研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    石田 晃彦(イシダ アキヒコ), イシダ アキヒコ

所属(マスター)

  • 工学研究院 応用化学部門 分子機能化学分野

所属(マスター)

  • 工学研究院 応用化学部門 分子機能化学分野

独自項目

syllabus

  • 2020, 応用生物化学A(生物計測化学), Applied Biochemistry A (Analytical Biochemistry), 修士課程, 総合化学院, 分子認識,酵素反応,免疫反応,生体分子間相互作用,生物計測化学,マイクロ分析・診断デバイス
  • 2020, 応用化学学生実験Ⅰ, Applied Chemistry Laboratory Ⅰ, 学士課程, 工学部, 実験技術、安全、酸化還元滴定、キレート滴定、酸塩基平衡、錯形成平衡、吸光光度法、クラジウス-クラペイロンの式、蒸発エンタルピー、電荷移動錯体、分子間相互作用、電極反応、計測制御、吸収・蛍光スペクトル、分子軌道論、反応速度、アレニウスの式
  • 2020, 化学英語, English for Chemistry, 学士課程, 工学部, 化学,英語,単語,表記法
  • 2020, 分析化学Ⅰ, Analytical Chemistry Ⅰ, 学士課程, 工学部, 化学量論,溶液内化学平衡,反応速度,分析試薬,分析反応場,滴定,沈殿,溶媒抽出,吸光・蛍光光度法,クロマトグラフィー,電気泳動,酵素分析法,免疫測定法,誤差,相関分析,回帰分析

researchmap

プロフィール情報

学位

  • 博士(工学)(東北大学)
  • 工学修士(東北大学)

プロフィール情報

  • 石田, イシダ
  • 晃彦, アキヒコ
  • ID各種

    200901062864071825

対象リソース

業績リスト

研究分野

  • ナノテク・材料 / 分析化学

委員歴

  • 2016年04月 - 2017年03月   日本分析化学会   ぶんせき編集委員
  • 2009年   日本分析化学会北海道支部   化学系学協会北海道支部2010冬季研究発表会実行委員   日本分析化学会北海道支部
  • 2007年   日本分析化学会   分析化学年会実行委員   日本分析化学会

論文

  • Keine Nishiyama, Kazuki Takahashi, Mao Fukuyama, Motohiro Kasuya, Ayuko Imai, Takumi Usukura, Nako Maishi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Kyoko Hida, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Biosensors & bioelectronics 190 113414 - 113414 2021年10月15日 
    Antibody detection methods for viral infections have received broad attention due to the COVID-19 pandemic. In addition, there remains an ever-increasing need to quantitatively evaluate the immune response to develop vaccines and treatments for COVID-19. Here, we report an analytical method for the rapid and quantitative detection of SARS-CoV-2 antibody in human serum by fluorescence polarization immunoassay (FPIA). A recombinant SARS-CoV-2 receptor binding domain (RBD) protein labeled with HiLyte Fluor 647 (F-RBD) was prepared and used for FPIA. When the anti-RBD antibody in human serum binds to F-RBD, the degree of polarization (P) increases by suppressing the rotational diffusion of F-RBD. The measurement procedure required only mixing a reagent containing F-RBD with serum sample and measuring the P value with a portable fluorescence polarization analyzer after 15 min incubation. We evaluated analytical performance of the developed FPIA system using 30 samples: 20 COVID-19 positive sera and 10 negative sera. The receiver operating characteristic curve drawn with the obtained results showed that this FPIA system had high accuracy for discriminating COVID-19 positive or negative serum (AUC = 0.965). The total measurement time was about 20 min, and the serum volume required for measurement was 0.25 μL. Therefore, we successfully developed the FPIA system that enables rapid and easy quantification of SARS-CoV-2 antibody. It is believed that our FPIA system will facilitate rapid on-site identification of infected persons and deepen understanding of the immune response to COVID-19.
  • Ayano Nakamura, Mitsutoshi Aoyagi, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    ACS Food Science & Technology 2021年09月20日
  • Takeshi Komatsu, Ryan Russel Gabatino, Harrienica Hofileña, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Journal of Chemical Education 2021年09月14日 [査読有り]
  • Akinori Yamaguchi, Hajime Miyaguchi, Akihiko Ishida, Manabu Tokeshi
    ACS Applied Bio Materials 2021年08月16日
  • Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Hideaki Hisamoto, Manabu Tokeshi
    ACS omega 6 12 8340 - 8345 2021年03月30日 
    Analytical methods with fluorescence detection are in widespread use for detecting low abundance analytes. Here, we report a simple method for fluorescence signal amplification utilizing a structure of an azide-unit pendant water-soluble photopolymer (AWP) in a microchannel. The AWP is a poly(vinyl alcohol)-based photocross-linkable polymer, which is often used in biosensors. We determined that the wall-like structure of the AWP (AWP-wall) constructed in a microchannel functioned as an amplifier of a fluorescence signal. When a solution of fluorescent molecules was introduced into the microchannel having the AWP-wall, the fluorescent molecules accumulated inside the AWP-wall by diffusion. Consequently, the fluorescence intensity inside the AWP-wall increased locally. Among the fluorescent molecules considered in this paper, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) (DDAO) showed the highest efficiency of fluorescence signal amplification. We prepared a calibration curve for DDAO using the fluorescence intensity inside the AWP-wall, and the sensitivity was 5-fold that for the microchannel without the AWP-wall. This method realizes the improved sensitivity of fluorescence detection easily because the fluorescence signal was amplified only by injecting the solution into the microchannel having the AWP-wall. Furthermore, since this method is not limited to only the use of microchannel, we expect it to be applicable in various fields.
  • Takeshi Komatsu, Ryoga Maeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS sensors 6 3 1094 - 1102 2021年03月26日 [査読有り]
     
    The development of low-cost, user-friendly paper-based analytical devices (PADs) that can easily measure target chemicals is attracting attention. However, most PADs require manipulation of the sample using sophisticated micropipettes for quantitative analyses, which restricts their user-friendliness. In addition, immobilization of detection molecules to cellulose fibers is essential for achieving good measuring ability as it ensures the homogeneity of color development. Here, we have described a dip-type PAD that does not require pipette manipulation for sample introduction and immobilization of detection molecules to cellulose fibers and its application to ascorbic acid (AA) and pH assays. The PAD consisted of a dipping area and two channels, each with two detection zones. The developed PADs show color distribution in the two detection zones depending on the sample flow from the dipping area. In comparison with a PAD that has one detection zone at the end of the channel, our developed device achieved higher sensitivity (limit of detection (LOD), 0.22 mg/mL) and reproducibility (maximum coefficient of variation (CV), 2.4%) in AA detection. However, in pH detection, the reproducibility of the PAD with one detection zone at the end of the channel (maximum CV, 21%) was worse than that with two zones (maximum CV, 11%). Furthermore, a dipping time over 3 s did not affect color formation or calibration curves in AA detection: LODs at 3 and 30 s dipping time were 18 and 5.8 μg/mL, respectively. The simultaneous determination of AA and pH in various beverages was performed with no significant difference compared to results of the conventional method.
  • Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Applied Bio Materials 4 2 1783 - 1793 2021年02月15日
  • Keine Nishiyama, Yohei Takeda, Kazuki Takahashi, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Haruko Ogawa, Manabu Tokeshi
    Analytical and bioanalytical chemistry 2021年02月05日 [査読有り]
     
    Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.
  • Takeshi Komatsu, Yuki Sato, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytica Chimica Acta 1144 85 - 95 2021年02月 [査読有り]
  • Niko Kimura, Masatoshi Maeki, Kosuke Sasaki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    RSC Advances 2021年
  • Keine Nishiyama, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    Sensors and Actuators B: Chemical 326 128982 - 128982 2021年01月
  • Taiga Ajiri, Takao Yasui, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Junji Nishii, Yoshinobu Baba, Manabu Tokeshi
    ACS Applied Nano Materials 3 9 8810 - 8816 2020年09月25日
  • Masatoshi Maeki, Sho Ito, Reo Takeda, Go Ueno, Akihiko Ishida, Hirofumi Tani, Masaki Yamamoto, Manabu Tokeshi
    Chemical science 11 34 9072 - 9087 2020年08月25日 
    Room-temperature (RT) protein crystallography provides significant information to elucidate protein function under physiological conditions. In particular, contrary to typical binding assays, X-ray crystal structure analysis of a protein-ligand complex can determine the three-dimensional (3D) configuration of its binding site. This allows the development of effective drugs by structure-based and fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein-ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein-ligand complex analysis, the simultaneous preparation of protein-ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein-ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein-ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin-ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.
  • Keine Nishiyama, Yohei Takeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Yutaka Yonezawa, Kunitoshi Imai, Haruko Ogawa, Manabu Tokeshi
    Sensors and Actuators B: Chemical 316 128160 - 128160 2020年08月 [査読有り][通常論文]
     
    A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIVhemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 μL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.
  • Masatoshi Maeki, Shohei Yamazaki, Reo Takeda, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Omega 5 28 17199 - 17206 2020年07月21日 [査読有り][通常論文]
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Applied Materials & Interfaces 12 30 34011 - 34020 2020年07月15日 [査読有り][通常論文]
     
    Microfluidic methodologies for preparation of lipid nanoparticles (LNPs) based on an organic solvent injection method enable precise size control of the LNPs. After preparation of LNPs, the organic solvent injection method needs some post-treatments, such as overnight dialysis or direct dilution with a buffer solution. LNP production using the microfluidic-based organic solvent injection method is dominated by kinetics rather than thermodynamics. Kinetics of ethanol removal from the inner and outer membranes of LNPs could induce a structural change in the membrane that could lead to fusion of LNPs. However, the effects of microfluidic post-treatment on the final size of LNPs have not been sufficiently understood. Herein, we investigated the effect of the post-treatment processes on the final product size of LNPs in detail. A simple baffle device and a model lipid system composed of a neutral phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and cholesterol were used to produce the LNPs. We demonstrated that flow conditions of the post-treatment diluting the remaining ethanol in the LNP suspension affected the final product size of LNPs. Based on the findings, we developed an integrated baffle device composed of an LNP production region and a post-treatment region for a microfluidic-based LNP production system; this integrated baffle device prevented the undesirable aggregation or fusion of POPC LNPs even for the high-lipid-concentration condition. Finally, we applied our concept to small interfering RNA (siRNA) delivery and confirmed that no significant effects due to the continuous process occurred on the siRNA encapsulation efficiency, biological distribution, and knockdown activity. The microfluidic post-treatment method is expected to contribute to the production of LNPs for practical applications and the development of novel LNP-based nanomedicines.
  • Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Sensors 5 5 1287 - 1294 2020年05月22日 [査読有り][通常論文]
  • Mazaafrianto DN, Ishida A, Maeki M, Tani H, Tokeshi M
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 35 11 1221 - 1226 2019年11月 [査読有り][通常論文]
     
