, Shusuke; Inoue, Yuuki; Kim, Yeji; Ikebukuro, Kazunori
  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
  MDPI
  Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.
  Dec. 2021, Research paper (scientific journal), joint, 22, 23, DOI(公開)(r-map)
• Continuous electrochemical monitoring of L-glutamine using redox-probe-modified L-glutamine-binding protein based on intermittent pulse amperometry
  Takamatsu, Shouhei; Lee, Jinhee; Asano, Ryutaro; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji
  SENSORS AND ACTUATORS B-CHEMICAL
  ELSEVIER SCIENCE SA
  The development of a continuous electrochemical monitoring system using a periplasmic binding protein (PBP), which changes its conformational dynamics upon ligand binding, is promising for in situ, real-time, continuous measurement technologies for use in effective biological production processes. This study focuses on a continuous L-glutamine biosensor based on Escherichia coli-derived L-glutamine-binding protein (QBP), a PBP that can speficically bind to L-glutamine. In this sensor, QBP was modified with a redox probe, amine-reactive phenazine ethosulfate (PES), and the conformational change in QBP through the recognition of L-glutamine was monitored electrochemically by intermittent pulse amperometry (IPA) based on the change in the access of QBP-modified PES to the electrode. This sensor exhibited a change in the current against logarithmic L-glutamine concentrations between 0.2-50 mu M. Continuous monitoring of L-glutamine, based on IPA measurements, revealed that continuous changes in L-glutamine concentration were observed for both increasing and decreasing concentrations. During continuous monitoring, L-glutamine concentration was monitored between 50 and 500 mu M, with a limit of detection of 50 mu M. This result is expected to address the future development of an electrochemical biosensor for in situ, real-time, continuous monitoring of various metabolites based on PBPs.
  01 Nov. 2021, Research paper (scientific journal), joint, 346, DOI(公開)(r-map)
• Rapid, convenient, and highly sensitive detection of human hemoglobin in serum using a high-affinity bivalent antibody-enzyme complex
  Miura, Daimei; Kimura, Hayato; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji; Asano, Ryutaro
  TALANTA
  ELSEVIER
  Human hemoglobin (Hb) is a biomarker of several diseases, and monitoring of Hb levels is required during emergent surgery. However, rapid and sensitive Hb detection methods are yet to be developed. The present study established a rapid, convenient, and highly sensitive detection method for Hb in human serum using a bivalent antibody-enzyme complex (AEC). AECs are promising sensing elements because of their ability to bind specific targets and their catalytic activity that produce signals. We recently reported a convenient and universal method to fabricate bivalent AECs with two antibody fragments, using the SpyCatcher/SpyTag system. The present study applied a bivalent AEC for highly sensitive and quantitative detection of human Hb. The bivalent anti-Hb AEC was successfully prepared by incubating both N- and C-terminus SpyCatcher-fused glucose dehydrogenase and SpyTag-fused anti-Hb single-chain variable fragments at 4 degrees C. As expected, the bivalent AEC for Hb with a multimeric structure showed higher affinity than the monovalent AEC, by means of avidity effects, unlike that for soluble epidermal growth factor receptor with a monomeric structure; this contributed to a great improvement in sensitivity. Finally, we established a rapid and wash-free homogeneous electrochemical detection system for Hb by integrating magnetic beads. The linear range of the system completely covered the clinically required Hb levels, even in human serum. This technology provides an ideal point-of-care test for Hb and other multimeric biomarkers.
  01 Nov. 2021, Research paper (scientific journal), joint, 234, 0039-9140, DOI(公開)(r-map)
• Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold
  Safitri, Fika Ayu; Tu, Anh Thi Tram; Hoshi, Kazuaki; Shobo, Miwako; Zhao, Dandan; Witarto, Arief Budi; Sumarsono, Sony Heru; Giri-Rachman, Ernawati Arifin; Tsukakoshi, Kaori; Ikebukuro, Kazunori; Yamazaki, Tomohiko
  BIOMOLECULES
  MDPI
  Guanine-quadruplex-based CpG oligodeoxynucleotides (G4 CpG ODNs) have been developed as potent immunostimulatory agents with reduced sensitivity to nucleases. We designed new monomeric G4 ODNs with an antiparallel topology using antiparallel type duplex/G4 ODNs as robust scaffolds, and we characterized their topology and effects on cytokine secretion. Based on circular dichroism analysis and quantification of mRNA levels of immunostimulatory cytokines, it was found that monomeric antiparallel G4 CpG ODNs containing two CpG motifs in the first functional loop, named G2.0.0, could maintain antiparallel topology and generate a high level of immunostimulatory cytokines in RAW264 mouse macrophage-like cell lines. We also found that the flanking sequence in the CpG motif altered the immunostimulatory effects. Gc2c.0.0 and Ga2c.0.0 are monomeric antiparallel G4 CpG ODNs with one cytosine in the 3 & PRIME; terminal and one cytosine/adenine in the 5 & PRIME; terminal of CpG motifs that maintained the same resistance to degradation in serum as G2.0.0 and improved interleukin-6 production in RAW264 and bone marrow-derived macrophages. The immunostimulatory activity of antiparallel G4 CpG ODNs is superior to that of linear natural CpG ODNs. These results provide insights for the rational design of highly potent CpG ODNs using antiparallel G4 as a robust scaffold.
  Nov. 2021, Research paper (scientific journal), joint, 11, 11, DOI(公開)(r-map)
• Artificial complementary chromatic acclimation gene expression system in Escherichia coli
  Ariyanti, Dwi; Ikebukuro, Kazunori; Sode, Koji
  MICROBIAL CELL FACTORIES
  BMC
  Background The development of multiple gene expression systems, especially those based on the physical signals, such as multiple color light irradiations, is challenging. Complementary chromatic acclimation (CCA), a photoreversible process that facilitates the control of cellular expression using light of different wavelengths in cyanobacteria, is one example. In this study, an artificial CCA systems, inspired by type III CCA light-regulated gene expression, was designed by employing a single photosensor system, the CcaS/CcaR green light gene expression system derived from Synechocystis sp. PCC6803, combined with G-box (the regulator recognized by activated CcaR), the cognate cpcG2 promoter, and the constitutively transcribed promoter, the P-trc Delta LacO promoter. Results One G-box was inserted upstream of the cpcG2 promoter and a reporter gene, the rfp gene (green light-induced gene expression), and the other G-box was inserted between the P-trc Delta LacO promoter and a reporter gene, the bfp gene (red light-induced gene expression). The Escherichia coli transformants with plasmid-encoded genes were evaluated at the transcriptional and translational levels under red or green light illumination. Under green light illumination, the transcription and translation of the rfp gene were observed, whereas the expression of the bfp gene was repressed. Under red light illumination, the transcription and translation of the bfp gene were observed, whereas the expression of the rfp gene was repressed. During the red and green light exposure cycles at every 6 h, BFP expression increased under red light exposure while RFP expression was repressed, and RFP expression increased under green light exposure while BFP expression was repressed. Conclusion An artificial CCA system was developed to realize a multiple gene expression system, which was regulated by two colors, red and green lights, using a single photosensor system, the CcaS/CcaR system derived from Synechocystis sp. PCC6803, in E. coli. The artificial CCA system functioned repeatedly during red and green light exposure cycles. These results demonstrate the potential application of this CCA gene expression system for the production of multiple metabolites in a variety of microorganisms, such as cyanobacteria.
  05 Jul. 2021, Research paper (scientific journal), joint, 20, 1, DOI(公開)(r-map)
• A self-powered glucose sensor based on BioCapacitor principle with micro-sized enzyme anode employing direct electron transfer type FADGDH
  Lee, Inyoung; Okuda-Shimazaki, Junko; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji
  JOURNAL OF PHYSICS-ENERGY
  IOP PUBLISHING LTD
  Diabetes mellitus is a disorder in which the body does not produce enough or respond normally to insulin; consequently, blood glucose levels increase to become abnormally high. Accordingly, the primary treatment of diabetes is to control glycemic levels continuously. To continuously control glycemic levels, several medical devices have been developed to monitor blood glucose levels, represented by sensors and monitors for the self-monitoring of blood glucose. The ultimate goal for those engaged in research to develop medical devices is to develop implantable biodevices, namely self-powered autonomously operated artificial pancreas systems. One of the most challenging issues in realizing an implantable artificial pancreas is the long-term continuous supply of electricity, which is currently dependent on rechargeable batteries, requiring periodical replacement. In this work, we report the development of a direct electron transfer type enzyme-based miniaturized self-powered glucose sensor based on the BioCapacitor principle with a micro-sized enzyme anode area (0.15 mm x 0.75 mm), which has only 0.1 mm(2) of electrode surface. As a result, a BioCapacitor utilizing a biofuel cell with a micro-sized enzyme anode was operated by self-power. In addition, the glucose concentration was detected within the range from 13 mM to 100 mM based on the frequency of charge/discharge cycles of the BioCapacitor. Although further improvement of the current density of the micro-sized anode is necessary to monitor a glucose concentration range lower than 13 mM, this self-powered glucose sensor with a micro-sized electrode based on the BioCapacitor principle was operated continuously for 6.6 h at 37 degrees C in 100 mM potassium phosphate buffer (pH 7.0). Our success indicates the potential to realize self-powered, autonomous, and implantable sensing modules for bio devices such as glucose-sensing systems for an artificial pancreas.
  Jul. 2021, Research paper (scientific journal), joint, 3, 3, 2515-7655, DOI(公開)(r-map)
• G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol
  Tsukakoshi, Kaori; Yamagishi, Yasuko; Kanazashi, Mana; Nakama, Kenta; Oshikawa, Daiki; Savory, Nasa; Matsugami, Akimasa; Hayashi, Fumiaki; Lee, Jinhee; Saito, Taiki; Sode, Koji; Khunathai, Kanjana; Kuno, Hitoshi; Ikebukuro, Kazunori
  NUCLEIC ACIDS RESEARCH
  OXFORD UNIV PRESS
  Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.
  21 Jun. 2021, Research paper (scientific journal), joint, 49, 11, 0305-1048, DOI(公開)(r-map), 6069, 6081
• G-quadruplex: Flexible conformational changes by cations, pH, crowding and its applications to biosensing
  Nishio, Maui; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  G-quadruplex (G4) is a non-canonical structure that is formed in G-rich sequences of nucleic acids. G4s play important roles in vivo, such as telomere maintenance, transcription, and DNA replication. There are three typical topologies of G4: parallel, anti-parallel, and hybrid. In general, metal cations, such as potassium and sodium, stabilize G4s through coordination in the G-quartet. While G4s have some functions in vivo, there are many reports of developed applications that use G4s. As various conformations of G4s could form from one sequence depending on varying conditions, many researchers have developed G4-based sensors. Furthermore, G4 is a great scaffold of aptamers since many aptamers folded into G4s have also been reported. However, there are some challenges about its practical use due to the difference between practical sample conditions and experimental ones. G4 conformations are dramatically altered by the surrounding conditions, such as metal cations, pH, and crowding. Many studies have been conducted to characterize G4 conformations under various conditions, not only to use G4s in practical applications but also to reveal its function in vivo. In this review, we summarize recent studies that have investigated the effects of surrounding conditions (e.g., metal cations, pH, and crowding) on G4 conformations and the application of G4s mainly in biosensor fields, and in others.
  15 Apr. 2021, Research paper (scientific journal), joint, 178, 0956-5663, DOI(公開)(r-map)
• Development of glycated peptide enzyme sensor based flow injection analysis system for haemoglobin A1c monitoring using quasi-direct electron transfer type engineered fructosyl peptide oxidase
  Hatada, Mika; Saito, Satomi; Yonehara, Satoshi; Tsugawa, Wakako; Asano, Ryutaro; Ikebukuro, Kazunori; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Haemoglobin A1e (hemoglobin A1e, HbA1c) is an important long-term glycemic control marker for diabetes. The aim of this study was to develop an enzyme flow injection analysis (FIA) system using engineered fructosyl peptide oxidase (FPOx) based on 2.5th generation principle for an HbA1c automated analytical system. FPOx from Phaeosphaeria nodorum (PnFPOx) was engineered by introducing a Lys residue at the R414 position, to be modified with amine reactive phenazine ethosulfate (arPES) in proximity of FAD. The engineered PnFPOx mutant with minimized oxidase activity, N56A/R414K, showed quasi-direct electron transfer (quasi-DET) ability after PES-modification. The FIA system was constructed by employing a PES-modified PnFPOx N56A/R414K and operated at 0 V against Ag/AgCl. The system showed reproducible responses with a linear range of 20-500 mu M for both fructosyl valine (FV) and fructosyl valylhistidine (FVH), with sensitivities of 0.49 nA mu AO and 0.13 nA mu M-1, and the detection limits of 1.3 mu M and 2.0 mu M for FV and FVH, respectively. These results indicate that the enzyme electrochemical FIA system covers the clinical range of HbA1c detection for more 200 consecutive measurements. Protease digested three different levels of HbA1c samples including healthy and diabetic range subjects were also measured with the FIA system. Thus, it will be possible to develop an integrated system consisting of sample pretreatment and sample electrochemical measurement based on an FIA system possessing quasi-DET type PnFPOx.
  01 Apr. 2021, Research paper (scientific journal), joint, 177, 0956-5663, DOI(公開)(r-map)
• Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors
  Ito, Kohei; Okuda-Shimazaki, Junko; Kojima, Katsuhiro; Mori, Kazushige; Tsugawa, Wakako; Asano, Ryutaro; Ikebukuro, Kazunori; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDHimmobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).
  15 Mar. 2021, Research paper (scientific journal), joint, 176, 0956-5663, DOI(公開)(r-map)
• Rational design of direct electron transfer type L-lactate dehydrogenase for the development of multiplexed biosensor
  Hiraka, Kentaro; Tsugawa, Wakako; Asano, Ryutaro; Yokus, Murat A.; Ikebukuro, Kazunori; Daniele, Michael A.; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  The development of wearable multiplexed biosensors has been focused on systems to measure sweat L-lactate and other metabolites, where the employment of the direct electron transfer (DET) principle is expected. In this paper, a fusion enzyme between an engineered L-lactate oxidase derived from Aerococcus viridans, AvLOx A96L/N212K mutant, which is minimized its oxidase activity and b-type cytochrome protein was constructed to realize multiplexed DET-type lactate and glucose sensors. The sensor with a fusion enzyme showed DET to a gold electrode, with a limited operational range less than 0.5 mM. A mutation was introduced into the fusion enzyme to increase K-m value and eliminate its substrate inhibition to construct b2LOxS. Together with the employment of an outer membrane, the detection range of the sensor with b2LOxS was expanded up to 10 mM. A simultaneous lactate and glucose monitoring system was constructed using a flexible thin-film multiplexed electrodes with b2LOxS and a DET-type glucose dehydrogenase, and evaluated their performance in the artificial sweat. The sensors achieved simultaneous detection of lactate and glucose without cross-talking error, with the detected linear ranges of 0.5-20 mM for lactate and 0.1-5 mM for glucose, sensitivities of 4.1 nA/mM.mm(2) for lactate and 56 nA/mM.mm(2) for glucose, and limit of detections of 0.41 mM for lactate and 0.057 mM for glucose. The impact of the presence of electrochemical interferants (ascorbic acid, acetaminophen and uric acid), was revealed to be negligible. This is the first report of the DET-type enzyme based lactate and glucose dual sensing systems.
  15 Mar. 2021, Research paper (scientific journal), joint, 176, 0956-5663, DOI(公開)(r-map)
• Rapid and homogeneous electrochemical detection by fabricating a high affinity bispecific antibody-enzyme complex using two Catcher/Tag systems
  Kimura, Hayato; Miura, Daimei; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji; Asano, Ryutaro
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Antibody-enzyme complexes (AECs) with binding ability to specific targets and catalytic activities to gain signals are known to be ideal sensing elements; however, AEC-based universal sensors applicable to point-of-care testing (POCT) have not yet been developed. Here, we achieved rapid and homogeneous electrochemical detection by fabricating a high-affinity bispecific AEC (bsAEC) using two Catcher/Tag systems. Recently, we reported a convenient and universal method to fabricate AECs using the SpyCatcher/SpyTag system. The resultant anti-epidermal growth factor receptor (anti-EGFR) AEC worked efficiently as a sensing element; however, the sensitivities did not meet the clinically required detection range of the soluble ectodomain of EGFR (sEGFR). To induce high affinity even to monomeric targets like sEGFR, we designed a convenient fabrication method for bsAEC using two Catcher/Tag systems, which did not express cross-reactivity. The anti-EGFR bsAEC was successfully prepared by constructing glucose dehydrogenase with two different catcher domains at the N- and C-terminus and by combining two corresponding Tag-fused anti-EGFR single-chain Fvs (scFvs), which recognize different epitopes on sEGFR. As expected, bsAEC showed a higher affinity than that of bivalent AEC with two identical anti-EGFR scFvs at low concentrations of sEGFR, and met the clinically required detection range of sEGFR. Further, by combining magnet beads, we established a rapid and wash-free homogeneous electrochemical detection method. This study offers new insights into the fabrication of universal POCT devices.
