Noriyuki Nakamura

National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

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Publications (24)110.43 Total impact

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    ABSTRACT: An atomic force microscope probe can be formed into an ultra-sharp cylindrical shape (a nanoneedle) using micro-fabrication techniques such as focused ion beam etching. This nanoneedle can be effectively inserted through the plasma membrane of a living cell to not only access the cytosol, but also to penetrate through the nuclear membrane. This technique shows great potential as a tool for performing intranuclear measurements and manipulations. Repeated insertions of a nanoneedle into a live cell were previously shown not to affect cell viability. However, the effect of nanoneedle insertion on the nucleus and nuclear components is still unknown. DNA is the most crucial component of the nucleus for proper cell function and may be physically damaged by a nanoneedle. To investigate the integrity of DNA following nanoneedle insertion, the occurrence of DNA double-strand breaks (DSBs) was assessed. The results showed that there was no chromosomal DNA damage due to nanoneedle insertion into the nucleus, as indicated by the expression level of γ-H2AX, a molecular marker of DSBs.
    No preview · Article · May 2013 · Journal of Bioscience and Bioengineering
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    ABSTRACT: A cell diagnosis technique was developed, which uses an Atomic Force Microscope (AFM) and an ultra-thin AFM probe sharpened to a diameter of 200nm (nanoneedle). Due to the high aspect ratio of the nanoneedle, it was successfully inserted into a living cell without affecting its viability. Furthermore, by functionalizing the nanoneedle with specific antibodies and measuring the unbinding forces ('fishing forces') during evacuation of the nanoneedle from the cell, it was possible to measure specific mechanical interactions between the antibody-functionalized nanoneedle and the intracellular contents of the cell. In this study, an anti-actin-antibody-functionalized nanoneedle was used to evaluate the actin cytoskeleton state in living cells. To examine the effect of cytoskeleton condition on the measured fishing forces, the cytoskeleton-disrupting drugs cytochalasin D (cytD) and Y-27632 were used, showing a marked decrease in the measured fishing forces following incubation with either of the drugs. Furthermore, the technique was used to measure the time course changes in a single cell during incubation with cytD, showing a gradual time-dependent decrease in fishing forces. Even minute doses of the drugs, the effects of which were hardly evident by optical and fluorescence methods, could be clearly detected by the measurement of nanoneedle-protein fishing forces, pointing to the high sensitivity of this detection method. This technique may prove beneficial for the evaluation of cytoskeleton conditions in health and disease, and for the selection of specific cells according to their intracellular protein contents, without the need for introduction of marker proteins into the cell.
    Full-text · Article · Jun 2012 · Biosensors & Bioelectronics
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    ABSTRACT: A nanoneedle, an atomic force microscope (AFM) tip etched to 200 nm in diameter and 10 μm in length, can be inserted into cells with the aid of an AFM and has been used to introduce functional molecules into cells and to analyze intracellular information with minimal cell damage. However, some cell lines have shown low insertion efficiency of the nanoneedle. Improvement in the insertion efficiency of a nanoneedle into such cells is a significant issue for nanoneedle-based cell manipulation and analysis. Here, we have formed nanofilms composed of extracellular matrix molecules on cell surfaces and found that the formation of the nanofilms improved insertion efficiency of a nanoneedle into fibroblast and neural cells. The nanofilms were shown to improve insertion efficiency even in cells in which the formation of actin stress fibers was inhibited by the ROCK inhibitor Y27632, suggesting that the nanofilms with the mesh structure directly contributed to the improved insertion efficiency of a nanoneedle.
    Full-text · Article · Mar 2012 · Biochemical and Biophysical Research Communications
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    ABSTRACT: We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.