    In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer having a dithiol-based anchor, which exhibited higher stability on a gold electrode than a monothiol-based aptamer because of its two anchors. The sensor was also based on a signal-on scheme that produces a signal current resulting from structure-switching of the aptamer upon interaction with OTA. For simple fabrication of this sensor, the non-covalent interaction of methylene blue with the aptamer was also employed as an electrochemical indicator. In this study, the performance of the sensor, including the dissociation constant of the aptamer-OTA complex, was characterized. The proposed sensor exhibited high reproducibility and enough sensitivity to detect the minimum amount of OTA required for the analysis of real food samples with a limit of detection of 113 pM.
  • Kenia Chávez Ramos, Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Toshihiro Kasama, Yoshinobu Baba, Manabu Tokeshi
    ACS Omega 4 15 16683 - 16688 2019年10月08日 [査読有り][通常論文]
     
    © 2019 American Chemical Society. Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23-100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.
  • Nishiyama Keine, Hoshikawa Koki, Maeki Masatoshi, Ishida Akihiko, Tani Hirofumi, Tokeshi Manabu
    Electroanalysis 31 9 1736 - 1743 Wiley 2019年09月06日 [査読有り][通常論文]
     
    A concentric ring array electrode that amplifies the current signal without redox cycling has been developed for highly sensitive electrochemical detection at a single potential in a microfluidic platform. Herein, the effect of ring-electrode width on the current and current density was examined. A ring-array electrode with widths that decrease from the inner to the outer ring was shown to exhibit the highest sensitivity. This electrode delivered a current density that was approximately 50 % higher than that of a conventionally used disc electrode. We used numerical simulations to further optimize this type of array electrode, which led to a limit of detection for catechol of 6.2 nmol/L. This ring array electrode has great potential for use in a variety of applications because it can be used to detect irreversible targets with a simple apparatus at a single potential and requires no electrode modification to achieve high sensitivity.
  • Keine Nishiyama, Toshihiro Kasama, Seiya Nakamata, Koya Ishikawa, Daisuke Onoshima, Hiroshi Yukawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    Analyst 144 15 4589 - 4595 2019年08月07日 [査読有り][通常論文]
     
    © 2019 The Royal Society of Chemistry. We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 μL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.
  • Wakao Osamu, Maeki Masatoshi, Ishida Akihiko, Tani Hirofumi, Hibara Akihide, Tokeshi Manabu
    SENSORS AND ACTUATORS B-CHEMICAL 285 418 - 422 2019年04月15日 [査読有り][通常論文]
     
    Fluorescence polarization (FP) is a one of the measurement techniques to study molecular interactions. We previously developed our own FP measurement system based on synchronization detection that uses a liquid crystal layer and an image sensor. The measurement cycle was fixed to 100 without any theoretical considerations, however, the influence of the synchrony and measurement cycles for FP values should be considered. In the present paper, we carried out an experimental and theoretical investigation into the influence of the synchrony between liquid crystal operation and image sampling for FP values of our system. When there was synchronization mismatch, the experimental FP values obtained using fluorescein ethylene glycol solution and the theoretical FP values changed according to the number of measurement cycles. Additionally, we measured the FP immunoassay for a physiologically active substance under synchronization mismatch. The synchronization mismatch influenced the measurement performance of the system, indicating that optimization of the number of image samplings was necessary to improve the measurement performance. For instance, the Mismatch 0.99 case, the measurement cycle should be 50 cycle judging from its dynamic range and R-square (R-2). From the investigation, we obtained theoretical and experimental knowledge on FP response to facilitate further applications of our FP system.
  • Osamu Wakao, Ken Satou, Ayano Nakamura, Polina A. Galkina, Keine Nishiyama, Ken Sumiyoshi, Fumio Kurosawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Mikhail A. Proskurnin, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Lab on a Chip 19 15 2581 - 2588 2019年 [査読有り][通常論文]
     
    © 2019 The Royal Society of Chemistry. High-throughput fluorescence polarization immunoassays (FPIAs) for mycotoxin were conducted using a portable FP analyzer with a microdevice. Simultaneous FPIA measurements for 8 different deoxynivalenol (DON) concentrations in 12 chambers (total of 96 samples) and high-throughput FPIA measurements for single DON concentrations in more than 500 chambers were conducted. The results indicated that simultaneous FPIAs for 96 independent samples and for 500 samples were possible by FP imaging. The FP analyzer has a size of 65 cm (W 35 cm × D 15 cm × H 15 cm) and costs less than $5000. The sample volume was 1 nL. Furthermore, it is expected that sample reaction and FP detection can be automatically conducted with the analyzer by changing the microdevice and the software. Its features such as low cost and portability will contribute to on-site measurement and point-of-care testing. Additionally, the high-throughput feature will contribute to the study of molecular interactions based on FP measurements.
  • Osamu Wakao, Ken Satou, Ayano Nakamura, Polina A. Galkina, Keine Nishiyama, Ken Sumiyoshi, Fumio Kurosawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Mikhail A. Proskurnin, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Lab on a Chip 19 15 2581 - 2588 Royal Society of Chemistry ({RSC}) 2019年 [査読有り][通常論文]
     
    © 2019 The Royal Society of Chemistry. High-throughput fluorescence polarization immunoassays (FPIAs) for mycotoxin were conducted using a portable FP analyzer with a microdevice. Simultaneous FPIA measurements for 8 different deoxynivalenol (DON) concentrations in 12 chambers (total of 96 samples) and high-throughput FPIA measurements for single DON concentrations in more than 500 chambers were conducted. The results indicated that simultaneous FPIAs for 96 independent samples and for 500 samples were possible by FP imaging. The FP analyzer has a size of 65 cm (W 35 cm × D 15 cm × H 15 cm) and costs less than $5000. The sample volume was 1 nL. Furthermore, it is expected that sample reaction and FP detection can be automatically conducted with the analyzer by changing the microdevice and the software. Its features such as low cost and portability will contribute to on-site measurement and point-of-care testing. Additionally, the high-throughput feature will contribute to the study of molecular interactions based on FP measurements.
  • Keine Nishiyama, Toshihiro Kasama, Seiya Nakamata, Koya Ishikawa, Daisuke Onoshima, Hiroshi Yukawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    The Analyst 144 15 4589 - 4595 2019年 [査読有り][通常論文]
     
    © 2019 The Royal Society of Chemistry. We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 μL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.
  • Donny N. Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    ACS Omega 3 12 16823 - 16830 2018年12月31日 [査読有り]
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Yusuke Note, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Omega 3 5 5044 - 5051 2018年05月09日 [査読有り][通常論文]
     
    The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.
  • Donny Nugraha Mazaafrianto, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Micromachines 9 5 2018年04月25日 [査読有り][通常論文]
     
    Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed, aptamers have made significant contributions as bio-recognition sensors. Microdevice systems allow for low reagent consumption, high-throughput of samples, and disposability. Due to these advantages, there has been an increasing demand to develop microfluidic-based aptasensors for analytical technique applications. This review introduces the principal concepts of aptasensors and then presents some advanced applications of microdevice-based aptasensors on several platforms. Highly sensitive detection techniques, such as electrochemical and optical detection, have been integrated into lab-on-a-chip devices and researchers have moved towards the goal of establishing point-of-care diagnoses for target analyses.
  • Osamu Wakao, Ken Satou, Ayano Nakamura, Ken Sumiyoshi, Masanori Shirokawa, Chikaaki Mizokuchi, Kunihiro Shiota, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Review of Scientific Instruments 89 2 024103  2018年02月01日 [査読有り][通常論文]
     
    Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementable in molecular interaction analysis. Previously we have developed a unique FP measurement system using a liquid crystal (LC) layer and an image sensor. The system is based on a principle of synchronized detection between the switching rate of the LC layer and the sampling rate of the CCD. The FP system realized simultaneous multiple sample detection however, the measurement precision was lower than that of the conventional FP apparatus. The main drawbacks were low light transmittance of the LC layer and insufficient synchronization between the LC layer and CCD. In this paper, we developed a new FP analyzer based on LC-CCD synchronization detection. By using a newly designed LC with high transmittance and improving synchronization, the performance of the system has been dramatically improved. Additionally, we reduced the cost by using an inexpensive CCD and an LED as the excitation source. Simultaneous FP immunoassay of multiple samples of prostaglandin E2 was performed. The error rate of the FP system is reduced from 16.9% to 3.9%, as comparable to the commercial conventional FP system.
  • Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 3 1404 - 1405 2018年 
    This paper reports a methodology for precise controlling the lipid nanoparticle (LNP) size by stepwise and rapid ethanol dilution using an integrated microfluidic device with baffle mixers. The integrated microfluidic device coupling the LNP synthesis and the post-treatment regions had better size controllability of LNPs than the conventional preparation methods. Additionally, 30 nm-sized siRNA-loaded LNPs prepared by the post-treatment process using the integrated microfluidic device showed great gene-silencing activity and specific intrahepatic biodistribution. The stepwise and rapid ethanol dilution methodology using the integrated microfluidic device provides LNPs with homogeneous size distribution for improving the efficacy of nanomedicines.
  • Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytical Sciences 34 1 39 - 44 2018年 [査読有り][通常論文]
     