  01 Mar. 2021, Research paper (scientific journal), joint, 175, 0956-5663, DOI(公開)(r-map)
• A Green Light-Regulated T7 RNA Polymerase Gene Expression System for Cyanobacteria
  Shono, Chika; Ariyanti, Dwi; Abe, Koichi; Sakai, Yuta; Sakamoto, Ippei; Tsukakoshi, Kaori; Sode, Koji; Ikebukuro, Kazunori
  MARINE BIOTECHNOLOGY
  SPRINGER
  In this study, we developed a green light-regulated T7 RNA polymerase expression system (T7 RNAP system), to provide a novel and versatile high-expression system for cyanobacteria without using any chemical inducer, realizing high expression levels comparable with previously reported for recombinant gene expression in cyanobacteria. The T7 RNAP system was constructed and introduced intoSynechocystissp. PCC6803. T7 RNAP was inserted downstream of thecpcG2promoter, which is recognized and activated by the CcaS/CcaR two-component green-light-sensing system, to compose a vector plasmid, pKT-CS01, to achieve the induction of T7 RNAP expression only under green light illumination, with repression under red light illumination. The reporter gene, superfolder green fluorescent protein (sfGFP), was inserted downstream of theT7promoter. Transcriptional analyses revealed that T7 RNAP was induced under green light but repressed under red light. Expression of thesfGFP protein derived from pKT-CS01 was observed under green light illumination and was approximately 10-fold higher than that in the control transformant, which expressedsfGFP directly under thecpcG2promoter, which is directly regulated by CcaS/CcaR, under green light illumination. Comparison with the strong promoter expression systems P(cpc560)and P(trc Delta lacO)revealed that the expression ofsfGFP by the T7 RNAP system was comparable with the levels obtained with strong promoters. These results demonstrated that the green light-regulated T7 RNAP gene expression system will be a versatile tool for future technological platform to regulate gene expression in cyanobacterial bioprocesses.
  Feb. 2021, Research paper (scientific journal), joint, 23, 1, 1436-2228, DOI(公開)(r-map), 31, 38
• Monomeric G-Quadruplex-Based CpG Oligodeoxynucleotides as Potent Toll-Like Receptor 9 Agonists
  Tu, Anh Thi Tram; Hoshi, Kazuaki; Ikebukuro, Kazunori; Hanagata, Nobutaka; Yamazaki, Tomohiko
  BIOMACROMOLECULES
  AMER CHEMICAL SOC
  Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) motifs trigger the immune response by stimulating endosomal Toll-like receptor (TLR) 9. Natural linear ODNs are susceptible to nuclease degradation, thereby limiting their clinical applications. Here, we designed monomeric G-quadruplex-based CpG ODNs (G4 CpG ODNs) containing CpG motifs in the central loop region of the G4 structure. The monomeric G4 CpG ODNs were more stable in serum than the linear ODNs. The monomeric G4 CpG ODNs containing two or three CpG motifs induced the production of immunostimulatory cytokines interleukin (IL)-6, IL-12, and interferon (IFN)-beta in mouse macrophage-like RAW264 cells. We also showed that the number of CpG motifs and the number of nucleotides between the CpG motif and G-tracts define the efficacy of the G4 CpG ODNs in activating TLR9. Incubating human peripheral blood mononuclear cells with G4 CpG ODNs promoted IL-6 and IFN-gamma production, confirming their stimulatory effects on human immune cells. Mice given intraperitoneal injections of G4 CpG ODNs produced higher plasma IL-6 compared with injections of linear ODNs. These findings provide further understanding of the parameters governing the immunostimulatory activity of G4 CpG ODNs, thereby providing insights into the rational design of highly potent G4 CpG ODNs for vaccine adjuvants.
  Sep. 2020, Research paper (scientific journal), joint, 21, 9, 1525-7797, DOI(公開)(r-map), 3644, 3657
• Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF
  Lee, Jinhee; Tatsumi, Atsuro; Tsukakoshi, Kaori; Wilson, Ellie D.; Abe, Koichi; Sode, Koji; Ikebukuro, Kazunori
  SENSORS
  MDPI
  Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.
  Jul. 2020, Research paper (scientific journal), joint, 20, 14, DOI(公開)(r-map)
• Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors
  Fitriana, Maya; Loew, Noya; Witarto, Arief Budi; Ikebukuro, Kazunori; Sode, Koji; Tsugawa, Wakako
  SENSORS
  MDPI
  In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 +/- 0.12 mu A/mM) and wide linear range (0-50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 degrees C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.
  May 2020, Research paper (scientific journal), joint, 20, 10, DOI(公開)(r-map)
• Rational engineering of Aerococcus viridans L-lactate oxidase for the mediator modification to achieve quasi-direct electron transfer type lactate sensor
  Hiraka, Kentaro; Kojima, Katsuhiro; Tsugawa, Wakako; Asano, Ryutaro; Ikebukuro, Kazunori; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  The L-lactate oxidase (LOx) based lactate sensors are widely used for clinical diagnostics, sports medicine, and food quality control. However, dissolved oxygen interference and electroactive interferent effects are inherent issues of current lactate sensors. In this paper, a quasi-direct electron transfer (quasi-DET) type lactate sensor was developed using rationally engineered Aerococcus viridans LOx (AvLOx) modified with amine-reactive phenazine ethosulfate (PES). Since the modification of wild type AvLOx by PES did not result quasi-DET, engineered AvLOx with additional Lys residue was designed. The additional Lys residue was introduced by substituting residue locating on the surface of AvLOx, and within 20 angstrom of the isoalloxazine ring of FMN. Among several constructed mutants, Ala96Leu/Asn212Lys double mutant showed the highest dye-mediated dehydrogenase activity with negligible oxidase activity, showing quasi-DET properties after PES modification, when the enzyme was immobilized on screen printed carbon electrode. The constructed electrode did not show oxygen interference in cyclic voltammetric analysis and distinct catalytic current with 20 mM L-lactate. The sensor performance of a chronoamperometric L-lactate sensor employing PES modified Ala96Leu/Asn212Lys AvLOx, marked with linear range between 0 and 1 mM, with sensitivity of 13 mu A/mM.cm(2), and a limit of detection of 25 mu M for L-lactate. By applying -200 mV vs. Ag/AgCl, L-lactate could be monitored with negligible interference from 170 mu M ascorbic acid, 1.3 mM acetaminophen, 1.4 mM uric acid or 20 mM glucose. These results indicated that a quasi-DET type lactate sensor was developed that did not suffer from the interference of oxygen and representative electroactive ingredient compounds.
  01 Mar. 2020, Research paper (scientific journal), joint, 151, 0956-5663, DOI(公開)(r-map)
• Ethanol Detection at the Parts per Billion Level with Single-Stranded-DNA-Modified Graphene Field-Effect Transistors
  Nozaki, Ryo; Ikuta, Takashi; Ueno, Kinuko; Tsukakoshi, Kaori; Ikebukuro, Kazunori; Maehashi, Kenzo
  PHYSICA STATUS SOLIDI B-BASIC SOLID STATE PHYSICS
  WILEY-V C H VERLAG GMBH
  For realizing the early diagnosis of diseases, detection of gases in exhaled breath in parts per billion (ppb) using compact devices is necessary. Herein, single-stranded-DNA-modified graphene field-effect transistors (FETs) are fabricated and the transfer characteristics are measured. The results reveal that shifts in the transfer characteristics are obtained by introducing ethanol gas at the ppb level, indicating that single-stranded-DNA-modified graphene FETs detect the gas at the ppb level. Moreover, by modifying DNA sequence 1 or sequence 2, the positive and negative voltage shifts of transfer characteristics are observed, respectively. In contrast, the shifts are hardly obtained using DNA sequence 3, which has a rigid conformation for adsorption of molecules. These differences in the behaviors of the transfer characteristics are attributed to the change in DNA conformation when gas is adsorbed. Therefore, single-stranded-DNA-modified graphene FETs have considerable potential to detect various gases at a low concentration depending on the design of DNA sequences, enabling their promising applications in disease diagnosis.
  Feb. 2020, Research paper (scientific journal), joint, 257, 2, 0370-1972, DOI(公開)(r-map)
• G-Quadruplex Structure Improves the Immunostimulatory Effects of CpG Oligonucleotides
  Hoshi, Kazuaki; Yamazaki, Tomohiko; Sugiyama, Yuuki; Tsukakoshi, Kaori; Tsugawa, Wakako; Sode, Koji; Ikebukuro, Kazunori
  NUCLEIC ACID THERAPEUTICS
  MARY ANN LIEBERT, INC
  Single-strand oligodeoxynucleotides (ODNs) containing unmethylated cytosine-phosphate-guanine (CpG) are recognized by the toll-like receptor 9, a component of the innate immunity. Therefore, they could act as immunotherapeutic agents. Chemically modified CpG ODNs containing a phosphorothioate backbone instead of phosphodiester (PD) were developed as immunotherapeutic agents resistant to nuclease degradation. However, they cause adverse side effects, and so there is a necessity to generate novel CpG ODNs. In the present study, we designed a nuclease-resistant nonmodified CpG ODN that forms G-quadruplex structures. G-quadruplex formation in CpG ODNs increased nuclease resistance and cellular uptake. The CpG ODNs designed in this study induced interleukin-6 production in a human B lymphocyte cell line and human peripheral blood mononuclear cells. These results indicate that G-quadruplex formation can be used to increase the immunostimulatory activity of CpG ODNs having a natural PD backbone.
  01 Aug. 2019, Research paper (scientific journal), joint, 29, 4, 2159-3337, DOI(公開)(r-map), 224, 229
• Generation of C5-desoxy analogs of tetrahydroisoquinoline alkaloids exhibiting potent DNA alkylating ability
  Tanifuji, Ryo; Tsukakoshi, Kaori; Ikebukuro, Kazunori; Oikawa, Hideaki; Oguri, Hiroki
  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
  PERGAMON-ELSEVIER SCIENCE LTD
  C5-desoxy analogs of tetrahydroisoquinoline (THIQ) alkaloids were designed and synthesized as hitherto unexplored structural variants for evaluation of their DNA alkylating activities. While chemical synthesis of the C5-desoxy analogs bearing a phenolic hydroxyl group in the A-ring of the saframycins was assumed to be laborious based on semi-synthetic modifications, a chemo-enzymatic approach allowed for concise access to the analogs. The C5-desoxy analog 7 exhibited greater DNA alkylating ability with a wider tolerance for the sequence variations compared to cyanosafracin B. The C5-desoxy A-ring having a C8 phenolic hydroxyl group, and a C1 substituent in the vicinity of the C21 aminonitrile responsible for DNA alkylation, were demonstrated to play pivotal roles in the interaction between the THIQ alkaloids and DNA.
  15 Jul. 2019, Research paper (scientific journal), joint, 29, 14, 0960-894X, DOI(公開)(r-map), 1807, 1811
• Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole
  Iida, Keisuke; Tsushima, Yamato; Ma, Yue; Masoud, Shadi Sedghi; Sakuma, Mai; Yokoyama, Tomomi; Yoshida, Wataru; Ikebukuro, Kazunori; Nagasawa, Kazuo
  BIOORGANIC & MEDICINAL CHEMISTRY
  PERGAMON-ELSEVIER SCIENCE LTD
  G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.
  15 Apr. 2019, Research paper (scientific journal), joint, 27, 8, 0968-0896, DOI(公開)(r-map), 1742, 1746
• Anti-Idiotype DNA Aptamer Affinity Purification-High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab
  Yamada, Tomohiro; Saito, Taro; Shimizu, Yutaka; Tsukakoshi, Kaori; Hayashi, Hideki; Mizuno, Hajime; Tsuji, Daiki; Yamamoto, Keisuke; Itoh, Kunihiko; Toyo'oka, Toshimasa; Ikebukuro, Kazunori; Todoroki, Kenichiro
  MOLECULES
  MDPI
  This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification-high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 mu g/mL and showed good correlation coefficients (r(2) > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 mu g/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.
  01 Mar. 2019, Research paper (scientific journal), joint, 24, 5, 1420-3049, DOI(公開)(r-map)
• High-Throughput Bioanalysis of Bevacizumab in Human Plasma Based on Enzyme-Linked Aptamer Assay Using Anti-Idiotype DNA Aptamer
  Yamada, Tomohiro; Saito, Taro; Hill, Yoshia; Shimizu, Yutaka; Tsukakoshi, Kaori; Mizuno, Hajime; Hayashi, Hideki; Ikebukuro, Kazunori; Toyo'oka, Toshimasa; Todoroki, Kenichiro
  ANALYTICAL CHEMISTRY
  AMER CHEMICAL SOC
  We propose a highly selective, sensitive, accurate, and high throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader. The calibration curve of bevacizumab ranged from 0.05 to 5.0 mu g/mL, and showed a good correlation coefficient (r(2) = 1.000). The limit of detection was 2.09 ng/mL. We also demonstrated both the possibility of highly sensitive detection using luminol chemiluminescence and the repeated use of affinity plates. The proposed method is applicable for planning optimal therapeutic programs and for an evaluation of the biological equivalencies in the development of biosimilars.
  19 Feb. 2019, Research paper (scientific journal), joint, 91, 4, 0003-2700, DOI(公開)(r-map), 3125, 3130
• Development of a third-generation glucose sensor based on the open circuit potential for continuous glucose monitoring
  Lee, Inyoung; Loew, Noya; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Continuous glucose monitoring (CGM) systems are most important in the current Type I diabetes care and as component for the development of artificial pancreas systems because the amount of insulin being supplied is calculated based on the CGM results. Therefore, to stably and accurately control the blood glucose level, CGM should be stable and accurate for a long period. We have been engaged in the biomolecular engineering and application of FAD dependent glucose dehydrogenase complex (FADGDH) which is capable of direct electron transfer. In this study, we report the development of the third-generation type open circuit potential (OCP) principle-based glucose sensor with direct electron transfer FADGDH immobilized on gold electrodes using a self-assembled monolayer (SAM). We developed a novel algorithm for OCP-based glucose sensors. By employing this new algorithm, high reproducibility of measurement and sensor preparation were achieved. In addition, the signal was not affected by the presence of acetaminophen and ascorbic acid in the sample solution. The thus optimized third-generation OCP-based glucose sensor could be operated continuously for more than 9 days without significant change in the signal, sensitivity and dynamic range, indicating its potential application for CGM systems.
  15 Jan. 2019, Research paper (scientific journal), joint, 124, 0956-5663, DOI(公開)(r-map), 216, 223
• Designer fungus FAD glucose dehydrogenase capable of direct electron transfer
  Ito, Kohei; Okuda-Shimazaki, Junko; Mori, Kazushige; Kojima, Katsuhiro; Tsugawa, Wakako; Ikebukuro, Kazunori; Lin, Chi-En; La Belle, Jeffrey; Yoshida, Hiromi; Sode, Koji
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b-binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b-binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words)
  01 Jan. 2019, Research paper (scientific journal), joint, 123, 0956-5663, DOI(公開)(r-map), 114, 123
• Esterification of PQQ Enhances Blood-Brain Barrier Permeability and Inhibitory Activity against Amyloidogenic Protein Fibril Formation
  Tsukakoshi, Kaori; Yoshida, Wataru; Kobayashi, Masaki; Kobayashi, Natsuki; Kim, Jihoon; Kaku, Toshisuke; Iguchi, Toshitsugu; Nagasawa, Kazuo; Asano, Ryutaro; Ikebukuro, Kazunori; Sode, Koji
  ACS CHEMICAL NEUROSCIENCE
  AMER CHEMICAL SOC
  Several neurodegenerative diseases have a common pathophysiology where selective damage to neurons results from the accumulation of amyloid oligomer proteins formed via fibrilization. Considering that the formation of amyloid oligomers leads to cytotoxicity, the development of chemical compounds that are able to effectively cross the blood-brain barrier (BBB) and inhibit this conversion to oligomers and/or fibrils is essential for neurodegenerative disease therapy. We previously reported that pyrroloquinoline quinone (PQQ) prevented aggregation and fibrillation of alpha-synuclein, amyloid beta(1-42) (A beta(1-42)), and mouse prion protein. To develop a novel drug against neurodegenerative diseases based on PQQ, it is necessary to improve the insufficient BBB permeability of PQQ. Here, we show that an esterified compound of PQQ, PQQ-trimethylester (PQQ-TME), has twice the BBB permeability than PQQ in vitro. Moreover, PQQ-TME exhibited greater inhibitory activity against fibrillation of alpha-synuclein, A beta(1-42), and prion protein. These results indicated that esterification of PQQ could be a useful approach in developing a novel PQQ-based amyloid inhibitor.
  Dec. 2018, Research paper (scientific journal), joint, 9, 12, 1948-7193, DOI(公開)(r-map), 2898, 2903
• Riboregulator elements as tools to engineer gene expression in cyanobacteria
  Ueno, Kinuko; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  SPRINGER
  Cyanobacteria are an ideal host for biofuel production. Although efforts have been made to genetically engineer cyanobacteria for efficient production of biofuels and other important chemicals, the tools that can be applied to cyanobacteria are still limited. A new gene regulation tool, riboregulator, has been examined for application in cyanobacteria. A riboregulator is a nature-inspired RNA tool, which is composed of two artificially designed RNA fragments. Owing to its high specificity and efficacy, it is suitable for metabolic engineering in cyanobacteria, and several studies have been done to optimize and improve the function of the riboregulator. In this review, we focus on the recent improvements made to riboregulators and compare them with other RNA-mediated gene regulation tools developed in cyanobacteria to investigate future applications of riboregulators.
  Sep. 2018, Research paper (scientific journal), joint, 102, 18, 0175-7598, DOI(公開)(r-map), 7717, 7723
• Self-powered, wireless continuous glucose sensing system based direct electron transfer
  Lee, Inyoung; Loew, Noya; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji
  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
  AMER CHEMICAL SOC
  19 Aug. 2018, Research paper (scientific journal), joint, 256, 0065-7727, DOI(公開)(r-map)
• Development of an electrochemical detection system for CpG methylation of human genome using methyl CpG binding domain and zinc finger protein
  Lee, Jinhee; Yoshida, Wataru; Hiraoka, Daisuke; Tatsumi, Atsuro; Abe, Koichi; Nakabayashi, Kazuhiko; Wakeda, Hironobu; Hata, Kenichiro; Marquete, Christophe; Blum, Loic; Sode, Koji; Ikebukuro, Kazunori
  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
  AMER CHEMICAL SOC
  19 Aug. 2018, Research paper (scientific journal), joint, 256, 0065-7727, DOI(公開)(r-map)
• Smart aptamers forming G-quadruplex: Structural and functional change of aptamers forming G-quadruplex in response to surrounding conditions and its regulation with the ligands
