詳細

森田　資隆 （モリタ　ヤスタカ）

  • 産業理工学部 生物環境化学科　教授
Last Updated :2024/04/23

コミュニケーション情報 byコメンテータガイド

  • コメント

    生物が持つ優れた生体機能性材料の研究をしています。特に、低温活性酵素、難分解性物質などの環境修復、さらにはペプチドを生理活性物質やバイオセンサーなどに利用する研究をしています。

研究者情報

学位

  • 博士（材料科学）

ホームページURL

J-Global ID

研究キーワード

  • バイオテクノロジー   酵素工学   ペプチド工学   バイオセンサー   バイオチップ   

現在の研究分野（キーワード）

    生物が持つ優れた生体機能性材料の研究をしています。特に、低温活性酵素、難分解性物質などの環境修復、さらにはペプチドを生理活性物質やバイオセンサーなどに利用する研究をしています。

経歴

  • 2015年 - 現在  近畿大学産業理工学部教授
  • 2008年  近畿大学産業理工学部准教授
  • 2005年  近畿大学産業理工学部講師

学歴

  •         - 1998年   北陸先端科学技術大学院大学   材料科学研究科   博士後期課程
  •         - 1995年   北陸先端科学技術大学院大学   材料科学研究科   博士前期課程
  •         - 1993年   東京農工大学   工学部   物質生物工学科

所属学協会

  • 日本農芸化学会   日本生物工学会   日本化学会   

研究活動情報

論文

  • Yasuhiro Shinkai; Shinichi Kashihara; Go Minematsu; Hirofumi Fujii; Madoka Naemura; Yojiro Kotake; Yasutaka Morita; Koichiro Ohnuki; Alesya A. Fokina; Dmitry A. Stetsenko; Vyacheslav V. Filichev; Masayuki Fujii
    NUCLEIC ACID THERAPEUTICS 27 3 168 - 175 2017年06月 [査読有り]
     
    Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptide beta 7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideb7 is a promising candidate for in vivo use and therapeutic applications.
  • Efficient transfection of siRNA by designed peptides.
    Kayano H; Takashina A; Naemura M; Kotake Y; Morita Y; Fujii M
    Proceedings of the 41st International Symposium on Nucleic Acids Chemistry. 374 - 375 2014年09月 [査読有り]
  • Yojiro Kotake; Yuichi Ozawa; Masanori Harada; Kyoko Kitagawa; Hiroyuki Niida; Yasutaka Morita; Kenji Tanaka; Takafumi Suda; Masatoshi Kitagawa
    GENES TO CELLS 18 11 999 - 1006 2013年11月 
    Y box binding protein 1 (YB1) has multiple functions associated with drug resistance, cell proliferation and metastasis through transcriptional and translational regulation. Increased expression of YB1 is closely related to tumor growth and aggressiveness. We showed that YB1 protein levels were decreased through replicative and premature senescence and were correlated with increased expression levels of p16(INK4A) tumor suppressor gene. Depletion of YB1 was associated with increased levels of p16 in human and murine primary cells. Forced expression of YB1 in mouse embryonic fibroblasts resulted in decreased expression of p16 and increased cell proliferation. Senescence-associated expression of -galactosidase was repressed in YB1-over-expressing cells. Chromatin immunoprecipitation assays showed that YB1 directly associates with the p16 promoter. Taken together, all our findings indicate that YB1 directly binds to and represses p16 transcription, subsequently resulting in the promotion of cell growth and prevention of cellular senescence.
  • 森田 資隆
    生物工学会誌 90 5 253 - 253 日本生物工学会 2012年
  • Toshifumi Sakaguchi; Taiki Nakano; Yuko Kimura; Shouhei Nogami; Ikue Kubo; Yasutaka Morita
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 111 4 443 - 447 2011年04月 
    Conjugative mating between the selenate-reducing bacterium Citrobacter sp. strain JSA and Escherichia coli S17-1 harboring the broad-host-range plasmid pKT230 or pKT240 (IncQ) allowed genetic transfer to strain JSA at a maximum frequency of 2.5 x 10(-5) (pKT230) and 5.1 x 10(-6) (pKT240) per recipient JSA cell. Kanamycin-resistant (selection marker of pKT230 and pKT240) transconjugants were routinely obtained with this method, and we confirmed that both vectors were also successfully transferred and replicated in strain JSA without alteration of the replicon. Furthermore, an electroporation method has also allowed transformation of JSA at a frequency of 10(-7) CO 10(-6) transformants per mu g vector DNA (per recipient cell), and PCR and hybridization analyses revealed that pKT230 and pKT240 are stably maintained in transformed JSA cells. These results indicated that both InQ plasmids can be used as vectors for gene transfer to selenate-reducing strain JSA. This is the first study to demonstrate an effective method for genetic transfer in a selenate-reducing Citrobacter bacterium and will aid in the elucidation of the selenium oxyanion reduction mechanism in this genus of environmental selenate-respiring isolates. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.
  • Toshifumi Sakaguchi; Masaki Kato; Naoki Kuriyama; Harutaka Niiyama; Shougo Hamada; Yasutaka Morita; Eiichi Tamiya
    CURRENT MICROBIOLOGY 59 1 88 - 94 2009年07月 
    Conjugal mating between selenate-reducing Citrobacter sp. strain JSA and Escherichia coli S17-1 bearing pSUP2021 allowed transposon mutagenesis and chromosomal transformation. Kanamycin-resistant transconjugants were obtained successfully by this method from a freshwater selenate-respiring Citrobacter sp. strain JSA. The maximum frequency of kanamycin-resistant Tn5 transconjugants was 3.6 x 10(-6) per recipient of this strain. Of these transconjugants, eight strains of selenate reduction-deficient transconjugants living by nitrate reduction were obtained in the strain JSA. Moreover, the same phenotype of deficient mutant was created by chemical mutagenesis with ethylmethanesulfonate. The results strongly indicate that selenate reducing anaerobic respiration was independent of nitrate reduction in the Citrobacter sp. isolate strain JSA.
  • Masatoshi Tsukamoto; Shu Taira; Shohei Yamamura; Yasutaka Morita; Naoki Nagatani; Yuzuru Takamura; Eiichi Tamiya
    ANALYST 134 10 1994 - 1998 2009年 
    We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 mu L/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.
  • Is Helianti; Takako Okubo; Yasutaka Morita; Eiichi Tamiya
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 74 1 107 - 112 2007年02月 
    This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95 degrees C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90 degrees C for 5.5 h and 67% of its activity after being incubated at 100 degrees C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).
  • Toshifumi Sakaguchi; Yasunori Morioka; Masahiro Yamasaki; Junpei Iwanaga; Kazuhiko Beppu; Hideaki Maeda; Yasutaka Morita; Eiichi Tamiya
    BIOSENSORS & BIOELECTRONICS 22 7 1345 - 1350 2007年02月 
    A BOD monitoring system based on a bio-chip which immobilized luminous bacterium in micrometer-order holes were arrayed and fabricated by micro-machine techniques, was developed. The acrylic chip (3 cm x 3 cm) comprises nine micro-holes (diameter: 700 mu m or 1 mm, depth: 100 mu m) arranged in a three by three array. Cells of the marine luminous bacterium, Photobacterium phosphoreum IFO 13896, which was grown at 15 degrees C for 15 h, were immobilized with 3% or 15% sodium alginate gel. BOD standard solutions or actual sample solution (approximately 10 ld) was fallen onto the cell-arrayed chip, and then the chip was incubated at 25 degrees C for 25 min. After incubation, biolummescence from the each hole was gray-scaled and measured by a chemi-imager or newly developed onsite-type-monitoring system using a digital camera and a mobile-type personal computer. BOD values less than 16 ppm could detect by the chip, in particular, linear relationship at the concentrations between 0 and 16ppm could be observed when luminous cells were immobilized with 3% sodium alginate gel. Steady bioluminescence was observed on the chip in the presence of BOD standard solution (GGA solution) which contained mineral elements. Furthermore, simultaneous detection of BOD values in various samples could be employed in the single chip. These results showed that the monitoring system with bio-chip could achieve highthrough-put and onsite BOD detection. Our newly developed onsite-type BOD detection system which was used a digital camera and a (mobile) laptop computer was applied to measure and detect organic pollution due to biodegradable substances in wastewater treatment system. The same performance as the chemi-imager system was obtained for data of bioluminescence. The obtained BOD values showed a similar correlation with that of the conventional method for BOD determination (BOD5). These results suggested for successful achievement of high-though-put and onsite detection of BOD in practical. (c) 2006 Elsevier B.V. All rights reserved.
  • Miyuki Chikae; Ryuzoh Ikeda; Kagan Kerman; Yasutaka Morita; Eiichi Tamiya
    BIORESOURCE TECHNOLOGY 97 16 1979 - 1985 2006年11月 
    The composting process of food wastes and tree cuttings was examined on four composting types composed from two kinds of systems and added mixture of microorganisms. The time courses of 32 parameters in each composting type were observed. The efficient composting system was found to be the static aerated reactor system in comparison with the turning pile one. Using the multiple regression analysis of all the data (159 samples) obtained from this study, some parameters were selected to predict the germination index (GI) value, which was adopted as a marker of compost maturity. For example, using the regression model generated from pH, NH4+ concentration, acid phosphatase activity, and esterase activity of water extracts of the compost, GI value was expressed by the multi-linear regression equation (p < 0.0001). High correlations between the measured GI value and the predicted one were made in each type of compost. As a result of these observations, the compost maturity might be predicted by only sensing of the water extract at the composting site without any requirements for a large-size equipment and skill, and this prediction system could contribute to the production of a stable compost in wide-spread use for the recycling market. (c) 2005 Elsevier Ltd. All rights reserved.
  • Yasutaka Morita; Kou Mamiya; Shohei Yamamura; Eiichi Tamiya
    BIOTECHNOLOGY PROGRESS 22 4 974 - 978 2006年08月 
    The P19 cell is a pluripotent stem cell of murine teratocarcinoma. When treated with retinoic acid, P19 cells can be differentiated along a neural cell lineage in culture. To isolate peptides that bind to the stem cell, we employed a phage display technology with undifferentiated P19 cells as the target. To reduce nonspecific binding of phages to the cell surface, the phage libraries were preadsorbed to the differentiated P19 cells before each selection on the undifferentiated P19 cells. After eight rounds of the selection, No. 28 phage displaying ALPSTSSQMPQL-peptide was isolated. Immunofluorescence analysis revealed that No. 28 phage selectively binds to the undifferentiated P19 cells but not to the differentiated P19 cells or SHSY cell line. The chemically synthesized peptide ALPSTSSQMPQL presented on the No. 28 phage efficiently inhibited the binding of No. 28 phage to the undifferentiated P19 cells. This result confirmed that No. 28 phage binding to the cell was mediated by the displayed peptide. The identified peptide may be targeted to a marker expressed on the stem cell and thus become a practical tool for the isolation of somatic stem cells.
  • T Endo; S Yamamura; N Nagatani; Y Morita; Y Takamura; E Tamiya
    SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 6 5 491 - 500 2005年07月 
    In recent years, label-free biosensors not requiring external modifications have been receiving intense attention. A label-free optical biosensor, which retains many of the desirable features of conventional surface plasmon resonance (SPR) reflectometry, namely, the ability to monitor the kinetics of biomolecular interactions in real-time without a label has been developed with several important advantages: the biosensor device is easy to fabricate, and simple to implement, requiring only an UV-Vis spectrophotometer or flatbed scanner. Importantly, the label-free optical biosensor can be easily multiplexed to enable high-throughput monitoring of biormolecular interactions in an array-based format. In this research, the development of a localized surface plasmon resonance (LSPR)-based label-free optical biosensor using a surface modified nanoparticle layer is aimed. This optical detection method promises to offer a massively parallel detection capability in a highly miniaturized package. The two-dimensional nanoparticle layer was formed by the surface modified silica nanoparticles. The optical properties and surface analysis of nanoparticle layer substrate were characterized through transmission measurements and atomic force microscopy (AFM). Simultaneously, the nanoparticle layer substrate was applied to the optical LSPR-based biosensor for label-free monitoring of the antigen-antibody reaction. The anti-fibrinogen antibody was immobilized onto the nanoparticle layer substrate surface. Different concentrations of fibrinogen were introduced to the anti-fibrinogen antibody immobilized nanoparticle layer substrate surface, and the change in the absorption spectrum, caused by the antigen-antibody reaction, was observed. By using this anti-fibrinogen antibody immobilized nanoparticle layer substrate; the detection limit of this optical LSPR-based biosensor was 10 ng/ml. (c) 2005 Elsevier Ltd. All rights reserved.
  • K Kerman; Y Morita; Y Takamura; E Tamiya
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 381 6 1114 - 1121 2005年03月 
    An electrochemical hybridization biosensor based on the intrinsic oxidation signals of nucleic acids and proteins has been designed, that makes use of the unique binding event between Escherichia coli single-strand binding protein ( SSB) and single- stranded DNA (ssDNA). The voltammetric signal from guanine oxidation significantly decreased upon binding of SSB to single-stranded oligonucleotides (probe), anchored on a single- walled carbon nanotube (SWCNT)-modified screen-printed carbon electrode (SPE). Simultaneously, oxidation of the tyrosine (Tyr) and tryptophan (Trp) residues of the SSB protein increased upon binding of the SSB protein to ssDNA and ss-oligonucleotides. After the hybridization, SSB did not bind to the double helix form, and the guanine signal could be observed along with the disappearance of the oxidation signal of the protein. The amplification of intrinsic guanine and protein oxidation signals by SWCNT, and a washing step with sodium dodecylsulfate, enabled the specific detection of a point mutation. Monitoring the changes in the guanine and protein signals upon hybridization greatly simplified the detection procedure. The detection limit of 0.15 mu g/ml target DNA can be applied to genetic assays. To the best of our knowledge, this is the first work that utilizes the monitoring of SSB-DNA interactions on a solid transducer for the electrochemical detection of DNA hybridization by using intrinsic oxidation signals.
  • Y Matsubara; K Kerman; M Kobayashi; S Yamamura; Y Morita; E Tamiya
    BIOSENSORS & BIOELECTRONICS 20 8 1482 - 1490 2005年02月 
    A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different ne targets. related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA rnicroarray,canner with associated software. This simple method of counting the high fluorescence signal released in rnicrochambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA. (C) 2004 Elsevier B.V. All rights reserved.
  • T Endo; A Okuyama; Y Matsubara; K Nishi; M Kobayashi; S Yamamura; Y Morita; Y Takamura; H Mizukami; E Tamiya
    ANALYTICA CHIMICA ACTA 531 1 7 - 13 2005年02月 
    Detection cf pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen-antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 mu g/ml. The regression analysis provided the correlation coefficients of r = 0.982-0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes. (c) 2004 Elsevier B.V. All rights reserved.
  • K Yuhki; Y Tomizawa; Y Morita; E Tamiya; Y Takamura
    Micro Total Analysis Systems 2004, Vol 2 297 294 - 296 2005年 [査読有り]
     