    We report on the effects of fabrication methods, photolithography, wax printing, screen printing, and craft cutting, on selected properties of microfluidic paper-based analytical devices (μPADs): cost, fabrication precision, wicking rate, and analytical accuracy. Photolithography requires numerous fabrication steps, and an oxygen plasma treatment is necessary when using an aqueous solution. Although the boundary between the hydrophobic and hydrophilic areas in the μPAD is sharpest, the obtained K-scale intensity in measuring of protein concentrations is lower than those of the devices by other methods. Wax printing offers the simplest and fastest fabrication, although solution leakage measures should be taken to improve the wicking rate and to prevent cross-contamination. Screen printing also offers easy fabrication. The screenprinted μPAD has a good wicking performance and shows a high detection intensity. Craft cutting allows automated fabrication of many μPADs at once. The craft cut μPAD has the fastest wicking rate among the four μPADs due to bare cellulose fibers. We consider that the detection intensity of this μPAD can be raised by optimizing the evaporation rate.
  • Masatoshi Maeki, Yuka Fujishima, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    PLOS ONE 12 11 2017年11月 [査読有り][通常論文]
     
    Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 mu m and 31 mu m, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 mu m chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60-80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15-25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment.
  • Taiga Ajiri, Takao Yasui, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    SENSORS AND ACTUATORS B-CHEMICAL 250 39 - 43 2017年10月 [査読有り][通常論文]
     
    Single-molecule detection of the biomolecules in a label-free manner has a tremendous impact for various fields. Recently, we developed a label-free detection method without any pretreatment procedures, which is based on optical diffraction derived from a nanofluidic channel array (in other words, a nanowall array). However, the single-molecule detection is hampered by the inherent sensitivity of the method. We propose a solution to improve the sensitivity of the method by adjusting the height of the nanowall array. Numerical simulations showed that a larger nanowall array height could provide better sensitivity, but a lower nanowall array height could provide better sensitivity difference, contrary to what we would intuitively expect. We used a 250 nm height nanowall array to achieve a label-free detection of 0.18 DNA molecules to verify the simulation prediction. These results demonstrate our method has a potential to be implemented in a highly sensitive refractometer with small sample consumption. (C) 2017 Elsevier B.V. All rights reserved.
  • Taiga Ajiri, Haruya Kasa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Junji Nishi, Manabu Tokeshi
    ANALYTICAL SCIENCES 33 10 1197 - 1199 2017年10月 [査読有り][通常論文]
     
    Recently, we developed a label-free detection method based on optical diffraction, and implemented it in on our fabricated micro- and nanofluidic device. This detection method is simple and useful for detecting biomolecules, but the device fabrication consists of complicated processes. In this paper, we propose a simple method for fabricating the micro- and nanofluidic device; the fabrication combines laser interference lithography with conventional photolithography. The performance of a device fabricated by the proposed method is comparable to the performance of the device in our previous study.
  • Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES 32 12 1359 - 1362 2016年12月 [査読有り][通常論文]
     
    We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an important marker for chronic kidney disease in cats, using immuno-pillar chips. The required amount of reagent solution is 200 times smaller than that for the conventional ELISA in the 96-well microplate (0.5 mu L versus 100 mu L). In addition, the total assay time in the proposed method is more than 12 times shorter than in the conventional method (20 min versus 240 min). The limit of detection in the new method of 3 ng mL(-1) is comparable to that of the conventional method (1 ng mL(-1)) and it is in the clinically relevant range.
  • Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    SENSORS AND ACTUATORS B-CHEMICAL 236 433 - 441 2016年11月 [査読有り][通常論文]
     
    This article describes the development of a simple, portable assay system using microfluidic paper-based analytical devices (mu PADs) coupled with colorimetric detection for rapid measurements. The properties of different paper substrates were first investigated to determine which type of paper would be the most suitable for the fabrication of the mu PADs. Simultaneous detection of horseradish peroxidase (HRP) utilizing a 5 mu L sample analytical volume was demonstrated using a single mu PAD. Hydrophilic test regions were separated by hydrophobic barriers, which were fabricated through photolithography. These test regions were immobilized with 10 mM of 3,3',5,5'-tetramethylbenzidine for HRP assay. The detection range obtained with the proposed system covered HRP concentrations from 0.37 to 124 fmol (or 31000 ng mL(-1)). The detection limit (blank + 3 sigma) for HRP was calculated to be 0.69 fmol (or 5.58 ng mL(-1)) through a 4-parameter logistic nonlinear regression using results obtained within a 15 min assay time. The findings obtained using the developed system suggest that mu PAD assay systems for simple but highly sensitive measurements can be designed to give on-site determinations of target compounds using peroxidase-conjugated molecules. ((c)) 2016 Elsevier B.V. All rights reserved.
  • Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 408 27 7559 - 7563 2016年11月 [査読有り][通常論文]
     
    A novel washing technique for microfluidic paper-based analytical devices (mu PADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow mu PADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model mu PAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 mu g mL(-1).
  • Lori Shayne Alamo Busa, Takeshi Komatsu, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES 32 8 815 - 818 2016年08月 [査読有り][通常論文]
     
    We report on the colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide using horseradish peroxidase on photolithography-fabricated (P-PAD) and wax-printed (W-PAD) paper-based analytical devices. Fabricating PADs via photolithography exposes the hydrophilic areas to polymers (photoresists) and solvents, not only reducing the hydrophilicity, but also affecting the TMB-H2O2 assay system with an unavoidable incomplete elimination of photoresist during fabrication. Detection signals are then observed in the presence of photoresist residues on the P-PAD, even at a blank HRP concentration.
  • Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    MICROMACHINES 7 5 2016年05月 [査読有り][通常論文]
     
    Food and water contamination cause safety and health concerns to both animals and humans. Conventional methods for monitoring food and water contamination are often laborious and require highly skilled technicians to perform the measurements, making the quest for developing simpler and cost-effective techniques for rapid monitoring incessant. Since the pioneering works of Whitesides' group from 2007, interest has been strong in the development and application of microfluidic paper-based analytical devices (PADs) for food and water analysis, which allow easy, rapid and cost-effective point-of-need screening of the targets. This paper reviews recently reported PADs that incorporate different detection methods such as colorimetric, electrochemical, fluorescence, chemiluminescence, and electrochemiluminescence techniques for food and water analysis.
  • Masatoshi Maeki, Shohei Yamazaki, Ashtamurthy S. Pawate, Akihiko Ishida, Hirofumi Tani, Kenichi Yamashita, Masakazu Sugishima, Keiichi Watanabe, Manabu Tokeshi, Paul J. A. Kenis, Masaya Miyazaki
    CRYSTENGCOMM 18 40 7722 - 7727 2016年 [査読有り][通常論文]
     
    Protein crystallization and subsequent X-ray diffraction analysis of the three-dimensional structure are necessary for elucidation of the biological functions of proteins and effective rational drug design. Therefore, controlling protein crystallization is important to obtain high resolution X-ray diffraction data. Here, a simple microfluidic method using chips with 10 and 50 mu m high crystallization chambers to selectively form suitable single protein crystals for X-ray analysis is demonstrated. As proof of concept, three different types of proteins: lysozyme, glucokinase from Pseudoalteromonas sp. AS-131 (PsGK), and NADPH-cytochrome P450 oxidoreductase-heme oxygenase complex were crystallized. We demonstrate that the crystal growth orientation depends on the height of the crystallization chamber regardless of the protein type. Our results suggest that the confined micro space induces the protein molecules to adhere to a specific crystal face and affects the growth kinetics of each crystal face. The present microfluidic-based protein crystallization method can reform a suitable single protein crystal for X-ray analysis from aggregates of needle-shaped protein crystals.
  • Takeshi Komatsu, Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST 141 24 6507 - 6509 2016年 [査読有り][通常論文]
     
    The combination of a microfluidic paper-based analytical device (mu PAD) and digital image analysis is widely used for quantitative analysis with mu PADs because of its easy and simple operation. Herein, we have demonstrated a quantitative analysis based on multiple color changes on a mu PAD. The CIE L*a*b* color system was employed to analyse the digital images obtained with the mu PAD. We made pH measurements using a universal pH-indicator showing multiple color changes for various pH values of aqueous test solutions. The detectable pH range of this method was wider than the typical grayscale-based image analysis, and we succeeded in the measurements for a wide pH range of 2-9.
  • Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST 141 24 6598 - 6603 2016年 [査読有り][通常論文]
     
    The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (mu PADs) is reported. The mu PADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3', 5,5'-tetra-methylbenzidine (TMB) was deposited at the control and test zones. mu PAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the mu PAD detection of biotin as a model compound with a detection limit of 0.10 mu g mL(-1). The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B-1, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL(-1). The mu PAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.
  • Akihiko Ishida, Mitsutaka Fujii, Takehiro Fujimoto, Shunsuke Sasaki, Ichiro Yanagisawa, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES 31 11 1163 - 1169 2015年11月 [査読有り][通常論文]
     
    A compact and lightweight liquid chromatography system is presented with overall dimensions of 26 cm width x 18 cm length x 21 cm height and weight of 2 kg. This system comprises a battery-operated compact electroosmotic pump, a manual injector, a microfluidic chip device containing a packed column and an electrochemical detector, and a USB bus-powered potentiostat. The pumping system was designed for microfluidic-based reversed-phase liquid chromatography in which an electroosmotically generated water stream pushes the mobile phase via a diaphragm for the output. The flow rate ranged from 0 to 10 mu L/min and had a high degree of precision. The pumping system operated continuously for over 24 h with dry batteries. The column formed in the microfluidic device was packed with 3-mu m ODS particles with a length of 30 mm and a diameter of 0.8 mm. The results presented herein demonstrate the performance of the pumping system and the column using alkylphenols, catecholamine, catechin, and amino acids.
  • Osamu Wakao, Yusaku Fujii, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    ANALYTICAL CHEMISTRY 87 19 9647 - 9652 2015年10月 [査読有り][通常論文]
     
    The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.
  • 西脇奈菜子, 笠間敏博, 石田晃彦, 谷博文, 馬場嘉信, 渡慶次学
    分析化学 64 5 329 - 335 2015年05月 [査読有り][通常論文]
     