  Tsukakoshi, Kaori; Nishio, Maui; Sasaki, Ikkei; Ma, Yue; Nagasawa, Kazuo; Kato, Yoshio; Nakamura, Chikashi; Sode, Koji; Ikebukuro, Kazunori
  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
  AMER CHEMICAL SOC
  19 Aug. 2018, Research paper (scientific journal), joint, 256, 0065-7727, DOI(公開)(r-map)
• Riboregulator elements as tools to engineer gene expression in cyanobacteria.
  Ueno, Kinuko; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  SPRINGER
  Cyanobacteria are one of the most attractive hosts for biofuel production; however, genetic approaches to regulate specific chromosomal genes in cyanobacteria remain limited. With the aim of developing a novel method to regulate chromosomal gene expression in cyanobacteria, we focused on riboregulatory technology. Riboregulators are composed of two RNA fragments whose interaction leads to target gene regulation with high specificity. In this study, we inserted a riboregulator sequence upstream of the chromosomal gene encoding AbrB-like transcriptional regulator, cyAbrB2, to investigate the utility of this tool. The inserted riboregulator was able to regulate cyabrB2 gene expression, with a high ON-OFF ratio up to approximately 50-fold. The transcription levels of several genes for which cyAbrB2 acts as a transcriptional upregulator were also decreased. Further, the cyAbrB2 expression-repressed mutant showed high glycogen accumulation, equivalent to that in the cyabrB2 deletion mutant (Delta cyabrB2). Phenotypic similarities between the cyabrB2 expression-repressed mutant and the Delta cyabrB2 mutant suggest that the riboregulator can potentially be used as a new chromosomal gene regulation tool in cyanobacteria.
  13 Jul. 2018, Research paper (scientific journal), joint, 102(18):, 18, DOI(公開)(r-map), 7717, 7723
• CpG Methylation Changes G-Quadruplex Structures Derived from Gene Promoters and Interaction with VEGF and SP1
  Tsukakoshi, Kaori; Saito, Shiori; Yoshida, Wataru; Goto, Shinichi; Ikebukuro, Kazunori
  MOLECULES
  MDPI
  G-quadruplex (G4) is a DNA/RNA conformation that consists of two or more G-tetrads resulting from four-guanine bases connected by Hoogsteen-type hydrogen bonds, which is often found in the telomeres of chromatin, as well as in the promoter regions of genes. The function of G4 in the genomic DNA is being elucidated and some G4-protein interactions have been reported; these are believed to play a role in vital cellular functions. In this study, we focused on CpG methylation, a well-known epigenetic modification of the genomic DNA, especially found in the promoter regions. Although many G4-forming sequences within the genomic DNA harbor CpG sites, the relationship between CpG methylation and the binding properties of associated proteins remains unclear. We demonstrated that the binding ability of vascular endothelial growth factor (VEGF) G4 DNA to VEGF165 protein was significantly decreased by CpG methylation. We identified the binding activity of G4 DNA oligonucleotides derived from gene promoter regions to SP1, a transcription factor that interacts with a G4-forming DNA and is also altered by CpG methylation. The effect of methylation on binding affinity was accompanied by changes in G4 structure and/or topology. Therefore, this study suggested that CpG methylation might be involved in protein binding to G4-forming DNA segments for purposes of transcriptional regulation.
  Apr. 2018, Research paper (scientific journal), joint, 23, 4, 1420-3049, DOI(公開)(r-map)
• Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand
  Yoshida, Wataru; Saikyo, Hiroki; Nakabayashi, Kazuhiko; Yoshioka, Hitomi; Bay, Daniyah Habiballah; Iida, Keisuke; Kawai, Tomoko; Hata, Kenichiro; Ikebukuros, Kazunori; Nagasawas, Kazuo; Karube, Isao
  SCIENTIFIC REPORTS
  NATURE PUBLISHING GROUP
  G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.
  15 Feb. 2018, Research paper (scientific journal), joint, 8, 2045-2322, DOI(公開)(r-map)
• Improving the induction fold of riboregulators for cyanobacteria,
  Sakamoto I, Abe K, Kawai S, Tsukakoshi K, Sakai Y, Sode K, Ikebukuro
  RNA Biol
  Feb. 2018, Research paper (scientific journal), joint, 1-6, doi: 10.1080/15476286.2017.1422470. [Epub ahead of print]
• Selection and Characterization of DNA Aptamers Against FokI Nuclease Domain.
  Nishio M, Yamagishi A, Tsukakoshi K, Kato Y, Nakamura C, Ikebukuro K.
  Methods Mol Biol.
  2018, Research paper (scientific journal), joint, 1867:, DOI(公開)(r-map), 165, 174
• Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production
  Hoshi, Kazuaki; Yamazaki, Tomohiko; Yoshikawa, C. Hiaki; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji
  INTERNATIONAL JOURNAL OF NANOMEDICINE
  DOVE MEDICAL PRESS LTD
  Background: Hemozoin, a chemical analog of a malarial pigment, is a crystal composed of heme dimers that can act as a potent Th1-type adjuvant, which strongly induces antibody production. However, the clinical applications of malarial hemozoin have limitations due to biosafety concerns and difficulties in the manufacturing process. Based on the premise that an analog of the heme polymer might display immunostimulatory effects, a hemin-containing polymer was developed as a novel immunostimulator. Materials and methods: To synthesize the copolymer containing hemin and N-isopropylacrylamide (NIPAM), this study employed a conventional radical polymerization method using 2,2'-azodiisobutyronitrile as the radical initiator; the synthesized copolymer was designated as NIPAM-hemin. Results: NIPAM-hemin was soluble and showed no cytotoxicity in vitro. The NIPAM-hemin copolymer induced the production of interferon (IFN)-gamma and interleukin (IL)-6 from peripheral blood mononuclear cells, although hemin and the NIPAM monomer individually did not induce the production of any cytokines. The production of IFN-gamma induced by NIPAM-hemin was independent of toll-like receptor 9 and the NLRP3 inflammasome pathway. Conclusion: Given that NIPAM-hemin induced IL-6 and IFN-gamma production in immune cells without any cytotoxic effects, NIPAM-hemin has potential therapeutic applications as a Th1-type adjuvant.
  2018, Research paper (scientific journal), joint, 13, 1178-2013, DOI(公開)(r-map), 4461, 4472
• Improving the induction fold of riboregulators for cyanobacteria
  Sakamoto, Ippei; Abe, Koichi; Kawai, Sumiya; Tsukakoshi, Kaori; Sakai, Yuta; Sode, Koji; Ikebukuro, Kazunori
  RNA BIOLOGY
  TAYLOR & FRANCIS INC
  Cyanobacteria are ideal cellular factories for biochemical production because of their ability to fix CO2 by photosynthesis and convert this molecule into biochemicals. Previously, we engineered a riboregulator that enables post-transcriptional gene regulation in the cyanobacterium Synechocystis sp. PCC 6803. Here, we improved the riboregulator by designing two RNA species, taRNA and crRNA, to enhance its induction fold. We inserted nucleotides into the crRNA loop to enhance intermolecular hybridization and successfully improved its induction fold. The engineered riboregulator exhibited a higher induction fold than the previously engineered riboregulator in both Escherichia coli and Synechocystis sp. PCC 6803. This improved riboregulator can be used to control gene expression over a wide dynamic range in cyanobacteria.
  2018, Research paper (scientific journal), joint, 15, 3, 1547-6286, DOI(公開)(r-map), 353, 358
• Development of aptamers against unpurified proteins
  Goto, Shinichi; Tsukakoshi, Kaori; Ikebukuro, Kazunori
  BIOTECHNOLOGY AND BIOENGINEERING
  WILEY
  SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples.
  Dec. 2017, Research paper (scientific journal), joint, 114, 12, 0006-3592, DOI(公開)(r-map), 2706, 2716
• Applying a riboregulator as a new chromosomal gene regulation tool for higher glycogen production in Synechocystis sp. PCC 6803,
  3. Ueno K, Sakai Y, Shono C, Sakamoto I, Tsukakoshi K, Hihara Y, Sode K, Ikebukuro K,
  Appl Microbiol Biotechnol
  Nov. 2017, Research paper (scientific journal), joint, 101, 23-24, 8465, 8474. doi: 10.1007/s00253-017-8570-4
• Development of HGF-binding aptamers with the combination of G4 promoter-derived aptamer selection and in silico maturation
  Yokoyama, Tomomi; Tsukakoshi, Kaori; Yoshida, Wataru; Saito, Taiki; Teramoto, Kentaro; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori
  BIOTECHNOLOGY AND BIOENGINEERING
  WILEY
  We describe the selection of aptamers based on bioinformatics-based approaches without Systematic Evolution of Ligands by EXponential enrichment (SELEX). SELEX is a potent method; however, it is time intensive and the PCR-amplification step, which is essential step for SELEX, leads to the loss of good aptamers. We have developed an aptamer-screening method, G4 promoter-derived aptamer selection (G4PAS), and an aptamer-improving method, in silico maturation (ISM). They are based on in silico sequence selection and computer assisted directed evolution, respectively. In this study, we succeeded in identifying new aptamers against hepatocyte growth factor (HGF) by G4PAS as well as improving the specificity of the HGF aptamers by ISM. Using ISM improved the specificity of the aptamer for HGF by up to 45-fold in comparison with the original aptamer. These methods enable easy and efficient identification of good aptamers, and the combination of G4PAS with ISM can thus serve as a potent approach for aptamer identification. Biotechnol. Bioeng. 2017;114: 2196-2203. (c) 2017 Wiley Periodicals, Inc.
  Oct. 2017, Research paper (scientific journal), joint, 114, 10, 0006-3592, DOI(公開)(r-map), 2196, 2203
• Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein
  Lee, Jinhee; Yoshida, Wataru; Abe, Koichi; Nakabayashi, Kazuhiko; Wakeda, Hironobu; Hata, Kenichiro; Marquette, Christophe A.; Blum, Loic J.; Sode, Koji; Ikebukuro, Kazunori
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 106 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3 h with a simple procedure.
  Jul. 2017, Research paper (international conference proceedings), joint, 93, 0956-5663, DOI(公開)(r-map), 118, 123
• DNA aptamers against FokI nuclease domain for genome editing applications
  Nishio, Maui; Matsumoto, Daisuke; Kato, Yoshio; Abe, Koichi; Lee, Jinhee; Tsukakoshi, Kaori; Yamagishi, Ayana; Nakamura, Chikashi; Ikebukuro, Kazunori
  BIOSENSORS & BIOELECTRONICS
  ELSEVIER ADVANCED TECHNOLOGY
  Genome editing with site-specific nucleases (SSNs) can modify only the target gene and may be effective for gene therapy. The main limitation of genome editing for clinical use is off-target effects; excess SSN5 in the cells and their longevity can contribute to off-target effects. Therefore, a controlled delivery system for SSN5 is necessary. Fold nuclease domain (FokI) is a common DNA cleavage domain in zinc finger nuclease (ZFN) and transcription activator-like effector nuclease. Previously, we reported a zinc finger protein delivery system that combined aptamer-fused, double-strand oligonucleotides and nanoneedles. Here, we report the development of DNA aptamers that bind to the target molecules, with high affinity and specificity to the FokI. DNA aptamers were selected in six rounds of systematic evolution of ligands by exponential enrichment. Aptamers F6#8 and #71, which showed high binding affinity to Fokl (K-d=82 nM, 74 nM each), showed resistance to nuclease activity itself and did not inhibit nuclease activity. We immobilized the ZFN-fused GFP to nanoneedles through these aptamers and inserted the nanoneedles into HEK293 cells. We observed the release of ZFN-fused GFP from the nanoneedles in the presence of cells. Therefore, these aptamers are useful for genome editing applications such as controlled delivery of SSNs.
  Jul. 2017, Research paper (international conference proceedings), joint, 93, 0956-5663, DOI(公開)(r-map), 26, 31
• , Development of HGF-binding aptamers with the combination of G4 promoter-derived aptamer selection and in silico maturation
  Yokoyama T, Tsukakoshi K, Yoshida W, Saito T, Teramoto K, Savory N, Abe K, Ikebukuro K
  Biotechnol Bioeng
  Jul. 2017, joint, 114, 10, 2196, 2203. doi: 10.1002/bit.26354.
• Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase
  Takano, Eri; Shimura, Nobuaki; Akiba, Takeshi; Kitayama, Yukiya; Sunayama, Hirobumi; Abe, Koichi; Ikebukuro, Kazunori; Takeuchi, Toshifumi
  MICROCHIMICA ACTA
  SPRINGER WIEN
  The authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis.
  Jun. 2017, Research paper (scientific journal), joint, 184, 6, 0026-3672, DOI(公開)(r-map), 1595, 1601
• Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands
  Bay, Daniyah H.; Busch, Annika; Lisdat, Fred; Iida, Keisuke; Ikebukuro, Kazunori; Nagasawa, Kazuo; Karube, Isao; Yoshida, Wataru
  BMC MOLECULAR BIOLOGY
  BIOMED CENTRAL LTD
  Background: G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. Results: When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex. Conclusion: Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.
  Jun. 2017, Research paper (scientific journal), joint, 18, 1471-2199, DOI(公開)(r-map)
• , Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase
  9. Takano E, Shimura N, Akiba T, Kitayama Y, Sunayama H, Abe K, Ikebukuro K, Takeuchi T
  Microchimica Acta
  Jun. 2017, joint, 184, 6, 1595, 1601.
• Detection of DNA methylation of G-quadruplex and i-motif-forming sequences by measuring the initial elongation efficiency of polymerase chain reaction
  Yoshida W, Yoshioka H, Bay DH, Iida K, Ikebukuro K, Nagasawa K, Karube I., ‘
  Anal Chem
  Sep. 2016, Research paper (scientific journal), joint, 88, 14, 7101, 7106.
• Structural regulation by a G-quadruplex ligand increases binding abilities of G-quadruplex-forming aptamers
  Tsukakoshi K, Ikuta Y, Abe K, Yoshida W, Iida K, Ma Y, Nagasawa K, Sode K, Ikebukuro K.,
  Chem Commun (Camb). 2016,
  Sep. 2016, Research paper (scientific journal), joint, 52, 85, 12646, 12649.
• Sensitive and Homogeneous Detection System with Aptamer-Based Biosensor,
  Tsukakoshi K, Ikebukuro K,
  Sens. Mater.
  Sep. 2016, joint, 28, 3, 1083, 1089.
• Methods for Improving Aptamer Binding Affinity, Molecules,
  Hasegawa H, Savory N, Abe K, Ikebukuro K.
  Molecules
  Mar. 2016, joint, 21, 4, E421.
• ATP-mediated Release of a DNA-binding Protein from a Silicon Nanoneedle Array
  1. Matsumoto, D, Nishio, M, Kato, Y, Yoshida, W, Abe, K, Fukazawa, K, Ishihara, K, Iwata, F, Ikebukuro, K, Nakamura, C,
  Electrochemistry
  Mar. 2016, Research paper (scientific journal), joint, 84, 5, 305, 307.
• アプタマーを用いた機能性電極
  李ジンヒ、池袋一典
  Electrochemistry
  Dec. 2015, joint, 2015, 12
• Inhibition of an allergen–antibody reaction related to Japanese cedar pollinosis using DNA sptamers sgainst the Cry j 2 allergen, 2015,
  Kazumasa Ogihara, Nasa Savory, Koichi Abe, Wataru Yoshida, Mitsuru Arakawa, Masahiko Asahi, Seika Kamohara, and Kazunori Ikebukuro,
  Nucleic acid therapeutics,
  Dec. 2015, Research paper (scientific journal), joint, 212, 57, 99, 105.
• DNA detection technology using zinc finger protein,
  Kumagai T., Abe K., Yoshida W., Ikebukuro K.,
  J. Microb. Biochem. Technol., in press
  Nov. 2015, joint, in press
• Inhibition of an allergen–antibody reaction related to Japanese cedar pollinosis using DNA sptamers sgainst the Cry j 2 allergen, 2015,
  K1. Saito T, Yoshida W, Yokoyama T, Abe K, Ikebukuro K, Identification of RNA Oligonucleotides Binding to Several Proteins from Potential G-Quadruplex Forming Regions in Transcribed Pre-mRNA,
  Molecules
  Nov. 2015, Research paper (scientific journal), joint, 20, 11, 20832, 20840.
• Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy
  Fukaya T, Abe K, Savory N, Tsukakoshi K, Yoshida W, Ferri S, Sode K, Ikebukuro K
  J Biotechnol.
  Oct. 2015, Research paper (scientific journal), joint, 212, 57, 99, 105.
• Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC 6803,
  Sakai Y, Abe K, Nakashima S, Ellinger JJ, Ferri S, Sode K, Ikebukuro K.
  Microbiologyopen
  Aug. 2015, Research paper (scientific journal), joint, 4, 4, 4881, 4884.
• Enzyme linking to DNA aptamers via a zinc finger as a bridge, 2015,
  Abe K, Murakami Y, Tatsumi A, Sumida K, Kezuka A, Fukaya T, Kumagai T, Osawa Y, Sode K, Ikebukuro K.,
  Chem Commun (Camb)
  Jul. 2015, Research paper (scientific journal), joint, 51, 57, 11467, 1149.
• Development of an automated direct blotting electrophoresis system for bioanalytical applications
  Goto S., Savory N , Abe K, Kinoshita H, Ikebukuro K.,
  Analytical Methods
  Jan. 2015, Research paper (scientific journal), joint, 7, 12, 4881, 4884.
• DNA aptamers against the Cry j 2 allergen of Japanese cedar pollen for biosensing applications.
  Ogihara K, Savory N, Abe K, Yoshida W, Asahi M, Kamohara S, Ikebukuro K
  Biosens Bioelectron
  Jan. 2015, Research paper (scientific journal), joint, 63, 159, 65
• Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX
  Savory N, Nzakizwanayo J, Abe K, Yoshida W, Ferri S, Dedi C, Jones BV, Ikebukuro K.
  J Microbiol Methods.
  Sep. 2014, Research paper (scientific journal), joint, 104, 2, 94, 100.
• The development of an autonomous self-powered bio-sensing actuator
  Hanashi, Takuya; Yamazaki, Tomohiko; Tanaka, Hiroshi, Ikebukuro, K, Tsugawa, W, Sode, K,
  Sensors and Actuators B-Chemical
  Jun. 2014, Research paper (scientific journal), joint, 196, 2, 429, 433
• Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein
  Lee, Jinhee, Tatsumi, Atsuro, Abe, Koichi, Yoshida, W . Sode, K, Ikebukuro, K, Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein, Abe K, Miyake K, Nakamura M, Kojima K, Ferri S, Ikebukuro K, Sode K. Abe K, Miyake K, Nakamura M, Kojima K, Ferri S, Ikebukuro K, Sode K
  Analytical Methods,
  Mar. 2014, Research paper (scientific journal), joint, 6, 14, 4991, 4994
• Electrochemical biosensors using aptamers for theranostics.
  Emerging techniques employed in aptamer-based diagnostic tests. Yoshida W, Abe K, Ikebukuro K
  Expert Rev Mol Diagn.
  Mar. 2014, joint, 14, 2, 143, 51
• Improving the gene-regulation ability of small RNAs by scaffold engineering in Escherichia coli.
  Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803. Abe K, Miyake K, Nakamura M, Kojima K, Ferri S, Ikebukuro K, Sode K. Abe K, Miyake K, Nakamura M, Kojima K, Ferri S, Ikebukuro K, Sode K
  Microb Biotechnol
  Mar. 2014, Research paper (scientific journal), joint, 7, 2, 177, 83
• Improving the gene-regulation ability of small RNAs by scaffold engineering in Escherichia coli.
  Sakai Y, Abe K, Nakashima S, Yoshida W, Ferri S, Sode K, Ikebukuro K.
  ACS Synth Biol
  Mar. 2014, Research paper (scientific journal), joint, 3, 3, 152, 62
• Simultaneous improvement of specificity and affinity of aptamers against Streptococcus mutans by in silico maturation for biosensor development
  10. Savory N, Takahashi Y, Tsukakoshi K, Hasegawa H, Takase M, Abe K, Yoshida W, Ferri S, Kumazawa S, Sode K, Ikebukuro K
  Biotechnol Bioeng
  Mar. 2014, Research paper (scientific journal), joint, 111, 3, 454, 6