    Extraction and purification of DNA/RNA from cells on chip is an important element for DNA-based micro total analysis system. Authors developed automated extraction and purification device using by taper shaped fluid cannel, electric and hydro drag force field, and valve switching system. It can manipulate extraction and purification of DNA from cells easily and rapidly.
  • Y Akagi; Rao, SR; Y Morita; Y Takamura; E Tamiya
    Micro Total Analysis Systems 2004, Vol 2 297 309 - 311 2005年 [査読有り]
     
    This study describes a high-throughput screening assay for novel neurotrophic factor using microarray based cell-chip and also the response of screened neurotrophic factor on Pheochromocytoma (PC12) cells neurosignaling pathway. High-throughput cell-based drug screening using microarray based chip formats play a key role in the development of novel and potential therapeutics at much faster rate with reduction in the cost of drug discovery. There have been reports on the use of nerve growth factor (NGF) in the prevention of the Alzheimer's disease in mice, rats, large primates. The screening of the novel neurotrophic factor was achieved adapting a combination of microarray based chips and combinatorial chemistry based peptide library synthesized on beads.
  • A Iiduka; Y Morita; E Tamiya; Y Takamura
    Micro Total Analysis Systems 2004, Vol 1 296 423 - 425 2005年 [査読有り]
     
    Novel microplasma for liquid sample optical emission spectrometry (OES) is reported, namely "liquid electrode plasma" or "bursting bubble plasma". The advantages of this method includes no requirement for plasma gas and nebulizer, and no contamination from electrodes is observed because of the low electric field around them, and the plasma does not interact directly with the electrodes. The microchip consisted of PDMS and SiO2. Solution samples were introduced into the channel, then direct current voltage (similar to 800 V) was applied from Pt electrodes inserted into the reservoirs at both sides, Typical spectra for NaCl, KCl, PBS, CuCl2 solutions were observed. In the case of blank aqueous solutions, different from conventional ICP-OES, no background peak was observed from plasma gases, such as Ar. The intensity ratio of K/Na showed a quantitative response, and the limit-of-detection from the OES data was estimated to be 300 ppb for Na.
  • Y Tomizawa; K Yuhki; Y Morita; E Tamiya; Y Takamura
    Micro Total Analysis Systems 2004, Vol 1 296 659 - 661 2005年 [査読有り]
     