    In order to realize ultra-early diagnosis of disease in practical applications, we have fabricated next-generation immuno-pillar devices with higher sensitivity. In the newly developed devices, capture antibodies were immobilized on affinity beads based on chemical bonding, while in the previous-generation ones, polystyrene beads were used for physical adsorption-based immobilization. To evaluate the sensitivity of the next-generation immuno-pillar device, we quantitatively analyzed C-reactive protein (CRP). The limit of detection was estimated to be 0.1 ng mL(-1) (total assay time, 23 mm), which was twoorders of magnitude lower than that obtained by using the previous-generation immuno-pillar device, and was low enough to perfoiin the CRP test. In addition, we investigated the storage stability of the immuno-pillar device, and confirmed that the device can retain its performance for over 9 months.
  • Saeed Mohammadi, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST 140 19 6493 - 6499 2015年 [査読有り][通常論文]
     
    This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 mu m, as obtained by this method. Fabricated microfluidic paper-based analytical devices (mu PADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 mu M, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for mu PADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.
  • Masatoshi Maeki, Tatsuyoshi Saito, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    RSC ADVANCES 5 57 46181 - 46185 2015年 [査読有り][通常論文]
     
    Formation behavior of lipid nanoparticles (LNPs) in microfluidic devices with a staggered herringbone micromixer (SHM) structure was investigated. The fundamental role for SHMs in LNP formation was demonstrated by determining such factors as the limiting SHM cycle numbers and the effect of flow rate. The SHM cycle numbers and the position of the first SHM were as significant as factors as the flow rate condition for producing the small-size LNPs.
  • Ryoko Kurishiba, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    LUMINESCENCE 29 78 - 78 2014年08月 [査読有り][通常論文]
  • Yusuke Nakatani, Chiaki Shido, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    LUMINESCENCE 29 83 - 84 2014年08月 [査読有り][通常論文]
  • Hirofumi Tani, Ai Masuyama, Akihiko Ishida, Manabu Tokeshi
    LUMINESCENCE 29 99 - 100 2014年08月 [査読有り][通常論文]
  • Tamio Kamidate, Masumi Maruya, Hirofumi Tani, Akihiko Ishida
    ANALYTICAL SCIENCES 25 9 1163 - 1166 2009年09月 [査読有り][通常論文]
     
    4-Iodophenol was applied to an enhancer in the direct detection of horseradish peroxidase (HRP) encapsulated in liposomes by using luminol chemiluminescence (CL). Luminol, 4-iodophenol and hydrogen peroxide permeate into the inner phase of liposomes containing HRP, resulting in the progress of 4-iodophenol-enhanced luminol CL catalyzed by HRP in liposomes. The CL intensity observed in liposomes was a factor of 150 greater than that observed in a lipid-free bulk solution. The detection limit in the direct detection of HR-P encapsulated in liposomes was sensitive by a factor of 30 compared with that in a lipid-free bulk solution. 4-Iodophenol effectively functioned as an enhancer in HRP-catalyzed luminol CL in liposomes.
  • Akihiko Ishida, Masamichi Natsume, Tamio Kamidate
    JOURNAL OF CHROMATOGRAPHY A 1213 2 209 - 217 2008年12月 [査読有り][通常論文]
     
    A microchip pressure-driven liquid chromatography (LC) with a packed column and an electrochemical flow cell has been developed by using polystyrene (PS) and poly(dimethylsiloxane) (PDMS). The cylindrical separation column with packed octadecyl silica particles was fabricated in the PS substrate. The three electrode system (working, reference, and counter electrode) for amperometric detection was fabricated onto the PS substrate, using the Au deposition, photolithography, and chemical etching. The detector flow cell was formed by sealing the electrode system with a PDMS chip containing a channel. In this flow cell, the effect of working electrode width (in the direction of flow) on chromatographic parameters, such as peak width and peak resolution were studied in electrode width ranging 50-5000 mu m. The effect of electrode width on sensitivity (current intensity. current density, and S/N ratio) was also examined. The sensitivity was discussed by simulating the concentration profile generated around the working electrode. The effects of the column packing size and the column size on the separation efficiency were examined. In this study, a good separation of three catechins was successfully achieved and the detection limits for (+)-catechin, epicarechin, and epigallocatechin gallate were 350, 450, and 160 nM, respectively. (c) 2008 Elsevier B.V. All rights reserved.
  • Akihiko Ishida, Yasuko Yamada, Tamio Kamidate
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY 392 5 987 - 994 2008年11月 [査読有り][通常論文]
     
    In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.
  • Tamio Kamidate, Kanako Komatsu, Hirofumi Tant, Akihiko Ishida
    ANALYTICAL SCIENCES 24 4 477 - 481 2008年04月 [査読有り][通常論文]
     
    Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H2O2 without lysis of liposomes. At a low concentration of H2O2, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H2O2. At a high concentration of H2O2, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H2O2 by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution.
  • Tamio Kamidate, Kanako Komatsu, Hirofumi Tani, Akihiko Ishida
    LUMINESCENCE 22 3 236 - 240 2007年05月 [査読有り][通常論文]
     
    The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H2O2 were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H2O2 permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H2O2. The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes. Copyright (C) 2007 John Wiley & Sons, Ltd.
  • Tamio Kamidate, Hirofumi Tani, Akihiko Ishida, Masaya Hayashi
    LUMINESCENCE 22 1 15 - 19 2007年01月 [査読有り][通常論文]
     
    Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (Phi(BL)) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in Phi(BL) was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and Phi(BL) to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and Phi(BL) in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of Phi(BL) Copyright (c) 2006 John Wiley & Sons, Ltd.
  • Akihiko Ishida, Takahiro Yoshikawa, Masamichi Natsume, Tamio Kamidate
    JOURNAL OF CHROMATOGRAPHY A 1132 1-2 90 - 98 2006年11月 [査読有り][通常論文]
     
    In micro total analysis systems, liquid chromatography (LC) works under pressure-driven flow is the essential analysis component. There were not, however, much works on microchip LC. Here we developed a microchip for reversed-phase LC using porous monolithic silica. The chip consisted of a double T-shaped injector and a similar to 40-cm serpentine separation channel. The octadecyl-modified monolithic silica was prepared in the specified part of the channel on the microchip using sol-gel process. Furthermore, the effect of geometry of turn sections on band dispersion at turns was examined under pressure-driven flow. High separation efficiencies of 15,000-18,000 plates/m for catechins were obtained using the LC chip. (c) 2006 Elsevier B.V. All rights reserved.
  • T Kamidate, K Yanashita, H Tani, A Ishida, M Notani
    ANALYTICAL CHEMISTRY 78 1 337 - 342 2006年01月 [査読有り][通常論文]
     
    Liposomes containing phosphatidylcholine (PC) and cholesterol (Chol) were applied to the enhancer for firefly bioluminescence (BL) assay for ATP in the presence of cationic surfactants using as an extractant for the release of ATP from living cells. Benzalkonium chloride (BAC) was used as an AT? extractant. However, BAC seriously inhibited the activity of luciferase, thus resulting in the remarkable decrease in the sensitivity of the BL assay for ATP. On the other hand, we found that BAC was associated with liposomes to form cationic liposomes containing BAC. The association rate of BAC with liposomes was faster than that of BAC with luciferase. As a result, the inhibitory effect of BAC on luciferase was eliminated in the presence of liposomes. In addition, cationic liposomes thus formed enhanced BL emission. BL measurement conditions were optimized in terms of liposome charge type, liposome size, and total concentration of PC and Chol. ATP can be sensitively determined without dilution of analytical samples by using liposomes. The detection limit of ATP with and without liposomes was 100 amol and 25 fmol in aqueous ATP standard solutions containing 0.06% BAC, respectively. The method was applied to the determination of ATP in Escherichia coli extracts. The BL intensity was linear from 4 x 10(4) to 1 x 10(7) cells mL(-1) in the absence of liposomes. On the other hand, the BL intensity was linear from 4 x 10(3) to 4 x 10(6) Cells mL(-1) in the presence of liposomes. The detection limit of ATP in E. coli extracts was improved by a factor of 10 via use of liposomes.
  • A Ishida, C Otsuka, H Tani, T Kamidate
    ANALYTICAL BIOCHEMISTRY 342 2 338 - 340 2005年07月 [査読有り][通常論文]
  • T Kamidate, N Kikuchi, A Ishida, H Tani
    ANALYTICAL SCIENCES 21 6 701 - 704 2005年06月 [査読有り][通常論文]
     
    Homogentisic acid gamma-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in p-iodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes.
  • T Kamidate, Y Ishida, H Tani, A Ishida
    BUNSEKI KAGAKU 54 6 569 - 572 2005年06月 [査読有り][通常論文]
     
    Fluorescein (FL) and H2O2 rapidly permeated into the inner phase of liposome which trapped horseradish peroxidase (HRP), to initiate HRP-catalyzed FL chemiluminescence (CL) with H2O2. The CL intensity was dependent on the concentration of H2O2. The optimum conditions of charge-type, composition and diameter of liposome in the assay of H2O2 were determined by measuring the CL intensity, to be maximal under optimum conditions. Anionic liposome containing phosphatidylcholine, dimyristoyl-glycero-phosphocholine and cholesterol (Chol) was effective for enhancing the CL intensity and stability of liposome. The CL intensity decreased with an increase in the content of Chol in liposome. The optimal content of Chol was thus determined to be 10 mol%. The effect of the liposome size on the CL intensity was examined by preparing liposomes with a different diameter. The CL intensity increased with an increase in the diameter of liposome. The optimal diameter of liposome was thus determined to be 1000 nm. The logarithmic calibration curve of H2O2 was linear over the range from the detection limit of 4.0 X 16(-8) M up to 1.0 X 10(-5) M. When HRP trapped in liposome was used as a catalyst, the CL intensity was greater than that observed by using HRP dissolved in the bulk solution in the range of 4.0 X 10(-7) M Up to 1.0 X 10(-5) M of H2O2.
  • T Kamidate, N Kikuchi, A Ishida, H Tani
    ANALYTICAL SCIENCES 21 6 701 - 704 2005年06月 [査読無し][通常論文]
     
