石田 晃彦 (イシダ アキヒコ)

工学研究院 応用化学部門 分子機能化学分野助教
Last Updated :2024/12/06

■研究者基本情報

学位

  • 博士(工学), 東北大学
  • 工学修士, 東北大学

Researchmap個人ページ

研究者番号

  • 20312382

研究分野

  • ナノテク・材料, 分析化学

■経歴

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, 学協会
  • 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, 学協会
  • 2007年
    日本分析化学会, 分析化学年会実行委員, 学協会

■研究活動情報

論文

  • Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.
    Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi
    ACS applied materials & interfaces, 16, 2, 2110, 2119, 2024年01月17日, [国際誌]
    英語, 研究論文(学術雑誌), RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.
  • Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations
    Masatoshi Maeki, Niko Kimura, Yuto Okada, Kazuki Shimizu, Kana Shibata, Yusuke Miyazaki, Akihiko Ishida, Kento Yonezawa, Nobutaka Shimizu, Wataru Shinoda, Manabu Tokeshi
    Nanoscale Advances, Royal Society of Chemistry (RSC), 2024年
    研究論文(学術雑誌), We investigated ethanol-induced structural changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations.
  • A portable liquid chromatography system based on a separation/detection chip module consisting of a replaceable ultraviolet-visible absorbance or contactless conductivity detection unit
    Akihiko Ishida, Takuma Nishimura, Kaito Koyama, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    Journal of Chromatography A, 1706, 464272, 464272, Elsevier BV, 2023年09月, [査読有り], [招待有り], [筆頭著者, 責任著者]
    英語, 研究論文(学術雑誌)
  • Mass production system for RNA-loaded lipid nanoparticles using piling up microfluidic devices
    Masatoshi Maeki, Yuto Okada, Shuya Uno, Kaisei Sugiura, Yuichi Suzuki, Kento Okuda, Yusuke Sato, Masao Ando, Hiroyuki Yamazaki, Masaki Takeuchi, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    Applied Materials Today, 2023年04月, [査読有り]
    研究論文(学術雑誌)
  • Microfluidic paper-based analytical devices for cancer diagnosis
    Ahmed A. Shalaby, Chia Wen Tsao, Akihiko Ishida, Masatoshi Maeki, Manabu Tokeshi
    Sensors and Actuators B: Chemical, 379, 2023年03月15日, [査読有り]
    研究論文(学術雑誌), Cancer is a leading cause of death worldwide. Early diagnosis of cancer is crucial for successful treatment which, in turn, will decrease mortality. The development of low-cost, accurate, and easy to operate point-of-need (PON) devices to be used for cancer diagnosis and treatment follow-up is a worldwide need, especially in developing countries. Paper-based analytical devices (PADs) are considered a key solution, as they provide a low-cost platform for developing PON biosensors for cancer biomarker detections. There are various types of 2D and 3D PADs according to the type of paper substrate (filter paper, chromatographic paper, nitrocellulose membranes, etc.), fabrication method (wax printing, screen printing, cutting, etc.), detection technique used (colorimetry, fluorescence, chemiluminescence, electrochemiluminescence, electrochemical, etc.), the assay principle and recognition element used (antibodies, aptamers, DNA, nanoparticles, enzymes, etc.). Controlling all these factors determines the performance, accuracy, and sensitivity of the developed devices. This review discusses all these factors in the different PADs used for detection of cancer biomarkers and summarizes the advantages and disadvantages of each one.
  • Effects of Addition of Blocking Agents on Fluorescence Polarization Immunoassay of Okadaic Acid Using a Microfluidic Device
    Shunsuke CHIDA, Kazuki TAKAHASHI, Mao FUKUYAMA, Motohiro KASUYA, Masatoshi MAEKI, Akihiko ISHIDA, Hirofumi TANI, Koji SHIGEMURA, Anatoly V. ZHERDEV, Sergei A. EREMIN, Akihide HIBARA, Manabu TOKESHI
    BUNSEKI KAGAKU, 72, 3, 133, 138, Japan Society for Analytical Chemistry, 2023年03月05日, [査読有り]
    日本語, 研究論文(学術雑誌)
  • ポータブル小型液体クロマトグラフ用の深紫外発光ダイオードを用いた吸光度検出モジュールの開発 ポータブル液体クロマト小型グラフのための深紫外発光漸ベースの吸光度検出モジュールの開発
    Kaito Koyama, Takuma Nishimura, Akihiko Ishida, Mitsue Hibino, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    分析化学, 72, 3, 125, 131, 2023年03月, [査読有り], [責任著者]
    日本語, 研究論文(学術雑誌)
  • A fabrication technique for paper-based analytical devices via two-sided patterning with thermal-transfer printer and laminator
    Takuya Monju, Manabu Hirakawa, Satoshi Kuboyama, Rikuro Saiki, Akihiko Ishida
    Sensors and Actuators B: Chemical, 375, 132886, 132886, Elsevier BV, 2023年01月, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌)
  • Effect of Organic Solvents on a Production of PLGA-Based Drug-Loaded Nanoparticles Using a Microfluidic Device
    Yi Bao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Omega, 7, 37, 33079, 33086, 2022年09月20日, [査読有り]
    研究論文(学術雑誌), The translation of nanoparticles (NPs) from laboratory to clinical settings is limited, which is not ideal. One of the reasons for this is that we currently have limited ability to precisely regulate various physicochemical parameters of nanoparticles. This has made it difficult to rapidly perform targeted screening of drug preparation conditions. In this study, we attempted to broaden the range of preparation conditions for particle size-modulated poly(lactic-co-glycolic-acid) (PLGA) NP to enhance their applicability for drug delivery systems (DDS). This was done using a variety of organic solvents and a glass-based microfluidic device. Furthermore, we compared the PDMS-based microfluidic device to the glass-based microfluidic device in terms of the possibility of a wider range of preparation conditions, especially the effect of different solvents on the size of the PLGA NPs. PLGA NPs with different sizes (sub-200 nm) were successfully prepared, and three different types of taxanes were employed for encapsulation. The drug-loaded NPs showed size-dependent cytotoxicity in cellular assays, regardless of the taxane drug used.
  • Preparation of size-tunable sub-200 nm PLGA-based nanoparticles with a wide size range using a microfluidic platform
    Yi Bao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    PLOS ONE, 17, 8, PUBLIC LIBRARY SCIENCE, 2022年08月