    Extraction of a specific molecule from a solution in microfluidic device is one of the important issues for micro total analysis system. At the previous microTAS, we reported a DNA trap employing mutually reverse electric force and hydro drag force field [1]. In this paper, we measured the trap probabilities of DNA as functions of DNA size and other conditions, and characterized this trap as a molecular filter in microfluidic device.
  • Kagan Kerman; Yasutaka Morita; Yuzuru Takamura; Eiichi Tamiya; Kenzo Maehashi; Kazuhiko Matsumoto
    Nanobiotechnology 1 1 65 - 70 2005年 
    We have developed a nanosensor array composed of carbon nanotube field-effect transistors (CNTFETs) on SiO2/Si substrates. Unlike previously reported CNTFETs where the recognition event occurred directly on the CNT, in this case, the reverse surface of the substrate was utilized for the biomolecular functionalization. A self-assembled monolayer (SAM) of peptide nucleic acid (PNA) probes associated with the tumor necrosis factor-α gene (TNF-α) was attached onto the gold electrode on the reverse side of the CNTFET device. A time-dependent conductance increase was monitored upon sequential introduction of wild-type (WT) DNA samples through a microfluidic channel of the poly(dimethylsiloxane) (PDMS) chip. High selectivity of PNA probes only toward the full-complementary WT DNA samples enabled rapid and simple discrimination against single-nucleotide polymorphism (SNP) or non-complementary (NC) DNA. Concentration-dependent measurements indicated a limit-of-detection (LOD) of 6.8 fM WT DNA. Our CNTFET-based biochip is a promising candidate for the development of an integrated, high-throughput, portable device for nucleic acid-based diagnostics. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved.
  • Takeshi Fukuda; Seizaburo Shiraga; Michiko Kato; Shohei Yamamura; Yasutaka Morita; Eiichi Tamiya; Teruo Hori; Shin-Ichiro Suye; Mitsuyoshi Ueda
    Nanobiotechnology 1 1 105 - 111 2005年 
    A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a "yeast cell chip." A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved.
  • Nanosystems for biosensing: multianalyte immunoassay on a protein chip
    E. Tamiya; 森田 資隆; Z.L. Zhi; Q. Hasan
    Methods Mol. Biol. 300 369 - 381 2005年
  • ZL Zhi; Y Morita; S Yamamura; E Tamiya
    CHEMICAL COMMUNICATIONS 19 19 2448 - 2450 2005年 
    A strategy for the high-sensitivity, high-selectivity, and multiplexed detection of oligonucleotide hybridizations has been developed with an encoded Ni microparticle random array that was manufactured by a ''top-down'' approach using micromachining and microfabrication techniques.
  • M Saito; M Kobayashi; SI Iwabuchi; Y Morita; Y Takamura; E Tamiya
    JOURNAL OF BIOCHEMISTRY 136 6 813 - 823 2004年12月 
    DNA condensation was only observed after the addition of Hoechst 33258 (H33258) among various types of DNA binding molecules. The morphological structural change of DNA was found to depend on the H33258 concentration. On comparison of fluorescence spectrum measurements with AFM observation, it was found that fluorescence quenching of DNA-H33258 complexes occurred after DNA condensation. Additionally, we showed that DNA condensation by H33258 was independent of sequence selectivity or binding style using two types of polynucleotides, i.e. poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC). Moreover, it was concluded that the condensation was caused by a strong hydrophobic interaction, because the dissolution of condensed DNA into its native form on dimethyl sulfoxide (DMSO) treatment was observed. This study is the first report, which defines the DNA condensation mechanism of H33258, showing the correlation between the single molecule scale morphology seen on AFM observation and the bulky scale morphology observed on fluorescence spectroscopy.
  • Matsubara, V; H Kerman; M Kobayashi; S Yamamura; Morita, Y; Y Takamura; E Tamiya
    ANALYTICAL CHEMISTRY 76 21 6434 - 6439 2004年11月 
    A novel method for multiplex TaqMan PCR in nanoliter volumes on a highly integrated silicon microchamber array is described. Three different gene targets, related to beta-actin, sex-determining region Y (SRY), and Rhesus D (RhD) were amplified and detected simultaneously on the same chip by using three different types of human genomic DNA as the templates. The lack of cross-contamination and carryover was shown using alternate dispensing of mineral oil-coated microchambers containing template and those without template. To confirm the specificity of our system to beta-actin, SRY, and RhD genes, we employed the larger volume PCR samples to a commercial real-time PCR system, SmartCycler. The samples were cycled with the same sustaining temperatures as with the microchamber array. Instead of the conventional method of DNA quantification, counting the number of the fluorescence released microchambers in consequence to TaqMan PCR was employed to our chip. This simple method of observing the end point signal had provided a dynamic quantitative range. Stochastic amplification of 0.4 copies/reaction chamber was achieved. The micro-fabricated PCR chip demonstrated a rapid and highly sensitive response for simultaneous multiple-target detection, which is a promising step toward the development of a fully integrated device for the "lab-on-a-chip" DNA analysis.
  • K Kerman; Y Morita; Y Takamura; M Ozsoz; E Tamiya
    ELECTROANALYSIS 16 20 1667 - 1672 2004年10月 
    Multi-walled carbon nanotubes (MWNTs) were used as nanowires, which combined DNA molecules to a carbon paste electrode (CPE). The attachment of MWNT on the electrode surface was controlled by a hybridization assay between adenine and thymine containing oligonucleotides. The appearance of guanine oxidation signal after hybridization with target DNA greatly simplified the specific sequence DNA detection mechanism. Combination of sidewall- and end-functionalization of MWNT provided a significant enhancement in the voltammetric signal of guanine oxidation in comparison with the signals obtained from only end-oxidized MWNT modified CPE and a bare CPE. A control experiment involving adenine containing polynucleotide (poly(A)) instead of adenine probe modified MWNT was performed. The effect of target and noncomplementary DNA concentration on the guanine signal was also monitored. Discrimination against single-base mismatch and noncomplementary DNA was achieved by surfactant containing washing solution. The promising conductivity of carbon nanotubes, and the creation of a larger surface area for DNA immobilization by sidewall- and end-oxidation of MWNT provided a detection limit down to 10 pg/mL, which is compatible with the demand of the genetic tests.
  • M. Chikae; 森田 資隆; Y. Hatano; R. Ikeda; Q. Hasan; E. Tamiya
    Environmental Toxicology and Pharmacology 18 1 9 - 12 2004年09月 
    The effects of bis(2-ethylhexyl) phthalate (DEHP), gamma-hexachlorocyclohexane (gamma-HCH), and 17beta-estradiol (E2) on the fry stage of medaka were investigated. The medaka fry were exposed to different concentrations (0.0 1, 0.1, 1, and 10 mug/L) of these chemicals for 3 weeks after hatching. Then, mortality, body weight, sex ratio, and gonadosomatic index (GSI) of the matured fish (after 5 months) were measured. Mortality was increased significantly in the 10 mug/L E2 group. Distortion of sex ratio was found in 1 and 10 mug/L E2 groups. DEHP treated groups showed the GSI reduction only in male fish. All the gamma-HCH and parts of the E2 treated groups showed the GSI reduction in both sexes. Exposure of DEHP, gamma-HCH, and E2 during the fry stage affected normal maturation of medaka at the concentrations which had no impact on mortality or sex ratio. (C) 2004 Elsevier B.V. All rights reserved.
  • T Kinpara; R Mizuno; Y Murakami; M Kobayashi; S Yamaura; Q Hasan; Y Morita; H Nakano; T Yamane; E Tamiya
    JOURNAL OF BIOCHEMISTRY 136 2 149 - 154 2004年08月 
    The completion of human genome sequencing has shifted the focus of research from genes to proteins. In this regard, a protein library chip has become a useful tool for cell-free protein synthesis. In this study, we attempted to make a highly-integrated protein chip from a DNA library using in vitro protein synthesis on a microchamber array fabricated by using PDMS (polydimethyl siloxane), a hydrophobic surface, and glass, a hydrophilic bottom substrate. These structural properties prevented cross-contamination among the chambers. The minimum volume capacity of the smallest chamber was about 1 pl. The total number of chambers per chip was 10,000 on one chip (capacity 150 pl) and 250,000 on two others (1 and 5 pl). Next, we attempted in vitro protein synthesis using this microchamber array. The fluorescence of Green Fluorescent Protein (GFP) expressed on the chamber was rapidly detected (within just 1 h). GFP expression was also successful using immobilized DNA molecules on polymer beads. DNA immobilized beads were added as the source to each microchamber. Protein was successfully synthesized from DNA immobilized beads, which allowed easy handling of the DNA molecules.
  • Y Morita; T Ohsugi; Y Iwasa; E Tamiya
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 28 4-6 185 - 190 2004年06月 
    A novel approach to develop a peptide, that can recognize fullerene (C60) is described for affinity selection of phage displayed peptides from a combinatorial peptide library. Biopanning was performed using cyclic 7-mer peptide library against C60 films deposited on silicon (Si) substrate, and eluted phages with organic solvent. The phage, that recognized C60 films deposited on Si substrate, were obtained from biopanning. The nucleotides of the phage, coding a cyclic 7-mer peptide, were sequenced by standard method. Seventeen kinds of peptide displayed phages were obtained. One kind of peptide (peptide No. 4) displayed phage recognized the C60 films deposited on Si substrate. Peptide No. 4 displayed no affinity towards the Si substrate. The recognition event was monitored by a fluorescent immunoassay. Additionally, peptide No. 4 phage could recognize C60 in powder form, but not the graphite powder. This recognition event in powder form was also observed by a fluorescent immunoassay. (C) 2004 Elsevier B.V. All rights reserved.
  • K Kerman; Y Morita; Y Takamura; M Ozsoz; E Tamiya
    ANALYTICA CHIMICA ACTA 510 2 169 - 174 2004年05月 
    The unique binding event between Escherichia coli single-stranded DNA binding protein (SSB) and single-stranded oligonucleotides conjugated to gold (Au) nanoparticles is utilized for the electrochemical detection of DNA hybridization. SSB was attached onto a self-assembled monolayer (SAM) of single-stranded oligonucleotide modified Au nanoparticle, and the resulting Au-tagged SSB was used as the hybridization label. Changes in the Au oxidation signal was monitored upon binding of Au tagged SSB to probe and hybrid on the electrode surface. The amplified oxidation signal of Au nanoparticles provided a detection limit of 2.17 pM target DNA, which can be applied to genetic diagnosis applications. This work presented here has important implications with regard to combining a biological binding event between a protein and DNA with a solid transducer and metal nanoparticles. (C) 2004 Elsevier B.V. All rights reserved.
  • M. Chikae; 森田 資隆; R. Ikeda; Q. Hasan; E. Tamiya
    Environmental Toxicology and Pharmacology 17 1 29 - 33 2004年05月 
    The effects of oral administration of tamoxifen, 17alpha-ethynylestradiol (EE2), flutamide, and methyltestosterone (MT), on plasma vitellogenin levels of male and female medaka were investigated. Medaka were fed diets containing different concentrations of these chemicals for 7 days, and these plasma vitellogenin levels were measured. Tamoxifen increased significantly the vitellogenin levels in male, but inhibited the normal vitellogenin induction in female in the high concentration groups. EE2 increased significantly vitellogenin levels in both sexes. Flutamide increased significantly the vitellogenin levels in female, but gave no effects on male. MT inhibited the normal vitellogenin induction in female, but increased slightly vitellogenin levels in male without a clear tendency. Administration of tamoxifen, EE2, flutamide, and MT showed the different pattern in vitellogenin levels in both sexes. (C) 2004 Elsevier B.V. All rights reserved.
  • Y Akagi; Rao, SR; Y Morita; E Tamiya
    SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 5 3 343 - 349 2004年05月 
    This study describes the optimization of fluorescent cell-based assays using microchamber array chip formats as well as using automatic nanoliter volumes of sample dispensing system for high-throughput screening analysis of anticancer drugs. Cell-based assays can be employed efficiently in the screening of potential anticancer drug candidates and bioactive compounds with distinct biological function. Identification and development of cell-based assays adapted to high-throughput screening requirements is important when screening chemicals for their potential anticancer properties. Cell-based screening assays using microchamber array chip formats and automatic nanoliter volumes of sample dispensing system requires an optimization as a prerequisite for parameters including assay liquid volume and number of cells per each chamber, and the total cell-based assay itself. Further, the anticancer effect of mitomycin C was studied as an example against human cervical carcinoma cell line-HeLa 229 using cell-based assay that was optimized on chamber array chip formats and determined the cytotoxicity of mitomycin C by measuring the cell proliferation of HeLa with Calcein-AM fluorescent dye. The cell-based screening assay that was performed using chamber array chip formats was compared with the conventional 96-well plate formats was discussed. The assay described in this study is rapid, simple and inexpensive that is desirable in selecting anticancer candidates. (C) 2004 Elsevier Ltd. All rights reserved.
  • K Kerman; Y Matsubara; Y Morita; Y Takamura; E Tamiya
    SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 5 3 351 - 357 2004年05月 