• Vascular Endothelial Growth Factor (VEGF) Detection Using an Aptamer and PNA-Based Bound/Free Separation System
  Mita, Chifuku, Abe, Koichi; Fukaya, Takahiro, Ikebukuro K.,
  Materials
  Feb. 2014, Research paper (scientific journal), joint, 7, 2, 1046, 1054
• Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression
  Abe K, Sakai Y, Nakashima S, Araki M, Yoshida W, Sode K, Ikebukuro K
  Biotechnol Lett
  Feb. 2014, Research paper (scientific journal), joint, 36, 2, 287, 94
• Electrochemical biosensors using aptamers for theranostics.
  6. Abe K, Yoshida W, Ikebukuro K
  Adv Biochem Eng Biotechnol. 2014; Review
  Jan. 2014, joint, 140:, 183, 202
• Automatic polymerase chain reaction product detection system for food safety monitoring using zinc finger protein fused to luciferase
  Yoshida W, Kezuka A, Murakami Y, Lee J, Abe K, Motoki H, Matsuo T, Shimura N, Noda M, Igimi S, Ikebukuro K.
  Anal Chim Acta
  Nov. 2013, Research paper (scientific journal), joint, 801, 78, 83
• Fluorescent-ligand-mediated screening of G-quadruplex structures using a DNA microarray
  Iida, K. Takahiro Nakamura, Wataru Yoshida, Masayuki Tera, Kazuhiko Nakabayashi, Kenichiro Hata, Kazunori Ikebukuro,* and Kazuo Nagasawa*
  Angew. Chem. Int. Ed.(inside cover)
  Nov. 2013, Research paper (scientific journal), joint, 52, 46, 12052, 12055
• Two-dimensional electrophoresis-based selection of aptamers against an unidentified protein in a tissue sample
  Savory, Nasa, Goto, Shinichi, Yoshida, Wataru, Unuma, Y, Nakamura, M , Abe, K , Ferri, S, Ikebukuro, K
  Analytical letters
  Oct. 2013, Research paper (scientific journal), joint, 46, 18, 2954, 2963
• In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis
  Savory N, Lednor D, Tsukakoshi K, Abe K, Yoshida W, Ferri S, Jones BV, Ikebukuro K
  Biotechnol Bioeng
  Oct. 2013, Research paper (scientific journal), joint, 110, 10, 2573, 2580
• Screening of Peptide ligands for pyrroloquinoline quinone glucose dehydrogenase using antagonistic template-based biopanning
  Abe K, Yoshida W, Terada K, Yagi-Ishii Y, Ferri S, Ikebukuro K, Sode K.,
  Int J Mol Sci
  Aug. 2013, Research paper (scientific journal), joint, 14, 12, 23244, 56
• Detection of histone modification by chromatin immunoprecipitation combined zinc finger luciferase-based bioluminescence resonance energy transfer assay
  Yoshida W, Kezuka A, Abe K, Wakeda H, Nakabayashi K, Hata K, Ikebukuro K
  Anal Chem. 2013 Jul 2;
  Jul. 2013, Research paper (scientific journal), joint, 85, 13, 6485, 90
• An Optical Biosensing System Based on Interference-Enhanced Reflection with Aptameric Enzyme Subunits of Thrombin
  W. Yoshida and K. Ikebukuro
  Analytical letters
  Feb. 2013, Research paper (scientific journal), joint, 46, 2, 242, 249
• Rapid cytotoxicity screening platform for amyloid inhibitors using a membrane-potential sensitive fluorescent probe
  Kim J, Sasaki Y, Yoshida W, Kobayashi N, Veloso AJ, Kerman K, Ikebukuro K, Sode K.
  Anal Chem. 2013
  Jan. 2013, Research paper (scientific journal), joint, 85, 1, 185, 92
• Aptamer selection based on g4-forming promoter region
  Yoshida W, Saito T, Yokoyama T, Ferri S, Ikebukuro K. (2013)
  PLoS One
  Jan. 2013, Research paper (scientific journal), joint, 8, 6, e65497.
• Affinity improvement of a VEGF aptamer by in silico maturation for a sensitive VEGF-detection system
  15. Yoshihiko Nonaka, Wataru Yoshida, Koichi Abe, Stefano Ferri, Holger Schulze, Till T. Bachmann and Ikebukuro K
  Analytical Chemistry
  Jan. 2013, Research paper (scientific journal), joint, 85, 2, 1132, 1137
• アミロイド形成蛋白質に結合する核酸リガンド，DNAアプタマーの開発の現状：アミロイドオリゴマー結合アプタマーの同定
  塚越かおり、阿部公一、早出広司、池袋一典
  Dementia Japan
  Sep. 2012, joint, 26, 3
• Selection of DNA aptamers that recognize α-synuclein oligomer using a competitive screening method,
  Kaori Tsukakoshi, Koichi Abe, Koji Sode, and Kazunori Ikebukuro
  Analytical Chemistry
  Jul. 2012, Research paper (scientific journal), joint, 84, 13, 5542, 5547
• BioLC-Oscillator: A Self-Powered Wireless Glucose-Sensing System with the Glucose Dependent Resonance Frequency
  Takuya Hanash, Tomohiko Yamazaki, Wakako Tsugawa, Kazunori Ikebukuro, Koji Sode
  Electrochemistry
  May 2012, Research paper (scientific journal), joint, 80, 5, 367, 370
• Electrochemical SNP Detection Using Glucose Dehydrogenase,
  Koichi Abe, Chiharu Kiyohara, Yoko Saito, Koji Sode, Kazunori Ikebukuro,
  Electrochemistry
  May 2012, Research paper (scientific journal), joint, 80, 5, 345, 347
• Electrochemical Detection of Vascular Endothelial Growth Factor by an Aptamer-Based Bound/Free Separation System,
  Koichi Abe, Hijiri Hasegawa, Kazunori Ikebukuro,
  Electrochemistry
  May 2012, Research paper (scientific journal), joint, 80, 5, 348, 352
• Electrochemical Detection of Vascular Endothelial Growth Factor with Aptamer Sandwich
  Yoshihiko Nonaka, Koichi Abe and Kazunori Ikebukuro,
  Electrochemistry
  May 2012, Research paper (scientific journal), joint, 80, 5, 363, 366
• BioRadioTransmitter: a self-powered wireless glucose-sensing system
  Takuya Hanash, Tomohiko Yamazaki, Wakako Tsugawa, Kazunori Ikebukuro, Koji Sode
  J. Diabetes Sci. Technol.
  Sep. 2011, Research paper (scientific journal), joint, 5, 5, 1030, 1035
• Control of aptamer function using radiofrequency magnetic field,
  Taira K, Abe K, Ishibasi T, Sato K, Ikebukuro K.,
  J Nucleic Acids. 2011;2011:
  Sep. 2011, Research paper (scientific journal), joint, 2011, 103872.
• Development of a novel biosensing system based on the structural change of a polymerized guanine-quadruplex DNA nanostructure,
  Morita Y, Yoshida W, Savory N, Han SW, Tera M, Nagasawa K, Nakamura C, Sode K, Kazunori Ikebukuro (2011)
  Biosens. Bioelectron., 26(12)
  Aug. 2011, Research paper (scientific journal), joint, 26, 12, 4837, 41
• Analysis of the unbinding force between telomestatin derivatives and human telomeric G-quadruplex by atomic force microscopy,
  Amemiya Y, Furunaga Y, Iida K, Tera M, Nagasawa K, Ikebukuro K, Nakamura C.
  Chem Commun (Camb)
  Jul. 2011, Research paper (scientific journal), joint, 47, 26, 7485, 7
• Aptameric sensors based on structural change for diagnosis
  Koichi Abe, Daisuke Ogasawara, Wataru Yoshida, Koji Sode, Kazunori Ikebukuro
  Faraday Discuss.
  Feb. 2011, Research paper (scientific journal), joint, 149, 93, 105
• A Non-label homogeneous protein detection based on laser interferometric photo-thermal displacement measurement using aptamers,
  Kaori Tsukakoshi, Daisuke Ogasawara, Eiji Takahashi, Ryo Katayama, Kazunori Ikebukuro
  Biotechnology Journal,10
  Jan. 2011, Research paper (scientific journal), joint, 6, 1, 101, 116.
• Selection of DNA aptamer against prostate specific antigen using a genetic algorithm and application to sensing,
  Nasa Savory, Koichi Abe, Koji Sode, Kazunori Ikebukuro
  Biosens. Bioelectron.
  May 2010, Research paper (scientific journal), joint, 26, 4, 1386, 1391.
• Constructing an improved pyrroloquinoline quinone glucose dehydrogenase binding aptamer for enzyme labeling
  Koichi Abe, Koji Sode, Kazunori Ikebukuro
  Biotechnol. Lett.
  May 2010, Research paper (scientific journal), joint, 32, 9, 1293, 1298.
• Visualization of G-quadruplexes by using a BODIPY-labeled macrocyclic heptaoxazole
  Masayuki Tera, Iida K, Ikebukuro K, Seimiya H, Shin-Ya K, Nagasawa K
  Org Biomol Chem,
  Feb. 2010, Research paper (scientific journal), joint, 8, 12, 2749, 2755
• Screening of DNA aptamer which binds to alpha-synuclein
  Kaori Tsukakoshi Ryuichi Harada, Koji Sode and Kazunori Ikebukuro
  Biotechnol. Lett.
  Feb. 2010, Research paper (scientific journal), joint, 32, 5, 643, 648.
• Screening and improvement of an anti-VEGF DNA aptamer,
  Yoshihiko Nonaka, Koji Sode and Kazunori Ikebukuro
  Molecule
  Feb. 2010, Research paper (scientific journal), joint, 15, 1, DOI(公開)(r-map), 215, 225
• Pyrroloquinoline quinone inhibits the fibrillation of amyloid proteins
  3. Jihoon Kim, Masaki Kobayashi, Makoto Fukuda, Daisuke Ogasawara, Natsuki Kobayashi, Sungwoong Han, Chikashi Nakamura, Masaki Inada, Chisato Miyaura, Kazunori Ikebukuro and Koji Sode
  Prion
  Feb. 2010, Research paper (scientific journal), joint, 4, 1, DOI(公開)(r-map), Epub ahead of print
• The effect of amino acid substitution in the imperfect repeat sequences of alpha-synuclein on fibrillation
  Ryuichi Haradaa, Natsuki Kobayashia, Jihoon Kima, Chikashi Nakamurab, Sung-Woong Hanb, Kazunori Ikebukuroa and Koji Sode (2009)
  Biochim Biophys Acta
  Oct. 2009, Research paper (scientific journal), joint, 1792, 10, DOI(公開)(r-map), 998, 1003.
• Kinetic mechanism and inhibitor characterization of WNK1 kinase
  Yukiko I. Yagi*, Koichi Abe, Kazunori Ikebukuro and Koji Sode
  Biochemistry
  Oct. 2009, Research paper (scientific journal), joint, 48, 43, DOI(公開)(r-map), 10255, 10266.
• Zn finger-based direct detection system for PCR products of Salmonella spp. and the influenza A virus
  Yuko Osawa, Hiroaki Motoki, Takafumi Matsuo, Michio Horiuchi, Koji Sode, Kazunori Ikebukuro
  Biotechnolgy Leterrs
  Feb. 2009, Research paper (scientific journal), joint, 31, 5, 725, 733
• An Aptamer-Based Bound/Free Separation System for Protein Detection, Electroanalysis
  Mieko Fukasawa, Wataru Yoshida, Hiroki Yamazaki, Koji Sode, and Kazunori Ikebukuro,
  Electroanalysis
  Jan. 2009, Research paper (scientific journal), joint, 21, 11, DOI(公開)(r-map), 1297, 1302.
• DNA aptamers that bind to PQQGDH as an electrochemical labeling tool
  Yuko Osawa, Madoka Takase, Koji Sode, Kazunori Ikebukuro
  Electroanalysis
  Jan. 2009, Research paper (scientific journal), joint, 21(11),, 11, DOI(公開)(r-map), 1303, 1308.
• BioCapacitor-A novel category of biosensor.
  Hanashi T, Yamazaki T, Tsugawa W, Ferri S, Nakayama D, Tomiyama M, Ikebukuro K, Sode K
  Biosensors & Bioelectronics
  Nov. 2008, Research paper (scientific journal), joint, E pub ahead of print, DOI(公開)(r-map)
• コンピューター内進化を用いたアプタマーの改良
  池袋一典
  日本化学会情報化学部会誌「CICSJ Bulletin」
  Oct. 2008, only, 26, 3, 5, 8
• Zinc finger protein-based detection system of PCR products for pathogen diagnosis
  Yuko Osawa, Hiroaki Motoki, Takafumi Matsuo, Michio Horiuchi, Koji Sode, Kazunori Ikebukuro
  Nucleic Acids Symp Ser (Oxf
  Sep. 2008, Research paper (scientific journal), joint, 2008, 52, 23, 24
• Detection system based on the conformational change in an aptamer and its application to simple bound/free separation
  Ogasawara D, Hachiya NS, Kaneko K, Sode K, Ikebukuro K
  Biosensors & Bioelectronics
  Sep. 2008, Research paper (scientific journal), joint, E pub ahead of print, DOI(公開)(r-map)
• Aggregation and fibrillation study of alpha-synuclein under applied voltage
  Osawa, Yuko, Ikebukuro, Kazunori, Kobayashi, Natsuki, Han, SungWoong, Nakamura, Chikashi, Sode, Koji
  ELECTROCHEMISTRY
  Aug. 2008, Research paper (scientific journal), joint, 76, 8, 614, 618
• Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing
  Wataru Yoshida, Eriko Mochizuki, Hiroki, Yamazaki, Koji Sode, Kazunori Ikebukuro
  Biosensors & Bioelectronics
  Jun. 2008, Research paper (scientific journal), joint, E pub ahead of print, DOI(公開)(r-map)
• The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins
  Yuko Osawa, Kazunori Ikebukuro, Hiroaki Motoki, Takafumi Matsuo, Michio Horiuchi, Koji Sode
  Nucleic acids research
  Apr. 2008, Research paper (scientific journal), joint, 36, 11, e68
• Improvement of aptamer affinity by dimerization
  Hijiri Hasegawa, Ken-Ichi Taira, Koji Sode, Kazunori Ikebukuro
  Sensors
  Feb. 2008, Research paper (scientific journal), joint, 8, 5, 1090, 1098
• Selection of DNA aptamers against VEGF(165) using a protein competitor and the aptamer blotting method
  Hijiri Hasegawa, Koji Sode, Kazunori Ikebukuro
  Biotechnology Letters
  Jan. 2008, Research paper (scientific journal), joint, 30, 5, 829, 834
• Screening of DNA aptamer against mouse prion protein by competitive selection
  Daisuke Ogasawara, Hijiri Hasegawa, Kiyotoshi Kaneko, Koji Sode, and Kazunori Ikebukuro
  Prion
  Dec. 2007, Research paper (scientific journal), joint, 1, 4, 248, 254
• Selection of DNA aptamers that inhibit enzymatic activity of PQQGDH and its application
  Kazunori Ikebukuro, Madoka Takase, and Koji Sode
  Nucleic Acids Symp Ser,
  Nov. 2007, Research paper (scientific journal), joint, 51:, 403, 404.
• Construction of target molecule sensing system using aptameric enzyme subunit based on PQQGDH activity
  Kazunori Ikebukuro, Yo Morita, Wataru Yoshida, and Koji Sode
  Nucleic Acids Symp Ser,
  Nov. 2007, Research paper (scientific journal), joint, 51:, 401, 402.
• Selection and characterization of DNA aptamers against VEGF165 with aptamer blotting method and its application
  Kazunori Ikebukuro, Hijiri Hasegawa, and Koji Sode
  Nucleic Acids Symp Ser,
  Nov. 2007, Research paper (scientific journal), joint, 51:, 399, 400.
• Specific detection of PCR product from Legionella pneumophila strainPhiladelphia1 using zinc finger protein Sp2
  Kazunori Ikebukuro, Takenori Kumagai, Hiroaki Motoki, Yuko Osawa, Takafumi Matuo, Michio Horiuchi, and Koji Sode
  Nucleic Acids Symp Ser,
  Nov. 2007, Research paper (scientific journal), joint, 51:, 285, 286
• Aptameric enzyme subunit for homogeneous protein sensing
  Wataru Yoshida, Koji Sode, and Kazunori Ikebukuro
  Nucleic Acids Symp Ser
  Nov. 2007, Research paper (scientific journal), joint, 51:, 99, 100.
• Peptide ligand screening of alpha-synuclein aggregation modulators by in silico panning
  Koichi Abe, Natsuki Kobayashi, Koji Sode and Kazunori Ikebukuro
  BMC bioinformatics
  Nov. 2007, Research paper (scientific journal), joint, 8, 45
• Label free homogeneous detection of IgE by aptameric enzyme subunit
  Wataru Yoshida, Kazunori Ikebukuro and Koji Sode
  Biotechnology Letters
  Nov. 2007, Research paper (scientific journal), joint, 30, 3, 829, 834
• Aptameric enzyme subunit for homogenious DNA sensing
  Kazunori Ikebukuro, Wataru Yoshida and Koji Sode
  Biotechnology Letters
  Oct. 2007, Research paper (scientific journal), joint, 30, 2, 243, 252
• Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples
  Chee GJ, Nomura Y, Ikebukuro K, Karube I.
  Biosens Bioelectron.
  Jan. 2007, Research paper (scientific journal), joint, 22, 2, 3092, 3098
• In silico panning for a non-competitive peptide inhibitor
  Yukiko Yagi , Kotaro Terada, Takahisa Noma , Kazunori Ikebukuro and Koji Sode
  BMC Bioinformatics
  Jan. 2007, Research paper (scientific journal), joint, 8, 1, 11
• Pyrroloquinoline quinone (PQQ) prevents fibril formation of alpha-synuclein
  Biochemical Biophysical Research Communication
  Oct. 2006, Research paper (scientific journal), joint, 349, 3, 1139, 1144
• Analysis of DNA and zinc finger interactions using mechanical force spectroscopy
  Yanyan Wang, Shin-ichiro Oyokawa, SungWoong Han, Wei Huang, Kazunori Ikebukuro, Chikashi Nakamura and Jun Miyake
  NanoBiotechnology
  Sep. 2006, Research paper (scientific journal), joint, 2, 3-4, 87, 94
• Homogeneous DNA sensing using enzyme-inhibiting DNA aptamers
  1. Wataru Yoshida, Koji Sode and Kazunori Ikebukuro
  Biochemical and Biophysical Research Communications
  Sep. 2006, Research paper (scientific journal), joint, 348, 1, 245, 252
• Analysis of the evolution of the thrombin-inhibiting DNA aptamers using a genetic algorithm
  Kazunori Ikebukuro, Wataru Yoshida, Takahisa Noma and Koji Sode
  Biotechnology Letters
  Sep. 2006, Research paper (scientific journal), joint, 28, 23, 1933, 1937
• Characterization and application of aptamers for Taq DNA polymerase selected using an evolution-mimicking algorithm
  Takahisa Noma, Koji Sode
  Biotechnology Letters
  Sep. 2006, Research paper (scientific journal), joint, 28, 23, 1939, 1944
• Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm
  Takahisa Noma
  Biochemical and Biophysical Research Communications
  Jul. 2006, Research paper (scientific journal), joint, 347, 1, 226, 231
• A screening method of DNA aptamers that bind to a specific protein in tissue,
  Takahisa Noma, Kazunori Ikebukuro, Koji Sode, Takuya Ohkubo, Yuji Sakasegawa, Naomi Hachiya and Kiyotoshi Kaneko
  Biotechnology Letters
  May 2006, Research paper (scientific journal), joint, 28, 17, 1377, 1381
• Aptameric enzyme subunit for biosensing based on enzymatic activity measurement,
  Wataru Yoshida, Koji Sode and Kazunori Ikebukuro
  Analytical Chemistry
  May 2006, Research paper (scientific journal), joint, 78, 10, 3296, 3303
• Development of a novel sensing probe using DNA aptamer inhibiting enzymatic activity
  Kazunori Ikebukuro, Wataru Yoshida, and Koji Sode
  Nucleic Acids Symp Ser (Oxf)
  Nov. 2005, Research paper (scientific journal), joint, 2005, 49, 83, 83
• Screening of DNA aptamers against multiple proteins in tissue
  14. Takahisa Noma, Kazunori Ikebukuro, Koji Sode, Takuya Ohkubo, Yuji Sakasegawa, Naomi Hachiya and Kiyotoshi Kaneko
  Nucleic Acids Symp Ser (Oxf)
  Nov. 2005, Research paper (scientific journal), joint, 2005, 49, 357, 358
• A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm
  Kazunori Ikebukuro, Yuji Okumura, Koichi Sumikura and Isao Karube
  Nucleic Acids Research
  Aug. 2005, Research paper (scientific journal), joint, 33, 12, e108
• Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand. ., 21(1), .
  Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro and Isao Karube
  Biosens Bioelectron
  Jun. 2005, Research paper (scientific journal), joint, 21, 7, 67, 73
• Development of a novel glucose enzyme fuel cell system employing protein engineered PQQ glucose dehydrogenase. .
  Noriko Yuhashi, Masamitsu Tomiyama, Junko Okuda, Satoshi Igarashi, Kazunori Ikebukuro, and Sode, Koji
  Biosens Bioelectron
  May 2005, Research paper (scientific journal), joint, 20, 10, 2145, 50
• Novel electrochemical sensor system for protein using the aptamers in sandwich manner
  Kazunori Ikebukuro, Chiharu Kiyohara and Koji Sode
  Biosens Bioelectron
  Feb. 2005, Research paper (scientific journal), joint, 20, 10, 2168, 2172
• アプタマーを分子認識素子とするバイオセンサの開発」
  池袋一典
  次世代センサ14号2巻、p5-8、 (2005)
  Jan. 2005, Research paper (scientific journal), only, 14, 2, 5, 8
• Electrochemical Detection of Protein Using a Double Aptamer Sandwich
  Kazunori Ikebukuro, Chiharu Kiyohara and Koji Sode
  Analytical Letters
  Dec. 2004, Research paper (scientific journal), joint, 37, 14, 2901, 2909
• Selection of DNA aptamers that inhibit Taq DNA polymerase by an algorithm mimicking evolution.
  Takahisa Noma, Kazunori Ikebukuro and Koji Sode
  Nippon Kessho Seicho Gakkaishi
  Dec. 2004, Research paper (scientific journal), joint, 31, 3, 190
• Development of Salmonella gyrB PCR product detection method using Zn finger protein
  Tomohiko Yabata, Kazunori Ikebukuro and Koji Sode
  Nippon Kessho Seicho Gakkaishi
  Dec. 2004, Research paper (scientific journal), joint, 31, 3, 189
• Development of a novel DNA sensing system using DNA aptamer inhibited enzymatic activity 1
  Kazunori Ikebukuro, Wataru Yoshida and Koji Sode
  Nucleic Acids Symp Ser (Oxf),
  Nov. 2004, Research paper (scientific journal), joint, 2004, 48, 231, 232
• Development of a novel DNA sensing system using DNA aptamer that inhibits enzymatic activity 2