    Homogentisic acid gamma-lactone (HAL) chemiluminescence (CL) was applied to the determination of horseradish peroxidase (HRP) encapsulated in liposomes. HRP was detected after the lysis of HRP-trapped liposomes with Triton X-100. CL response rate, detection limit and linear range of calibration curve for HRP in HAL CL were compared with those in p-iodophenol (p-IP)-enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in p-IP enhanced luminol CL, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in p-IP-enhanced luminol CL. The linear range of calibration curve for HRP in HAL CL was improved by a factor of 50 compared with that in p-IP-enhanced luminol CL. From these results, it was found that HAL CL were superior to p-IP-enhanced luminol CL for the determination of HRP encapsulated in liposomes.
  • 上舘民夫, 石田良樹, 谷博文, 石田晃彦
    分析化学 54 6 569 - 572 2005年06月 [査読無し][通常論文]
     
    Fluorescein (FL) and H2O2 rapidly permeated into the inner phase of liposome which trapped horseradish peroxidase (HRP), to initiate HRP-catalyzed FL chemiluminescence (CL) with H2O2. The CL intensity was dependent on the concentration of H2O2. The optimum conditions of charge-type, composition and diameter of liposome in the assay of H2O2 were determined by measuring the CL intensity, to be maximal under optimum conditions. Anionic liposome containing phosphatidylcholine, dimyristoyl-glycero-phosphocholine and cholesterol (Chol) was effective for enhancing the CL intensity and stability of liposome. The CL intensity decreased with an increase in the content of Chol in liposome. The optimal content of Chol was thus determined to be 10 mol%. The effect of the liposome size on the CL intensity was examined by preparing liposomes with a different diameter. The CL intensity increased with an increase in the diameter of liposome. The optimal diameter of liposome was thus determined to be 1000 nm. The logarithmic calibration curve of H2O2 was linear over the range from the detection limit of 4.0 × 10-8 M up to 1.0 × 10-5 M. When HRP trapped in liposome was used as a catalyst, the CL intensity was greater than that observed by using HRP dissolved in the bulk solution in the range of 4.0 × 10-7 M up to 1.0 × 10-5 M of H2O2. © 2005 The Japan Society for Analytical Chemistry.
  • N Nakata, A Ishida, H Tani, T Kamidate
    ANALYTICAL SCIENCES 19 8 1183 - 1185 2003年08月 [査読有り][通常論文]
     
    Cationic liposomes composed of two components, diethylaminoethyl-carbamoyl cholesterol and phosphatidylcholine, were applied to an enhancer for a firefly bioluminescent (BL) assay of bacterial ATP in the presence of an ATP extractant. Trichloroacetic acid (TCA), which inhibits the activity of luciferase, was used as an ATP extractant. Cationic liposomes enhanced the BL intensity as long as luciferase was active. The detection limits for cell numbers of Escherichia coli extracts in the presence of cationic liposomes and in water alone were 199 and 897 colony forming units ml(-1), respectively. The sensitivity for bacterial ATP in the presence of cationic liposomes was improved by a factor of 2.5 times compared to that in the presence of diethylaminoethyl-dextran.
  • A Ishida, T Yoshikawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY 316 1 127 - 130 2003年05月 [査読有り][通常論文]
     
    The firefly bioluminescence (BL)(1) technique for measuring biomass, cell status, and activity of adenosine 5'-triphosphate (ATP)-related enzymes and reporter protein has been widely used because of its rapidity, high sensitivity, and robustness [1-9]. This technique is based on the reaction including the oxidative decarboxylation of luciferin by firefly luciferase in the presence of ATP and Mg2+, resulting in the production of light. Recently this BL assay has received considerable attention in the food industry because this method can be used as a rapid monitoring system for the safety of food products and hygiene of food contact surfaces at critical control points of food processing [10-14]. ATP is present in all living cells and the amount of ATP per cell is fairly constant. The presence of ATP in the food products and manufacturing environment suggests contamination of microbes and food residues. The firefly BL method is subject to lowering of sensitivity because of the inhibition of luciferase by ATP extractants and various salts. The ATP extractants are essential compounds for release of ATP from living cells, such as dilute acids, surfactants, boiling buffers, and organic solvents [15,16]. Among these extractants, trichloroacetic acid (TCA), which is suitable for a variety of types of cells, is the most widely used. This extractant, however, is a rather potent inhibitor of luciferase [2,17,18]. In addition to the ATP extractant, several anions such as perchlorate, nitrate, and halide ions inhibit the enzyme. Chloride ion is contained as a. form of sodium chloride in a variety of foods. Although bacteria can barely survive at a high concentration of NaCl, the presence of bacteria is often detected in foods containing NaCl. Therefore, there should be a considerable loss in sensitivity of the BL assay for monitoring bacterial contamination of those food products because of the inhibition by NaCl and TCA. Previously, we have found that diethylaminoethyldextran (DEAE-Dx) enhanced the light emission from the BL reaction [19] and have successively applied it to a highly sensitive BL assay of ATP in the presence of TCA or Triton X-100 [20]. On the other hand, the effect of a variety of ions on the BL reaction was studied and the possible mechanisms of the inhibition by several inorganic anions were presented [21-23]. Furthermore the inhibitory effects of inorganic ions in real samples on the BL assay were investigated [24-26]. However, there was no study on improving the sensitivity of the BL assay in the presence of those inhibitors. In this report, we describe the highly sensitive BL assay under severe conditions, in which, in addition to TCA, chloride ion inhibits luciferase, based on the enhancement effect of DEAE-Dx on the BL emission.
  • A Ishida, T Yoshikawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY 316 1 127 - 130 2003年05月 [査読無し][通常論文]
     
    The firefly bioluminescence (BL)(1) technique for measuring biomass, cell status, and activity of adenosine 5'-triphosphate (ATP)-related enzymes and reporter protein has been widely used because of its rapidity, high sensitivity, and robustness [1-9]. This technique is based on the reaction including the oxidative decarboxylation of luciferin by firefly luciferase in the presence of ATP and Mg2+, resulting in the production of light. Recently this BL assay has received considerable attention in the food industry because this method can be used as a rapid monitoring system for the safety of food products and hygiene of food contact surfaces at critical control points of food processing [10-14]. ATP is present in all living cells and the amount of ATP per cell is fairly constant. The presence of ATP in the food products and manufacturing environment suggests contamination of microbes and food residues. The firefly BL method is subject to lowering of sensitivity because of the inhibition of luciferase by ATP extractants and various salts. The ATP extractants are essential compounds for release of ATP from living cells, such as dilute acids, surfactants, boiling buffers, and organic solvents [15,16]. Among these extractants, trichloroacetic acid (TCA), which is suitable for a variety of types of cells, is the most widely used. This extractant, however, is a rather potent inhibitor of luciferase [2,17,18]. In addition to the ATP extractant, several anions such as perchlorate, nitrate, and halide ions inhibit the enzyme. Chloride ion is contained as a. form of sodium chloride in a variety of foods. Although bacteria can barely survive at a high concentration of NaCl, the presence of bacteria is often detected in foods containing NaCl. Therefore, there should be a considerable loss in sensitivity of the BL assay for monitoring bacterial contamination of those food products because of the inhibition by NaCl and TCA. Previously, we have found that diethylaminoethyldextran (DEAE-Dx) enhanced the light emission from the BL reaction [19] and have successively applied it to a highly sensitive BL assay of ATP in the presence of TCA or Triton X-100 [20]. On the other hand, the effect of a variety of ions on the BL reaction was studied and the possible mechanisms of the inhibition by several inorganic anions were presented [21-23]. Furthermore the inhibitory effects of inorganic ions in real samples on the BL assay were investigated [24-26]. However, there was no study on improving the sensitivity of the BL assay in the presence of those inhibitors. In this report, we describe the highly sensitive BL assay under severe conditions, in which, in addition to TCA, chloride ion inhibits luciferase, based on the enhancement effect of DEAE-Dx on the BL emission.
  • T Kamidate, Y Ishida, H Tani, A Ishida
    CHEMISTRY LETTERS 32 4 402 - 403 2003年04月 [査読無し][通常論文]
     
    A method for direct detection of horseradish peroxidase (HRP) as a marker molecule trapped in liposomes by the use of HRP-catalyzed fluorescein chemiluminescence (CL) with hydrogen peroxide has been developed. Maximum CL emission in the direct detection of HRP in liposomes increased by a factor of 13 times compared with that in the detection of HRP dissolved in lipid-free buffer solution.
  • Akihiko Ishida, Manabu Ikemoto, Yoshiki Ishida, Tamio Kamidate
    Bulletin of the Chemical Society of Japan 76 5 985 - 989 2003年 [査読有り][通常論文]
     
    The preconcentrations of epinephrine (EP) and norepinephrine (NE) into phosphatidylcholine liposomes with imposed pH gradient across the membrane were investigated by adding the catecholamine (CA) to the external medium of liposomes. To determine the CAs entrapped into liposomes, a simple method was also developed, based on the adsorption of CAs onto alumina and chemiluminescence detection. The uptake of the CAs into the liposomes was examined over a wide ΔpH range of 0-5 units (internal pH 5.0). The maximum uptakes of EP and NE were 70% and 88%, respectively, around an external pH of 9 (ΔpH4), where zwitterionic species of EP and NE are dominant. The final concentrations of EP and NE in internal volumes were 34- and 43-fold greater than the initial concentrations of EP and NE, respectively. The uptake of the CAs was studied by using a mathematical model, which indicates that the uptake is contributed by the molar fraction of the zwitterion species in the external volume as well as the pH gradient. The uptake of EP was found to suffer from a steric hindrance due to a methyl group substituted for the hydrogen of the amino group. This study proves the capability of the liposomes for accumulation media of CAs.
  • Kamidate, T., Ishida, Y., Tani, H., and Ishida, A.:"Direct Detection of Horseradish Peroxidase as a Marker Molecule Encapsulated in Liposomes via Use of Fluorescein Chemiluminescence", Chemistry Letters,32:402-403 (2003)*
    2003年 [査読無し][通常論文]
  • Ishida, A., Ikemoto, M., Ishida, Y., Kamidate, T.: "Preconcentration of Catecholamines into Liposomes with Imposed pH Gradients", Bulletin of the Chemical Society of Japan, 76:985-989 (2003).*
    2003年 [査読無し][通常論文]
  • A Ishida, T Yoshikawa, T Nakazawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY 305 2 236 - 241 2002年06月 [査読有り][通常論文]
     