    The specific binding of peptide nucleic acid (PNA) to its complementary DNA target is combined with magnetic separation to enable discrimination against single nucleotide polymorphisms (SNP). PNA probes with biotin label at 5'-end were attached to strepavidin coated superparamagnetic iron oxide beads. PNA modified beads were then challenged with non-complementary, SNP containing and perfect-match DNA targets. PNA probe showed no affinity towards non-complementary DNA. The non-specific binding of SNP containing DNA target was suppressed by the washing step of the beads by using sodium dodecylsulfate in blank buffer solution. Then, an electro-active intercalator, 7-dimethyl-amino-1,2-benzophenoxazinium salt (Meldola's blue, MDB) was introduced to the beads. MDB intercalated between the double-helix of the hybrid molecules on the beads. After removing the excessive MDB, the beads were collected from the solution by immersing a biotin modified carbon paste electrode into the solution. Specific hybridization between PNA probe and DNA target was determined by monitoring the voltammetric peak of MDB. Numerous factors affecting the MDB signal, such as target DNA concentration, intercalator concentration and accumulation time were investigated. MDB signal indicated a detection limit of 2 pM in connection with 20 min hybridization time. (C) 2004 Elsevier Ltd. All rights reserved.
  • M. Chikae; 森田 資隆; Y. Hatano; R. Ikeda; Q. Hasan; E. Tamiya
    Environmental Toxicology and Pharmacology 16 3 141 - 145 2004年04月 
    Effects of two widely found chemical pollutants, bis(2-ethylhexyl) phthalate (DEHP) and benzo[a]pyrene (BaP), on the embryos of Japanese medaka were investigated. The embryos were exposed to different concentrations (0.01, 0.1, 1, and 10 mug/l) of DEHP and BaR The following were investigated: (1) hatching time and hatching rate in embryos, (2) mortality, sex ratio, body weight and gonadosomatic index (GSI) in adulthood. These two chemicals delayed the hatching time without dose-dependence, but these chemicals had no effect on hatching rate. Mortality was raised and body weight was reduced by DEHP and BaP-treatment; distortion of sex ratio appeared at the lowest concentration of DEHP tested. GSI was decreased because of the BaP-treatment. DEHP and BaP negatively affected Japanease medaka embryos, and the influences of the effects continued into adult stage. Moreover, the effects did not appear to be necessarily dose-dependent. (C) 2003 Elsevier B.V. All rights reserved.
  • M Kobayashi; KB Takashi; M Saito; S Kaji; M Oomura; S Iwabuchi; Y Morita; Q Hasan; E Tamiya
    ELECTROCHEMISTRY COMMUNICATIONS 6 4 337 - 343 2004年04月 
    A novel DNA quantification method by using a redox-active molecule, Hoechst 33258, 2'-(4-hydroxyphenyl)-5-(4-methyl-l-piperazinyl)-2,5'-bi(lH-benzimidazole), is reported. Hoechst 33258 was interacted with DNA in solution without immobilization on the electrode surface, thus the time-consuming probe immobilization step was eliminated. The changes in the anodic current signal of Hoechst 33258 at 0.50 V was monitored in the presence and absence of solution-phase DNA by using a bare glassy carbon electrode (GCE) in connection with linear sweep voltammetry (LSV). The interaction of DNA with several small molecules was also monitored by using cyclic voltammetry (CV) at a bare GCE in order to determine the most effective molecule for DNA aggregation. Hoechst 33258 was found to form an aggregate in the presence of DNA, and this phenomenon was confirmed by using atomic force microscopy (AFM). The aggregation of DNA-Hoechst 33258 complex led to a decrease in the voltammetric signal in proportion to the quantity of DNA in a wide linear range between 1.5 and 25 mg/l. The interaction of Hoechst 33258 with polynucleotides was also monitored to determine its sequence specificity. DNA amplification by polymerase chain reaction (PCR), and the detection of DNA sequences related to Salmonella enteritidis, Streptococcus sobrinus, and hepatitis B virus (HBV) were demonstrated by using DNA-Hoechst 33258 aggregation system. (C) 2004 Elsevier B.V. All rights reserved.
  • K Kerman; M Saito; Y Morita; Y Takamura; M Ozsoz; E Tamiya
    ANALYTICAL CHEMISTRY 76 7 1877 - 1884 2004年04月 
    Rapidly increasing information about the human genome requires a fast and simple method for the detection of single-nucleotide polymorphisms (SNPs). To date, the conventional SNP detection technologies have been unable to identify all possible SNPs and needed further development in cost, speed, and sensitivity. Here we describe a novel method to discriminate and code all possible combinations. SNPs were coded by monitoring the changes in the electrochemical signal of the monobase-modified colloidal gold (An) nanoparticles. First, a chitosan layer was formed on the alkanethiol self-assembled monolayer-modified Au nanoparticle. The monobases were then attached onto the chitosan-coated An nanoparticles through their 5' phosphate group via the formation of a phosphoramidate bond with the free amino groups of chitosan. The size of the surface-modified Au nanoparticle was found to be 8.46 +/- 1.53 nm by using atomic force microscopy. If there is a SNP in DNA and the mismatched bases are complementary to the mono-base, An nanoparticles accumulate on the electrode surface in the presence of DNA polymerase I (Klenow fragment), thus resulting in a significant change in the An oxide wave. In this report, monobase-modified Au nanoparticles show not only the presence of a SNP, but also identify which bases are involved within the pair. Especially, the identification of a transversion SNP, which contains a couple of the same pyrimidine or purine bases, is greatly simplified. A model study was performed by using a synthetic 21-base DNA probe related to tumor necrosis factor (TNF-alpha) along with its all possible mutant combinations. This versatile nanoparticle-based electrochemical protocol is a promising candidate for coding all mutational changes.
  • Y Matsubara; Y Murakami; M Kobayashi; Y Morita; E Tamiya
    BIOSENSORS & BIOELECTRONICS 19 7 741 - 747 2004年02月 
    In this report, the development of a microfluidic cell chip for monitoring allergic response is described. A rat basophilic leukemia cell line (RBL-2H3). a tumor analog of rat mucosal mast cells, has been used as a model to observe its allergic response upon antigen stimulus. The cells were cultivated on a poly(dimethylsiloxane) (PDMS) chip, the surface of which was modified by several methods. The PDMS chip, which comprised a cell cultivation chamber and microfluidic channels, was fabricated by conventional molding methods. In order to detect the allergic response, a fluorescent dye, quinacrine, was introduced inside the cell compartment that included histamine. The cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with anti-DNP IgE. When exocytosis events occurred, the microfluidic system detected the fluorescent signal of quinacrine, which was released from RBL-2H3 cells by using a photomultiplier tube (PMT) fitted onto a microscope. (C) 2003 Elsevier B.V. All rights reserved.
  • Peptide nucleic acid modified magnetic beads for intercalator based electrochemical detection of DNA hybridization
    K.Kerman; Y. Morita; Y.Takamura; E.Tamiya
    Science and Technology of Advancecd Materials 5 3 351 - 357 2004年 [査読有り]
  • M Chikae; R Ikeda; Q Hasan; Y Morita; E Tamiya
    ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 15 1 33 - 36 2003年12月 
    The effect that oral administration of four alkylphenols, (1) bisphenol A (BPA), (2) p-t-octylphenol (OP), (3) p-nonylphenol (NP) and (4) p-n -nonylphenol (n- NP), as well as 17alpha-ethynylestradiol (EE2) had on male medaka fish vitellogenin was investigated. The male medaka was fed diets containing different concentrations of these chemicals for 7 days, after which their plasma vitellogenin levels were measured. Vitellogenin levels up to approximate to 10(7) ng/ml were found. This value is close to that of the normal estrous female medaka. The median effective concentration (EC50) values resulting from BPA, OP, NP and EE2 in the diet were calculated as 1600, 2600 940 and 0.37 mug/g diet, respectively. (C) 2003 Elsevier B.V. All rights reserved.
  • T Sakaguchi; K Kitagawa; T Ando; Y Murakami; Y Morita; A Yamamura; K Yokoyama; E Tamiya
    BIOSENSORS & BIOELECTRONICS 19 2 115 - 121 2003年11月 
    A biochemical oxygen demand (BOD) sensing system based on bacterial luminescence from recombinant Escherichia coli containing lux A-E genes from Vibrio fischeri has been developed. It was possible to use frozen cells of luminescent recombinants of E. coli as the bacterial reagents for measurement. Steady bioluminescence was observed during the incubation time between 90 and 150 min in the presence of a sole carbon source such as glucose, acetate, L-glutamate and BOD standard solution (GGA solution). This disposable bacterial reagent was applied to measure and detect organic pollution due to biodegradable substances in various wastewaters. The obtained values of this study showed a similar correlation with that of the conventional method for BOD determination (BOD5). Bacterial luminescence that was visualized with an imaging system using a charge coupled device (CCD) camera and a photomulti-counter demonstrated that this method could also be used for multi-sample detection of organic pollution due to biodegradable substances by using a microtiter plate. These results suggested for successful achievement of high-though-put detection of BOD in practical. (C) 2003 Elsevier B.V. All rights reserved.
  • K Kerman; Y Morita; Y Takamura; E Tamiya
    ELECTROCHEMISTRY COMMUNICATIONS 5 10 887 - 891 2003年10月 
    A label-free electrochemical detection protocol for DNA hybridization is reported for the first time by using a gold electrode (AuE). The oxidation signal of guanine was monitored at +0.73 V by using square wave voltammetry (SWV) on self-assembled L-cysteine monolayer (SAM) modified AuE. The electrochemical determination of hybridization between an inosine substituted capture probe and native target DNA was also accomplished. 6-mer adenine probe was covalently attached to SAM via its amino link at 5' end. Then, 6-mer thymine-tag of the capture probe was hybridized with the adenine probe, thus left the rest of the oligonucleotide available for hybridization with the target. The dependence of the guanine signal upon the concentration of the target was observed. Probe modified AuE was also challenged with non-complementary and mismatch containing oligonucletides. Label-free detection of hybridization on AuE is greatly advantageous over the existing carbon and mercury electrode materials, because of its potential applicability to microfabrication techniques. Performance characteristics of the genosensor are described, along with future prospects. (C) 2003 Published by Elsevier B.V.
  • Y Akagi; A Hashigasako; P Degenaar; S Iwabuchi; Q Hasan; Y Morita; E Tamiya
    JOURNAL OF BIOCHEMISTRY 134 3 353 - 358 2003年09月 
    This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 muM. We used two types of Ca2+ channel inhibitors, MK-801 and omega-Conotoxin GVIA, which act to suppress Ca2+ transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when omega-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca2+ channels in the presynapse. Glutamate release and Ca2+ flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.
  • ZL Zhi; Y Morita; Q Hasan; E Tamiya
    ANALYTICAL CHEMISTRY 75 16 4125 - 4131 2003年08月 
    Micromachining techniques, which originated in the microelectronics industry, have been employed to manufacture microparticles bearing an engraved dot-type signature for biomolecular encoding. These metallic microstructures are photolithographically defined and manufactured in a highly reproducible manner. In addition, the code introduced on the particle face is a straightforward visible feature that is easily recognizable with the use of optical microscopy. The number of distinct codes theoretically could be many thousands, depending on the coding element numbers. Such microparticles are, thus, with appropriate surface organic functionalizations, ideal for encoding biomolecular libraries and serving as a platform for developing high-throughput multiplexed bioassay schemes based on suspension array technology. As proof of this statement, we demonstrated that encoded microparticles tagged with antibodies to human immunoglobulin classes are capable, using imaging detection as the interrogating approach, of high sensitivity and high specificity, as well as multiplexed detection of the respective antigens in a microliter-sample volume.
  • ZL Zhi; Y Murakami; Y Morita; Q Hasan; E Tamiya
    ANALYTICAL BIOCHEMISTRY 318 2 236 - 243 2003年07月 
    This paper describes the random fluidic self-assembly of metallic particles into addressable two-dimensional microarrays and the use of these arrays as a platform for constructing a biochip useful for bioassays. The basic units in the assembly were the micro-fabricated particles carrying a straightforward visible code and the corresponding array template patterned on a glass substrate. The particles consisted of a hydrophobic and magnetic Ni-polytetrafluoroethylene (PTFE) composite layer on one face, and on the other face a gold layer that was modified for biomolecular attachment. An array template was photoresist-patterned with spatially discrete microwells in which an electrodeposited Ni-PTFE hydrophobic composite layer and a hydrophobic photo-adhesive coating were deposited. The particles, after biontaterial attachment and binding processes in bulk, were self-assembled randomly onto the lubricated bonding sites on the chip substrate, driven by a combination of magnetic, hydrophobic, and capillary interactions. The encoding symbol carried by the particles was used as the signature for the identification of each target/assay attached to the particle surface. We demonstrate here the utility of microfabricated-encoded particle arrays for conducting multianalyte immunoassays in a parallel fashion with the use of imaging detection. (C) 2003 Published by Elsevier Science (USA).
  • Yuzuru Takamura; Yasutaka Morita; Eiichi Tamiya
    Digest of Papers - Microprocesses and Nanotechnology 2003 - 2003 International Microprocesses and Nanotechnology Conference, MNC 2003 316  2003年 [査読有り]
     