    No preview · Article · Oct 2011 · Biosensors & Bioelectronics
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    ABSTRACT: In this study, we have developed a method of mechanical force detection for ligands bound to receptors on a cell surface, both of which are involved in a signal transduction pathway. This pathway is an autocrine pathway, involving the production of insulin-like growth factor-II (IGF-II) and activation of the IGF-I receptor, involved in myoblast differentiation induced by MyoD in C3H10T1/2 mouse mesenchymal stem cells. Differentiation of C3H10T1/2 was induced with the DNA demethylation agent 5-azacytidine (5-aza). The etched AFM tip used in the force detection had a flat surface of which about 10 µm(2) was in contact with a cell surface. The forces required to rupture the interactions of IGF-IIs on a cell and anti mouse IGF-II polyclonal antibody immobilized on an etched AFM tip were measured within 5 days of induction of differentiation. The mean unbinding force for a single paired antibody-ligand on a cell was about 81 pN, which was measured at a force loading rate of about 440 nN/s. The percentage of unbinding forces over 100 pN increased to 32% after 2 days from the addition of 5-aza to the medium. This method could be used in non-invasive and successive evaluation of a living cell's behavior.
    No preview · Article · Jan 2011 · Journal of Molecular Recognition
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    ABSTRACT: We developed a novel nanobiosensor for monitoring mRNA expression in a single living cell by using an atomic force microscope (AFM) equipped with a nanoprobe. The nanoprobe was constructed by immobilizing a biotin-modified molecular beacon onto an ultrathin needle (nanoneedle) via neutravidin. In order to evaluate the effectiveness of the nanoprobe, we selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the target. A single HeLa cell contained approximately 1000 copies of GAPDH mRNA. The nanoprobe was directly inserted into living HeLa cells, and it reacted with the intrinsic target mRNA within the cells. The nanoprobe could be renatured by pulling it out of the cells. Further, we successfully used the nanoprobe for continuous detection of GAPDH mRNA in multiple cells, i.e., the nanoprobe was highly specific and sensitive for the detection of intrinsic mRNA in single living cells. mRNAs are thought to be highly condensed because of the large number of organelles and complexes present in cells and the limited space available for distribution. Therefore, direct analysis of intrinsic mRNAs in living cells would be advantageous, and our novel nanoprobe is highly suitable for monitoring the RNAs in living cells.
    No preview · Article · Dec 2010 · Biosensors & Bioelectronics
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    ABSTRACT: Previously, we reported that a nanoneedle of 200 nm diameter manipulated by an atomic force microscope apparatus could be inserted into a living cell. The insertion probabilities varied according to cell type. However, the nanoneedle was never successfully inserted into artificial liposomes. In the current study, we found that the stress fibers and actin filaments comprising the plasmalemmal undercoat are important, determining factors as to whether a nanoneedle can be successfully inserted into a cell. Depolymerization of microtubules increased both the number of stress fibers and insertion efficiency in NRK cells. These results indicate that the insertion efficiency of a nanoneedle (200 nm in diameter) into a cell with a smaller actin meshwork in its plasmalemmal undercoat is enhanced and the formation of stress fibers obviously contributes to this incremental enhancement. These facts are not only important as technical information to improve the efficiency of cell manipulation but also as observations of the mechanical properties of the native cell cortex.
    No preview · Article · Aug 2010 · Cytoskeleton
  • Sung-Woong Han · Chikashi Nakamura · Yosuke Imai · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: In this study, we have evaluated a sensor system for a hormonal drug effect in a single cell level using a novel low invasive single cell DNA delivery technology using a nanoneedle. An estrogen responsive GFP reporter vector (pEREGFP9) was constructed and its estrogenic response activity was confirmed in breast cancer cells (MCF-7) using lipofection as the means of transferring the vector to the cells. The pEREGFP9 vector was delivered to a single MCF-7 using a nanoneedle and the effect of ICI 182,780, which is an antagonist of estrogen, was observed using the GFP expression level. By ICI 182,780 treatment, the fluorescence intensity of the GFP was decreased by 30-50% within 24h. This technology is the very first trial of single cell diagnosis and we are looking forward to applying it to precious single cell diagnosis in medical fields.