    英語, 研究論文(学術雑誌), The realization of poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) from laboratory to clinical applications remains slow, partly because of the lack of precise control of each condition in the preparation process and the rich selectivity of nanoparticles with diverse characteristics. Employing PLGA NPs to establish a large range of size-controlled drug delivery systems and achieve size-selective drug delivery targeting remains a challenge for therapeutic development for different diseases. In this study, we employed a microfluidic device to control the size of PLGA NPs. PLGA, poly (ethylene glycol)-methyl ether block poly (lactic-co-glycolide) (PEG-PLGA), and blend (PLGA + PEG-PLGA) NPs were engineered with defined sizes. Blend NPs exhibit the widest size range (40-114 nm) by simply changing the flow rate conditions without changing the precursor (polymer molecular weight, concentration, and chain segment composition). A model hydrophobic drug, paclitaxel (PTX), was encapsulated in the NPs, and the PTX-loaded NPs maintained a large range of controllable NP sizes. Furthermore, size-controlled NPs were used to investigate the effect of particle size of sub-200 nm NPs on tumor cell growth. The 52 nm NPs showed higher cell growth inhibition than 109 nm NPs. Our method allows the preparation of biodegradable NPs with a large size range without changing polymer precursors as well as the nondemanding fluid conditions. In addition, our model can be applied to elucidate the role of particle sizes of sub-200 nm particles in various biomedical applications, which may help develop suitable drugs for different diseases.
  • Non-competitive fluorescence polarization immunosensing for CD9 detection using a peptide as a tracer.
    Kazuki Takahashi, Shunsuke Chida, Thanawat Suwatthanarak, Mikiko Iida, Min Zhang, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Takao Yasui, Yoshinobu Baba, Akihide Hibara, Mina Okochi, Manabu Tokeshi
    Lab on a chip, 22, 16, 2971, 2977, 2022年06月17日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), This paper is the first report of a non-competitive fluorescence polarization immunoassay (NC-FPIA) using a peptide as a tracer. The NC-FPIA can easily and quickly quantify the target after simply mixing them together. This feature is desirable for point-of-need applications such as clinical diagnostics, infectious disease screening, on-site analysis for food safety, etc. In this study, the NC-FPIA was applied to detect CD9, which is one of the exosome markers. We succeeded in detecting not only CD9 but also CD9 expressing exosomes derived from HeLa cells. This method can be applied to various targets if a tracer for the target can be prepared, and expectations are high for its future uses.
  • Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device.
    Masatoshi Maeki, Yuto Okada, Shuya Uno, Ayuka Niwa, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Journal of visualized experiments : JoVE, 181, 2022年03月22日, [国際誌]
    英語, 研究論文(学術雑誌), The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.
  • Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device
    Masatoshi Maeki, Yuto Okada, Shuya Uno, Ayuka Niwa, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Journal of Visualized Experiments, 2022, 181, 2022年03月
    研究論文(学術雑誌), The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.
  • Electrochemical enzyme-based blood ATP and lactate sensor for a rapid and straightforward evaluation of illness severity
    Keine Nishiyama, Ryohei Mizukami, Shizuka Kuki, Akihiko Ishida, Junji Chida, Hiroshi Kido, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    Biosensors and Bioelectronics, 113832, 113832, Elsevier {BV}, 2022年02月, [査読有り], [責任著者]
    研究論文(学術雑誌)
  • Facile and rapid detection of SARS-CoV-2 antibody based on a noncompetitive fluorescence polarization immunoassay in human serum samples.
    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, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Determination of Deoxynivalenol in Wheat, Barley, Corn Meal, and Wheat-Based Products by Simultaneous Multisample Fluorescence Polarization Immunoassay Using a Portable Analyzer
    Ayano Nakamura, Mitsutoshi Aoyagi, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    ACS Food Science & Technology, 1, 9, 1623, 1628, American Chemical Society ({ACS}), 2021年10月15日, [査読有り]
    研究論文(学術雑誌)
  • Using a Paper-Based Analytical Device Designed for Remote Learning Environments to Achieve Simple Quantitative Colorimetry without Micropipettes
    Takeshi Komatsu, Ryan Russel Gabatino, Harrienica Hofileña, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Journal of Chemical Education, 98, 9, 3050, 3054, American Chemical Society ({ACS}), 2021年09月14日, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌)
  • Paper-Based Analytical Device for the On-Site Detection of Nerve Agents
    Akinori Yamaguchi, Hajime Miyaguchi, Akihiko Ishida, Manabu Tokeshi
    ACS Applied Bio Materials, 4, 8, 6512, 6518, American Chemical Society ({ACS}), 2021年08月16日, [査読有り]
    研究論文(学術雑誌)
  • Simple Approach for Fluorescence Signal Amplification Utilizing a Poly(vinyl alcohol)-Based Polymer Structure in a Microchannel
    Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Hideaki Hisamoto, Manabu Tokeshi
    ACS Omega, 6, 12, 8340, 8345, American Chemical Society ({ACS}), 2021年03月30日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Dip-Type Paper-Based Analytical Device for Straightforward Quantitative Detection without Precise Sample Introduction
    Takeshi Komatsu, Ryoga Maeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Sensors, 6, 3, 1094, 1102, American Chemical Society ({ACS}), 2021年03月26日, [査読有り], [責任著者], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • One-Step Production Using a Microfluidic Device of Highly Biocompatible Size-Controlled Noncationic Exosome-like Nanoparticles for RNA Delivery
    Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Applied Bio Materials, 4, 2, 1783, 1793, American Chemical Society ({ACS}), 2021年02月15日
    英語, 研究論文(学術雑誌), Size-controlled lipid nanoparticle (LNP)-based DNA/RNA delivery is a leading technology for gene therapies. For DNA/RNA delivery, typically, a cationic lipid is used to encapsulate DNA/RNA into LNPs. However, the use of the cationic lipid leads to cytotoxicity. In contrast, noncationic NPs, such as exosomes, are ideal nanocarriers for DNA/RNA delivery. However, the development of a simple one-step method for the production of size-controlled noncationic NPs encapsulating DNA/RNA is still challenging because of the lack of electrostatic interactions between the cationic lipid and negatively charged DNA/RNA. Herein, we report a microfluidic-based one-step method for the production of size-controlled noncationic NPs encapsulating small interfering RNA (siRNA). Our microfluidic device, named iLiNP, enables the efficient encapsulation of siRNA, as well as control over the NP size, by varying the flow conditions. We applied this method to produce size-controlled exosome-like NPs. The siRNA-loaded exosome-like NPs did not show in vitro cytotoxicity at a high siRNA dosage. In addition, we investigated the effect of the size of the exosome-like NPs on the target gene silencing and found that the 40-50 nm-sized NPs suppressed target protein expression at a dose of 20 nM siRNA. The iLiNP-based one-step production method for size-controlled noncationic-NP-encapsulated RNA is a promising method for the production of artificial exosomes and functionally modified exosomes for gene and cell therapies.