    We describe some novel integrated analysis methods for DNA / protein or cells employing micro chamber array.
  • ZL Zhi; Y Morita; E Tamiya
    MEMS-03: IEEE THE SIXTEENTH ANNUAL INTERNATIONAL CONFERENCE ON MICRO ELECTRO MECHANICAL SYSTEMS 411 - 414 2003年 [査読有り]
     
    Micromachining techniques have been employed to manufacture metallic microparticles bearing an engraved bit-type signature for bimolecular encoding. The well-defined metallic microparticles are, with proper organic derivatization of the surfaces, used for coupling antibodies and served as a platform in the carrying out high-throughput multiplexed binding assays based on suspension array technology. As proof-of-concept, we demonstrated that encoded microparticles immobilized with anti-IgA, anti-IgG, and anti-IgM antibodies, could be used to sensitively and specifically detect the corresponding antigens in a parallel manner using imaging detection technique.
  • MEMS-based biosensors for environemntal monitoring
    T.Endo; Y.Morita; E.Tamiya
    SPIE Proceedings Environmental Monitoring and Remediation III 5270 101 - 110 2003年 [査読有り]
  • T Fukumori; Y Morita; E Tamiya; K Yokoyama
    ANALYTICAL SCIENCES 19 1 181 - 183 2003年01月 
    A novel molecular tool for double-stranded (ds) DNA detection using synthetic peptide is described. The peptide was designed based on the DNA binding domain of the lambda phage CRO repressor (CRO). The designed peptides contain helix-turn-helix (HTH), which is DNA binding motif. A cyclic peptide and a mutant peptide based on CRO were also designed, and the resulting affinity for dsDNA was increased. Furthermore, native amino acids of the peptide were replaced with arginine to increase the affinity for dsDNA. The affinity of these peptides for DNA binding was assessed by surface plasmon resonance (SPR) technique.
  • S Taira; Y Morita; E Tamiya; K Yokoyama
    ANALYTICAL SCIENCES 19 1 177 - 179 2003年01月 
    We developed DNA-conjugated polymer for DNA chip fabrication. A 30 mer probe DNA and disulfide bridges were covalently attached to the polymer side chain. The DNA-conjugated polymer can be specifically adsorbed on a gold substrate surface by a self-assembly technique. The interaction between fully matched DNA and DNA-conjugated polymer was investigated by surface plasmon resonance (SPR) technique. The DNA-conjugated polymer-modified gold surface highly recognized fully matched DNA, rather than unmatched DNA. Therefore, DNA-conjugated polymer can be used for novel DNA chip fabrication.
  • Eiichi Tamiya; Masato Saito; Shinichirou Iwabuchi; Yasutaka Morita
    SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS 4 1 61 - 67 2003年 
    Scanning near-field optical/atomic-force microscopy (SNOAM) provided us with simultaneous topographic and fluorescence images of human chromosomes. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic force microscopy cantilever. Optical resolution was approximately 50-100 nm in fluorescence mode. Conventional karyotyping information was linked with SNOAM topographic analyses such as location of centromere and length of individual chromosomes. The height profile clearly indicated higher teromere regions. The SNOAM fluorescence images were different shapes from topographic images probably due to results from the combination of fluorescence dye and chromosome DNA. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • Y Matsubara; M Kobayashi; Y Morita; E Tamiya
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 65 5 481 - 488 2002年12月 
    We recently developed a microchamber array chip for DNA amplification by adopting semiconductor microfabrication technology; a polymerase chain reaction (PCR) was performed in the microchamber array, and the amplified DNA was detected using a fluorescent dye. In order to manipulate a single cell or sample into each microchamber individually in this system, the chip was directly sealed with a cover glass slip which impeded the retrieval of the products from each chamber. The present study was therefore carried out to improve the system by developing methods for covering the microchambers and introducing the reaction solution. First, we fabricated a microchamber array chip, and the oil layer was coated on the whole chip instead of the cover glass slip. The solution for DNA amplification was introduced into each chamber through an oil layer using a nano-liter dispenser. Following this, the microarray chip was placed onto the thermal cycling system for DNA amplification, and the amplified DNA was subsequently detected by fluorescence microscopy. In this system, the products were easily retrieved using a micromanipulator for further analysis.
  • S Iwabuchi; T Mori; K Ogawa; K Sato; M Saito; Y Morita; T Ushiki; E Tamiya
    ARCHIVES OF HISTOLOGY AND CYTOLOGY 65 5 473 - 479 2002年12月 
    The present study was performed to introduce a novel chromosome dissection method employing atomic force microscopy (AFM) in a dynamic force mode for the chemical or molecular biological analysis of tiny chromosomal fragments. After AFM observation of human chromosomes prepared for light microscopy, a region of interest was dissected by increasing the loading force in a series of single-line scans of the target portion by controlling it with the amplitude reference of the tip in a dynamic force mode. The marker gene of the nucleolar organizing region (NOR) was amplified by our designed primers for 5.8S ribosomal DNA. After the dissection, topographic profiles in the section were then obtained with a carbon nanotube (CNT) probe in ambient condition. These results are discussed in relation to a fundamental technology for chromosomal analysis.
  • Y Nakamura; T Sawada; Y Morita; E Tamiya
    BIOCHEMICAL ENGINEERING JOURNAL 12 1 79 - 86 2002年10月 
    The identification of a psychrotrophic bacterium, which produced a violet pigment, isolated from the organic residue of a water tank keeping rainbow trout and the antibacterial effect of the violet pigment were investigated experimentally. The psychrotrophic bacterium was found to be a new species very closely related to Janthinobacterium lividum. H-1 NMR, C-13 NMR, and Fr-MS spectra analyses results showed that the chemical structure of the violet pigment was a mixture of violacein and deoxyviolacein. The antibacterial effect of the violet pigment was confirmed for putrefactive bacteria such as Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Pseudomonas aeruginosa. The growth-inhibitory and lethal effects of the violet pigment on the putrefactive bacteria were evaluated by increasing the concentration of the violet pigment, ranging from 5 to 20 mg dm(-3). It was found that higher concentrations of the violet pigment caused not only growth inhibition but also the death of the putrefactive bacteria. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Helianti, I; Y Morita; Y Murakami; K Yokoyama; E Tamiya
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 59 4-5 462 - 466 2002年08月 
    Two recombinant Aeropyrum pernix glutamate dehydrogenase (GDH) enzymes with different length N-termini were cloned and expressed in Escherichia coli: sGDH begins with the amino acid sequence of the extracted native enzyme (M-Q-P-T D-P-L-E-E), whereas lGDH begins with the sequence of the predicted ORF (M-E-V-L-A-L-Q-P-T D) and is longer than sGDH by five amino acids (M-E-V-L-A). Purified recombinant lGDH was more stable than sGDH, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant lGDH. This stabilising effect of extending the N-terminal sequence on an oligomeric enzyme such as GDH is novel; factors affecting stabilisation have previously only been discussed in the context of the contribution of internal amino acids.
  • S Yamamura; Y Morita; Q Hasan; Rao, SR; Y Murakami; K Yokoyama; E Tamiya
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 93 6 595 - 600 2002年06月 
    A keratin-degrading bacterium was isolated from soil containing deer fur. An axenic culture of the keratin-degrading bacterium was obtained in liquid culture using a keratin enrichment technique. The isolated bacterium was gram negative and catalase- and oxidase-positive. Transmission electron microscopic observations showed that the bacterium was rod-shaped, 1.0-1.3 mum long and 0.7 mum in diameter. Phylogenetic analysis of 16S rDNA revealed that the new isolate has only 90.6% homology with Stenotrophomonas nitritireducens. Hence, this new bacterium was designated as Stenotrophomonas sp. D-1. The optimum temperature was determined to be 20degreesC for maximum growth and keratinolytic enzyme production. Amino acid data, obtained after treating keratin powder with the supernatant culture, suggest that the major free amino acids resulting from keratin degradation are phenylalanine, tyrosine and valine. In addition, native chicken feather was degraded completely at 20degreesC in 2.5 d by this bacterium.
  • S Yamamura; Y Morita; Q Hasan; K Yokoyama; E Tamiya
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 294 5 1138 - 1143 2002年06月 
    A novel keratin-degrading bacterium Stenotrophomonas sp. strain D-1, isolated from deer fur, produced two types of extracellular proteins: proteolytic and disulfide bond-reducing. The results on the biochemical properties suggest that this protease belongs to the serine protease, and the disulfide bond-reducing protein could be the disulfide reductase type. None of these enzymes showed keratinolytic activity independently. However, after mixing of the two enzymes, the keratinolytic activity was increased tremendously (more than 50-fold) over that of the protease only. This keratinolytic activity was more than 2-fold higher than that of the combination with proteinase K (also known for its high keratinolytic activity). Since the two enzymes discovered in this study acted cooperatively and resulted in higher keratinolytic activity, a new mechanism of keratin degradation has been revealed. To our knowledge, this is the first report on the cooperative action of two enzymes resulting in the effective degradation of keratin. (C) 2002 Elsevier Science (USA). All rights reserved.
  • Shohei Yamamura; Yasutaka Morita; Quamrul Hasan; Kenji Yokoyama; Eiichi Tamiya
    Biochemical and Biophysical Research Communications 295 4 1034  2002年 [査読有り]
  • M Murahashi; Y Ishimori; K Kawano; T Kase; M Mouri; Y Morita; Y Murakami; K Yokoyama; E Tamiya
    ADVANCED ENVIRONMENTAL SENSING TECHNOLOGY II 4576 255 - 262 2002年 [査読有り]
     
    In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages followed by I)Eco-sensor, 2)Prediction of plume propagation using a computer simulation technique, and 3)Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated the automatic BLMs preparation device. An automatic BLMs preparation was remarkably improved. The sensitivity to volatile organic chlorinated compounds such as cis-1,2-dichloroethylene was in the order of 10ppb using the monoolein BLMs even in real underground water. We also have been developing a smaller sized eco-sensor for the practical use.
  • E Tamiya; Y Morita
    6TH WORLD MULTICONFERENCE ON SYSTEMICS, CYBERNETICS AND INFORMATICS, VOL VI, PROCEEDINGS 441 - 444 2002年 [査読有り]
     
    The micro-chamber array used to run PCR was fabricated on silicon wafer using photolithography and anisotropic etching. The amplification of a fragment of Green fluorescence protein (GFP) gene by PCR was monitored by a technique using energy transfer of the fluorescent dyes in micro-chamber array. The fluorescence following the amplification of the GFP gene fragment was observed by a CCD camera through a microscope. A micro-chamber array for PCR was developed by semiconductor micro-fabrication techniques; the volume of each chamber was 85 pL. Single cell PCR was done using jurkat cell and ribosomal DNA in micro-chamber array. PDMS chip that had 10,000 wells (2,500 well/cm(2)) was made and in vitro protein synthesis was gone on the chip. GFP gene was expressed in micro-chamber array using the ribosome system based on the lysate from Escherichia coli. GFP expression was observed with fluorescent intensity.
  • Y.Morita; Y.Murakami; Y. Yokoyama; E.Tamiya
    Biological Systems Engineering 830 210 - 219 2002年 [査読有り]
     
    Dioxins and endocrine disrupting chemicals are toxic and bad for health, and they become a one of social problem. The combinatorial synthesis of chemical libraries by the split-and-mix strategy makes it easy to synthesize a large number of peptide libraries and possible to select an optimal peptide that binds with chemicals. In this study, the screening and the characterization of peptide ligands that bind with high affinity to 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-TCDD) were carried out. Combinatorial peptide libraries were synthesized by solid phase synthesis and they consisted of hepta-peptide sequences which bind to resin with the C-terminal of the peptide. Five peptides which bound with 2,3,7-TCDD were screened from peptide library, and amino acid sequences of these peptides were investigated. One of the peptides, designed as peptide A, as the concentration of 2,3,7-TCDD increased, the fluorescence decreased almost proportionally in the competitive binding assay. This indicated that the analysis system could be used for detection of 2,3,7-TCDD.
  • M Kobayashi; T Mizukami; Y Morita; Y Murakami; K Yokoyama; E Tamiya
    ELECTROCHEMISTRY 69 12 1013 - 1016 2001年12月 
    Thirty-two microelectrodes were fabricated onto a glass chip and used for an integrated DNA sensor. Several probe DNAs consisting of mercaptohexyl moiety at their 5' end were immobilized on the gold electrodes by a DNA arrayer. Then target DNAs were hybridized and reacted with Hoechst 33258, which was a DNA minor groove binder and redox-active dye. Linear sweep voltammetry or cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. It was derived from Hoechst 33258 concentrated at the electrode surface through association with formed hybrid. The DNA sensor obtained by microfabricated electrodes with DNA probes and redox-active DNA intercalator was able to detect 0.1 nM of target sequence and 100 nM of single-base mismatched DNA. Sixteen electrodes that immobilized HIV probes gave higher response in thirty-two electrodes, which immobilized four kinds of DNA probes. These result shows this method can detect target DNA specifically.
  • P Degenaar; B Le Pioufle; L Griscom; A Tixier; Y Akagi; Y Morita; Y Murakami; K Yokoyama; H Fujita; E Tamiya
    JOURNAL OF BIOCHEMISTRY 130 3 367 - 376 2001年09月 
    In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic force microscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-day-old chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.
  • Helianti, I; Y Morita; A Yamamura; Y Murakami; K Yokoyama; E Tamiya
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 56 3-4 388 - 394 2001年08月 
    Glutamate dehydrogenase (GDH) was purified and characterized from an aerobic hyperthermophilic archaeon Aeropyrum pernix (A. pernix) K1. The enzyme has a hexameric structure with a native molecular mass of about 285 +/- 15 kDa. It was specific for NADP and thermostable (74% activity was remained after 5 h incubation at 100 degreesC). The activity of the enzyme increased in the presence of polar water-miscible organic solvents such as acetonitrile, methanol, and ethanol. The N-terminal sequence of GDH is Met-Gln-Pro-Thr-Asp-Pro-Leu-Glu-Glu-Ala. This sequence, except for the methionine, corresponds to amino acids 7-15 of the open reading frame (ORF) encoding the predicted GDH (ORF APE 1386). In the ORF nucleotide sequence, the codon TTG appears at the position of the methionine, suggesting that the leucine codon might be recognized as an initiation codon and translated to methionine in A. pernix GDH.
  • H Nagai; Y Murakami; Y Morita; K Yokoyama; E Tamiya
    ANALYTICAL CHEMISTRY 73 5 1043 - 1047 2001年03月 
    A microchamber array for PCR was developed by semiconductor microfabrication technology. The microchambers were designed to be of picoliter quantity. To optimize fluid retention, the surface states of the substrate and the inner walls were examine for four different types of microchamber. The substrate was silicon, while silicon dioxide was selected for the inner walls. PCR was performed in the microchamber array, and the amplification of DNA was detected using a technique based on the energy transfer of fluorescent dyes. The lower volume limit for PCR was investigated using various sizes of microchambers. Microchambers with volume greater than 86 pL gave successful PCR. In addition, the system was improved in order to take up the PCR product, To prevent mixing of the samples, the samples were dried after PCR using a membrane that permeates only vapor.
  • M Kobayashi; M Oomura; T Kusakawa; Y Morita; Y Murakami; K Yokoyama; E Tamiya
    TRANSDUCERS '01: EUROSENSORS XV, DIGEST OF TECHNICAL PAPERS, VOLS 1 AND 2 334 - 337 2001年 [査読有り]
     