  Kazunori Ikebukuro, Wataru Yoshida, Takahisa Noma and Koji Sode
  Nucleic Acids Symp Ser (Oxf)
  Nov. 2004, Research paper (scientific journal), joint, 2004, 48, 309, 310
• 英語学術論文　 その他31報　合計103報 英語著書　合計４編
  Oct. 2004, only
• Molecular engineering of PQQGDH and its applications
  Satoshi Igarashi, Junko Okuda, Kazunori Ikebukuro and Koji Sode
  Archives of Biochemistry and Biophysics
  Sep. 2004, Research paper (scientific journal), joint, 428, 1, 52, 63
• Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique,
  Hirotaka Miyachi, Kazunori Ikebukuro, Kazuyoshi Yano, Hiroyuki Aburatani and Isao Karube
  Biosens. Bioelectron.
  Jul. 2004, Research paper (scientific journal), joint, 20, 2, 184, 189
• Development of an enzymatic flow-injection chemiluminescence system for determining inorganic pyrophosphate ion,
  Hideaki Nakamura, Ruriko Yamazaki, Takayuki Shirai, Hisae Sano, Yu Nakami, Kazunori Ikebukuro, Kazuyoshi Yano, Yoko Nomura, Yasushi Hasebe, Yoshiko Arikawa, Yuzo Masuda, and Isao Karube
  ANALYTICA CHIMICA ACTA
  Mar. 2004, Research paper (scientific journal), joint, 518, 45, 49.
• PCR-Based Ribosomal DNA Detection Technique for Microalga (Heterosigma carterae) Causing Red Tide and Its Application to a Biosensor Using Labeled Probe,
  Ryoichi Asai, Kozue Ootani, Yoko Nomura, Chikashi Nakamura, Kazunori Ikebukuro, , Yoshiko Arikawa, Jun Miyake and Isao Karube
  Marine Biotechnology.
  Nov. 2003, Research paper (scientific journal), joint, 5, 5, 417, 423
• Screening of DNA aptamers inhibiting Taq DNA polymerase using algorithm mimicking evolution,
  Kazunori Ikebukuro and Noma, Takahisa
  Nucleic Acids Research Supplement 3(3rd International Symposium on Nucleic Acids Chemistry [and] 30th Symposium on Nucleic Acids Chemistry in Japan, 2003),
  Nov. 2003, Research paper (scientific journal), only, 2003, 309, 310.
• Surface plasmon resonance probe with a novel integrated reference sensor surface,
  Takuo Akimoto, Kazunori Ikebukuro and Isao Karube
  Biosensors & Bioelectronics, 18(12), .
  Oct. 2003, Research paper (scientific journal), joint, 18, 12, 1447, 1453
• A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine,
  8. Ryoichi Asai, Keijoro Nakanishi, Chikashi Nakamura, Jun Miyake, Isao Karube
  Phycological Research,
  Oct. 2003, Research paper (scientific journal), joint, 51, 2, 118, 125
• Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance
  Kazuyoshi Yano, Masato Shionoya, Tetsuya Yoshino, Shinichi Y. Sawata, Kazunori Ikebukuro and Isao Karube
  Biosensors & Bioelectronics, 18(10), 1201-1207
  Oct. 2003, Research paper (scientific journal), joint, 18, 10, 1201, 1207
• Improvement of a CL-FIA system using maltose phosphorylase for the detemination of phosphate-ion in fresh water
  Analytical Letters,
  Sep. 2003, Research paper (scientific journal), joint, 36, 9, 1805, 1817
• A bioassay to detect contaminant-induced messenger RNA using a transcriptomic approach: Detection of RT-PCR-amplified single-stranded DNA based on the SPR sensor in cyanobacteria
  Analytical Letters, 36(8), 1475-1491.
  Aug. 2003, Research paper (scientific journal), joint, 36, 8, 1475, 1491
• (2003) Novel strategy for DNA aptamers inhibiting enzymatic activity using algorithm mimicking evolution,
  Kazunori Ikebukuro, Yuji Okumura, Koichi Sumikura and Isao Karube
  Nucleic Acids Research Supplement 3(3rd International Symposium on Nucleic Acids Chemistry [and] 30th Symposium on Nucleic Acids Chemistry in Japan, 2003)
  Jul. 2003, Research paper (scientific journal), joint, 2003, 205, 206
• Amperometric glucose sensor using thermostable co-factor binding glucose dehydrogenase
  Yukie Nakazawa, Tomohiko Yamazaki, Wakako Tsugawa, Kazunori Ikebukuro and Koji Sode
  IEEJ Transactions on Sensors and Micromachinines, 123(6), 185-189
  Jun. 2003, joint
• Electrochemical DNA sensor using genetically engineered thermostable pyrroloquinoline quinone glucose dehydrogenase
  Kazunori Ikebukuro, Yoko Saito, Satoshi Igarashi and Koji Sode
  Electrochemistry, 71(6), 490-495.
  Jun. 2003, joint
• Molecular engineering of PQQGDH and its applications,
  Satoshi Igarashi, Junko Okuda, Kazunori Ikebukuro and Koji Sode
  Arch Biochem Biophys
  May 2003, Research paper (scientific journal), joint, 428, 1, 52, 63
• Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film
  Kojima K, Hiratsuka A, Suzuki H, Yano K, Ikebukuro K, Karube
  Analytical Chemistry
  May 2003, Research paper (scientific journal), joint, 75, 5, 1116, 1122
• Development of a reactor type bio-sensor for trichloroethylene
  Tae-Sung Han, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Kitayama Atsushi, Teruyuki Nagamune and Isao Karube
  Analytical Letters 36(3), 539-547.
  Feb. 2003, joint
• Exploration of structural features of monomeric helical peptides designed with a genetic algorithm
  9. Wuming. Zhang, Michael. G. Loughran, Shu-ichi. Kanna, Kazuyoshi. Yano, Kazunori. Ikebukuro, Yohei Yokobayashi, Ryoko Kuroda and Isao Karube
  Proteins: Structure, Function, and Genetics, 53(2), 193-200.
  Jan. 2003, Research paper (scientific journal), joint, 53, 2, 193, 200.
• コンピューター内進化を利用した酵素阻害DNAアプタマーの探索
  化学と教育、51号1巻、p26-27
  Jan. 2003, only
• Improved substrate specificity of water-soluble pyrroloquinoline quinone glucose dehydrogenase by a peptide ligand
  Hiromi Yoshida, Yukiko Yagi, Kazunori Ikebukuro and Koji Sode
  Biotechnology Letters, 25(4), 301-305.
  Jan. 2003, joint
• Flow injection microbial trichloroethylene sensor
  Tae-Song Han, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Atsushi Kitayama, Teruyuki Nagamune, Isao Karube
  Talanta, 57(2), 271-276.
  Dec. 2002, joint
• A simple nitrate sensor system using titanium trichloride and an ammonium electrode
  Seung-Jin Cho, Satoshi Sasaki, Kazunori Ikebukuro, Isao Karube
  Sensors and Actuators, B: Chemical, 85(1-2), 120-125.
  Aug. 2002, joint
• A flow method with photocatalytic oxidation of dissolved organic matter using a solid-phase (TiO₂) reactor followed by amperometric detection of consumed oxygen
  Yoon-Chang Kim, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Kazuhiko Hashimoto, Isao Karube
  Analytical Chemistry, 74(15), 3858-3864.
  Feb. 2002, Research paper (scientific journal), joint
• Detection technique of asymmetric RT-PCR-based amplified single-stranded DNA and its application to biosensor for detection of mRNA for cyanobacteria, Anabaena variabilis
  Roichi Asai, Chikashi Nakamura, Kazunori Ikebukuro, Isao Karube and Jun Miyake
  Biotechnology Letters, 24(20), 1677-1682.
  Feb. 2002, joint
• Amperometric DNA sensor using the pyrroquinoline quinone glucose dehydrogenase ? avidin conjugate
  Kazunori Ikebukuro, Yumiko Kohiki and Koji Sode
  Biosensors and Bioelectronics
  Jan. 2002, joint, 17, 11-12, 1075, 1080
• Detection of phycobilin pigments and their seasonal change in Lake Kasumigaura using a sensitive in situ fluorometric sensor
  Asai R, Horiguchi Y, Yoshida A, McNiven S, Tahira P, Ikebukuro K, Uchiyama S, Masuda Y, Karube I
  Analytical Letters
  Nov. 2001, Research paper (scientific journal), joint, 34, 14, 2521, 2533
• Dioxin detection based on immunoassay using a polyclonal antibody against octa-chloricated dibenzo-p-dioxin (OCDD)
  Mifumi shimomura, Yoko Nomura, Kyong-Hoon Lee, Kazunori Ikebukuro and Isao Karube
  Analyst,
  Nov. 2001, joint, 126, 1207, 1209.
• Microbial sensor for trichloroethylene determination
  Tae-Song Han, Yoon-Chang Kim, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Atsushi Kitayama, Teruyuki Nagamune, Isao Karube
  Analytica Chimica Acta, 431(2), 225-230.
  Aug. 2001, joint, 431, 2, 225, 230
• Simple and rapid detection method using surface plasmon resonance for dioxins, polycholorinated biphenylx and atrazine
  Mifumi shimomura, Yoko Nomura, Wei Zhang, Masato Sakino, Kyong-Hoon Lee, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta,
  Jul. 2001, joint, 434, 223, 230
• A fluorescent nitrate sensing system using a reaction cartridge and titanium trichloride
  Seung-Jin Cho, Satoshi Sasaki, Kazunori Ikebukuro, Isao Karube
  Talanta,
  Jun. 2001, joint, 54, 5, 903, 911.
• Photocatalytic sensor for the determination of chemical oxygen demand using flow injection analysis
  Yoon-Chang Kim, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Kazuhiko Hashimoto, Isao Karube
  Analytica Chimica Acta,
  May 2001, joint, 432, 1, 59, 66
• Biosensor for the evaluation of biochemical oxygen demand using photocatalytic pretreatment
  Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro and Isao Karube
  Sensors and Actuators, B: Chemical,
  Apr. 2001, joint, 80,, 15, 20.
• Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques.
  Atsunori Hiratsuka, Kenichi Kojima, Hiroaki Suzuki, Hitoshi Muguruma, Kazunori Ikebukuro and Isao Karube
  The Analyst
  Mar. 2001, joint, 126, 5, 658, 663
• Detection of toxic chemicals with high sensitivity by measuring the quantity of induced P450 mRNAs based on surface plasmon resonance.
  Masaaki Oyama, Takeshi Ikeda, Tae-Kyu Lim, Kazunori Ikebukuro, Yuzo Masuda and Isao Karube
  Biotechnol. Bioeng
  Jan. 2001, joint, 71, 3, 217, 222
• Relationship between theoretical oxygen demand and photocatalytic chemical oxygen demand for specific classes of organic chemicals.
  Yoon-Chang Kim, Satoshi Sasaki, Kazuyoshi Yano, Kazunori Ikebukuro, Kazuhiko Hashimoto and Isao Karube
  The Analyst, 125 (11), 1915-1918.
  Oct. 2000, joint
• Disposable type chemical oxygen demand sensor using a microfabricated Clark-type oxygen electrode with a TiO2 suspension solution.
  Kyong-Hoon Lee, Yoon-Chang Kim, Hiroaki Suzuki, Satoshi Sasaki, Kazunori Ikebukuro, Kazuhiko Hashimoto and Isao Karube
  Electroanalysis, 12 (16), 1334-1338.
  Sep. 2000, joint
• Development of a fluorometric sensor for the measurement of phycobilin pigment and application to freshwater phytoplankton.
  Ryoichi Asai, Scott McNiven, Kazunori Ikebukuro, Isao Karube, Yasuo Horiguchi, Shuichi Uchiyama, Akira Yoshida and Yuzo Masuda
  Field Analytical Chemistry and Technology, 4 (1), 53-61.
  Aug. 2000, joint
• Effect of incident angle of a light on sensitivity and detection limit for antibody layer with surface plasmon resonance spectroscopy
  Takuo Akimoto, Satoshi Sasaki, Kazunori Ikebukuro and Isao Karube
  Biosensors & Bioelectronics, 15 (7-8), 355-362.
  Jul. 2000, joint
• Estimation of sensitivity for refractive index and immunoreaction in a surface plasmon resonance sensor probe
  Takuo Akimoto, Satoshi Sasaki, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 417 (2), 125-131.
  Jun. 2000, joint
• Photocatalytic sensor for chemical oxygen demand determination based on oxygen electrode.
  Yoon-Chang Kim, Kyong-Hoon Lee, Satoshi Sasaki, Kazuhiko Hashimoto, Kazunori Ikebukuro and Isao Karube
  Analytical Chemistry, 72 (14), 3379-3382.
  May 2000, joint
• Optical fiber biosensor for the determination of low biochemical oxygen demand.
  Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro and Isao Karube
  Biosensors & Bioelectronics, 15 (7-8), 371-376.
  May 2000, joint
• Application of polymer-embedded proteins to fabrication of DNA array.
  Hirotaka Miyachi, Atsunori Hiratsuka, Kazunori Ikebukuro, Kazuyoshi Yano, Hitoshi Muguruma andIsao Karube
  Biotechnology and Bioengineering. 69 (3),323-329.
  Mar. 2000, joint
• Bioassay of bile acids using an enzyme-linked DNA aptamer.
  Teru Kato, Kazuyoshi Yano, Kazunori Ikebukuro and Isao Karube
  The Analyst, 125, 1371-1373.
  Feb. 2000, joint
• Increasing the sensitivity of piezoelectric odor sensors based on molecularly imprinted polymers.
  Hong-Seok Ji, Scott McNiven, Takashi Saito, Kazunori Ikebukuro, Isao Karube
  Biosensors & Bioelectronics, 15 (7-8), 403-409.
  Feb. 2000, joint
• Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (PCR).
  Eriko Kai, Kazunori Ikebukuro, Sadayori Hoshina, Haruo Watanabe and Isao Karube
  FEMS Immunology and Medical Microbiology., 29 (4), 283-288.
  Feb. 2000, joint
• In vitro selection of DNA aptamers which bind to cholic acid.
  Teru Kato, Taro Takemura, Kazuyoshi Yano, Kazunori Ikebukuro and Isao Karube
  Biochimica et Biophysica Acta, gene structure and expression, 1493, 12-18.
  Jan. 2000, Research paper (scientific journal), only
• Interaction of three-way DNA junctions with steroids.
  Teru Kato, Kazuyoshi Yano, Kazunori Ikebukuro and Isao Karube
  Nucleic Acids Research, 28(9), 1963-1968.
  Jan. 2000, Research paper (scientific journal), joint
• Application of chimeric RNA-DNA oligonucleotides to the detection of pathogenic microorganisms using surface plasmon resonance
  Hirotaka Miyachi, Kazuyoshi Yano, Midori Kono, Sadayori Hoshina, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 407 (1-2), 1-10.
  2000, joint, 407, 1-2, 1, 10
• Highly sensitive detection of atrazine based on the SPR determination of P450 mRNA levels in Saccharomyces cerevisiae.
  Tae-Kyu Lim, Masaaki Oyama, Kazunori Ikebukuro and Isao Karube
  Analytical Chemistry, 72 (13), 2856-2860.
  2000, joint
• 遺伝的アルゴリズムによる機能性分子の探索
  生物工学会誌、日本生物工学会、72巻、3号、p210、(1999)
  Oct. 1999, only
• Highly sensitive chemiluminescence flow-injection detection of the red tide phytoplankton Heterosigma carterae.
  Ryoichi Asai, Ritsuko Matsukawa, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 390 (1-3), 237-244.
  Aug. 1999, joint
• Refractive-index and thickness sensitivity in surface plasmon resonance spectroscopy.
  Takuo Akimoto, Satoshi Sasaki, Kazunori Ikebukuro and Isao Karube
  Applied Optics, 38 (19), 4058-4064.
  Jul. 1999, joint
• Selective piezoelectric odor sensors using molecularly imprinted polymers.
  Hong-Seok Ji, Scott McNiven, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 390 (1-3), 93-100.
  May 1999, joint
• Highly sensitive trilayer piezoelectric odor sensor.
  Hong-Seok Ji, Scott McNiven, Kazuyoshi Yano, Kazunori Ikebukuro, Uwe T. Bornscheuer, Rolf D. Schmid and Isao Karube
  Analytica Chimica Acta, 387 (1), 39-45.
  May 1999, joint
• Detection of Escherichia coli O157 : H7 DNA using two fluorescence polarization methods.
  Bang-Ce Ye, Kazunori Ikebukuro and Isao Karube
  Fresenius J. Analytical Chemistry., 365(5) 452-457.
  Apr. 1999, joint
• Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by the polymerase chain reaction.
  Shinya Sawata, Eriko Kai, Kazunori Ikebukuro, Tetsuya Iida, Takeshi Honda and Isao Karube
  Biosensors and Bioelectronics, 14 (4), 397-404.
  Mar. 1999, joint
• 1999) Development of highly sensitive BOD sensor and its evaluation using preozonation.
  Gab-Joo Chee, Yoko Nomura, Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 394, 65-71.
  Feb. 1999, joint
• An automatic flow-injection analysis system for determining phosphate ion in river water using pyruvate oxidase G (from Aerococcus viridans).
  Hideaki Nakamura, Hiroko Tanaka, Mami Hasegawa, Yuzo Masuda, Yoko Nomura, Yoshiko Arikawa, Ritsuko Matsukawa, Kazunori Ikebukuro and Isao Karube
  Talanta, 50(4), 799-807.
  Feb. 1999, joint
• A novel method for detecting PCR products in solution using surface plasmon resonance.
  Eriko Kai, Shinya Sawata, Kazunori Ikebukuro, Tetsuya Iida, Takeshi Honda and Isao Karube
  Analytical Chemistry., 71 (4), 796-800.
  Feb. 1999, Research paper (scientific journal), joint
• Development of a highly sensitive chemiluninescent flow-injection analysis sensor for phosphate ion detection using maltose phosphorylase.
  Hideaki Nakamura, Mami Hasegawa, Yoko Nomura, Yoshiko Arikawa, Ritsuko Matsukawa, Kazunori Ikebukuro and Isao Karube
  Journal of Biotechnology, 75(2-3), 127-133.
  Jan. 1999, joint
• (1999) A glucose sensor with a plasma-polymerized thin film by dry processes.
  Atsunori Hratsuka, Hitoshi Muguruma, Kazunori Ikebukuro and Isao Karube
  Electroanalysis, 11(15), 1098-1100.
  Jan. 1999, joint
• Chemiluminescence flow-injection system for rapid detection of red tide phytoplankton, Chattonella antiqua.
  Ryoichi Asai, Ritsuko Matsukawa, Kazunori Ikebukuro and Isao Karube
  Analytical Letters, 31 (13), 2279-2288.
  Nov. 1998, joint
• Direct measurement of etophenprox using surface plasmon resonance.
  Satoshi Sasaki, Eriko Kai, Hirotaka Miyachi, Hitoshi Muguruma, Kazunori Ikebukuro, Hidero Ohkawa and Isao Karube
  Analytica Chimica Acta, 363(2-3), 229-233.
  Nov. 1998, joint
• Development of an odorant sensor using polymer-coated quartz crystals modified with unusual lipids.
  Kazuyoshi Yano, Uwe T. Bornscheuer, Rolf D. Schmid, Hiroshi Yoshitake, Hong-Seok Ji, Kazunori Ikebukuro, Yuzo Masuda and Isao Karube
  Biosensors & Bioelectronics, 13(3-4), 397-405.
  Oct. 1998, joint
• Quantitative analysis of polymerase chain reaction using anisotropy ratio and relative hydrodynamic volume of fluorescence polarization method.
  Bang-Ce Ye Kazunori Ikebukuro and Isao Karube
  Nucleic Acids Research, 26 (15), 3614-3615.
  Oct. 1998, Research paper (scientific journal), joint
• Application of a linear alkylbenzene sulfonate biosensor to river water monitoring.
  Yoko Nomura, Kazunori Ikebukuro, Kenji Yokoyama, Toshihumi Takeuchi, Yoko Arikawa, Sizue Ohno and Isao Karube.
  Biosensors & Bioelectronics, 13, 1047-1053.
  Sep. 1998, joint
• Purification of single stranded DNA from asymmetric PCR product using the SMART^TM system.
  Eriko Kai, Koichi Sumikura Kazunori Ikebukuro　and Isao Karube
  Biotechnology Techniques, 12 (12), 935-939.
  Sep. 1998, joint
• Cloning and expression of gene encoding cyanidase from Pseudomonas stutzeri AK61.
  Atsushi Watanabe, Kazuyoshi Yano Kazunori Ikebukuro　and Isao Karube
  Applied Microbiology and Biotechnology, 50 (1), 93-97.
  Aug. 1998, joint
• Purification and characterization of a cyanide-degrading enzyme from Pseudomonas stutzeri AK61.
  Atsushi Watanabe, Kazuyoshi Yano Kazunori Ikebukuro　and Isao Karube
  Microbiology, 144(6), 1677-1682.
  Jun. 1998, joint
• Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis.
  Atsushi Watanabe, Kazuyoshi Yano　Kazunori Ikebukuro Isao Karube
  Biochimica et Biophysica Acta, 1382(1), 1-4.
  May 1998, Research paper (scientific journal), joint
• Photogeneration of NADPH by oligothiophenes coupled with Ferredoxin-NADPH reductase.
  Young-Sug Kim, Kazunori Ikebukuro, Hitoshi Muguruma and Isao Karube
  Journal of Biotechnology, 59(3), 213-220.
  Apr. 1998, joint
• Molecularly imprinted polymers which mimic multiple hydrogen bonds between nucleotide bases.
  Kazuyoshi Yano, Koichiro Tanabe, Toshifumi Takeuchi, Jun Matsui　Kazunori Ikebukuro and Isao Karube
  Analytica Chimica Acta, 363(2-3), 111-117.
  Apr. 1998, joint
• Selective recognition of 2,4 -dichlorophenoxyacetic acid using a molecularly imprinted polymer.
  Yoko Nomura, Hitoshi Muguruma, Kazuyoshi Yano, Akimitsu Kugimiya, Scott McNiven and Kazunori Ikebukuro　Isao Karube
  Analytical Letters, 31(6), 973-980.
  Mar. 1998, joint
• Reagentless phosphate ion sensor system for environmental monitoring.
  Suzuki Masayasu, Hironobu Kurata, Yoshihiro Inoue, Izumi Kubo, Hideaki Nakamura Kazunori Ikebukuro and Isao Karube
  DENKI KAGAKU, 66(6) 579-583.
  Feb. 1998, Research paper (scientific journal), joint