  • Non-competitive fluorescence polarization immunoassay for detection of H5 avian influenza virus using a portable analyzer.
    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, 413, 18, 4619, 4623, 2021年02月05日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Rapid, sensitive universal paper-based device enhances competitive immunoassays of small molecules
    Takeshi Komatsu, Yuki Sato, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytica Chimica Acta, 1144, 85, 95, Elsevier BV, 2021年02月, [査読有り], [責任著者], [国際誌]
    英語, 研究論文(学術雑誌), Competitive immunoassays comprise the standard means of detecting small molecules. However, conventional methods using microwells are difficult to apply during point-of-care tests (POCT) because they require complicated handling and are time consuming. Although paper-based analytical devices (PAD) have received considerable focus because of their rapid and straightforward operation, only a few devices have been proposed for competitive immunoassays. Herein, we describe a novel universal PAD format with a 3-dimensional configuration for competitive immunoassays that rapidly and sensitively detects small molecules. The proposed device comprised a layered structure with uniform color formation and high capture efficiency between antigen and antibody that results in rapid and reproducible results. The device rapidly (90 s) assayed biotin as a model target, with a limit of detection (LOD) of 5.08 ng mL-1, and detected progesterone with an LOD of 84 pg mL-1 within 5 min. Moreover, sample volumes and reagent consumption rates were minimized. Thus, our device could be applied to competitive immunoassays of various small molecules in POCT.
  • Three-dimensional, symmetrically assembled microfluidic device for lipid nanoparticle production
    Niko Kimura, Masatoshi Maeki, Kosuke Sasaki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    RSC Advances, 11, 3, 1430, 1439, ROYAL SOC CHEMISTRY, 2021年
    英語, 研究論文(学術雑誌), Sub 100 nm-sized lipid nanoparticles (LNPs) have been widely used in drug delivery systems (DDSs). The size of the LNPs is an important parameter for the DDS performance, such as biodistribution and gene silencing using siRNAs. However, the LNPs prepared by the conventional preparation method show a wide size distribution. To improve the LNP size distribution, we developed a microfluidic device, named the iLiNP (TM) device, in a previous study. This device could produce LNPs in the size range of 20 to 150 nm, but the size distribution of the large-sized LNPs needs to be further improved. From the viewpoint of the LNP formation process, a homogeneous and slow rate dilution of ethanol plays an important role in improving the large-size LNP size distribution. In this study, we developed a three-dimensional, symmetrically assembled microfluidic device named the 3D-iLiNP device with the aim of precise size control of large-sized LNPs. We designed the 3D-iLiNP device using a computational fluid dynamics simulation and demonstrated that the 3D-iLiNP device can improve the LNP size distribution. The gene silencing activity of four kinds of siRNA-loaded LNPs was investigated via in vitro and in vivo experiments to elucidate the effect of the LNP size distribution. The results revealed that the LNPs with a size between 90 and 120 nm showed higher gene silencing activity than those with other sizes. The 3D-iLiNP device is expected to improve DDS performance by precisely controlling the size of LNPs.
  • One-step non-competitive fluorescence polarization immunoassay based on a Fab fragment for C-reactive protein quantification
    Keine Nishiyama, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    Sensors and Actuators B: Chemical, 326, 128982, 128982, Elsevier {BV}, 2021年01月
    研究論文(学術雑誌)
  • Silica Nanopillar Arrays for Monitoring Diffraction-Based Label-Free Biomolecule Separation
    Taiga Ajiri, Takao Yasui, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Junji Nishii, Yoshinobu Baba, Manabu Tokeshi
    ACS Applied Nano Materials, 3, 9, 8810, 8816, American Chemical Society ({ACS}), 2020年09月25日
    英語, 研究論文(学術雑誌), Recent studies on nanopillar arrays, one type of nanofluidic device, have demonstrated various tools for bioanalysis. When carrying out nanopillar array-based separation, it is indispensable to observe biomolecules, such as DNA, proteins, and extracellular vesicles, that have fluorescence labeling; however, fluorescence labeling influences the biomolecular characteristics. Here, we have proposed label-free monitoring of biomolecule separation by using diffracted light derived from the nanopillar array that was fabricated inside a microchannel by combining laser interference lithography with general photolithography techniques. Using an electrophoresis approach, we demonstrated that our diffraction-based label-free method possessed high sensor initialization ability, and the nanopillar array device successfully monitored DNA separation without labeling bias. Results obtained using our label-free monitoring of DNA separation confirmed, for the first time, that the molecular dynamics of DNA molecules in the nanopillar array were changed in the presence or absence of fluorescent labeling. The presented concept will provide a useful tool for nonbiased monitoring of label-free biomolecule analysis in nanofluidic channels.
  • Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein–ligand complex structure analysis
    Masatoshi Maeki, Sho Ito, Reo Takeda, Go Ueno, Akihiko Ishida, Hirofumi Tani, Masaki Yamamoto, Manabu Tokeshi
    Chemical Science, 11, 34, 9072, 9087, Royal Society of Chemistry ({RSC}), 2020年08月25日, [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer
    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, Elsevier {BV}, 2020年08月, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Development of a Microfluidic-Based Post-Treatment Process for Size-Controlled Lipid Nanoparticles and Application to siRNA Delivery