    We have developed a novel electrochemical method for gene detection. This method uses an intercalater without immobilization of probe DNA. Moreover, we have also fabricated a microchip, couples a PCR chamber with a chamber for electrochemical gene detection. This configuration allows on-chip DNA amplification and detection. The configuration is simple and versatile, enabling easy accommodation of different chip designs. In this report, we demonstrated the gene detection of Hepatitis B Virus (HBV) sequence in a plasmid.
  • M. Kobayashi; 森田 資隆; T. Mizukami; Y. Murakami; K. Yokoyama; E. Tamiya
    New Technology Japan 29 6 11 - 17 2001年
  • Y Murakami; K Idegami; H Nagai; T Kikuchi; Y Morita; A Yamamura; K Yokoyama; E Tamiya
    MATERIALS SCIENCE & ENGINEERING C-BIOMIMETIC AND SUPRAMOLECULAR SYSTEMS 12 1-2 67 - 70 2000年08月 [査読有り]
     
    Novel methods to construct a multianalyte biosensing chip are described. A method is two-step immobilization in which materials are not immobilized directly but indirectly via small support materials. Biomaterials are immobilized on a certain support particle in the first step. The particles are placed or stuck on a chip in the second step. The other method is random fluidic self-assembly of the microsupports for reducing the complication in the second step on the chip. The combination of the methods enables us to immobilize various kinds of biomaterials densely on a chip. The ways to avoid possible problems due to random distribution are discussed. Several examples are also described. (C) 2000 Elsevier Science S.A. All rights reserved.
  • Y Murakami; H Nagai; T Kikuchi; A Yamamura; K Idegami; M Yanase; YS Choi; Y Morita; E Tamiya
    1ST ANNUAL INTERNATIONAL IEEE-EMBS SPECIAL TOPIC CONFERENCE ON MICROTECHNOLOGIES IN MEDICINE & BIOLOGY, PROCEEDINGS 29 - 32 2000年 [査読有り]
     
    This paper describes a random distribution method to bring biomaterials into an array of sites on a chip. A mixture of biomolecules or a mixed suspension of particles immobilized with biomaterials; was simply poured on an array and the components were randomly distributed. A microchamber array for PCR was developed by semiconductor microfabrication techniques. Amplification of pGFP was confirmed using a technique based on energy transfer of fluorescent dyes. Addition of BSA in PCR mixture enhances PCR efficiency. Furthermore, employing a special membrane made it possible to pick up the PCR products. Random distribution method is also applied to the construction of biosensor arrays. We first employed a two-step immobilization in which materials are not immobilized directly but indirectly via small support materials. In this case random distribution is called random fluidic self-assembly. This arranges the microparticles reducing the complication in the second step on the chip.
  • Y Murakami; T Kikuchi; M Yanase; H Nagai; Y Morita; E Tamiya
    MICRO TOTAL ANALYSIS SYSTEMS 2000, PROCEEDINGS 191 - 194 2000年 [査読有り]
     
    This paper describes the reduction of the size of enzyme immunoassay (EIA) utilizing microchemical reaction in a microchamber array. The microchamber array was fabricated by micromachining of a silicon chip. Glass beads of 100 mum were immobilized with antibody, and put in the microchambers of 150 mum. As a competitive ELISA, sample solution mixed with HRP-conjugated antigens was allowed to react in the microchamber. As a sandwich assay, sample solution and HRP-conjugated antibody were sequentially added to the chamber. The addition of buffer, hydrogen peroxide, and fluorogenic substrate produced fluorescent dye, and a fluorescence microscope observed its fluorescence.
  • 早川和一; 内海彰子; 鹿角昌平; 鳥羽陽; 木津良一; 阪口利文; 山村晃; 森田資隆; 民谷栄一
    水環境学会誌 23 11 731 - 736 日本水環境学会 = Japan Society on Water Environment 2000年 [査読有り]
     
    金沢大学医薬保健研究域薬学系
  • A Koizumi; T Morita; Y Murakami; Y Morita; T Sakaguchi; K Yokoyama; E Tamiya
    ANALYTICA CHIMICA ACTA 399 1-2 63 - 68 1999年11月 [査読有り]
     
    Antibody (Ab) labeled with alkaline phosphatase was separated and detected by a capillary electrophoresis system equipped with a laser induced fluorescence detection system. The combination of B/F separation and the LIF detection of the label enzyme is potentially useful for a highly sensitive detection of antigen (Ag). After B/F separation by capillary electrophoresis (CE), label enzymes catalyze the hydrolysis of fluorescein diphosphate in the running buffer for several minutes, producing fluorescein. Experiments with various ratios of Ab to Ag revealed that the faster peak was the complex of Ab and Ag, and the other was free Ab. (C) 1999 Elsevier Science B.V. All rights reserved.
  • S Iwabuchi; A Hashigasako; Y Morita; T Sakaguchi; Y Murakami; K Yokoyama; E Tamiya
    SCANNING AND FORCE MICROSCOPIES FOR BIOMEDICAL APPLICATIONS, PROCEEDINGS OF 3607 102 - 107 1999年 [査読有り]
     
    Scanning near-field optical/atomic-force microscopy (SNOAM) was first applied to detect fluorescence hybridization of DNA immobilized on nano-particle media. Hybridization can also be used to determine the sequence of unknown DNA. 100-nm in diameter of polystyrene sphere carboxylate was used as the nano-particle media. Template DNA including target sequence was chemically modified with animo group at the 5'-end of single-stranded DNA. Amine-coupling reaction made covalent bond between template DNA and carboxyl group on the surface of the media. Single-stranded DNA of specific base sequence labeled either fluorescent dye, that is used to detect the complementary base sequence by hybridization. Simultaneous imaging the colloidal particles showed us topography and near-field fluorescence images of them. All particles were observed in the topographic image, however, some particles were realized in the fluorescence image. This result indicated that fluorescent hybridized DNAs on the surface of the media were visualized specifically. High density arrays or integration of media is a fast and effective means of accessing the gene variation. However, in this study, detection of hybridized fluorescent DNA conjugated with particle is a major purpose rather than arrangement technique.
  • E Tamiya; S Iwabuchi; A Hashigasako; Y Murakami; T Sakaguchi; Y Morita; K Yokoyama
    SCANNING AND FORCE MICROSCOPIES FOR BIOMEDICAL APPLICATIONS, PROCEEDINGS OF 3607 42 - 51 1999年 [査読有り]
     
    A scanning near-field optical/atomic-force microscope(SNOAM) system was applied to simultaneous topographic and fluorescence imaging of biological samples in air and liquid. The SNOAM uses a bent optical fiber simultaneously as a dynamic mode atomic-force microscopy(AFM) cantilever and a scanning near-field optical microscopy(NSOM) probe. The SNOAM system used 458 or 488 nm from Ar ion laser multiline fo excitation of green fluorescent protein(GFP), since a native GFP has been known to give a maximum at 395 nm and a broad absorption spectrum until 500 nm. Topographic and fluorescence images of recombinant E. coli were obtained simultaneously with a high spatial resolution which was apparently better than that of a conventional confocal microscope. Nanoscopic GFP fluorescence spectrum was obtained by positioning the optical fiber probe above the bright area of the E. coli cells. Comparing topographic and fluorescence images, individual E. coli cells expressed different fluorescent intensity. Fluorescence obtained by SNOAM indicated GFP oxidation possibly occurred near cell surface. SNOAM also provided us with simultaneous topographical and optical images of human chromosomes. Native chromosomes were spread out onto a coverslip using the surface-spreading whole-mount method. Topographic images clearly indicated duplicated structure on metaphase chromosome, while fluorescence images were a different shape probably because it depended on the combination of SYBR(TM) Green I and chromosome DNA. Atomic force images have some artefacts, however they can be corrected by comparison with the fluorescence image. Topography and fluorescence images of RBL-2H3 mast cells surface: were determined with/without DNP-BSA stimulation by SNOAM system. Near-field fluorescence images were obtained from the granules stained with quinacrine. Fluorescence profiles and intensties were largely changed after allergen stimulation. Exocytotic events of granules were specially discussed based on SNOAM.
  • 村上裕二; 井手上公太郎; 永井秀典; 森田資隆; 山村晃; 阪口利文; 横山憲二; 民谷栄一
    電気学会論文誌E 119 42591 436 - 442 1999年 [査読有り]
     
    We propose a novel method for individual immobilization of biomaterial for biosensor application. Various kinds of biomaterial were first immobilized on certain supports such as glass beads or microfabricated metal particles of which size is almost the same as the size of transducer element. Then the suspension mixture of the various support was arranged on the transducer array by the fluidic self-assembly method at random. Glass beads immobilized with glucose oxidase and/or peroxidase were successfully arranged by this method using gravity, as a short-range force required in self-assembly. The beads gave chemiluminescence with the addition of luminol and its substrate. The metal particles consisted of nickel and/or gold layers formed by electroplating and evaporation on a photolithographically patterned chip. The coin-shaped particles were arranged on a nickel dot array by magnetic force interaction as the short-range force. The direction of binding of the particles were also controllable using the multilayer structure. © 1999, The Institute of Electrical Engineers of Japan. All rights reserved.
  • Y Morita; Q Hasan; T Sakaguchi; Y Murakami; K Yokoyama; E Tamiya
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 50 6 669 - 675 1998年12月 [査読有り]
     
    Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 degrees C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 degrees C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The N-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate.
  • Y Morita; Q Hasan; T Sakaguchi; Y Murakami; K Yokoyama; E Tamiya
    ENZYME ENGINEERING XIV 864 300 - 304 1998年 [査読有り]
  • Y Morita; K Kondoh; Q Hasan; T Sakaguchi; Y Murakami; K Yokoyama; E Tamiya
    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY 74 11 1377 - 1383 1997年11月 [査読有り]
     
    Protease activity was detected in the culture medium of Serratia marcescens AP3801 grown at 10 degrees C, which was isolated from soil collected from the top of a mountain. The enzyme, designated as CP-58 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 58 kDa, and its isoelectric point was close to 6.0. Maximal activity toward azocasein was observed at 40 degrees C and from pH 6.5 to 8.0. The activity was strongly inhibited by 1,10-phenanthroline, suggesting that the enzyme is a metalloprotease. The N-terminal amino acid sequence was Ser-Leu-Asn-Gly-Lys-Thr-Asn-Gly-Trp-Asp-Ser-Val-Asn-Asp-Leu-Leu-Asn-Tyr-His-Asn-Arg-Gly-Asn (or Asp)-Gly-Thr-Ile-Asn-Asn-Lys-Pro-Ser-Phe-Asp-Ile-Ala. A search through databases for sequence homology aligned CP-58 protease with metalloprotease. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-58 protease has a broader specificity than other proteases against the peptide substrate.
  • Y Morita; T Nakamura; Q Hasan; Y Murakami; K Yokoyama; E Tamiya
    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY 74 4 441 - 444 1997年04月 [査読有り]
     
    The properties of amylase, lipase and protease, excreted by newly isolated bacteria from snow-covered soil, salmon intestine and crab intestine, have been investigated. One amylase, one lipase, and three proteases have been characterized by shifts in their apparent optimal activities toward low temperatures and by reductions in their activation energy values. The discovered enzymes were rapidly inactivated at temperatures above the optimum (30 to 40 degrees C). These results suggest that the enzymes are cold-active. The best cold-active protease producer, isolated from salmon intestine, has been identified as Flavobacterium balustinum by the analysis of 16S rRNA. The optimal growth temperature of this bacterium was 20 degrees C, but a higher amount of protease activity was present at 10 degrees C.