    A highly sensitive ATP biolumineseence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli. (C) 2002 Elsevier Science (USA).
  • T Kamidate, Y Hashimoto, H Tani, A Ishida
    ANALYTICAL SCIENCES 18 3 273 - 276 2002年03月 [査読有り][通常論文]
     
    The uptake Of Cull was investigated using various types of liposomes composed of phosphatidylcholine (PC), cholesterol (Chol) and dicethylphosphate (DCP). DCP played a role as a ligand for Cu2+. Multilamellar vesicles (MLVs) were more effective for the uptake of Cull compared to unilamellar vesicles prepared by the extrusion technique. The uptake efficiency of MLVs for Cull was dependent on the molar ratio of DCP in MLVs. The uptake percent of Cull was 92% using MLVs having a PC:DCP:Chol molar ratio of 4:33; 95% of the total vesicle Cull was bound to DCP of the outer membrane surface of the MLVs, and the remaining 5% of the total Cull was distributed into the interior side of the MLVs. MLVs having a PC:DCP:Chol molar ratio of 4:3:3 were also effective as separation media for Mn2+ CO2+, Ni2+ and Zn2+. The uptake efficiency of the MLVs for the transition-metal ions increased in the order Co2+ < Zn2+ < Ni2+ < Mn2+ < Cu2+.
  • Akihiko Ishida
    Bunseki Kagaku 49 1 71 - 72 2000年 [査読有り][通常論文]
     
    The importance of simple analytical methods that require no sophisticated instruments is increasing. For simple measurements, visual methods based on the perception of color change and the recognition of figure are more suitable. The author has developed simple methods for the visual determination of trace metal ions, based on construction of the following chemical systems. First, for the visual sensitive detection of trace aluminium, a simple concentration method was developed. After a drop of solution are evaporated on a hydrophobic substrate, components, such as several pH buffers or poly(vinyl alcohol), form a ring-like solid phase. Aluminium ion was concentrated into the ring as a fluorescent chelate of 2,2′-dihydroxyazobenzene and distinctly detected. This method has been successively applied to the determination of ppb levels of aluminium in tealeaves. Second, a color-forming reaction system has been developed, which makes it possible to visualize the concentration of metal ions by using a color property of the Xylenol Orange (XO)-metal complex system. The Xylenol Orange-Fe(III) system gives yellow, red, and purple blue along with an increase in the iron concentration, owing to a sequential formation of FeIII(xo) and FeIII2(xo). A field test for iron leached from rock samples was developed by using the color reaction, and was applied to the prediction of water quality in aquifers.
  • A Ishida, E Kaneko, T Yotsuyanagi
    CHEMISTRY LETTERS 28 4 351 - 352 1999年04月 [査読有り][通常論文]
     
    A field test for iron leached from rocks has been developed by using a purple blue iron(III)-Xylenol Orange (XO) complex formed at the iron(III) concentrations above the molar concentration of XO. The test is performed in an aqueous drop on a Tefron(R) plate. When the iron content exceeds a criterion, a distinct color change from yellow to purple blue appears in the drop containing a 10-mg rock sample, HCl, XO, and (NH4)(2)S2O8. This method was applied to the prediction of water quality in aquifers.

MISC

  • 小松雄士, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学 日本化学会春季年会講演予稿集(CD-ROM) 100th 2020年
  • 前田陵我, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 42nd (Web) 2020年
  • 清水一樹, 真栄城正寿, 真栄城正寿, 石田晃彦, 谷博文, 西井準治, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 42nd (Web) 2020年
  • 真栄城正寿, 真栄城正寿, 岡田悠斗, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 日本DDS学会学術集会プログラム予稿集 36th 2020年
  • 岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 42nd (Web) 2020年
  • 竹田怜央, 真栄城正寿, 真栄城正寿, 伊藤翔, 伊藤翔, 上野剛, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 42nd (Web) 2020年
  • UNDERSTANDING THE LIPID NANOPARTICLES STRUCTURE DYNAMICS USING A TIME-RESOLVED SAXS MEASUREMENT
    Masatoshi Maeki, Niko Kimura, Kazuki Shimizu, Kento Yonezawa, Nobutaka Shimizu, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi Proc. Micro TAS 2019 1478 -1479 2019年10月 [査読有り][通常論文]
  • PORTABLE FLUORESCENCE POLARIZATION ANALYZER FOR ON-SITE MULTISAMPLE IMMUNOASSAY
    Ayano Nakamura, Osamu Wakao, Ken Satou, Mitsutoshi Aoyagi, Kazuhiko Nishimura, Chikaaki Mizokuchi, Ken Sumiyoshi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi Proc. Micro TAS 2019 1393 -1394 2019年10月 [査読有り][通常論文]
  • DEVELOPMENT OF A THREE-DIMENSIONAL MICROMIXER DEVICE FOR PRODUCTION OF VARIOUS LIPID-BASED NUCLEIC ACID NANOCARRIERS
    Niko Kimura, Masatoshi Maeki, Yusuke Sato, Kosuke Sasaki, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi Proc. Micro TAS 2019 368 -369 2019年10月 [査読有り][通常論文]
  • HIGH-THROUGHPUT X-RAY CRYSTALLOGRAPHY BASED ON THE PROTEIN CRYASTAL ARRAY
    Reo Takeda, Masatoshi Maeki, Sho Ito, Go Ueno, Kunio Hirata, Akihiko Ishida, Hirofumi Tani, Masaki Yamamoto, Manabu Tokeshi Proc. Micro TAS 2019 191 -192 2019年10月 [査読有り][通常論文]
  • FLUORESCENCE SIGNAL AMPLIFICATION FOR SENSITIVE ENZYME IMMUNOASSAY UTILIZING AN IMMUNO-WALL
    Keine Nishiyama, Toshihiro Kasama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi Proc. Micro TAS 2019 689 -690 2019年10月 [査読有り][通常論文]
  • 真栄城正寿, 真栄城正寿, 木村笑, 清水一樹, 石田晃彦, 谷博文, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 40th 2019年
  • 西山慶音, 武田洋平, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 小川晴子, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 40th 2019年
  • 竹田怜央, 真栄城正寿, 真栄城正寿, 伊藤翔, 伊藤翔, 上野剛, 平田邦夫, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 39th 2019年
  • 木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 39th 2019年
  • 真栄城正寿, 木村笑, 石田晃彦, 谷博文, 渡慶次学 日本分析化学会年会講演要旨集(Web) 68th 2019年
  • 石田晃彦, 西村卓馬, 真栄城正寿, 谷博文, 渡慶次学 日本分析化学会年会講演要旨集(Web) 68th 2019年
  • MICROSCOPIC REAL-TIME MEASUREMENT OF PROTEIN CRYSTAL GROWTH IN MICROFLUIDIC DEVICES
    Masatoshi Maeki, Shohei Yamazaki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi Proc. Micro TAS 2018 2192 -2193 2018年11月 [査読有り][通常論文]
  • Microfluidic stepwise and rapid ethanol dilution for precise size control of lipid nanoparticles
    Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima Proc. Micro TAS 2018 1404 -1405 2018年11月 [査読有り][通常論文]
  • ナノピラーデバイスを用いたDNAのサイズ分離と無標識検出
    阿尻 大雅, 安井 隆雄, 笠 晴也, 真栄城 正寿, 石田 晃彦, 谷 博文, 西井 準治, 馬場 嘉信, 渡慶次 学 日本分析化学会講演要旨集 67年会 125 -125 2018年08月 [査読無し][通常論文]
  • 木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 渡慶次学, 渡慶次学, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 37th 2018年
  • A Small-Sized Lipid Nanoparticles Production Method Using Microfluidic Devices with Baffle Structures
    N. Kimura, M. Maeki, Y. Sato, A. Ishida, H. Tani, H. Harashima, M. Tokeshi Proc. Micro TAS 2017 965 -966 2017年10月 [査読無し][通常論文]
  • High-Throughput Fluorescence Polarization Measurement System Towards Molecular Interaction Analysis
    O. Wakao, M. Maeki, A. Ishida, H. Tani, A. Hibara, M. Tokeshi Proc. Micro TAS 2017 557 -558 2017年10月 [査読有り][通常論文]
  • A Microfluidic-Based Technique for Creating Amyloid Nanostructures and Its Application to Enzyme Reaction
    M. Maeki, S. Sato, A. Ishida, H. Tani, M. Tokeshi Proc. Micro TAS 2017 1279 -1280 2017年10月 [査読有り][通常論文]
  • 木村笑, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学 化学とマイクロ・ナノシステム学会研究会講演要旨集 35th 2017年
  • 真栄城正寿, 木村笑, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学 化学工学会秋季大会研究発表講演要旨集(CD-ROM) 49th 2017年
  • 木村笑, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学 化学とマイクロ・ナノシステム 16 (2) 2017年
  • Development of a New Technique for Washing Steps in Multistep Assays Using Microfluidic Paper-based Analytical Devices
    Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi Proceedings of MicroTAS 2016 970 -971 2016年10月 [査読有り][通常論文]
  • Fluorescence Polarization-based Multiple Samples Detection Using Microchamber Array Towards High-throughput Molecular Interaction Analysis
    Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi Proceedings of MicroTAS 2016 1400 -1401 2016年10月 [査読有り][通常論文]
  • Effect of the Grooved Structures and the Ethanol Concentration on the Small-sized Lipid Nanoparticles Formation
    Yuka Fujishima, Masatoshi Maeki, Yusuke Sato, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi Proceedings of MicroTAS 2016 1412 -1413 2016年10月 [査読有り][通常論文]
  • がん診断のためのエクソソーム分析法の開発
    阿尻 大雅, 安井 隆雄, 真栄城 正寿, 石田 晃彦, 谷 博文, 馬場 嘉信, 渡慶次 学 日本分析化学会講演要旨集 65年会 165 -165 2016年08月 [査読無し][通常論文]
  • Label-free detection of extracellular vesicles for cancer diagnosis
    Taiga Ajiri, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi Micro Total Analysis Systems 2015 1789-1791 2015年10月27日 [査読有り][通常論文]
  • Fluorescence Polarization Imaging for Multisample Immunoassay
    若尾摂, 真栄城正寿, 石田晃彦, 谷博文, 火原彰秀, 渡慶次学 Proceedings of MicroTAS 2015 1816 -1818 2015年10月 [査読有り][通常論文]
  • Microfluidic Approach for Production of Lipid Nanoparticles-Based Nano Medicine
    真栄城正寿, 斎藤竜亮, 佐藤悠介, 安井隆雄, 加地範匡, 石田晃彦, 谷博文, 馬場嘉信, 原島秀吉, 渡慶次学 Proceedings of MicroTAS 2015 838 -840 2015年10月 [査読有り][通常論文]
  • LCD-CCD Synchronization Detection for Fluorescece Polarization Immunoassay
    Osamu Wakao, Yusaku Fujii, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi Proceedings of the Micro TAS 2014 Symposium 2271 -2273 2014年10月 [査読有り][通常論文]
  • DEVELOPMENT OF 3RD GENERATION IMMUNO-PILLAR DEVICE FOR HIGH SENSITIVE DETECTION OF DISEASE MARKERS
    N. Nishiwaki, T. Kasama, A. Ishida, H. Tani, Y. Baba, M. Tokeshi Micro Total Analysis Systems 2013 1164-1166 2013年10月28日 [査読有り][通常論文]
  • Portable Liquid Chromatography System Based on Battery-Powered Electroosmotic Pump and Microchip with Packed Column and Electrochemical Detector
    Akihiko Ishida, Takehiro Fujimoto, Satoshi Yokokawa, Hirofumi Tani, Manabu Tokeshi, Ichiro Yanagisawa Proceedings of the MicroTAS 2012 Symposium 1183 -1185 2012年10月 [査読有り][通常論文]
  • On-Chip Bioluminescence Assay of ATP and Kinases Using Immobilized Firefly Luciferase in Three-Dimensional Microfluidic Chip
    Hirofumi Tani, Atsuki Morisaki, Akihiko Ishida, Manabu Tokeshi Proceedings of the MicroTAS 2012 Symposium 1618 -1620 2012年10月 [査読有り][通常論文]
  • 日本分析化学会北海道支部編「水の分析(第5版)」<共著>
    化学同人 2005年 [査読無し][通常論文]
  • 「リポソーム応用の新展開」~人工細胞の開発に向けて~<共著>
    エヌ・ティー・エス 2005年 [査読無し][通常論文]
  • A Ishida, E Kaneko, T Yotsuyanagi CHEMISTRY LETTERS (3) 217 -218 1999年03月 [査読無し][通常論文]
     