    Niko Kimura, Masatoshi Maeki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Applied Materials & Interfaces, 12, 30, 34011, 34020, American Chemical Society ({ACS}), 2020年07月29日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Real-Time Measurement of Protein Crystal Growth Rates within the Microfluidic Device to Understand the Microspace Effect
    Masatoshi Maeki, Shohei Yamazaki, Reo Takeda, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Omega, 5, 28, 17199, 17206, American Chemical Society ({ACS}), 2020年07月21日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Preparation of high-quality protein crystals is a major challenge in protein crystallography. Natural convection is considered to be an uncontrollable factor of the crystallization process at the ground level as it disturbs the concentration gradient around the growing crystal, resulting in lower-quality crystals. A microfluidic environment expects an imitated microgravity environment because of the small Gr number. However, the mechanism of protein crystal growth in the microfluidic device was not elucidated due to limitations in measuring the crystal growth process within the device. Here, we demonstrate the real-time measurement of protein crystal growth rates within the microfluidic devices by laser confocal microscopy with differential interference contrast microscopy (LCM-DIM) at the nanometer scale. We confirmed the normal growth rates in the 20 and 30 μm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 μm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.
  • Paper-Based Device for the Facile Colorimetric Determination of Lithium Ions in Human Whole Blood
    Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS Sensors, 5, 5, 1287, 1294, American Chemical Society ({ACS}), 2020年05月22日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Lithium carbonate is an effective medicine for the treatment of the bipolar disorder, but the concentration of lithium in the patient's blood must be frequently monitored because of its toxicity. To date, no colorimetric methods of lithium ion detection in whole blood without pretreatment have been reported. Here, we report a colorimetric paper-based device that allows point-of-care testing in one step. This device is composed of two paper-based elements linked to each other: a blood cell separation unit and a colorimetric detection unit. After a portion of whole blood has been placed on the end of the separation unit, plasma in the sample is automatically transported to the detection unit, which displays a diagnostic color. The key feature of this device is its simple, user-friendly operation. The limit of detection is 0.054 mM and the coefficient of variance is below 6.1%, which are comparable to those of conventional instruments using the same colorimetric reaction. Furthermore, we achieved high recovery (>90%) and reproducibility (<9.8%) with spiked human blood samples. Thus, the presented device provides an alternative method for the regular monitoring of lithium concentrations in the treatment of bipolar disorder by augmenting the coefficient of variation (maximum value, 6.1%).
  • Development of a Paper-based Analytical Chip for the Detection of Bacterial 16S rRNA in Wastewater Samples
    Meri Nakajima, Akihiko Ishida, Manabu Tokeshi, Hisashi Satoh
    BUNSEKI KAGAKU, 69, 12, 715, 722, JAPAN SOC ANALYTICAL CHEMISTRY, 2020年
    日本語, 研究論文(学術雑誌), Analysis of bacteria in the sewage wastewater treatment process is essential for process stabilization and upgrading. Although bacteria are currently being analyzed by molecular biology techniques targeting the 16S rRNA gene, there is a problem that they are time-consuming and labor-intensive. In this study, we developed a paper-based analytical chip by using two types of DNA molecules that specifically bind to bacterial 16S rRNA. We optimized the fabrication method of the detection probe and the paper-based analytical chip, and then detected synthetic DNA having a nucleotide sequence that hybridizes with the designed DNA molecules and bacterial 16S rRNA extracted from an activated sludge sample. We evaluated the amount of nucleic acids quantitatively by taking images of the detection line on the paper-based analytical chip with a smartphone and analyzing its brightness with an open-source image processing program, ImageJ. Our method was able to detect 85 nM of bacterial 16S rRNA concentration in the extract. Nucleic acids that did not hybridize with either of the designed DNA molecules were not detected, demonstrating high selectivity of our method.
  • An aprotic polar solvent assisted size-tuning method for microfluidic production of lipid-based drug nanocarriers with various sizes
    Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1021, 1022, 2020年
    研究論文(国際会議プロシーディングス), This paper reports a microfluidic methodology for precise size-tuning of lipid nanoparticles (LNPs) with various sizes by using lipid solvents composed of a protic or an aprotic or mixed solvents. This method expanded the size-tunable range of the iLiNP device while kept the precise size controllability and the mass productivity. In addition, the produced siRNA-loaded LNPs with various sizes were evaluated in vitro experiments and confirmed effective gene-silencing activity and intracellular uptake depending on the LNP sizes. This LNP size-tuning methodology is expected to be a breakthrough in the current limited size-tunability of LNPs.
  • Novel format of a paper-based device for competitive immunoassays
    Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 494, 495, 2020年
    研究論文(国際会議プロシーディングス), We report a new microfluidic paper-based analytical device (μPAD) that allows competitive immunoassays without washing operations. This device consists of three layers and has the following features: (1) uniform color development, (2) high controllability of wicking rate, (3) rapid and highly sensitive measurement. The performance of the device was evaluated by the biotin/anti-biotin antibody assay, and then was successfully applied to the sensitive measurement of progesterone (female sex hormone).
  • Detection of avian influenza virus and its antibody by fluorescence polarization immunoassay
    Keine Nishiyama, Yohei Takeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Haruko Ogawa, Manabu Tokeshi
    MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1226, 1227, 2020年