書籍

  • コンビナトリアルペプチドを用いたバイオセンシング、バイオセンサーの先端科学技術と応用（普及版）
    森田 資隆 2014年10月
  • siRNAの細胞導入技術、遺伝子治療・診断の最先端技術と新しい医薬品・診断薬の開発、技術情報協会
    高科あゆみ; 神武洋二郎; 森田資隆; 山田康枝; 藤井政幸 2014年05月
  • 薬物細胞アッセイと細胞スクリーニング技術、動物実験代替のためのバイオマテリアル開発（普及版）
    森田 資隆 2014年04月
  • ペプチド、バイオセンサの先端科学技術と新製品への応用開発、技術情報協会、第３章第１節（６）
    森田 資隆 2014年04月
  • コンビナトリアルバイオエンジニアリングの最前線（普及版）, ファージディスプレイ法による幹細胞認識
    森田 資隆; 民谷栄一 (担当:共著範囲:)シーエムシー出版 2010年
  • コンビナトリアルバイオエンジニアリングの最前線（普及版）, 細胞チップを用いた薬剤スクリーニング
    赤木良教; 森田 資隆; 民谷栄一 (担当:共著範囲:)シーエムシー出版 2010年
  • バイオセンサーの先端科学技術と応用, コンビナトリアルペプチドを用いたバイオセンシング
    森田 資隆; 民谷栄一 (担当:共著範囲:)シーエムシー出版 2007年
  • ナノバイオ大事典, 糖鎖チップ
    森田 資隆; 民谷栄一 (担当:共著範囲:)テクノシステム 2007年
  • 動物実験代替のためのバイオマテリアル開発, 薬物細胞アッセイと細胞スクリーニング技術
    森田 資隆; 民谷栄一 (担当:共著範囲:)シーエムシー出版 2007年
  • チップ技術とコンビナトリアル機能分子設計、コンビナトリアルサイエンスの展開（普及版）
    森田資隆; 金原健; 村上裕二; 横山憲二; 民谷栄一 (担当:共著範囲:)2007年
  • コンビナトリアルバイオエンジニアリングの最前線, 細胞チップを用いた薬剤スクリーニング
    赤木良教; 森田 資隆; 民谷栄一 (担当:共著範囲:)2004年
  • コンビナトリアルバイオエンジニアリングの最前線, ファージディスプレイ法による幹細胞認識
    森田 資隆; 民谷栄一 (担当:共著範囲:)2004年
  • 抗体エンジニアリングの最前線, バイオセンサーチップと抗体エンジニアリング
    民谷栄一; 森田 資隆; 山村昌平; 鈴木正康; 岸裕幸; 村口篤 (担当:共著範囲:)2004年
  • Heavy oil spilled from Russian tanker “Nakhodka” in 1997, Edited by K. Tazaki, 21st Century COE Kana, Isolation and characterization of oil degradable microorganisms from heavy oil-spilled marine shores
    T. Sakaguchi; 森田 資隆; E. Tamiya (担当:共著範囲:)2003年
  • 21世紀版薄膜作製応用ハンドブック, バイオセンサ
    民谷栄一; 森田 資隆 (担当:共著範囲:)2003年
  • コンビナトリアル・バイオエンジニアリング-情報から機能の創造をめざして-, チップテクノロジー-ハイスループットスクリーニングの新展開-
    森田 資隆; 村上裕二; 金原健; 民谷栄一 (担当:共著範囲:)2003年
  • Biological systems engineering, Synthesis and analysis of peptide ligand for biosensor application using combinatorial chemistry
    森田 資隆; Y. Murakami; K. Yokoyama; E. Tamiya (担当:共著範囲:)2002年
  • Micro total Analysis Systems 2002, In vitro protein synthesis on a high-integrated microchamber chip with low DNA molecules
    T. Kinpara; 森田 資隆; H. Nakano; T. Yamane; E. Tamiya (担当:共著範囲:)2002年
  • Biological Systems Engineering, Sensor peptide based on fluorescence resonance energy transfer
    K. Yokoyama; 森田 資隆; M. Matsumoto; H. Ishikawa; E. Tamiya (担当:共著範囲:)2002年
  • Micro total Analysis Systems 2002, High-throughput screening of anticancer drug using microarray based cell chip
    S. R. Rao; 森田 資隆; Y. Akagi; E. Tamiya (担当:共著範囲:)2002年
  • Peptides: the wave of the future, Screening and design of hybrid peptide that binds with glucose oxidase
    K. Yokoyama; 森田 資隆; T. Sakai; H. Ishikawa; E. Tamiya (担当:共著範囲:)2002年
  • 生命化学のセントラルドグマ-テーラーメイド・バイオケミストリーのめざすもの, バイオセンサー-新規バイオセンサー創成に向けてのチャレンジ-
    民谷栄一; 森田 資隆; 村上裕二; 横山憲二 (担当:共著範囲:)2002年
  • BIO INDUSTRY BIO INDUSTRY, チップ技術とコンビナトリアル機能分子設計
    森田 資隆; 金原健; 村上裕二; 横山憲二; 民谷栄一 (担当:共著範囲:)2001年
  • Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries 2000, Synthesis and analysis of peptide ligand for biosensor application using combinatorial chemistry
    森田 資隆; K. Yokoyama; E. Tamiya (担当:共著範囲:)2001年
  • Microchamber Array for Immunosensor Applications, "microTAS 2000", Kluwer Academic Publications
    Y. Murakami; T. Kikuchi; M. Yanase; H. Nagai; Y. Morita; E. Tamiya 2000年
  • Random distribution of biomaterials as a handling method on microarray applied to PCR, biosensors and high through-put screening, 1st annual international IEEE-EMBS special topic conference on microtechnologies in Medicine & Biology
    Y. Murakami; H. Nagai; T. Kikuchi; A. Yamamura; K. Idegami; M. Yanase; Y.S. Choi; Y. Morita; E. Tamiya 2000年
  • Scanning near-field optical/atomic force microscope (SNOAM) for biomedical applications, Proceedings of scanning and force microscopies for biomedical applications, Society of Photo-optical Instrumentation Engineers - The International Society for Optical
    E. Tamiya; S. Iwabuchi; A. Hashigasako; Y. Murakami; T. Sakaguchi; Y. Morita; K. Yokoyama 1999年
  • Advanced imaging for DNA analysis based on scaning near-field optical/atomic-force microscopy (SNOAM), Proceedings of scanning and force microscopies for biomedical applications. Society of Photo-optical Instrumentation Engineers - The International Socie
    S. Iwabuchi; A. Hashigasako; Y. Morita; T. Sakaguchi; Y. Murakami; K. Yokoyama; E. Tamiya 1999年
  • Extracellular proteinases from extremophiles. Enzyme Engineering XIV, Annals of the New York Academy of Sciences
    Y. Morita; Q. Hasan; T. Sakaguchi; Y. Murakami; K. Yokoyama; E. Tamiya 1998年