    By constructing a self-ordered nearly perfect ring system, a new type of visual test method has been developed. Poly(vinyl alcohol) forms a sharp ring-like solid phase (10.6 mm diameter) when a 100-mu l drop of its aqueous solution was evaporated on a poly(vinyl chloride) plate. Aluminium ion of 1-30 ppb has been determined visually as a fluorescent 1:1 chelate of 2,2'-dihydroxyazobenzene concentrated into the ring.

書籍等出版物

  • マイクロ流体分析
    渡慶次 学, 真栄城 正寿, 佐藤 記一, 佐藤 香枝, 火原 彰秀, 石田 晃彦 (担当:共著範囲:Chapter5 マイクロチップクロマトグラフィー)
    2020年10月
  • 山本, 重夫 
    シーエムシー出版 2016年08月 (ISBN: 9784781311166) x, 265p
  • 山本, 重夫 (担当:分担執筆範囲:第IV編第1章色で見分ける細菌汚染スクリーニング法~新しいキシレノールオレンジ-鉄錯体法の技術~)
    シーエムシー出版 2010年07月 (ISBN: 9784781302478) x, 265p
  • 日本分析化学会北海道支部, 厚谷, 郁夫, 田中, 俊逸 (担当:分担執筆範囲:4.7 フローインジェクション分析(FIA))
    化学同人 2005年06月 (ISBN: 9784759809916) x, 472p
  • 秋吉, 一成, 辻井, 薫, 奥, 直人, 久保井, 亮一, 宝谷, 紘一 (担当:分担執筆範囲:第4節リポソームを利用した高感度計測法)
    エヌ・ティー・エス 2005年06月 (ISBN: 4860430859) 4, 10, 683, 10, 12p, 図版14p

担当経験のある科目(授業)

  • 全学教育科目 英語演習 中級
  • 分析化学
  • 化学英語

所属学協会

  • 電気化学会   化学センサ研究会   日本分析化学会北海道支部   日本分析化学会   日本化学会   The Japan Society for Analytical Chemistry   The Chemical Society of Japan   American Chemical Society   

Works(作品等)

  • 北海道地域3大学新技術説明会にて成果発表
    2012年
  • 第24回国際計量計測展にて研究成果の発表と試作品の展示
    2010年
  • 第3回次世代医療システム産業化フォーラム2010にて技術発表
    2010年
  • 北海道大学・科学技術振興機構主催の北海道大学 新技術説明会にて技術発表
    2009年
  • 北海道大学工学系イノベーションブリッジ2007にて研究成果発表
    2007年

共同研究・競争的資金等の研究課題

  • 育種・染色体操作を用いたサケ科魚類の魚卵アレルゲン性低減化の試み
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 清水 裕, 笹岡 友季穂, 藤本 貴史, 平松 尚志, 石田 晃彦, 渡慶次 学, 佐伯 宏樹
  • ハイブリッド型電気化学マイクロセルの構築と微量生体試料分析への応用
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 石田 晃彦
     
    前年度に作製した同心円型アレイ電極を組み込んだウォールジェットセルの応答挙動を明らかにするため電流値と流量の関係を検討した。様々な流量でモデル物質の溶液をセルに導入して電流値を測定したところ,電流値と流量の両対数プロットの傾きから,電流値は流量の2/3乗に比例することが明らかとなった。これは,円盤型電極を組み込んだ従来のウォールジェットセル(3/4乗に比例)やチャンネルフローセル(1/3乗に比例)とは異なる挙動であった。このことは,本セルの応答挙動の特徴を示すものであるが,理論的考察については今後の課題とした。
    次に,このセルを小型HPLCシステムの検出器に応用した。小型HPLCシステムは,我々がこれまでに開発したもので,その分離部(小型カラム)および検出部を小型基板に組み込んで一つの小型モジュールとしているところに特徴がある。本研究では,セルをこのモジュールに組み込んだHPLCシステムを構築し,いくつかのモデル物質で良好に分離と検出ができることを確認した。 つづいて,上記のシステムを用いてドーパミン,エピネフリン,ノルエピネフリンの分離・検出を試みたところ,すべての成分はベースライン分離され,ドーパミンの検出限界は,0.8 nM (S/N = 3)であった。この検出限界は血中濃度と同等であることから,本研究で開発したセルは,特別な前濃縮を必要としない優れた感度をもつことを示している。
    本研究では,独自の着想に基づいて,同心円型アレイ電極からなるウォールジェット式電気化学セルを開発し,これが微量生体成分のHPLC分析に有用であることを実証した。生体成分には,電気化学的に検出できるものが多いことから,検出セルに関する本研究の成果が小型HPLCシステムと融合することで,近い将来,医療現場でのポイントオブケア検査に大いに貢献することが期待される。
  • 2018年度 現場での即時検査が可能な高性能可搬型分析装置の開発 補助事業
    JKA:研究補助開発研究
    研究期間 : 2018年04月 -2019年03月 
    代表者 : 石田 晃彦
  • ポータブル分析装置の高感度なマイクロ検出器の開発
    科学技術振興機構:研究成果展開事業(研究成果最適展開支援プログラム(A-STEP))フィージビリティスタディ(FS)ステージ 探索タイプ
    研究期間 : 2015年01月 -2015年12月 
    代表者 : 石田 晃彦
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2012年05月 -2015年03月 
    代表者 : 渡慶次 学, 湯澤 由紀夫, 秋山 真一, 谷 博文, 石田 晃彦
     
    糖尿病性腎症診断のための、パネル化診断チップの開発を行った。糖尿病性腎症のバイオマーカーのパネルとして、MCP-1、L-FABP、Angiotensinogen、CTGFおよびCollagen IVを選定した。標準試料および患者検体を用いて、開発したパネル化診断チップの性能評価を行ったところ、わずか12分の測定時間で、試料・試薬量0.5 μLで、従来のELISA法と同様な結果を得ることに成功した。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2011年 -2013年 
    代表者 : 石田 晃彦
     
    本研究では,複数の微細電極を適切な間隔で配列した単一くし形電極を考案し,電極の形状因子などを詳細に検討することで,より微量の物質を電気化学的に検出することが可能となった。本電極は,その形状の工夫によって1個の電極単独で高感度化を達成しているところに特色があり,多くの物質に適用でき,比較的安価な測定器を用いることができる点で有用である。今後,本電極は医療診断の分野で活用が見込まれているマイクロ流体分析デバイスの有力な検出器としての利用が期待される。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2006年 -2007年 
    代表者 : 石田 晃彦
     