    研究論文(国際会議プロシーディングス), This paper reports a high-throughput and rapid method for a fluorescence polarization immunoassay (FPIA) of H5-avian influenza virus (H5-AIV) and anti-H5-AIV antibody using a portable FP analyzer with a microdevice. For both assays, a fluorescein-labeled recombinant fragment of H5-hemagglutinin was prepared. Although the labeled fragment had low molecular-weight, it served as a tracer for the FPIA. Also, a microdevice having multiplex microchannels was designed, allowing simultaneous multiplex analysis at a small sample volume. Consequently, the virus and the antibody in several samples were successfully detected rapidly in the same format.
  • An Electrochemical Sensor Based on Structure Switching of Dithiol-modified Aptamer for Simple Detection of Ochratoxin A.
    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, JAPAN SOC ANALYTICAL CHEMISTRY, 2019年11月, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌), 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.
  • Rapid, Sensitive, and Selective Detection of H5 Hemagglutinin from Avian Influenza Virus Using an Immunowall Device
    Kenia Chávez Ramos, Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Toshihiro Kasama, Yoshinobu Baba, Manabu Tokeshi
    ACS Omega, 4, 15, 16683, 16688, American Chemical Society ({ACS}), 2019年10月08日, [査読有り]
    研究論文(学術雑誌), © 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.
  • A Concentric Ring Electrode for a Wall‐jet Cell in a Microfluidic Device
    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], [責任著者]
    英語, 研究論文(学術雑誌), 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.
  • Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification
    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]
    研究論文(学術雑誌), © 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.
  • Sensitive fluorescent polarization immunoassay by optimizing synchronization mismatch condition
    Wakao Osamu, Maeki Masatoshi, Ishida Akihiko, Tani Hirofumi, Hibara Akihide, Tokeshi Manabu
    SENSORS AND ACTUATORS B-CHEMICAL, 285, 418, 422, ELSEVIER SCIENCE SA, 2019年04月15日, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer
    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]
    研究論文(学術雑誌), © 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.
  • High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer
    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]
    研究論文(学術雑誌), © 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.
  • Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification
    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, Royal Society of Chemistry ({RSC}), 2019年, [査読有り]
    研究論文(学術雑誌), © 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.
  • Label-Free Electrochemical Sensor for Ochratoxin A Using a Microfabricated Electrode with Immobilized Aptamer
    Donny N. Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    ACS Omega, 3, 12, 16823, 16830, American Chemical Society (ACS), 2018年12月31日, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌), Ochratoxin A (OTA) is one of the most abundant food-contaminating mycotoxins that is also a potential carcinogen and responsible for many diseases affecting humans. Consequently, a sensitive, portable device for the detection of OTA is highly desirable. In this study, a miniaturized electrochemical aptamer-based sensor was developed for the label-free, sensitive detection of OTA. For the construction of the sensor, a gold thin-film three-electrode system was fabricated using standard microfabrication techniques on a polystyrene substrate (25 mm x 25 mm). Subsequently, the thiol-modified linker, 6-mercaptohexanol, DNA aptamer, and methylene blue (MB) were sequentially applied to the working electrode to construct a sensing layer. MB served as a redox indicator that interacted with the aptamer via the guanine bases and phosphate backbone to form complexes. The addition of OTA to the sensor induced the folding of the aptamer, which was accompanied by the release of the aptamer-MB-OTA complex from the sensor. Thus, the amount of MB decreased with increasing concentration of OTA. Differential pulse voltammetry was used for monitoring the highly sensitive detection. The standard curve for OTA exhibited a wide linearity ranging from 0.1 to 300 ng mL(-1) with a detection limit of 78.3 pg mL(-1) (S/N = 3). The selectivity test confirmed that the aptamer had high affinity only for the target. The OTA recoveries with the proposed sensor in commercial samples of coffee and beer were 86.4-107%.
  • 血中ATPと乳酸を指標とする重症度診断のための電気化学酵素センサーの開発               
    水上 良平, 真栄城 正寿, 石田 晃彦, 谷 博文, 渡慶次 学
    日本分析化学会講演要旨集, 67年会, 287, 287, (公社)日本分析化学会, 2018年08月
    日本語
  • Development of the iLiNP Device: Fine Tuning the Lipid Nanoparticle Size within 10 nm for Drug Delivery
    Niko Kimura, Masatoshi Maeki, Yusuke Sato, Yusuke Note, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Omega, 3, 5, 5044, 5051, American Chemical Society, 2018年05月09日, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Recent microdevice-based aptamer sensors
    Donny Nugraha Mazaafrianto, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Micromachines, 9, 5, MDPI AG, 2018年04月25日, [査読有り]
    英語, 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.
  • A compact fluorescence polarization analyzer with high-transmittance liquid crystal layer
    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, American Institute of Physics Inc., 2018年02月01日, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • A post-treatment methodology for precise size control of lipid nanoparticles by stepwise and rapid ethanol dilution
    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
    研究論文(国際会議プロシーディングス), 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.
  • Characteristics of microfluidic paper-based analytical devices fabricated by four different methods
    Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytical Sciences, 34, 1, 39, 44, Japan Society for Analytical Chemistry, 2018年, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Understanding the formation mechanism of lipid nanoparticles in microfluidic devices with chaotic micromixers
    Masatoshi Maeki, Yuka Fujishima, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    PLOS ONE, 12, 11, PUBLIC LIBRARY SCIENCE, 2017年11月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Optimization of the nanofluidic design for label-free detection of biomolecules using a nanowall array