講演・口頭発表等

  • 未分化なP19細胞に結合するペプチドの結合特異性と細胞マーカー分子の探索
    角大輔; 益拓海; 玉田英之; 森田資隆
    第17回バイオテクノロジー部会シンポジウム 2023年09月
  • 未分化なP19細胞に結合するペプチドの結合特異性と細胞マーカー分子の探索
    角大輔; 益拓海; 玉田英之; 森田資隆
    第１１回近畿大学院生サミット 2023年08月
  • 未分化なP19細胞に結合するペプチドの結合特異性と細胞マーカー分子の探索  [通常講演]
    角大輔; 益拓海; 玉田英之; 森田資隆
    第６０回化学関連支部合同九州大会 2023年07月
  • 未分化なP19細胞に特異的に結合するペプチドの探索  [通常講演]
    角大輔; 益拓海; 玉田英之; 森田資隆
    第５９回化学関連支部合同九州大会 2022年07月
  • 新規低温菌BS-c株が産生する低温活性リパーゼの特性評価  [通常講演]
    佐伯友啓; 森田 資隆; 檜谷 孝; 長田真裕
    サイエンスネットワーク・院生サミット2013 2013年11月 近畿大学産業理工学部 サイエンスネットワーク・院生サミット2013
  • 低温菌BS-c株が産生する低温活性リパーゼの特性評価  [通常講演]
    佐伯友啓; 森田 資隆; 檜谷 孝; 長田真裕
    第12回近畿大学環境科学研究会 2013年08月 近畿大学産業理工学部 第12回近畿大学環境科学研究会
  • 新規低温菌BS-c株が産生する低温活性リパーゼの特性評価  [通常講演]
    佐伯友啓; 森田 資隆; 桧谷孝; 長田真裕
    第50回化学関連支部合同九州大会 2013年07月 北九州国際会議場 第50回化学関連支部合同九州大会
  • Application of the phage display technology to screening and detecting of an undifferentiated embryonal carcinoma stem cell and a nerve growth factor  [通常講演]
    森田 資隆
    American Chemical Society 2012年08月 Philadelphia, Pennsylvania, USA American Chemical Society
  • 新規低温性鉄還元菌の簡易同定と還元特性  [通常講演]
    穴井順也; 森田 資隆; 竹入 裕
    第48回化学関連支部合同九州大会 2011年07月 北九州国際会議場 第48回化学関連支部合同九州大会
  • 新規低温性ケラチン分解酵素の簡易精製と特性評価  [通常講演]
    田端克哉; 森田 資隆; 松山裕子
    第48回化学関連支部合同九州大会 2011年07月 北九州国際会議場 第48回化学関連支部合同九州大会
  • ケラチン分解菌が産生する２つの酵素によるケラチンの協同的な分解  [通常講演]
    田端克哉; 森田 資隆; 松山裕子
    第47回化学関連支部合同九州大会 2010年 北九州国際会議場 第47回化学関連支部合同九州大会
  • 新規な低温性鉄還元菌の探索とその温度特性に関する評価  [通常講演]
    森田 資隆; 穴井順也; 竹入 裕
    第47回化学関連支部合同九州大会 2010年 北九州国際会議場 第47回化学関連支部合同九州大会
  • 新規鉄還元菌の探索と還元特性に関する研究  [通常講演]
    竹入裕; 森田 資隆; 森敬之
    第9回近畿大学環境科学研究会 2010年 近畿大学薬学部 第9回近畿大学環境科学研究会
  • ケラチン分解菌が産生する低温活性酵素の探索とその特性評価  [通常講演]
    水之江庄士郎; 森田 資隆; 桧谷 孝
    第47回化学関連支部合同九州大会 2010年 北九州国際会議場 第47回化学関連支部合同九州大会
  • 低温菌BS-c株が産生する新規低温活性リパーゼの特性評価  [通常講演]
    森田 資隆; 桧谷孝; 長田真裕
    第9回近畿大学環境科学研究会 2010年 近畿大学薬学部 第9回近畿大学環境科学研究会
  • 新規低温性リパーゼの探索と特性評価  [通常講演]
    森田 資隆; 桧谷孝
    第46回化学関連支部合同九州大会 2009年 第46回化学関連支部合同九州大会
  • Undifferentiated embryonal carcinoma stem cell and nerve growth factor screenings using phage display technology  [通常講演]
    森田 資隆
    238th ACS National Meeting 2009年 238th ACS National Meeting
  • ケラチン分解菌が産生する複合酵素の粗酵素特性  [通常講演]
    森田 資隆; 松山裕子
    第８回近畿大学環境科学研究会 2009年 第８回近畿大学環境科学研究会
  • ケラチン分解菌が産生する複合酵素の粗酵素特性  [通常講演]
    森田 資隆; 松山裕子
    第46回化学関連支部合同九州大会 2009年 第46回化学関連支部合同九州大会
  • 新規鉄還元菌の探索と特性評価  [通常講演]
    森田 資隆; 竹入裕
    第46回化学関連支部合同九州大会 2009年 第46回化学関連支部合同九州大会
  • 新規低温性リパーゼの探索と特性評価  [通常講演]
    森田 資隆; 桧谷孝
    第８回近畿大学環境科学研究会 2009年 第８回近畿大学環境科学研究会
  • 新規鉄還元菌の探索と特性評価  [通常講演]
    森田 資隆; 竹入裕
    第８回近畿大学環境科学研究会 2009年 第８回近畿大学環境科学研究会
  • 低温性ケラチン分解菌由来ジスルフィドレダクターゼの特性評価  [通常講演]
    森田 資隆; 松山裕子
    第45回化学関連支部合同九州大会 2008年 第45回化学関連支部合同九州大会
  • 低温性ケラチン分解菌由来ジスルフィドレダクターゼの特性評価  [通常講演]
    森田 資隆; 松山裕子
    第７回近畿大学環境科学研究会 2008年 第７回近畿大学環境科学研究会
  • A screening of phage displayed peptides for the recognition of fullerene  [通常講演]
    森田 資隆
    236th ACS National Meeting 2008年 236th ACS National Meeting
  • Isolation and characterization of phage displayed peptides for the recognition of P19 embryonic carcinoma stem cells  [通常講演]
    森田 資隆; E. Tamiya
    Engineering Cell Biology: The Cell in Context 2007年 Engineering Cell Biology: The Cell in Context
  • Selection and properties for the recognition of P19 embryonic carcinoma stem cells by the peptide  [通常講演]
    森田 資隆; E. Tamiya
    232nd American Chemical Society National Meeting, Poster presentation 2006年 232nd American Chemical Society National Meeting, Poster presentation
  • A screening of phage displayed peptides for the recognition of P19 embryonic carcinoma  [通常講演]
    森田 資隆; K. Mamiya; E. Tamiya
    2005 International Chemical Congress of Pacific Basin Societies 2005年 2005 International Chemical Congress of Pacific Basin Societies
  • A cooperative action of two enzymes in keratin degradation  [通常講演]
    森田 資隆; S. Yamamura; E. Tamiya
    Enzyme Engineering XVII 2003年 Santa Fe, USA Enzyme Engineering XVII
  • Real-time monitoring of on-chip DNA hybridization with evanescent microscope  [通常講演]
    森田 資隆; E. Tamiya
    Japan-European Workshop on Chem Micro Mechatronics System and Biochips 2003年 Paris, France Japan-European Workshop on Chem Micro Mechatronics System and Biochips
  • Development of cell chip for bio-sensing tool  [通常講演]
    森田 資隆; E. Tamiya
    Japan-European Workshop on Chem Micro Mechatronics System and Biochips 2003年 Paris, France Japan-European Workshop on Chem Micro Mechatronics System and Biochips
  • Design of fullerene binding peptide using combinatorial bioengineering  [通常講演]
    森田 資隆; T. Ohsugi; Y. Murakami; K. Yokoyama; E. Tamiya
    Biosensor 2002 2002年 Kyoto, Japan Biosensor 2002
  • Highthroughput screening of functional molecule using combinatorial bioengineering and chip technology  [通常講演]
    森田 資隆; E. Tamiya
    The 6th World Multiconference on Systemics, Cybernetics and Informatics 2002年 Orlando, Florida, USA The 6th World Multiconference on Systemics, Cybernetics and Informatics
  • Biodegradation of alkane by psychrotrophic bacteria for cold environment  [通常講演]
    森田 資隆; H. Onishi; S.R. Rao; T. Sakaguchi; Y. Murakami; K. Yokoyama; E. Tamiya
    Enzyme Engineering XVI 2001年 Potsdam, Germany Enzyme Engineering XVI

MISC

産業財産権

  • 特願2008-228700:発光微生物固定化チップによる有機汚濁計測方法  2008年
    阪口利文, 森田資隆
  • 2007-68531:残留農薬検出方法、農薬検出キット、及び農薬検出ストリップ  2007年
    民谷栄一, 森田資隆
  • 特願2006-30229:残留農薬検出方法、農薬検出キット、及び農薬検出ストリップ  2006年
    民谷栄一, 森田資隆
  • 2005-179257:新規胚性幹細胞化誘導物質  2005年
    民谷栄一, 森田資隆, S.R. Rao
  • 2005-102628:細胞の固定化および採取方法  2005年
    民谷栄一, 森田資隆, S.R. Rao, 山村昌平
  • 特開2004-033062:細菌を利用したカロテノイド類の新規合成法とその方法により得られるカロテノイド類  2004年
    民谷栄一, 森田資隆, 山村昌平, S.R. Rao
  • 特許第3041423号:集積化されたマイクロウェルを用いたポリメラーゼ連鎖反応装置    2003年
    民谷栄一, 横山憲二, 村上裕二, 阪口利文, 森田資隆, 永井秀典, 井出上公太郎
  • 特開2003-070459:ケラチナーゼ活性の高い新規細菌株  2003年
    民谷栄一, 横山憲二, 山村昌平, 森田資隆
  • 特開2003-079364:新規多環式芳香族炭化水素分解菌，それを含有する新規多環式芳香族炭化水素分解剤  2003年
    民谷栄一, S.R. Rao, 森田資隆, 村上裕二, 横山憲二, 阪口利文
  • 特開2003-310255:新規ケラチン分解酵素  2003年
    民谷栄一, 山村昌平, 森田資隆, 横山憲二
  • 特開2003-079364:新規多環式芳香族炭化水素分解菌，それを含有する新規多環式芳香族炭化水素分解剤  2003年
    民谷栄一, 森田資隆, S.R. Rao, 村上裕二, 横山憲二, 阪口利文
  • 特開2003-070459:ケラチナーゼ活性の高い新規細菌株  2003年
    民谷栄一, 森田資隆, 横山憲二, 山村昌平
  • 特開2003-310255:新規ケラチン分解酵素  2003年
    民谷栄一, 森田資隆, 山村昌平, 横山憲二
  • 特開2005-179257:新規胚性幹細胞化誘導物質  
    民谷栄一, 森田資隆, S.R. Rao, 山村昌平
  • 特開2005-102628:細胞の固定化および採取方法  
    民谷栄一, 森田資隆, S.R. Rao, 山村昌平

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2003年 -2005年 
    代表者 : 民谷 栄一; 高村 禅; 中野 秀雄; 岸 裕幸; 森田 資隆
     
    申請者らは、微細加工技術によりピコおよびナノリットルレベルでの微量容積を有するチャンバーを多数配置したマイクロチャンバーアレイチップを設計・作製し、このチップ上で一分子および一細胞レベルでの検出および解析が可能なシステムの開発を行った。まず、免疫細胞の網羅的解析システムの開発として、ナノリソグラフィー技術であるLIGAプロセスを用いて、20万個以上のチャンバーウェル(直径10μm)を有するポリスチレン製マイクロアレイチップを作製し、チップ上にマウス脾リンパ球細胞溶液を展開した結果、各チャンバーウェル中に80%以上の導入効率にて一細胞を配置することに成功した。このチップ上にて、抗原刺激を行った後、マイクロアレイスキャナーにより、Ca^<2+>シグナルを指標として活性化Bリンパ球細胞集団の検出および解析を行うことができた。次に、一分子および一細胞レベルでの遺伝子解析システムとして、微細加工技術によりナノリッターチャンバー(容量約50nL)を1248個集積化したシリコン製マイクロチャンバーアレイチップを作製し、本チップ上で病原性微生物やヒト白血病細胞Tリンパ腫のJurkat細胞をナノリッターディスペンサーによって、1つのマイクロチャンバーにつき一細胞を導入されるように分注し、一細胞からの遺伝子増幅することに成功した。したがって、一枚のチップ上で、一細胞および一分子レベルで生体試料を分析し、かつ網羅的に解析する新規デバイスシステムを構築できたことから、今後、医療、診断などの新しいツールして期待できると思われる。その他にも、DNAやプロテインライブラリー、細胞スクリーニング技術、ナノ材料などを用いた高感度検出法などを組み合わせて独自の集積型チップデバイスを作製し、迅速かつ高感度な生体試料測定系の構築にも成功している。以上より、疾病診断用デバイスや治療・創薬デバイスの確立に繋がる各種バイオセンシングデバイスの基盤研究を行い、これらのデバイスの創成と応用に向けて研究を遂行できた。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2001年 -2001年 
    代表者 : 横山 憲二; 森田 資隆
     
    これまでに我々は、ファージ提示系ランダムペプチドライブラリーを用いることにより、グルコースオキシダーゼ(GOx)の52-58番目および197-203番目のアミノ酸配列に対し親和性を示す12merペプチドを取得している。本研究では、さらに強い非共有結合によりタンパク質を認識するペプチドポリマーの創成を試みた。 1.ペプチドモノマーの固相合成 GOxを認識するペプチド配列を持つアミノ酸側鎖保護基付ビニルモノマーPM1(GOxの52-58番目を認識)、PM2(同197-203番目)をFmoc固相法により合成した。N末端αアミノ基にはリンカー6-aminohexanoic acidを介してアクリロイルを固相上で導入し、側鎖保護基を残したままクリベージした。次に、逆相HPLCにて分取し、MALDI-TOF質量分析にて生成物を確認した。 2.プチドポリマーの合成と親和性の評価 PM1またはPM2と両親媒性のN, N-ジエチルアクリルアミドをコモノマーとし、ラジカル共重合反応を行った。次にトリフルオロ酢酸を主成分とするクリベージカクテルでペプチド側鎖を脱保護した後、分画分子量1万の限外ろ過膜で低分子量成分を分離し、目的のペプチドポリマーを得た。合成したペプチドポリマーとGOxとの親和性は、表面プラズモン共鳴測定装置により評価した。その結果、いずれのペプチドポリマーの場合も基板上に固定化したGOxに対し高い親和性を示したのに対し、ヘモグロビンや牛血清アルブミンに対してはほとんど結合しなかった。また、単独のペプチドとペプチドポリマーのGOxに対する親和性を比較した。その結果、いずれも単独のペプチドにくらべ、同等あるいはそれ以上の親和性を示した。以上から、ペプチドを高分子化することにより、ペプチドが持つ親和性をさらに上昇させることに成功した。

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