    No preview · Article · Aug 2008 · Biosensors & Bioelectronics
  • Chikashi NAKAMURA · Hideki KAMIISHI · Noriyuki NAKAMURA · Jun MIYAKE
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    ABSTRACT: An influx of calcium ions into a living cell occurs when the cell is deformed mechanically by compressing, indenting or stretching. The calcium ions are taken up through mechanosensitive channels sensing deforming signals. The calcium ion is a key trigger for the reformation of cytoskeletal structure. Indentation of a capillary to a cell during microinjection causes influx of calcium ions, as does a typical AFM tip. A nanoneedle, which is a sharpened needle shaped AFM tip of 200 nm in diameter does not cause any influx of calcium ions when it is inserted into a cell. The mechanical stress from the nanoneedle insertion is negligible and its invasiveness is low.
    No preview · Article · Aug 2008 · Electrochemistry -Tokyo-
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    ABSTRACT: An antibody column in tandem with a fluorescent dye entrapped liposome column was developed for highly sensitive detection of an endocrine disruptor, bisphenol A (BPA). Anti-BPA antibody was immobilized in a protein G column with orientation control. A derivative of BPA was conjugated to phospholipase A2 (PLA2). BPA sample solutions mixed with the BPA-PLA2 conjugates were injected on to the anti-BPA antibody column and competitive binding occurred in the antibody column. The amount of the free conjugate was proportional to the concentration of the BPA sample. The eluted conjugates were injected on to the second column gel on which calcein-entrapped liposomes were immobilized and the PLA2-catalyzed hydrolysis of liposomal phospholipids causing fluorescent dye leakage as a signal amplification. In this system, the mixture of BPA and BPA-PLA2 conjugate were incubated for 60 min in the anti-BPA column, and then the collected solution was applied to the liposome column. The BPA detection range of 0.02-140 ng mL(-1) was wider than 0.03-6.6 ng mL(-1) obtained by the method of competitive ELISA using the same antibody. Moreover, this system could be adapted to an HPLC system resulting in almost the same detection limit in online detection. The method could be applied to environmental samples, river water and soil extracts. The BPA concentration of 0.1 ng mL(-1) and 10 ng g(-1) was detectable in water and soil extract, respectively.
    Full-text · Article · Oct 2006 · Analytica chimica acta
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    ABSTRACT: The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.
    No preview · Article · Jan 2006 · Analytical Chemistry
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    Ikuo Obataya · Chikashi Nakamura · SungWoong Han · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: We have developed a tool for directly inserting proteins into living cells by using atomic force microscopy (AFM) and an ultrathin needle, termed a nanoneedle. The surface of the nanoneedle was modified with His-tagged proteins using nickel chelating nitrilotriaceticacid (NTA). The fluorescent proteins, DsRed2-His6 and EGFP-His6, could be attached to and detached from the surface of the nanoneedle. These results suggest that the Ni-NTA modified nanoneedle can successfully be used for specific delivery of proteins. The nanoneedle modified with DsRed2-His6 was able to penetrate the surface of a living HeLa cell, as confirmed by laser scanning fluorescence microscopy and monitoring an exerting force on the nanoneedle using AFM. Force curves using the nanoneedle indicated that the needle was able to penetrate at displacement speeds of 0.10–10 µm/s. These results suggest that this technique can be used to directly insert proteins into living cells and is applicable for modulation or regulation of single cell activity.
    Preview · Article · Nov 2005 · Nanobiotechnology
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    ABSTRACT: Phospholipase A2 (PLA2)-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, 3,3-bis[N,N-di(carboxymethyl)aminomethyl]fluorescein (calcein). This enzymatic reaction was applied as signal amplification for biosensor detection of low concentrations of polychlorinated biphenyls (PCBs). In order to increase the fluorescent signal for improvement of PCBs detection, the effect of BSA on optimal lipid composition for PLA2-catalyzed membrane leakage from fluorescent liposomes has been investigated in this report. Various kinds of calcein-entrapped liposomes were immobilized in Sephacryl S1000 gel beads using avidin-biotin binding. In a contrast, free calcein was removed by size exclusion chromatography on Sephacryl S300 for free liposome suspensions. The PLA2-catalyzed membrane leakage was detected both in these gel-bead-immobilized liposomes and in free liposome suspensions. In both systems, the fluorescent release from the liposomes by PLA2 hydrolytic action significantly increased with increasing albumin concentration. The most rapid and greatest membrane leakage by PLA2 hydrolysis was found in anionic liposomes in the presence of albumin, both in free liposome suspensions and gel-bead-immobilized liposomes. Finally, the stabilities of various free liposomes and gel-bead-immobilized liposomes were monitored. Immobilized 1-palmitoy-2-oleoylphosphatidylcholine (POPC)/1-palmitoy-2-oleoylphosphatidylglycerol (POPG) liposome gel was chosen due to its excellent stability and large dye leakage by PLA2. A concentration of PCBs as low as 0.1 ng/mL was detectable using this tandem column system.