Books and Other Publications

• CHEMICAL, GAS, AND BIOSENSORS FOR INTERNET OF THINGS AND RELATED APPLICATIONS, Aptameric sensors utilizing its property as DNA
  Kinuko Ueno, Kaori Tsukakoshi and Kazunori Ikebukuro
  Elsevier
  The biosensors using aptamer are reviewed and its cutting edge technology was explained.
  2019

  URL(公開)(r-map)
• 「アプタマーの医療応用　」CSJカレントレビュー24 化学で医療・診断・創薬の革新を目指す
  長谷川聖、池袋一典
  医療･診断･創薬の化学　-医療分野に挑む革新的な化学技術-、日本化学会編、化学同人(2017)、p55-63.
  Jul. 2017
• "History and Development of Nucleic Acid Aptamers" in Aptamers: Tools for Targeted Nanotherapy and Molecular ImagingApplications of Metaheuristics in Process Engineering,
  Savory, N., Abe, K., Ikebukuro, K.
  Pan Stanford publication
  Nov. 2015
• In silico Maturation: Processing Sequences to Improve Biopolymer Functions Based on Genetic Algorithms, Applications of Metaheuristics in Process Engineering,
  Savory, N., Abe, K., Yoshida, W., & Ikebukuro, K.
  Springer
  Feb. 2014
• 化学便覧応用化学編第7版　(2014/01)、8.5.4項「リボザイムとアプタマー」、
  吉田亘、阿部公一、池袋一典
  丸善出版株式会社
  Jan. 2014
• “Electrochemical Biosensors Using Aptamers for Theranostics” in “Advances in Biochemical Engineering/Biotechnology”, 2013, Edited by Man Bock Gu,
  Koichi Abe, Wataru Yoshida and Kazunori IKEBUKURO
  Springer-Verlag
  Jan. 2014
• 「バイオテクノロジー」、機械工学ハンドブック
  池袋一典
  朝倉書店
  Jul. 2011
• “Aptamer sensors combined with enzymes for highly sensitive detection” in “Biosensors - Emerging Materials and Applications”
  Koichi Abe and Kazunori IKEBUKURO
  InTech
  Jul. 2011
• Biosensors using Aptameric enzyme subunit; application of aptamers to allosteric control of enzymes” in “Analytial Applications of Aptamers”
  Kazunori Ikebukuro, Wataru Yoshida and Koji Sode
  Wiley
  Jan. 2009
• Dictionary of biosensors and chemical sensors, "aptamer sensor"
  Isao Karube
  Technosystem Co., Ltd
  Jul. 2007
• 「アプタマーを分子認識素子とするバイオセンサの開発」　ユビキタスバイオセンシング
  三林浩二監修
  Jan. 2006
• 和文論文　合計５報 和文総説　合計５編 和文著書　合計３編
  Jan. 2005
• 医学書院医学大辞典
  共著者3400名超
  医学書院
  Mar. 2003
• “19. Cyanides” in “Handbook for Water Analysis”
  共著者６０名
  Oct. 2000
• Enzymes and Microbial Biosensors: Techniques and Protocols in “Methods in Biotechnology”, vol. 6
  共著者４０名
  Jul. 1998