    Taiga Ajiri, Takao Yasui, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    SENSORS AND ACTUATORS B-CHEMICAL, 250, 39, 43, ELSEVIER SCIENCE SA, 2017年10月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Using Laser Interference Lithography in the Fabrication of a Simplified Micro and Nanofluidic Device for Label-free Detection
    Taiga Ajiri, Haruya Kasa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Junji Nishi, Manabu Tokeshi
    ANALYTICAL SCIENCES, 33, 10, 1197, 1199, JAPAN SOC ANALYTICAL CHEMISTRY, 2017年10月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • 尿中シュウ酸分析のための単一くし形電極を組み込んだ液体クロマトグラフィーチップの開発               
    藤井 大地, 真栄城 正寿, 石田 晃彦, 谷 博文, 渡慶次 学
    日本分析化学会講演要旨集, 66年会, 24, 24, (公社)日本分析化学会, 2017年08月
    日本語
  • マイクロ流体デバイスを用いた化学発光イムノアッセイの高感度化               
    菊地 優仁, 真栄城 正寿, 石田 晃彦, 谷 博文, 渡慶次 学
    日本分析化学会講演要旨集, 66年会, 52, 52, (公社)日本分析化学会, 2017年08月
    日本語
  • 血中ATPと乳酸測定のための電気化学センサーの開発               
    水上 良平, 西山 慶音, 真栄城 正寿, 石田 晃彦, 谷 博文, 渡慶次 学
    日本分析化学会講演要旨集, 66年会, 134, 134, (公社)日本分析化学会, 2017年08月
    日本語
  • Rapid Detection of Cat Cystatin C (cCys-C) Using Immuno-Pillar Chips
    Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES, 32, 12, 1359, 1362, JAPAN SOC ANALYTICAL CHEMISTRY, 2016年12月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Simple and sensitive colorimetric assay system for horseradish peroxidase using microfluidic paper-based devices
    Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    SENSORS AND ACTUATORS B-CHEMICAL, 236, 433, 441, ELSEVIER SCIENCE SA, 2016年11月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Novel concept of washing for microfluidic paper-based analytical devices based on capillary force of paper substrates
    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, SPRINGER HEIDELBERG, 2016年11月, [査読有り]
    英語, 研究論文(学術雑誌), 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).
  • 血中リチウム濃度測定のためのペーパーデバイスの開発               
    小松 雄士, 真栄城 正寿, 石田 晃彦, 谷 博文, 渡慶次 学
    日本分析化学会講演要旨集, 65年会, 163, 163, (公社)日本分析化学会, 2016年08月
    日本語
  • 3,3 ',5,5 '-Tetramethylbenzidine Oxidation on Paper Devices for Horseradish Peroxidase-based Assays
    Lori Shayne Alamo Busa, Takeshi Komatsu, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES, 32, 8, 815, 818, JAPAN SOC ANALYTICAL CHEMISTRY, 2016年08月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Advances in Microfluidic Paper-Based Analytical Devices for Food and Water Analysis
    Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    MICROMACHINES, 7, 5, MDPI AG, 2016年05月, [査読有り]
    英語, 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.
  • A microfluidic-based protein crystallization method in 10 micrometer-sized crystallization space
    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, ROYAL SOC CHEMISTRY, 2016年, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Image analysis for a microfluidic paper-based analytical device using the CIE L(star)a(star)b(star) color system
    Takeshi Komatsu, Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST, 141, 24, 6507, 6509, ROYAL SOC CHEMISTRY, 2016年, [査読有り], [責任著者]
    英語, 研究論文(学術雑誌), 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.
  • A competitive immunoassay system for microfluidic paper-based analytical detection of small size molecules
    Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST, 141, 24, 6598, 6603, ROYAL SOC CHEMISTRY, 2016年, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • A Portable Liquid Chromatograph with a Battery-operated Compact Electroosmotic Pump and a Microfluidic Chip Device with a Reversed Phase Packed Column
    Akihiko Ishida, Mitsutaka Fujii, Takehiro Fujimoto, Shunsuke Sasaki, Ichiro Yanagisawa, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES, 31, 11, 1163, 1169, JAPAN SOC ANALYTICAL CHEMISTRY, 2015年11月, [査読有り], [筆頭著者, 責任著者]
    英語, 研究論文(学術雑誌), 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.
  • Fluorescence Polarization Measurement System Using a Liquid Crystal Layer and an Image Sensor
    Osamu Wakao, Yusaku Fujii, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    ANALYTICAL CHEMISTRY, 87, 19, 9647, 9652, AMER CHEMICAL SOC, 2015年10月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • イムノピラーデバイスの高性能化:抗体固定化担体の改良
    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, JAPAN SOC ANALYTICAL CHEMISTRY, 2015年05月, [査読有り]
    日本語, 研究論文(学術雑誌), 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.
  • An instrument-free, screen-printed paper microfluidic device that enables bio and chemical sensing
    Saeed Mohammadi, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYST, 140, 19, 6493, 6499, ROYAL SOC CHEMISTRY, 2015年, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • A strategy for synthesis of lipid nanoparticles using microfluidic devices with a mixer structure
    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, ROYAL SOC CHEMISTRY, 2015年, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Bioluminescence Assay Based on Controlled Inhibitory Effect on Firefly Luciferase
    Ryoko Kurishiba, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    LUMINESCENCE, 29, 78, 78, WILEY-BLACKWELL, 2014年08月, [査読有り]
    英語
  • Bioluminescence immunoassay using luciferase-encapsulated liposome as a label
    Yusuke Nakatani, Chiaki Shido, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    LUMINESCENCE, 29, 83, 84, WILEY-BLACKWELL, 2014年08月, [査読有り]
    英語
  • Effect of reductants on oscillatory chemiluminescence of fluorescein catalyzed by horseradish peroxidase
    Hirofumi Tani, Ai Masuyama, Akihiko Ishida, Manabu Tokeshi
    LUMINESCENCE, 29, 99, 100, WILEY-BLACKWELL, 2014年08月, [査読有り]
    英語
  • Application of 4-Iodophenol-enhanced Luminol Chemiluminescence to Direct Detection of Horseradish Peroxidase Encapsulated in Liposomes
    Tamio Kamidate, Masumi Maruya, Hirofumi Tani, Akihiko Ishida
    ANALYTICAL SCIENCES, 25, 9, 1163, 1166, JAPAN SOC ANALYTICAL CHEMISTRY, 2009年09月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Microchip reversed-phase liquid chromatography with packed column and electrochemical flow cell using polystyrene/poly(dimethylsiloxane)
    Akihiko Ishida, Masamichi Natsume, Tamio Kamidate
    JOURNAL OF CHROMATOGRAPHY A, 1213, 2, 209, 217, ELSEVIER SCIENCE BV, 2008年12月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation
    Akihiko Ishida, Yasuko Yamada, Tamio Kamidate
    ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 392, 5, 987, 994, SPRINGER HEIDELBERG, 2008年11月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Direct determination of horseradish peroxidase encapsulated in liposomes by using luminol chemiluminescence