    No preview · Article · Oct 2005 · Journal of Chromatography A
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    SungWoong Han · Chikashi Nakamura · Ikuo Obataya · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: We have previously demonstrated a new cell manipulation technology by using an atomic force microscope (AFM) and ultrathin needles, named nanoneedles. The nanoneedle is an AFM tip etched by a focused ion beam (FIB) and is sharpened from 200 to 800 nm in diameter. In this study, we have evaluated the proper diameter of a needle required for insertion into human cells over a long period without causing cell death, and achieved highly efficient gene expression method for human cells using a nanoneedle and an AFM.
    Preview · Article · Aug 2005 · Biochemical and Biophysical Research Communications
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    ABSTRACT: We developed a new low invasive cell manipulation and gene or molecule transfer system in a single living cell by using an atomic force microscope (AFM) and ultra thin needle, a nanoneedle. DNA was immobilized on the surface of the nanoneedle by covalent bonding and avidin-biotin affinity binding. Immobilization of DNA on the nanoneedle was confirmed by measuring the unbinding force between avidin and biotin. The DNA-immobilized nanoneedle was successfully inserted into HEK293 cells. Though TO-PRO-3 iodide staining experiments using confocal microscopy, we observed the immobilized DNA on the surface of the nanoneedle, which was retained after 10 times insertions to and evacuations from a living cell.
    Preview · Article · May 2005 · Biosensors & Bioelectronics
  • Ikuo Obataya · Chikashi Nakamura · SungWoong Han · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: Mechanical responses during insertion of a silicon nanoneedle into a living melanocyte were observed by using an atomic force microscope (AFM). In order to study the dependence of the mechanical response on the shape of the nanoneedle, we prepared various shapes of silicon AFM tips by focused-ion beam (FIB) etching. The force curves showed increases up to 0.65-1.9 nN after contact on the cell surface, and then the force dropped corresponding with the penetration of the needle through the cell membrane. The force required for penetration was significantly smaller than that using a normal pyramidal tip. The force curves with a cylindrical tip showed a shorter indenting distance before penetration than that with the cone-shaped tip. It is considered that the information about the geometry of penetrating material leads to the development of more suitable micro- and nano-materials to insert into a living cell for cell surgery.
    No preview · Article · Mar 2005 · Biosensors & Bioelectronics
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    Ikuo Obataya · Chikashi Nakamura · Sungwoong Han · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: We have developed a tool for performing surgical operations on living cells at nanoscale resolution using atomic force microscopy (AFM) and a modified AFM tip. The AFM tips are sharpened to ultrathin needles of 200-300 nm in diameter using focused ion beam etching. Force-distance curves obtained by AFM using the needles indicated that the needles penetrated the cell membrane following indentation to a depth of 1-2 microm. The force increase during the indentation process was found to be consistent with application of the Hertz model. A three-dimensional image generated by laser scanning confocal microscopy directly revealed that the needle penetrated both the cellular and nuclear membranes to reach the nucleus. This technique enables the extended application of AFM to analyses and surgery of living cells.