Presentations

• “Computer aided evolution of the desired functions of peptide/DNA aptamers”
  The third International Workshop on Symbiosis of Biology and Nanodevices
  The computer aided evolution method for the function of aptamer is explained and some successful examples were shown.
  26 Jun. 2019, Oral presentation(invited, special)
• Smart aptamer: the aptamer changing its structure and function depending on the environmental condition
  CSIRO-NIMS Symposium: Materials and technologies for life science applications
  Smart aptamer sensing chemical environment and changing its structure and function was first reported and its application was shown.
  19 Mar. 2019, Oral presentation(invited, special)
• 「グアニン四重鎖構造を形成するDNAのナノ構造変化を利用したセンシング」
  日本学術振興会第174委員会第62回研究会
  周囲の環境で構造と機能が変わるDNAアプタマーの紹介とこれを用いた応用について最先端の研究を紹介した。
  27 Jun. 2018, Oral presentation(invited, special)
• BINDING PROPERTIES OF AMYLOID OLIGOMER-BINDING DNA APTAMERS AGAINST AMYLOID BETA OLIGOMERS
  13th International Conference on Alzheimer’s and Parkinson’s Diseases (AD/PD 2017)
  29 Mar. 2017, Poster presentation
• 標的DNA検出に向けたアルカリフォスファターゼ融合ジンクフィンガー蛋白質の開発
  電気化学第84回大会
  25 Mar. 2017, Other
• Development of the electrochemical detection system of thrombin activity
  PepCon-2017
  22 Mar. 2017, Poster presentation
• アミロイドオリゴマー結合アプタマーを用いた、核酸増幅に基づく水溶性アミロイドβオリゴマーの検出
  日本化学会第97回春季年会
  16 Mar. 2017, Other
• 非天然塩基の導入によるトロンビン結合アプタマーの結合能の改良
  日本化学会第97回春季年会
  16 Mar. 2017, Other
• シトシンメチル化がG-quadruplexとタンパク質の結合に及ぼす影響の評価
  第39回 日本分子生物学会年会
  01 Dec. 2016, Poster presentation
• 抗体医薬品を特異的に認識する抗イディオタイプアプタマーの探索とその特性評価
  第39回 日本分子生物学会年会
  01 Dec. 2016, Poster presentation
• Development of the Electrochemical Detection System Using the Combination of Aptamer and Enzyme
  PRiME 2016
  02 Oct. 2016, Oral presentation(general)
• Development of a detection system for epigenetic modifications using enzyme fused zinc finger protein
  PRiME 2016
  02 Oct. 2016, Oral presentation(general)
• Construction of photo regulation system of protein expression in Synechocystis sp. PCC 6803
  The 43rd International Symposium on Nucleic Acids Chemistry.
  28 Sep. 2016, Poster presentation
• Effect of G-quadruplex ligand on the topology of G-quadruplex forming aptamer and its affinity to the target molecules
  The 43rd International Symposium on Nucleic Acids Chemistry.
  28 Sep. 2016, Poster presentation
• Screening and characterization of aptamer for myoglobin
  The 43rd International Symposium on Nucleic Acids Chemistry.
  28 Sep. 2016, Poster presentation
• Development of DNA aptamers against FokI nuclease domain for genome editing
  The 43rd International Symposium on Nucleic Acids Chemistry.
  28 Sep. 2016, Poster presentation
• グアニン四重鎖特異的リガンドを用いたDNAアプタマーの構造制御
  第10回バイオ関連化学シンポジウム
  08 Sep. 2016, Oral presentation(general)
• 高感度検出への応用を目指したアルカリホスファターゼ阻害アプタマーの探索
  第10回バイオ関連化学シンポジウム
  08 Sep. 2016, Poster presentation
• リボレギュレーターを用いた内在性遺伝子cyabrB2 の転写および発現制御
  第10回バイオ関連化学シンポジウム
  08 Sep. 2016, Poster presentation
• タンパク質とG-quadruplexとの結合にシトシンのメチル化が与える影響の解析
  第10回バイオ関連化学シンポジウム
  08 Sep. 2016, Poster presentation
• Improvement of binding affinity of G-quadruplex forming aptamers
  Biosensor2016
  26 May 2016, Poster presentation
• Interaction between G-quadruplex (G4)-forming aptamer and heme protein
  Biosensor2016
  26 May 2016, Poster presentation
• Development of a detection system for epigenetic modifications using enzyme fused zinc finger protein
  Biosensor2016
  26 May 2016, Oral presentation(general)
• Selection and characterization of DNA aptamers against FokI nuclease domain for genome editing
  Biosensor2016
  26 May 2016, Oral presentation(general)
• Development of multivalent aptamers for high-sensitive detection of target proteins
  Biosensor2016
  26 May 2016, Oral presentation(general)
• シトシンのメチル化がG-quadruplex構造とタンパク質との結合に及ぼす影響の解析
  日本化学会第96回春季年会
  26 Mar. 2016, Other
• Synechocystis sp. PCC 6803におけるリボレギュレーターを用いた転写因子cyAbrB2の発現制御
  日本化学会第96回春季年会
  26 Mar. 2016, Other
• Development of Light inducible riboregulator for Escherichia coli
  2nd International Workshop on Cyanofactory
  05 Mar. 2016, Poster presentation
• Screening method to obtain aptamers binding different sites of the target molecule
  Pacifichem 2015
  17 Dec. 2015, Poster presentation
• Characterization of aptamer forming G-quadruplex structure and its binding to target molecules in hydrated ionic liquid
  Pacifichem 2015
  17 Dec. 2015, Poster presentation
• Investigation of the effects of G-quadruplex ligand (7OTD) on the expression of cell transcriptome
  Pacifichem 2015
  16 Dec. 2015, Poster presentation
• “RNA-based gene regulators for controlling gene expression in cyanobacteria”
  Pacifichem 2015
  16 Dec. 2015, Other
• Engineering of a wide dynamic range green-light regulation system for gene expression in Synechocystis sp. PCC 6803
  Pacifichem 2015
  16 Dec. 2015, Poster presentation
• G-quadruplex (G4) 形成アプタマーの構造制御及び結合能向上に向けたG4 リガンドの応用
  第38回 日本分子生物学会年会・第88回日本生化学会大会 合同大会
  03 Dec. 2015, Poster presentation
• ゲノム編集技術への応用を目指したFokIヌクレアーゼドメインに結合するDNAアプタマーの探索及び特性評価
  第38回 日本分子生物学会年会・第88回日本生化学会大会 合同大会
  03 Dec. 2015, Poster presentation
• Identification of DNA aptamers from G-quadruplex sequence in human genome”
  1st International Symposium on Functional Nucleic Acids: From Laboratory to Targeted Molecular Therapy”
  27 Nov. 2015, Oral presentation(general)
• 「シアノバクテリアで機能するリボレギュレーターのエンジニアリング」
  藍藻の分子生物学2015
  27 Oct. 2015, Oral presentation(invited, special)
• Synechocystis sp. PCC 6803由来RpaBの発現を抑制する人工small RNAの開発」
  藍藻の分子生物学2015
  27 Oct. 2015, Poster presentation
• 二次構造予測に基づくsmall RNAの機能改良法の開発」
  第67回日本生物工学会大会
  27 Oct. 2015, Poster presentation
• FokIヌクレアーゼドメインに対するDNAアプタマーの構造及び機能解析
  第67回日本生物工学会
  27 Oct. 2015, Poster presentation
• G-quadruplex (G4) 形成アプタマーの構造及び結合能に対するG4リガンドの影響の解析
  第67回日本生物工学会
  27 Oct. 2015, Poster presentation
• Analysis of interaction between aptamers and target molecules in hydrated ionic liquid
  The 42nd International Symposium on Nucleic Acids Chemistry.
  24 Sep. 2015, Poster presentation
• Detection of anti-VEGF antibody bevacizumab using DNA aptamer
  The 42nd International Symposium on Nucleic Acids Chemistry.
  24 Sep. 2015, Poster presentation
• 熱応答性磁性ナノ粒子と DNA アプタマーを用いた 標的タンパク質検出システムの開発
  pre-mRNA中に存在するG-quadruplex構造形成配列に着目したアプタマー探索
  11 Sep. 2015, Oral presentation(general)
• 熱応答性磁性ナノ粒子と DNA アプタマーを用いた 標的タンパク質検出システムの開発
  第9回バイオ関連化学シンポジウム
  11 Sep. 2015, Poster presentation
• FokIヌクレアーゼドメインに対するDNAアプタマーの探索
  第9回バイオ関連化学シンポジウム
  11 Sep. 2015, Poster presentation
• G4リガンドがVEGF結合アプタマーの構造及び結合能に与える影響の解析
  第9回バイオ関連化学シンポジウム
  11 Sep. 2015, Poster presentation
• 水和イオン液体中でのアプタマーと標的分子の相互作用解析
  2015年電気化学秋季大会
  11 Sep. 2015, Other
• Investigation of the effect of G-quadruplex ligand on the expression and splicing of VEGF mRNA.
  5th International Meeting on Quadruplex Nucleic Acids: G4thering in Bordeaux
  27 May 2015, Poster presentation
• 大腸菌で機能する光誘導型リボレギュレーターシステムの開発
  日本化学会第95回春季年会
  26 Mar. 2015, Other
• 水和イオン液体中でのトロンビン-トロンビン結合アプタマーの結合
  日本化学会第95回春季年会
  26 Mar. 2015, Other
• 抗VEGF抗体bevacizumab(Avastin) のanti-idiotype aptamerの探索
  日本化学会第95回春季年会
  26 Mar. 2015, Other
• Synechocystis sp. PCC6803内における緑色光誘導型リボレギュレーターの特性評価
  日本化学会第95回春季年会
  26 Mar. 2015, Other
• DNA 四重鎖構造の安定性に着目したメチル化 DNA 簡易検出法の開発
  日本化学会第95回春季年会
  26 Mar. 2015, Other
• Biosensing based on the change of nano-structure of aptamer by Kazunori Ikebukuro
  Sweden-Japan seminar on Nanomaterials and Nanotechnology
  11 Mar. 2015, Oral presentation(invited, special)
• Improvement of aptamer's fuction with in silico maturation
  第 10 回理研「バイオものづくり」シンポジウム
  06 Mar. 2015, Oral presentation(invited, special)
• Engineering artificIn silico maturation: improvement of aptamer function based on genetic algorithms employing in vitro functional screening process
  The 41th International Symposium on Nucleic Acids Chemistry
  05 Nov. 2014, Oral presentation(general)
• Engineering artificial small RNAs to control post-transcriptional gene expression in Synechocystis sp. PCC6803
  The 41th International Symposium on Nucleic Acids Chemistry
  05 Nov. 2014, Poster presentation
• グルコース脱水素酵素融合ジンクフィンガー蛋白質の電気化学検出への応用
  2014年度電気化学会秋季大会
  27 Sep. 2014, Other
• RNA結合タンパク質Hfqを利用したSynechocystis sp. PCC 6803内で機能するリボレギュレーターの遺伝子発現制御能の向上
  第8回バイオ関連化学シンポジウム
  11 Sep. 2014, Oral presentation(general)
• Engineering the scaffold region of natural and artificial small RNAs to enhance their gene regulation abilities PCC6803
  RNA2014
  05 Jun. 2014, Poster presentation
• DNA aptamers against the Cry j 2 allergen of Japanese cedar pollen
  Biosensor2014
  28 May 2014, Poster presentation
• In silico maturation: Advanced aptamer development for biosensing applications
  Biosensor2014
  28 May 2014, Poster presentation
• RNA aptamer screening focusing on G-quadruplex forming region of pre-mRNA
  Biosensor2014
  28 May 2014, Poster presentation
• Detection of epigenetic modifications using enzyme fused zinc finger protein
  Biosensor2014
  28 May 2014, Poster presentation
• Screening and improvement of DNA aptamers by in silico approachesspecificity
  Biosensor2014
  28 May 2014, Poster presentation
• A novel RNA based genetic control system for metabolic gene regulation PCC6803
  4th International Conference on Algal Biomass, Biofuels & Bioproducts
  15 May 2014, Other
• Loop-engineering of riboregulator to improve its gene regulation in Synechocystis sp. PCC 6803
  The 10th Asia-Pacific Marine Biotechnology Conference
  05 May 2014, Other
• 櫛型電極を用いた電気化学的なトロンビン活性の検出法の開発
  電気化学会第82回大会
  16 Mar. 2014, Other
• 櫛型電極を用いた電気化学的なトロンビン活性の検出法の開発
  電気化学会第82回大会
  15 Mar. 2014, Other
• スギ花粉アレルゲンCry j 2に結合するDNAアプタマーの開発
  第36回日本分子生物学会年会
  03 Dec. 2013, Poster presentation
• Aptamer selection based on putative G-quadruplexsequence in genome
  The 40th International Symposium on Nucleic Acids Chemistry
  14 Nov. 2013, Oral presentation(general)
• Screening and Improvement of DNA Aptamers against Hepatocyte Growth Factor by in silico Approaches
  The 40th International Symposium on Nucleic Acids Chemistry
  13 Nov. 2013, Poster presentation
• Detection of DNA methylation and histone modification by enzyme fused zinc finger protein
  The 40th International Symposium on Nucleic Acids Chemistry
  13 Nov. 2013, Poster presentation
• Improving gene regulation ability of bacterial small RNAs by scaffold engineering
  The 40th International Symposium on Nucleic Acids Chemistry
  13 Nov. 2013, Poster presentation
• 酵素融合ジンクフィンガー蛋白質を用いた遺伝子検出システムの開発
  電気化学会秋季大会
  27 Sep. 2013, Other
• Zinc-finger nucleaseを細胞内で特異的に放出する表面修飾法の検討
  第7回バイオ関連化学シンポジウム
  26 Sep. 2013, Poster presentation
• Hfq結合配列の改良によるsmallRNAの遺伝子発現制御能の向上
  第7回バイオ関連化学シンポジウム
  26 Sep. 2013, Other
• In silico 配列解析及び操作によるアプタマーの探索と改良
  第7回バイオ関連化学シンポジウム
  26 Sep. 2013, Poster presentation
• ループ領域改変によるリボレギュレーターの遺伝子発現能の向上
  第7回バイオ関連化学シンポジウム
  26 Sep. 2013, Poster presentation
• リボレギュレーターの光制御
  第7回バイオ関連化学シンポジウム
  26 Sep. 2013, Poster presentation
• 藍藻で機能するリボレギュレータの改変
  第65回日本生物工学会大会
  18 Sep. 2013, Oral presentation(general)
• ゲノム中のG-quadruplex形成配列に着目したアプタマー探索法
  第65回日本生物工学会大会
  18 Sep. 2013, Poster presentation
• Identification of HGF and HBEGF aptamers by “G4 Promoter-derived Aptamer Selection (G4PAS)”
  The Sixth International Meeting on Synethetic Biology
  10 Jul. 2013, Poster presentation
• Engineering Hfq binding sequence to control gene regulation of bacterial small RNA
  The Sixth International Meeting on Synethetic Biology
  10 Jul. 2013, Poster presentation
• Selection of DNA aptamers targeted to Proteus mirabilis membrane-associated proteins.
  SfAM Summer Conference 2013
  04 Jul. 2013, Poster presentation
• Aptamer selection based on genomic information; focusing on G-quadruplex forming sequence
  4th International Meeting on Quadruplex Nucleic Acids
  04 Jul. 2013, Poster presentation
• 微生物ゲノムDNAの電気化学的検出法に向けたGDH融合ジンクフィンガー蛋白質の開発
  電気化学会創立第80周年記念大会
  30 Mar. 2013, Other
• アプタマーとペプチド核酸を用いた血管内皮細胞増殖因子の検出システムの開発
  電気化学会創立第80周年記念大会
  30 Mar. 2013, Other
• ノロウイルスの外殻タンパク質VP1に結合するDNAアプタマーの探索
  日本化学会第94回春季年会
  27 Mar. 2013, Other
• Synechocystis sp. PCC6803において高い遺伝子発現制御能を持つリボレギュレータへの改良
  日本化学会第94回春季年会
  27 Mar. 2013, Other
• GDH 融 合 ジンクフィンガー蛋白質を用いた微生物ゲノムの電気化学的検出法の開発
  日本化学会第94回春季年会
  25 Mar. 2013, Other
• ループ構造改変によるリボレギュレータの改良
  日本化学会第94回春季年会
  25 Mar. 2013, Other
• RNA結合蛋白質を利用したリボレギュレータの改良
  日本化学会第94回春季年会
  25 Mar. 2013, Oral presentation(general)
• αシヌクレインの凝集線維化におけるランタニド系金属の影響
  日本化学会第94回春季年会
  24 Mar. 2013, Other
• CpGアイランドから見出されたグアニン四重鎖形成配列と低分子化合物の相互作用解析
  日本化学会第94回春季年会
  24 Mar. 2013, Other
• 特 異 的 蛍 光 リガンドと DNA マイクロアレイを基盤とした新規グアニン四重鎖形成配列の網羅的検出法の開発
  日本化学会第94回春季年会
  24 Mar. 2013, Oral presentation(general)
• ゲノム中のG-quadruplex形成配列情報に基づいたバイオマーカー結合アプタマーの探索
  日本化学会第94回春季年会
  24 Mar. 2013, Other
• ゲノム中のG-quadruplex構造形成配列に着目したアプタマー探索法の開発
  日本化学会第94回春季年会
  24 Mar. 2013, Other
• 二次元電気泳動装置システムを利用した、アプタマーのスクリーニング方法の開発
  第35回日本分子生物学会年会
  13 Dec. 2012, Poster presentation
• VEGF detection using aptamer and PNA-based Bound/Free separation system
  The 39th International Symposium on Nucleic Acids Chemistry
  16 Nov. 2012, Poster presentation
• Post-transcriptional gene regulation by artificial riboregulator in cyanobacteria
  The 39th International Symposium on Nucleic Acids Chemistry
  16 Nov. 2012, Poster presentation
• In silico selection of aptamers based on genomic information
  The 39th International Symposium on Nucleic Acids Chemistry
  16 Nov. 2012, Other
• アミロイドオリゴマー結合アプタマーの Aβオリゴマーに対する認識特異性
  認知症学会
  26 Oct. 2012, Poster presentation
• リボレギュレーターによるシアノバクテリアの遺伝子発現制御
  第64回日本生物工学会大会
  24 Oct. 2012, Other
• 遺伝子的アルゴリズムを適用したompCプロモーターの改良
  第64回日本生物工学会大会
  24 Oct. 2012, Other
• ルシフェラーゼ融合ジンクフィンガータンパク質を用いたヒストン修飾解析法の開発
  第64回日本生物工学会大会
  24 Oct. 2012, Other
• Cell-SELEXによる尿路感染症起因菌に結合するDNAアプタマーの探索
  第64回日本生物工学会大会
  24 Oct. 2012, Other
• 藍藻で機能するリボレギュレータのデザイン
  第64回日本生物工学会大会
  24 Oct. 2012, Other
• Aptameric Sensor for Detection of VEGF Based on Labeling Technique Using GDH Fused Zinc Finger Protein
  電気化学会秋季大会（Prime2012)
  11 Oct. 2012, Other
• VEGF結合アプタマーのin silico maturation
  第6回バイオ関連化学シンポジウム2012
  26 Sep. 2012, Poster presentation
• アミロイドオリゴマー結合DNAアプタマーのオリゴマー結合特異性の解析
  第6回バイオ関連化学シンポジウム2012
  26 Sep. 2012, Poster presentation
• In silico アプタマー探索法の開発
  第6回バイオ関連化学シンポジウム2012
  26 Sep. 2012, Other
• 二次元電気泳動装置「Auto2D」を利用した、DNAアプタマーのスクリーニング方法の開発
  日本プロテオーム学会2012年大会
  26 Jul. 2012, Poster presentation
• Construction of the Autoaggregation and Autolysis System Controlled by Riboregulator
  The 9th Asia-Pacific Marine Biotechnology Conference
  13 Jul. 2012, Other
• Directed evolution of ompC promoter by applying genetic algorithm
  The 9th Asia-Pacific Marine Biotechnology Conference
  13 Jul. 2012, Other
• Design of Riboregulators that Function in Cyanobacteria
  The 9th Asia-Pacific Marine Biotechnology Conference
  13 Jul. 2012, Other
• Construction of novel RNA tools for controlling synthetic cyanobacterial bioprocess
  The 9th Asia-Pacific Marine Biotechnology Conference
  13 Jul. 2012, Other
• Construction of the autoaggregation and autolysis system controlled by riboregulator
  The 2nd International Conference on Algal Biomass, Biofuels and Bioproducts
  10 Jun. 2012, Other
• Design of riboregulators that function in cyanobacteria
  The 2nd International Conference on Algal Biomass, Biofuels and Bioproducts
  10 Jun. 2012, Other
• Cell-SELEX for selection of anti-Proteus mirabilis DNA aptamers and in silico maturation to improve binding-specificity
  Biosensor2012
  15 May 2012, Other
• Improvement of Affinities of VEGF-binding Aptamers for Biosensor by in silico maturation
  Biosensor2012
  15 May 2012, Other
• Development of DNA aptamers that specifically bind to amyloid protein oligomer and flow cytometric detection of amyloid beta oligomer
  Biosensor2012
  15 May 2012, Other
• Quantitaive DNA methylation level measurement using MBD and Zinc finger fused luciferase
  Biosensor2012
  15 May 2012, Other
• Riboregulator for controlling synthetic cyanobacterial bioprocess
  Biosensor2012
  15 May 2012, Other
• セラノスティックスとバイオセンサー」
  特別企画「生物電気化学・バイオエンジニアリング領域の新展開」、電気化学会第79回大会
  31 Mar. 2012, Oral presentation(invited, special)
• 微生物ゲノムの電気化学的検出法に向けたGDH融合ジンクフィンガー蛋白質の開発
  電気化学会創立第80周年記念大会
  29 Mar. 2012, Other
• アプタマーとペプチド核酸を用いた血管内皮細胞増殖因子の検出システムの開発
  電気化学会創立第80周年記念大会
  29 Mar. 2012, Other
• GDH 融合ジンクフィンガー蛋白質を用いた微生物ゲノムの電気化学的検出法の開発
  日本化学会第93回春季年会
  23 Mar. 2012, Other
• ループ構造改変によるリボレギュレータの改良
  日本化学会第93回春季年会
  23 Mar. 2012, Other
• ゲノム中の G-quadruplex 形成配列情報に基づいたバイオマーカー結合アプタマーの探索
  日本化学会第93回春季年会
  23 Mar. 2012, Other
• ゲノム中の G-quadruplex 構造形成配列に着目したアプタマー探索法の開発
  日本化学会第93回春季年会
  23 Mar. 2012, Other
• RNA 結合蛋白質を利用したリボレギュレータの改良
  日本化学会第93回春季年会
  23 Mar. 2012, Other
• 「アミロイドオリゴマーを認識する核酸リガンドDNAアプタマーの開発」
  トピックス討論「認知症基礎研究のホットトピックス徹底討論」、第30回日本認知症学会学術集会
  12 Nov. 2011, Oral presentation(invited, special)
• Alpha fetoproteinに結合するDNAアプタマーの探索とその改良
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• ジンクフィンガー蛋白質融合ルシフェラーゼを用いた複数病原性微生物の検出システムの開発
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• 磁性ビーズを用いたDNAメチル化レベル評価法の開発
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• In silico maturation法を用いたVEGF結合アプタマーの改良
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• EcoTanker - 光アクチュエーターで操縦する大腸菌・マイクロ・タンカー
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• Proteus mirabilisに結合するDNAアプタマーの探索及びin silico maturation法による特異性の改良
  第63回日本生物工学会大会
  26 Sep. 2011, Other
• Detection of VEGF using aptamer labeled with FADGDH fused zinc finger protein
  62nd Annual Meeting of the International Society of Electrochemistry
  14 Sep. 2011, Other
• IN SILICO MATURATION OF VEGF-BINDING APTAMER FOR ELECTROCHEMICAL VEGF-DETECTION SYSTEM WITH APTAMER SANDWICH
  62nd Annual Meeting of the International Society of Electrochemistry
  12 Sep. 2011, Poster presentation
• VEGF検出用Capturable aptamerのin silico maturationによる改良
  2011年　電気化学秋季大会
  11 Sep. 2011, Other
• GDH標識アプタマーを用いたVEGFの電気化学的検出
  2011年　電気化学秋季大会
  11 Sep. 2011, Other
• Development of DNA aptamers against Proteus mirabilis by whole-cell SELEX and in silico maturation
  International Union of Microbiological Societies 2011 Congress
  07 Sep. 2011, Poster presentation
• Selection of DNA aptamers against Proteus mirabilis using whole-cell SELEX
  Society for General Microbiology, Spring Conference 2011
  14 Apr. 2011, Poster presentation
• In silico maturationによるVEGF結合DNAアプタマーの改良
  電気化学会第78回大会
  29 Mar. 2011, Other
• GDH標識アプタマーの構造変化を利用したVEGFの電気化学的測定
  電気化学会第78回大会
  29 Mar. 2011, Other
• アミロイド蛋白質オリゴマーに結合するDNA アプタマーの開発と応用
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• キメラEnvZ/OmpR系Two-somponent systemを用いた転写制御系
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• GDH標識DNAアプタマーを用いたVEGF検出系の開発
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• Alpha-fetoproteinに結合するDNAアプタマーの探索
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• ジンクフィンガー融合ルシフェラーゼを用いた病原性微生物の自動検出法の開発
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• 磁性ビーズを用いたDNAメチル化レベル評価法の開発
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• VEGFに結合するDNAアプタマーの探索と疾病診断用センサー素子としての改良
  日本化学会第91回春季年会
  29 Mar. 2011, Other
• 進化分子工学を利用した新規分子認識素子の開発
  東京バイオマーカー・イノベーション技術研究組合、第1回研究交流フォーラム
  08 Mar. 2011, Oral presentation(invited, special)
• メチル化CpG 結合タンパク質を用いた新規メチル化頻度評価法の開発3
  第62回日本生物工学会大会
  29 Oct. 2010, Poster presentation
• アプタマー酵素サブユニット(AES)を用いた高感度疾患マーカーの検出」
  シンポジウムⅠ　ナノテクノロジーが拓く未来の臨床検査、日本臨床検査自動化学会第42回大会
  09 Oct. 2010, Oral presentation(invited, special)
• アプタマーの進化法：in silico maturation
  イノベーション･ジャパン2010　新技術説明会
  01 Oct. 2010, Oral presentation(general)
• Aptameric sensors based on structural change for diagnosis
  Faraday discussion 149:Analysis for Healthcare Diagnostics and Theranostics
  07 Sep. 2010, Oral presentation(general)
• アプタマーを用いた電気化学的VEGF測定法の開発
  2010年電気化学秋季大会
  03 Sep. 2010, Other
• C反応性蛋白質に結合するアプタマーの改良
  2010年電気化学秋季大会
  03 Sep. 2010, Other
• 前立腺特異抗原(PSA)に結合するDNAアプタマーの探索及び改良
  2010年電気化学秋季大会
  03 Sep. 2010, Other
• 細胞障害性オリゴマー検出に向けたαシヌクレイン結合DNAアプタマーの探索
  2010年電気化学秋季大会
  02 Sep. 2010, Other
• Screening of the aptamers recognizing the marker protein for disease and those applications to sensing
  新技術説明会
  22 Jun. 2010, Oral presentation(general)
• Structural control of aptamer inserted polymerized G-quadruplex DNA nanostructure (Gq-switch) by small molecule and its application to biosensing
  Biosensors 2010
  28 May 2010, Oral presentation(general)
• The development of an autonomous self-powered bio-sensing actuator
  Biosensors 2010
  27 May 2010, Oral presentation(keynote)
• The development of a novel theranostic platform for cytotoxicity evaluation of amyloid-forming neurodegenerative disease causative proteins
  Biosensors 2010
  26 May 2010, Oral presentation(general)
• AptaDevelopment of aptameric sensor using conformational change of glucosedehydrogenase binding aptamer
  Biosensors 2010
  26 May 2010, Poster presentation
• 酵素融合ジンクフィンガー蛋白質を用いた標的分子検出法の開発
  日本化学会第90春季年会
  29 Mar. 2010, Other
• 染色体DNAの操作を目的としたテロメスタチン誘導体とヒトテロメア配列の力学的相互作用解析
  日本化学会第90春季年会
  29 Mar. 2010, Other
• メチル化DNA結合タンパク質MBD1を用いた新規メチル化頻度測定系の開発
  日本化学会第90春季年会
  29 Mar. 2010, Other
• アミロイド蛋白質細胞毒性バイオセンシングのセラノスティスクへの応用
  日本化学会第90春季年会
  28 Mar. 2010, Other
• 腫瘍マーカー検出用DNAアプタマーの探索
  日本化学会第90春季年会
  28 Mar. 2010, Other
• 前立腺特異抗原（PSA）に結合するDNAアプタマーの探索
  日本化学会第90春季年会
  28 Mar. 2010, Other
• センサー素子にアプタマーを用いた高感度測定系の構築を目的としたVEGFアプタマーの改良
  2009年電気化学会秋季大会
  11 Sep. 2009, Other
• ホタル由来ルシフェラーゼに結合するDNAプタマーの探索
  2009年電気化学会秋季大会
  11 Sep. 2009, Other
• ルシフェラーゼ融合ジンクフィンガーを用いたサルモネラ属の検出
  2009年電気化学会秋季大会
  11 Sep. 2009, Other
• C-reactive protein及びthyroglobulinに結合するDNAアプタマーの探索と改良
  2009年電気化学会秋季大会
  11 Sep. 2009, Other
• PQQGDHアプタマーを用いたaptamer sensorの開発
  2009年電気化学会秋季大会
  11 Sep. 2009, Other
• ジンクフィンガー蛋白質を用いた機能性蛋白質と機能性DNA分子の融合
  2009年電気化学会秋季大会
  10 Sep. 2009, Other
• α-シヌクレインに結合するDNAアプタマーの探索と測定への応用
  2009年電気化学会秋季大会
  10 Sep. 2009, Other
• Biosensors Based on Aptamer's Structural Change
  AsiaSense2009, July 31st, 2009,
  31 Jul. 2009, Oral presentation(keynote)
• Selection of DNA aptamers agaianst firefly luciferase
  First World Congress of International Academy of Nanomedicine(IANM), June 12, 2009, Hinan, China
  12 Jun. 2009, Poster presentation
• Improvement of DNA aptamers against C-reactive protein and thyroglobulin
  First World Congress of International Academy of Nanomedicine(IANM), June 12, 2009, Hinan, China
  12 Jun. 2009, Poster presentation
• Detection method of PCR products from Salmonella using fusion protein of zinc　finger protein & firefly luciferase
  First World Congress of International Academy of Nanomedicine(IANM), June 12, 2009, Hinan, China
  12 Jun. 2009, Poster presentation
• Aptamers and zinc finger proteins as nanotools for diagnosis
  First World Congress of International Academy of Nanomedicine(IANM), June 12, 2009, Hinan, China
  12 Jun. 2009, Oral presentation(invited, special)
• 蛍光偏向解消法を用いたαシヌクレインオリゴマーの検出
  蛋白質科学会
  22 May 2009, Poster presentation
• ルシフェラーゼ融合ジンクフィンガー蛋白質を用いたアプタマー標識法の開発
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• 血管内皮細胞増殖因子(VEGF165)を認識するDNAアプタマーの改良
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• アミロイド形成タンパク質の新規細胞毒性バイオセンシング系の開発
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• αシヌクレインに結合するDNAアプタマーの探索
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• アミロイド形成蛋白質αシヌクレイン中に見出される繰り返し配列の構造形成における役割
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• Thyroglobulin に結合するDNA アプタマーの改良の探索
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• C-reactive proteinに結合するDNAアプタマーの探索
  日本化学会第89春季年会
  28 Mar. 2009, Oral presentation(general)
• Thyroglobulinに結合するDNAアプタマーの探索および改良
  日本分子生物学会
  09 Dec. 2008, Poster presentation
• 酵素センサーへの応用を目指したFADGDHに結合するDNAアプタマーの探索
  日本分子生物学会
  09 Dec. 2008, Poster presentation
• C-reactive proteinに結合するDNAアプタマーの探索および改良
  日本分子生物学会
  09 Dec. 2008, Poster presentation
• BioCapacitor ~ A stand alone, self-powered wireless glucose sensing system~ and Firefly Luciferase fusion protein
  PACIFIC RIM MEETING 2008 on electrochemical and solid-state science
  16 Oct. 2008, Other
• FADグルコース脱水素酵素の遺伝的アルゴリズムによる基質特異性の改良
  日本生物工学会
  28 Aug. 2008, Poster presentation
• Detection of PCR Products from Legionella pneumophila using Zinc Finger Protein and Firefly Luciferase fusion protein
  Biosensors 2008, THe 10th World congress on Biosensors
  14 May 2008, Other
• BioCapacitor ~ A Novel Category Of Biosensor ~
  Biosensors 2008, THe 10th World congress on Biosensors
  14 May 2008, Other
• Bound/Free separation based on structural change of aptamers and its application to biosensing
  Biosensors 2008, THe 10th World congress on Biosensors
  14 May 2008, Other
• The Novel Detection System of PCR Products from Bacterial Genome using Zn Finger Proteins
  Biosensors 2008, THe 10th World congress on Biosensors
  14 May 2008, Other
• Aptameric enzyme subunit;novel recognition biosensing element for allosteric enzymecontrol
  Biosensors 2008, THe 10th World congress on Biosensors
  14 May 2008, Oral presentation(keynote)
• ルシフェラーゼに結合するDNA アプタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• Thyroglobulinに結合するDNAアプタマーの探索Aptameric Enzyme Subunit への応用を目指したFADGDH 活性に影響を及ぼすDNA アプタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• Thyroglobulinに結合するDNAアプTau タンパク質に結合するDNA アプタマーのスクリーニングタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• 標的分子認識に伴い構造変化するアプタマーのデザインとそのB/F分離への応用ルシフェラーゼに結合するDNA アプタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• 標的分子認識に伴い構造変化するアプタマーのデザインとそのB/F分離への応用
  日本化学会第88春季年会
  27 Mar. 2008, Other
• C-reactive proteinに結合するDNAアプタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• Thyroglobulinに結合するDNAアプタマーの探索
  日本化学会第88春季年会
  27 Mar. 2008, Other
• APTAMERIC ENZYME SUBUNIT (AES) BASED BIOSENSING
  THIRD INTERNATIONAL WORKSHOP ON "BIOSENSORS FOR FOOD SAFETY AND ENVIRONMENTAL MONITORING"
  07 Oct. 2007, Oral presentation(general)
• Homogeoeous protein detection using aptamer based on laser interferometric photo-thermal displacement measurement
  10th International Conference BIODETECTION TECHNOLOGIES 2007
  05 Oct. 2007, Poster presentation
• Detection of interaction between aptamer and protein based on laser interferometric photo-thermal displacement measurement
  3rd Annual Meeting of the Oligonucleotide Therapeutics Society
  05 Oct. 2007, Poster presentation
• Development of simple and rapid detection method of pathogenic bacteria using Zn finger protein
  10th International Conference BIODETECTION TECHNOLOGIES 2007
  14 Jul. 2007, Poster presentation
• Development of biosensing method using aptamers
  第5回　細胞工学技術研究会
  09 Jul. 2007, Oral presentation(invited, special)
• Aptameric biosensors
  Japan- American Frontier of Science symposium
  08 Dec. 2006, Oral presentation(general)
• Screening of peptide ligands by in silico panning
  Japan-China-Korea Joint Symposium on Enzyme Engineering
  30 Oct. 2006, Oral presentation(general)
• Decreasing the fibrillation and cytotoxicity of alpha-synuclein, a causative factor of Parkinson's disease
  Japan-China-Korea Joint Symposium on Enzyme Engineering
  30 Oct. 2006, Oral presentation(general)
• Aptameric enzyme subunit； novel analytical nanotool
  2nd ICBN int'l conference of Bio-nanointerface
  10 Oct. 2006, Poster presentation
• 電磁場による金ナノ粒子修飾トロンビンアプタマーの機能制御
  第９回日本化学会バイオテクノロジー部会シンポジウム
  28 Sep. 2006, Poster presentation
• マウスプリオンアプタマーの探索とそのセンシングへの応用
  第９回日本化学会バイオテクノロジー部会シンポジウム
  28 Sep. 2006, Oral presentation(general)
• DNA固定化担体によりB/F分離を行うタンパク質検出用アプタマーセンサーの開発
  第９回日本化学会バイオテクノロジー部会シンポジウム
  28 Sep. 2006, Oral presentation(general)
• Znフィンガータンパク質を用いた病原性微生物の特異的検出法の開発
  第58回日本生物工学会大会
  11 Sep. 2006, Other
• ピロロキノリンキノンによるヒト由来alpha-シヌクレインの凝集・線維化への影響
  第58回日本生物工学会大会
  11 Sep. 2006, Other
• Evolution of thrombin-inhibiting DNA aptamers based on inhibitory activity using a genetic algorithm
  XVIIinternational roundtable on Nucleosides, Nucleotides and Nucleic acids
  05 Sep. 2006, Poster presentation
• Aptameric enzyme subunit as novel molecular element for biosensing
  XVIIinternational roundtable on Nucleosides, Nucleotides and Nucleic acids
  05 Sep. 2006, Poster presentation
• Aptameric biosensors; novel sensing systems based on aptamers’ properties
  3rd International Symposium on Innovative BioPhysio Sensor Technology, July 7, 2006, Jeju
  07 Jul. 2006, Oral presentation(invited, special)
• Aptameric enzyme subunit: Novel biosensing element based on enzyme-inhibiting aptamer
  Biosensor 2006
  09 May 2006, Poster presentation
• Development of aptamer based protein detection system using DNA immobilized support for bound/free separation
  Biosensor 2006
  09 May 2006, Oral presentation(general)
• DNA固定化担体を用いた電気化学的タンパク質検出システムの開発
  電気化学会第73回大会
  02 Apr. 2006, Oral presentation(general)
• 金属ナノ粒子修飾トロンビン阻害DNAアプタマーの作製と特性検討
  電気化学会第73回大会
  02 Apr. 2006, Oral presentation(general)
• 電位を印加することによるa-シヌクレインの凝集の加速および凝集体の評価
  電気化学会第73回大会
  02 Apr. 2006, Oral presentation(general)
• 国際学会口頭発表２５件以上
  Apr. 2006, Other
• 国内学会口頭発表　２５件以上
  Apr. 2006, Other
• ジンクフィンガー蛋白質Sp2を用いたLegionella pneumophila strain philadelphia 1の迅速・簡便な検出法の開発
  日本化学会第86春期年会
  27 Mar. 2006, Oral presentation(general)
• 変異αシヌクレインの細胞毒性評価
  日本化学会第86春期年会
  27 Mar. 2006, Oral presentation(general)
• 複数標的蛋白質に対するDNAアプタマーの同時探索法の開発
  日本化学会第86春期年会
  27 Mar. 2006, Oral presentation(general)
• Aptamer blottingによる細胞中の蛋白質に対するDNAアプタマーの探索（２）
  日本化学会第86春期年会
  27 Mar. 2006, Oral presentation(general)
• Aptamer blottingによる細胞中の蛋白質に対するDNAアプタマーの探索（１）
  日本化学会第86春期年会
  27 Mar. 2006, Oral presentation(general)
• Aptameric enzyme subunit； novel analytical nanotool
  NSF/MEXT U.S. Young scientists symposium on bionanotechnology
  09 Dec. 2005, Oral presentation(invited, special)
• Aptameric enzyme subunit as a novel nano sensing probe
  NSF/MEXT U.S. Young scientists symposium on bionanotechnology
  15 Mar. 2005, Oral presentation(invited, special)
• Technology transfer from the University to the industry
  6th Japan-EU biotechnology policy working group meeting (Brussels, Belgium)
  12 Feb. 2001, Oral presentation(invited, special)
• Development of DNA sensor for rapid detection
  計測連合シンポジウム 先端計測2000
  18 May 2000, Oral presentation(invited, special)
• Biosensors for environmental monitoring
  An approarch to environmental sensing, Biomolecular mechanism & design workshop '99 (Tsukuba, Japan)
  Jun. 1999, Oral presentation(invited, special)
• Recent achievements in biosensors development in Japan
  New trends in biosensor development, NATO advanced research workshop (Kiev, Ukraine)
  Jul. 1998, Oral presentation(invited, special)
• Molecular imprinting technique for biosensing
  New biomolecular recognition elements for analytical biotechnology (Luckenwalde, Germany)
  Jun. 1998, Oral presentation(invited, special)
• DNA sensor
  New biomolecular recognition elements for analytical biotechnology (Luckenwalde, Germany)
  Jun. 1998, Oral presentation(invited, special)
• バイオセンサーと環境センシングの新展開
  国際環境バイオ・エコ技術講演会 (tokyo, Japan)
  Jan. 1997, Oral presentation(invited, special)