    Tamio Kamidate, Kanako Komatsu, Hirofumi Tant, Akihiko Ishida
    ANALYTICAL SCIENCES, 24, 4, 477, 481, JAPAN SOC ANALYTICAL CHEMISTRY, 2008年04月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes
    Tamio Kamidate, Kanako Komatsu, Hirofumi Tani, Akihiko Ishida
    LUMINESCENCE, 22, 3, 236, 240, JOHN WILEY & SONS LTD, 2007年05月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes
    Tamio Kamidate, Hirofumi Tani, Akihiko Ishida, Masaya Hayashi
    LUMINESCENCE, 22, 1, 15, 19, JOHN WILEY & SONS LTD, 2007年01月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Reversed-phase liquid chromatography on a microchip with sample injector and monolithic silica column
    Akihiko Ishida, Takahiro Yoshikawa, Masamichi Natsume, Tamio Kamidate
    JOURNAL OF CHROMATOGRAPHY A, 1132, 1-2, 90, 98, ELSEVIER SCIENCE BV, 2006年11月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes
    T Kamidate, K Yanashita, H Tani, A Ishida, M Notani
    ANALYTICAL CHEMISTRY, 78, 1, 337, 342, AMER CHEMICAL SOC, 2006年01月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Fluorescein chemiluminescence method for estimation of membrane permeability of liposomes
    A Ishida, C Otsuka, H Tani, T Kamidate
    ANALYTICAL BIOCHEMISTRY, 342, 2, 338, 340, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2005年07月, [査読有り], [筆頭著者, 責任著者]
    英語
  • Determination of peroxidase encapsulated in liposomes using homogentisic acid gamma-lactone chemiluminescence
    T Kamidate, N Kikuchi, A Ishida, H Tani
    ANALYTICAL SCIENCES, 21, 6, 701, 704, JAPAN SOC ANALYTICAL CHEMISTRY, 2005年06月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Determination of hydrogen peroxide by fluorescein chemiluminescence catalyzed with horseradish peroxidase encapsulated in liposome
    T Kamidate, Y Ishida, H Tani, A Ishida
    BUNSEKI KAGAKU, 54, 6, 569, 572, JAPAN SOC ANALYTICAL CHEMISTRY, 2005年06月, [査読有り]
    日本語, 研究論文(学術雑誌), 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,: Determination of Peroxidase Encapsulated in Liposomes Using Homogentisic Acid γ-Lactone Chemiluminescence, Anal. Sci., 21, 701-704 (2005)*
    T Kamidate, N Kikuchi, A Ishida, H Tani
    ANALYTICAL SCIENCES, 21, 6, 701, 704, JAPAN SOC ANALYTICAL CHEMISTRY, 2005年06月
    英語, 研究論文(学術雑誌), 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.
  • リポソームに内封したペルオキシダーゼを触媒に用いるフルオレセイン化学発光による過酸化水素の定量
    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
    日本語, 研究論文(学術雑誌), 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.
  • Cationic liposomes enhanced firefly bioluminescent assay of bacterial ATP in the presence of an ATP extractant
    N Nakata, A Ishida, H Tani, T Kamidate
    ANALYTICAL SCIENCES, 19, 8, 1183, 1185, JAPAN SOC ANALYTICAL CHEMISTRY, 2003年08月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • Firefly bioluminescence assay of ATP using diethylaminoethyl-dextran as an enhancer in the presence of sodium chloride and ATP extranctant
    A Ishida, T Yoshikawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY, 316, 1, 127, 130, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2003年05月, [査読有り]
    英語, 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.
  • Firefly bioluminescence assay of ATP using diethylaminoethyl-dextran as an enhancer in the presence of sodium chloride and ATP extranctant
    A Ishida, T Yoshikawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY, 316, 1, 127, 130, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2003年05月
    英語, 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.
  • Direct detection of horseradish peroxidase as a marker molecule encapsulated in liposomes via use of fluorescein chemiluminescence
    T Kamidate, Y Ishida, H Tani, A Ishida
    CHEMISTRY LETTERS, 32, 4, 402, 403, CHEMICAL SOC JAPAN, 2003年04月
    英語, 研究論文(学術雑誌), 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.
  • Preconcentration of catecholamines into liposomes with imposed pH gradients
    Akihiko Ishida, Manabu Ikemoto, Yoshiki Ishida, Tamio Kamidate
    Bulletin of the Chemical Society of Japan, 76, 5, 985, 989, Chemical Society of Japan, 2003年, [査読有り]
    英語, 研究論文(学術雑誌), 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
  • 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
  • Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran
    A Ishida, T Yoshikawa, T Nakazawa, T Kamidate
    ANALYTICAL BIOCHEMISTRY, 305, 2, 236, 241, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2002年06月, [査読有り]
    英語, 研究論文(学術雑誌), 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).
  • Uptake of transition metal ions using liposomes containing dicetylphosphate as a ligand
    T Kamidate, Y Hashimoto, H Tani, A Ishida
    ANALYTICAL SCIENCES, 18, 3, 273, 276, JAPAN SOC ANALYTICAL CHEMISTRY, 2002年03月, [査読有り]
    英語, 研究論文(学術雑誌), 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+.
  • Development of simple methods for the visual determination of trace metal ions by using characteristic color-forming reaction systems of metal chelates
    Akihiko Ishida
    Bunseki Kagaku, 49, 1, 71, 72, Japan Society for Analytical Chemistry, 2000年, [査読有り]
    日本語, 研究論文(学術雑誌), 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.
  • Field screening test for iron leached from well bore rock samples by using color development of iron(III)-xylenol orange system
    A Ishida, E Kaneko, T Yotsuyanagi
    CHEMISTRY LETTERS, 28, 4, 351, 352, CHEMICAL SOC JAPAN, 1999年04月, [査読有り]
    英語, 研究論文(学術雑誌), 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.
  • A New Confined Spot Test with Hydrophobic Filter Paper and Its Application to the Visual Fluorometric Determination of Trace Aluminium with 2,2'-Dihydroxyazobenzene
    KANEKO Emiko, ISHIDA Akihiko, DEGUCHI Yuji, YOTSUYANAGI Takao
    Chemistry letters, 1994, 9, 1615, 1618, The Chemical Society of Japan, 1994年09月05日
    英語, A new sensitive and simple spot test has been developed for the visual determination of trace element in water, based on the formation of a size confined colored ring on hydrophobic filter paper. When 0.1 cm3 of aqueous solution is spotted on the water-repelling surface and evaporated in an oven, a small fleck of 6 mm diameter edged with a colored analyte is formed. This method has been successfully applied to the visual fluorometric determination of trace aluminium(III) ion with 2,2′-dihydroxyazobenzene (DHAB or H2L). The 1 : 1 chelate, [AlL]+, becomes localized in a concentric ring zone at pH 6.5. The detection limit is 2 × 10−8 mol dm−3 (0.5 ppb) by visual fluorometry with a UV lamp in the dark.