    Preview · Article · Feb 2005 · Nano Letters
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    ABSTRACT: The purpose of this study was to screen for peptides that bind herbicides with a chlorinated aniline chemical structure. A tetrapeptide library was constructed using a solid phase split synthesis approach. Peptide beads were suspended in a buffer containing fluorescent-labeled dichloroaniline (DCA) as the bait. Eighteen fluorescent peptide beads were selected which bound to the bait after two rounds of staining screenings. The beads were then stained and suspended in a solution containing an excess of DCA and five quenched peptide beads were subsequently selected that recognized the DCA moiety. The screened peptides had many sequence similarities. The binding affinity of the screened peptides to herbicides was analyzed using surface plasmon resonance (SPR). N′-(3,4-dichlorophenyl)-N,N-dimethylurea [3-(3,4-dichlorophenyl)-1,1-dimethylurea] solution was injected over the peptide immobilized SPR chip. The SPR signal was found to increase in proportion to the DCMU concentration, whereas no signal was obtained from the negative control, 2-(2-methyl-4-chlorophenoxy) propionic acid (MCPP). From these results it is suggested that the screened peptide selectively recognizes the chemical structure of DCA.
    No preview · Article · Jun 2004 · Journal of Molecular Catalysis B Enzymatic
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    ABSTRACT: Cationic poly(viologen) derivatives [(V2+)m] and anionic polymer [poly(styrenesulfonic acid-co-maleic acid), sodium salt] were assembled on electrode surfaces by layer-by-layer (LBL) and Langmuir–Blodgett (LB) methods. The assembling process was in situ investigated by quartz crystal microbalance (QCM) technique. Based on the frequency change, the average surface coverage of poly(butylviologen) in the LBL multilayers is in the range of 1.6∼3.3×10−10 mol/cm2. The mass change for one-layer LB film of poly(dodecylviologen)-anionic polymer is about 44, 96 and 263 ng/cm2 for the films deposited at 5, 10 and 15 mN/m, respectively. The assembled LBL and LB multilayers show well reversible redox characteristics of the viologen. For poly(viologen) with short alkyl chains, the cyclic voltammograms (CVs) show two redox couples of (V2+)m ↔ (V+)m and (V+)m ↔ (0.5(V+)2)m when the last layer is poly(viologen), or one redox couple of (V2+)m ↔ (V+)m when the last layer is anionic polymer. While for poly(viologen) with long alkyl chains, the CVs show only one couple of (V2+)m ↔ (V+)m. The LBL multilayers show much higher stability than the self-assembled monolayers and LB films in electrolyte solution.
    No preview · Article · Apr 2004 · Electrochimica Acta
  • Chikashi Nakamura · Chie Miyamoto · Ikuo Obataya · Noriyuki Nakamura · Jun Miyake
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    ABSTRACT: Scanning probe microscopy has been applied in many studies to manipulate atoms or molecules. In particular, force spectroscopy using an atomic force microscope (AFM) is a powerful tool to elucidate intermolecular or intramolecular interactions and provide mechanical information. If enzymes could retain their activity when immobilized on probes, not only could enzyme-substrate interactions be investigated but also the probes could be used for precise biomolecular manipulation at the nano-scale. In our study, a method based on "Enzymatic Nanolithography" was successfully performed in a buffered solution using Staphylococcal serine V8 protease and AFM. To estimate the fabricating activity of the protease immobilized on the AFM tip to peptides immobilized on a substrate, we designed and synthesized peptides that showed enzymatic action specific to the protease. When the protease digested the reporter peptide a quencher residue was released from the main flame of the peptide and resulted in fluorescence. In the designed 9 mer peptides, TAMRA functioned as a good quencher for FAM. After contact of the protease-immobilized tip to the reporter peptide layer, a fluorescent area was observed by microscopic imaging.
    No preview · Article · Jan 2004 · Proceedings of SPIE - The International Society for Optical Engineering

Publication Stats

687 Citations
110.43 Total Impact Points


  • 2002-2013
    • National Institute of Advanced Industrial Science and Technology
      • • Biomedical Research Institute
      • • Research Center for Stem Cell Engineering
      Tsukuba, Ibaraki, Japan
  • 2005-2012
    • Tokyo University of Agriculture and Technology
      • • Division of Biotechnology and Life Science
      • • Graduate School of Engineering
      Edo, Tōkyō, Japan