External funds

• DNAアプタマーに関する指導
  commissioned research, From 01 Jul. 2016, To 31 Mar. 2017
• 酵素阻害アプタマーを用いた高感度簡易迅速疾病診断法の開発
  commissioned research, From 01 Apr. 2016, To 31 Mar. 2017
• 酵素阻害アプタマーを用いた高感度簡易迅速疾病診断法の開発
  commissioned research, From 16 Nov. 2015, To 31 Mar. 2016
• アプタマーを用いた各種測定法の習得
  commissioned research, From 08 Jul. 2015, To 09 Jul. 2015
• 反応内臓チップによる小型遺伝子定量装置の実用化開発
  commissioned research, From 01 Apr. 2014, To 31 Mar. 2015
• 反応内臓チップによる小型遺伝子定量装置の実用化開発
  commissioned research, From 01 Apr. 2013, To 31 Mar. 2014
• 反応内臓チップによる小型遺伝子定量装置の実用化開発
  commissioned research, From 01 Oct. 2012, To 31 Mar. 2013
• 食品分析用バイオセンサの試作評価
  commissioned research, From 01 Oct. 2012, To 31 Mar. 2013
• マイクロ流路チップ・フローサイトメーターとアプタマーを用いたアルツハイマー病早期診断法の開発
  commissioned research, From 01 Apr. 2012, To 30 Sep. 2012
• アプタマーを利用した電気化学的VEGF高感度・迅速検出システムの開発
  commissioned research, From 01 Apr. 2012, To 31 Jul. 2012
• アプタマーを利用した電気化学的VEGF高感度・迅速検出システムの開発
  commissioned research, From 01 Dec. 2011, To 31 Mar. 2012
• マイクロ流路チップ・フローサイトメーターとアプタマーを用いたアルツハイマー病早期診断法の開発
  commissioned research, From 01 Oct. 2011, To 31 Mar. 2012
• 標的タンパク質認識アプタマーの分子進化
  cooperative research, From 01 Apr. 2010, To 31 Mar. 2011
• 平成20年度東京都重点戦略プロジェクト支援事業｢超高齢化都市のモデルに対応する有害微生物・ウイルスの複数・迅速・検出装置｣
  cooperative research, From 01 Apr. 2010, To 28 Feb. 2011
• 光熱変換とアプタマーを用いた標的タンパク質検出法の開発
  cooperative research, From 18 May 2009, To 31 Mar. 2010
• 平成20年度東京都重点戦略プロジェクト支援事業 「超高齢化都市のモデルに対応する有害微生物・ウイルスの複数・迅速・検出装置」
  cooperative research, From 10 Dec. 2008, To 31 Mar. 2009
• 光熱変換とアプタマーを用いた標的タンパク質検出法の開発
  cooperative research, From 22 Oct. 2008, To 31 Mar. 2009
• 変形アプタマーによる酵素捕捉を利用した超高感度センシングシステムの開発
  commissioned research, From 17 Jun. 2008, To 31 Mar. 2009
• 臨床診断技術に関する研究
  cooperative research, From 22 May 2008, To 21 May 2009
• ペプチドリガンドの新規探索法in silico panning法の開発
  commissioned research, From 20 Jul. 2007, To 31 Mar. 2008
• レーザー干渉光熱変換法とアプタマーを用いた標的分子迅速検出技術の開発
  commissioned research, From 01 Apr. 2007, To 31 Oct. 2007
• レーザー干渉光熱変換法とアプタマーを用いた標的分子迅速検出技術の開発
  commissioned research, From 01 Nov. 2006, To 31 Mar. 2007
• 干渉増幅反射法とアプタマーを利用した新規センシングシステムに関する研究
  cooperative research, From 17 Jan. 2006, To 31 Mar. 2006
• 光導波路測定装置を用いたレジオネラ簡便検出用DNAセンシングシステムの開発
  commissioned research, From 2006, To 2007
• レーザ干渉光熱変換法とアプタマーを用いた標的分子迅速検出技術の開発
  commissioned research, From 2006, To 2007
• 標的タンパク質に結合するアプタマー材料の探索
  cooperative research, From 26 Apr. 2005, To 31 Mar. 2006
• リガンドの探索及び探索手法開発研究
  cooperative research, From 01 Apr. 2005, To 31 Mar. 2006
• 光導波路測定装置を用いた蛋白質の凝集・綿維化の解析
  cooperative research, From 01 Apr. 2005, To 31 Mar. 2006
• in silico 進化によるリガンドペプチドの探索
  cooperative research, From 10 Feb. 2005, To 31 Mar. 2005
• 干渉増幅反射法を応用したバイオセンサーの開発
  cooperative research, From 18 Oct. 2004, To 31 Mar. 2005
• 標的タンパク質に結合するアプタマー材料の探索
  cooperative research, From 13 Sep. 2004, To 31 Mar. 2005
• 生体分子設計方法の開発研究
  cooperative research, From 22 Jun. 2004, To 31 Mar. 2005
• Research and Development of the method for designing biomolecules
  cooperative research, From 2004, To 2004
• Screening of the aptamers binding to the target protein
  cooperative research, From 2004, To 2004
• Development of the protein detection system using the Photo-wave guide spectrofluorometer and DNA aptamer
  cooperative research, From 2003, To 2004
• 干渉増幅反射法による分子間相互作用を応用したバイオセンサーの構築
  cooperative research, From 2003, To 2004
• 微生物によるバイオ燃料電池変換技術の開発
  commissioned research, From 2002, To 2004
• 実用型SNP測定用チップの開発
  commissioned research, From 1999, To 2000
• Development of technology for analysis of protein expression and interaction
  cooperative research, From 1999, To 2000
• Development of biosensors for environmental monitoring
  cooperative research, From 1998, To 2000
• Development of the novel detection system for protein included in micrometer size of tissue slice
  cooperative research, From 0000, To 0000

Committee Memberships

• Biosensors and Bioelectronics
  Associate editor
  From 20190501, To 20290430
• 日本核酸化学会
  評議員
  From 20181001, To 20280331
• 日本化学会
  化学と工業編集委員
  From 2000, To 0000

Media Coverage

• 東京農工大、迅速簡便ウイルス検出・ウイルス汚染箇所可視化ツールーPULSERAAーを開発
  農工大らの国際共同研究チームは、ウイルスを迅速簡便に検出し、さらに検出試薬を噴射するのみでウイルス汚染箇所を可視化する基盤技術を開発した、と掲載される。
  日経バイオテク
  From 15 Oct. 2024, To 15 Oct. 2024
• Researchers uncover novel amyloidosis　α-S1-casein forms amyloid by overexpression and N-terminal truncation in canine mammary tumor
  A collaboration led by scientists at Tokyo University of Agriculture and Technology (TUAT), Japan, has discovered a novel amyloid protein from canine mammary tumors.
  EurekAlert!／News-Medical.Net／Phys.Org
  From 23 Jan. 2023, To 24 Jan. 2023
• G-quadruplex-forming DNA molecules enhance enzymatic activity of myoglobin
  Dr. Ikebukuro and his group have developed many DNA aptamers that bind proteins, especially enzymes.
  PHYS ORG
  From 01 Jul. 2021, To 01 Jul. 2021
• 東京農工大など、ルミノール反応を300倍以上に増強するDNAアプタマーを開発
  東京農工大学の池袋一典教授らの研究チームが、ミオグロビン蛋白質のルミノールに対する酸化反応の活性を300倍以上に増強するDNAアプタマーを開発したと発表したことが紹介される。
  日経バイオテク
  From 11 Jun. 2021, To 11 Jun. 2021
• DNAに“設計図”以外の新機能を発見　酵素活性を強める効果
  東京農工大学大学院が、DNAに「生命の設計図」以外の機能として、酵素活性を強める効果を見つけたと発表したことが紹介される。
  yahooニュース／ITmedia／gooニュース／msnニュース／ニコニコニュース
  From 09 Jun. 2021, To 09 Jun. 2021
• 運転中の心筋梗塞、予兆を早期検知…デンソーなどDNAの機能を発見
  デンソーと東京農工大学、理化学研究所、ノースカロライナ大学は、特殊な構造（グアニン四重鎖構造）を持つDNAがミオグロビンタンパク質の持つ酵素活性を増強する機能があることを発見したことが紹介される。
  yahooニュース／Response／msnニュース
  From 08 Jun. 2021, To 08 Jun. 2021
• 新型コロナウイルスに対する安価、短時間、高感度の抗原検査法を開発
  JNCと東京農工大学の池袋一典教授が、新型コロナウイルスの迅速‐高感度検出に成功したことが紹介される。
  MONOist／yahooニュース
  From 14 Dec. 2020, To 14 Dec. 2020
• JNCなど　新型コロナ迅速検出　DNAアプタマー・検体混合 検出感度も向上
  JNCと東京農工大学の池袋一典教授が、新型コロナウイルスの迅速‐高感度検出に成功したことが紹介される。
  日刊工業新聞／化学工業日報／石油化学新聞日刊通信／日刊ケミカルニュース
  From 25 Nov. 2020, To 25 Nov. 2020
• JNC　コロナを迅速かつ高感度に検出する技術を共同開発　JNC , 東京農工大学 , 迅速-高感度免疫診断技術AptⅠa（アプティア）法
  JNCと東京農工大学の池袋一典教授が、新型コロナウイルスの迅速‐高感度検出に成功したことが紹介される。
  日刊ケミカルニュース／Jchem-News
  From 25 Nov. 2020, To 25 Nov. 2020

Professional Memberships

• 日本化学会
  From 2000
• 電気化学会 生物工学研究会
• The Society for Biotechnology
• 日本分子生物学会

Awards

• Elsevier and Biosensors 2008
  The Elsevier Biosensors & Bioeletronics Award 2008, 2nd Prize
  Selection of DNA aptamers against insulin and construction of an aptameric enzyme subunit for insulin sensing
  1000件中の2位
  15 May 2008
• Marquis who's who
  Listed in 25th Silver Aniversary edition of Marquis who's who in the world
  research activities
  2007
• Marquis Who's Who in Asia 2007に掲載
  2006
• British council fellowship
  1991