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    エヌ・ティー・エス, 2005年06月, 4860430859, 4, 10, 683, 10, 12p, 図版14p, 日本語, [分担執筆]

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
    科学研究費助成事業 基盤研究(C)
    2020年04月01日 - 2023年03月31日
    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
    進捗状況を3項目に分けて述べる。
    1.不妊魚の作出:ニジマスの卵と凍結保存したブラウントラウトあるいはサクラマスの精子を受精し、第二極体放出阻止処理により作出された雑種三倍体候補(A:ニジマス×ブラウントラウト、B:ニジマス×サクラマス)の倍数性を調査した。その結果、候補Aでは全25個体で、候補Bでは1個体を除く30個体が三倍体であった。これらの個体はPITタグで標識し、現在も継続して飼育している。(藤本)
    2.魚卵アレルゲン検知系の構築:引き続き、ニジマス卵アレルゲンであるβ’-component(BC)のペーパー免疫分析デバイスの構築に取り組んだ。デバイスは濾紙にインクを印刷して加熱する常法により作製し、インクで囲まれた領域を分析反応ゾーンとした。分析は、反応ゾーンに新規作製した抗BC抗体を固定化して試料、酵素標識抗BC抗体、発色試薬を順に加えて行う手順とした。本手法では、従来並みの感度(検出感度:約1 ng/mL)の分析が従来の1/100の時間(約20分)で可能となった。(渡慶次、石田)
    加えて、交雑種に対応した検知系に使用する抗体の作成のため、サクラマス排卵からBCを精製し、これを家兎に免疫し抗血清を作製した。(平松)
    3.不妊化魚の魚卵アレルゲン性の調査:全23個体の不妊化三倍体ニジマスの内臓組織に含まれるBCを測定したが、全個体の筋肉には魚卵アレルギー発症リスクが認められなかった。しかし、5個体において生殖線から少量のBCが検出されたが、その内3個体は目視にて明確な生殖腺の発達が観られた。目視で生殖線の発達が確認できなかった20個体について、生殖腺の組織切片を作製・観察し、その発達状況を確認した。その結果、3個体で発達中の卵母細胞が散在しているのが確認され、生殖腺の外観だけでアレルギー発症リスクの有無を見分けるのは困難であることが判明した。(清水、佐伯、笹岡、平松)
    日本学術振興会, 基盤研究(C), 北海道大学, 20K05903
  • 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
    科学研究費助成事業 基盤研究(C)
    2017年04月01日 - 2020年03月31日
    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。
    日本学術振興会, 基盤研究(C), 北海道大学, 17K05892
  • 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               
    研究補助開発研究
    2018年04月 - 2019年03月
    石田 晃彦
    JKA, 研究代表者, 競争的資金
  • ポータブル分析装置の高感度なマイクロ検出器の開発               
    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
    2015年01月 - 2015年12月
    石田 晃彦
    科学技術振興機構, 研究代表者, 競争的資金
  • 次世代診断チップの開発とその臨床診断への応用
    科学研究費助成事業 基盤研究(A)
    2012年05月31日 - 2015年03月31日
    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, 基盤研究(A), 北海道大学, 24245013
  • 単一くし形微小電極を組み込んだ高感度マイクロ電気化学検出システムの構築とその応用
    科学研究費助成事業 基盤研究(C)
    2011年 - 2013年
    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, 基盤研究(C), 北海道大学, 研究代表者, 競争的資金, 23550087
  • 刺激応答性ベシクルを利用する高感度電気化学検出バイオ分析チップの開発
    科学研究費助成事業 若手研究(B)
    2006年 - 2007年
    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, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 18750056
  • リポソームを利用する高感度な化学発光分析用ナノリアクターの開発
    科学研究費助成事業 基盤研究(B)
    2006年 - 2007年
    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, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 18350037
  • 化学発光速度を指標とするリポソームの膜透過性の評価法の開発
    科学研究費助成事業 萌芽研究
    2005年 - 2006年
    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, 萌芽研究, 北海道大学, 連携研究者, 競争的資金, 17655028
  • 液体クロマトグラフィーの高性能マイクロシステムの構築とその生体分子分析への応用
    科学研究費助成事業 若手研究(B)
    2004年 - 2005年
    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, 若手研究(B), 北海道大学, 研究代表者, 競争的資金, 16750056
  • 標識体をナノリアクターに利用する高感度な化学発光分析システムの構築
    科学研究費助成事業 基盤研究(B)
    2003年 - 2004年
    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, 基盤研究(B), 北海道大学, 連携研究者, 競争的資金, 15350038
  • 高輝度光微細加工による生体内物質分析用微小電気化学反応セル創製に関する研究
    科学研究費助成事業 萌芽研究
    2003年 - 2004年
    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。
    日本学術振興会, 萌芽研究, 北海道大学, 連携研究者, 競争的資金, 15656177
  • 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
    科学研究費助成事業 特別研究員奨励費
    1998年 - 1998年
    石田 晃彦
    日本学術振興会, 特別研究員奨励費, 東北大学, 97J06075
  • マイクロチップを利用する化学分析システムの開発               
    競争的資金

産業財産権

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    特許5935696
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    特許権, 石田 晃彦, 山田 泰子, 上舘 民夫, 国立大学法人 北海道大学
    JP2006308601, 2006年04月25日
    WO2006-118093, 2006年11月09日
    特許第4940432号
    2012年05月30日
    200903081314391360
  • 細胞中ATPの定量法
    特許権, 上舘 民夫, 谷 博文, 石田 晃彦, 三菱化学メディエンス株式会社
    特願2004-061585, 2004年03月05日
    特開2005-245342, 2005年09月15日
    特許第4546115号
    2010年07月09日
    201103016902185272
  • イオン性検体のスポットテスト法
    特許権, 四ツ柳 隆夫, 金子 恵美子, 宮川 貴行, 石田 晃彦, 株式会社東北テクノアーチ
    特願平6-048632, 1994年03月18日
    特開平7-260693, 1995年10月13日
    特許第3393701号
    2003年01月31日
    201103034197155383
  • 試料中成分溶出量の簡易判定方法及びその簡易判定用キット
    特許権, 石田 晃彦, 金子 恵美子, 四ツ柳 隆夫, 株式会社東北テクノアーチ
    特願平10-177968, 1998年05月21日
    特開平11-326309, 1999年11月26日
    200903018923337891

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