    本研究では,微小流路内においてベシクル内から漏出させた電気化学活性物質をアンペロメトリーにより高感度に検出することが重要な課題である。電流値は電極面内に形成する測定物質の濃度勾配の積算値であるから,感度を上げるには作用電極の面積または濃度勾配を大きくすることが必要である。これまでに,電極面積を大きくすることは,S/N比を低下させるため有効ではないことを微小流体デバイスを用いて示した。これは,物質が電極反応により消費されながら流れる結果,電極の下流ほど電極垂直方向の濃度勾配が小さくなるためである。そこで,与えられた面積の中で濃度勾配を増加させることが必要になる。そのため,本研究では一本の電極を複数に分割して間隔を置いて配置するくし状の電極を検討した。電極がない部分で試料が補充されるので,下流側での濃度勾配の減少を抑えることができるからである。さらに十分な間隔があれば,分割したどの電極上でも同様の濃度勾配が形成されると推察される。くし形の電極については,これまでに2対の電極を用いて酸化還元を繰り返して感度を上げる検討は行われていたが,くし形電極単独での詳細な検討はなかった。そこで,ポリマー基板およびポリジメチルシロキサンを用いて,間隔をあけた2本の作用電極をもつ微小流体デバイスを作製し,このアプローチの妥当性を検証した。その際,微小流体デバイスで用いられる流速範囲で,物質の種類を変えることにより電流応答の拡散係数依存性を調べた。その結果,どの拡散係数およびどの流速でも間隔の増加とともに電流値は増加し,200μm近辺で最大一定となった。また,電流値を様々な拡散係数および流速で数値シミュレーションしたところ実験結果と一致した。以上から,このアプローチが妥当であり,微小流体デバイスで用いられる流速で様々な電気活性物質に適用できることを明らかにした。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2006年 -2007年 
    代表者 : 上舘 民夫, 谷 博文, 石田 晃彦
     
    1.ペルオキシダーゼ内封リポソームの調製法の検討 押出し法により、フォスファチジルコリン(PC)、フォスファチジルグリセロール(PG)及びコレステロール(Chol)からなるリポソームに西洋わさび由来のペルオキシダーゼ(HRP)を封入した。その結果、内封されたHRPの分子数は約1200であった。一方、リポソームに内封するHRPの分子数を増大するため、凍結融解法によるHRP内封リポソームの調製を試みた。その結果、約3600分子のHRPが内封でき、押出し法と比較して、内封するHRPの分子数を約3倍増大することに成功した。 2.リポソーム中のHRPの高感度検出法の開発 標準系およびリポソーム系について、最適濃度条件でHRPの定量下限を求めたところ、標準系では3.0x10^<-9>M、リポソーム系では1.0x10^<-9>Mとなり、リポソームの内水相をナノリアクターに用いると、より高感度にHRPの検出ができることが明らかになった。 3.HRP内封リポソームの抗体への結合と発光強度の比較 HRP内封リポソームを免疫測定法の標識体に応用するため、リポソームを抗体に結合することを試みた。その結果、HRP内封リポソーム標識化抗体当りの発光強度は、ビオチン-アビジン結合HRP標識体と比較して125倍増大することが明らかになった。
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2005年 -2006年 
    代表者 : 上舘 民夫, 谷 博文, 石田 晃彦
     
    1.エオシンYを用いる膜透過性の評価 リボソームの内水相にペルオキシダーゼ(POD)を封入し、その外水相にエオシンYと過酸化水素を添加すると、エオシンYと過酸化水素は迅速に膜を透過し、内水相においてPODを触媒とする化学発光反応が進行した。そこで、リン脂質であるフォスファチジルコリンに対して30〜45%のコレステロール含量を有するリボソームにPODを封入し、発光応答曲線を測定した。その結果、コレステロール含量が増大するほど発光初期速度は減少した。この結果から、エオシンYの発光初期速度が膜透過性を反映することがわかった。 2.ピレン法による膜流動性の評価 コレステロール含量が増大するほど発光初期速度は遅くなる原因として、コレステロール含量が増大するほどリボソームの膜流動性が減少することが考えられる。そこで、30〜45%のコレステロール含量を有するリボソームに蛍光プローブであるピレンを加え、膜流動性を評価した。その結果、コレステロール含量が増大するほど、膜流動性が減少した。したがって、エオシンYの発光初期速度を用いる方法が膜透過性の評価法として利用できることが明らかになった。 3.膜透過性の速度論的解析 リボソーム内へのエオシンYと過酸化水素の透過と化学発光反応を考慮して、発光初期速度を表す速度式を解析した。その結果、発光初期速度は膜透過速度定数、反応速度定数および基質初濃度で表された。また、反応速度定数および基質初濃度が一定のとき、発光初期速度は膜透過速度定数に比例することがわかった。エオシンYの膜透過速度定数を求めたところ、3.02x10^<-3> s^<-1>の値になった。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2004年 -2005年 
    代表者 : 石田 晃彦
     
    本課題の目的は,液体クロマトグラフィー(LC)の要素をガラスやポリマーチップ上に集積化したLCチップを作製し,これを生体分子の分離分析に適用してその有用性を実証することである。本年度は検出部である電極の面積が分離性能および感度(S/N比)に与える影響を検討した。その際,LCチップ間で生じる分離カラムの性能誤差が応答に影響しないよう同一の分離カラムで検討するために,分離カラムから出たバンドを分岐させ,それぞれ異なる電極で同時に検出するLCチップを作製した。電極は金をチップ上に蒸着することにより作製した。分岐部でバンドが等分されることについては蛍光顕微鏡法により確認した。このLCチップを用いて検討した結果,分離度は電極面積に依存しないことを確認した。これは分離カラム(2.5cm長,約10000段/m)から出た試料のバンド幅が電極幅よりも十分に大きいためである。バンド幅と理論段の関係を考慮すると,この結果は小さな分離カラムをもつLCチップ全般にあてはまるといえる。一方,S/N比に関しては電極面積が大きいほど減少した。この理由を電極表面上の試料の流体・拡散シミュレーションに基づいて考察した。すなわち,試料の電極反応が電極上流部で終了するため,電極面積が大きいほど下流部で面積過剰となり電極単位面積当たりに得られる電流値が減少するためと考察した。電極幅は50μm幅が最も有効であった。最後に,医学的な効果が報告されているカテキン類((+)-カテキン,エピカテキン,没食子酸エピガロカテキン)の分離を試みた。その結果,ベースライン分離を達成し,それぞれの検出限界は数百nMレベルであった。また,市販の各種茶飲料のカテキン分析に応用したところ,各成分の分離に加えて,茶葉の発酵の程度が各試料のクロマトグラムに反映していることが確認でき,実試料に対しても良好に適用できることを明らかにした。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2003年 -2004年 
    代表者 : 上舘 民夫, 谷 博文, 石田 晃彦
     
    リポソームの内水相をナノリアクターとして応用するため、西洋わさび由来のペルオキシターゼ(horseradish peroxidase:HRP)を内封したリポソームを調製した。リポソームはフォスファチジルコリン(PC)、フォスファチジルグリセロール(PG)およびコレステロール(Chol)の3成分からなり、押出し法により調製した。3成分の組成はPC:PG:Chol=8:1:1とした。また、HRP内封リポソームの平均粒子径は600nmとした。 つぎに、リポソーム内水相においてHRPを触媒とする化学発光(CL)反応を行った。CL試薬にはフルオレセイン(FL)、ルミノールおよびホモゲンチジン酸γ-ラクトン(HAL)を用いた。実験はHRP内封リポソームの外水相にCL試薬と過酸化水素を加え、それらの物質がリポソーム膜を透過し、リポソーム内でHRPの触媒作用により放出される発光を観測した。FL、ルミノールおよびHALとも、リポソーム膜を迅速に透過し、添加後、直ちにCL反応が進行した。しかしながら、用いるHRP内封リポソーム量が少なくなると、触媒であるHRP量が減少するため、HALを除いて発光速度は著しく減少した。 つぎに、HALを用いてHRP内封リポソームの使用量の影響をみた。発光量は使用量に依存し、調製したHRP内封リポソームを約8000倍希釈しても、HAL-CL反応はリポソーム内で迅速に進行した。HAL-CL反応系は従来使用されてきたルミノール系と比較して、測定時間および検量線の直線範囲において優れていた。 最後に、HRP内封リポソームをイムノアッセイの標識体に応用するため、測定容器に対する非特異的吸着について検討した。その結果、HRP未封入リポソームがマスキング剤として有用であった。以上の結果から、HRP内封リポソームをイムノアッセイの標識体に応用できることが明らかになった。
  • 日本学術振興会:科学研究費助成事業 萌芽研究
    研究期間 : 2003年 -2004年 
    代表者 : 坂入 正敏, 石田 晃彦
     
    本研究課題は、高輝度光(集光したパルスYAGレーザー照射)表面微細加工法と電気化学的手法により、酵素反応によって生体内で生成される過酸化水素などの、生体微量物質分析用微小電気化学セルをアルミニウム上に作製すること、さらに、対極などを取り付け微小電気化学分析システムにして、その性能を評価することを目的としている。 昨年度は、アルミニウムを用いてアノード酸化、レーザー加工、電気めっきを用いて、2電極型システムを作製でき、K_3Fe(CN)_6/K_4Fe(CN)_6の混合溶液(約1.5μl)を用いてCV測定を行ったところ、アノードピーク電流の走査速度依存性や濃度依存性を確認できたが、ピーク形状および液漏れの問題があった。そこで、本年度は、流路などの形状の改良および作製工程の改良を行い、より完成度の高い生体微量物質分析用微小電気化学セルの作製およびその電気化学的特性評価を行った。 微細溝および微細貫通孔には、レーザー照射後、再度アノード酸化することで、保護性の皮膜を形成し、電極部にはめっきにより金を析出させた。これら、再アノード酸化および金めっきにより、各種溶液で安定に動作可能な、理論内容量1.5μlの微小電気化学セルを作製できた。作製したセルを用いて、電気化学測定を行った結果、従来のマクロ電極と同様のサイクリックボルタモグラムが測定できた。すなわち、アノードピーク電流は電位走査速度の平方根に比例して増加し、その傾きから電極面積を見積もった結果、妥当な値を得ることができた。
  • 自己組織化システムを用いる高性能サイズ制御スポットテスト法
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 1998年 -1998年 
    代表者 : 石田 晃彦
  • マイクロチップを利用する化学分析システムの開発

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