Poly(butylene succinate) reinforced by small amount of grafted nanofibrillated bacterial cellulose: Toughness variability based on nanocomposites preparation method
Hamidah binti Hashim, Xiaochao Xia, Hiroshi Kani, Shuichiro Seno, Feng Li, Takuya Isono, Takuya Yamamoto, Hirofumi Tani, Toshifumi Satoh, Kenji Tajima
Composites Part A: Applied Science and Manufacturing, 185, 108341, 108341, Elsevier BV, Oct. 2024
Scientific journal

Application of bacterial-derived long cellulose nanofiber to suspension culture of mammalian cells as a shear protectant
Eiichiro Kaneko, Haruto Tsujisaki, Masashi Fujiwara, Hidenori Ando, Yasushi Sato, Tatsuhiro Ishida, Hirofumi Tani, Kenji Tajima
International Journal of Biological Macromolecules, 135938, 135938, Elsevier BV, Sep. 2024
Scientific journal

Elucidating the structural changes of cellulose molecules and dynamics of Na ions during the crystal transition from cellulose I to II in low temperature and low concentration NaOH solution
Yuki Kugo, Satoshi Nomura, Takuya Isono, Shin-ichiro Sato, Masashi Fujiwara, Toshifumi Satoh, Hirofumi Tani, Tomoki Erata, Kenji Tajima
Carbohydrate Polymers, 332, 121907, 121907, Elsevier BV, May 2024
Scientific journal

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, Sep. 2023
Scientific journal

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, Apr. 2023, [Peer-reviewed]
Scientific journal

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, 05 Mar. 2023, [Peer-reviewed]
Japanese, Scientific journal

Development of an absorbance detection module using a deep UV light-emitting diode for a portable miniaturized liquid chromatograph
Kaito Koyama, Takuma Nishimura, Akihiko Ishida, Mitsue Hibino, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
BUNSEKI KAGAKU, 72, 3, 125, 131, Mar. 2023
Japanese, Scientific journal

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, 20 Sep. 2022, [Peer-reviewed]
Scientific journal, 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, Aug. 2022
English, Scientific journal, 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, 17 Jun. 2022, [International Magazine]
English, Scientific journal, 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, 22 Mar. 2022, [International Magazine]
English, Scientific journal, 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, Mar. 2022
Scientific journal, 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, Nov. 2021
Scientific journal

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}), 15 Oct. 2021
Scientific journal

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 and Bioelectronics, 190, 113414, 113414, Elsevier BV, Oct. 2021
Scientific journal

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}), 14 Sep. 2021
English, Scientific journal, The majority of chemical experiments conducted during educational programs are carried out in laboratories because they require instructors with special skills, in addition to large, expensive instruments. Recently, there has been an increasing demand for chemical experiments that can be carried out anywhere. Herein, we propose a novel type of paper-based analytical device (PAD) for enabling quantitative analysis without the requirement for a micropipette, since the PAD features a large sample loading zone and a waste zone, enabling accurate volume control of a liquid sample. We initially employed a micropipette to evaluate the PAD and demonstrate the quantitative analysis of ascorbic acid (AA) and pH using different loading volumes. Finally, we determined the AA concentrations and pH values of commercially available beverages using disposable plastic droppers, and the obtained results were in good agreement with those obtained through conventional methods. This PAD format can therefore be used as a novel educational tool for conducting certain chemical analyses in remote learning environments.

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, SPRINGER HEIDELBERG, Jul. 2021
English, Scientific journal, 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 mu L 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.

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}), 30 Mar. 2021
English, Scientific journal, 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}), 26 Mar. 2021
English, Scientific journal, 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 mu g/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}), 15 Feb. 2021
English, Scientific journal, 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.

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, Feb. 2021
English, Scientific journal, 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. (C) 2020 Elsevier B.V. All rights reserved.

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
English, Scientific journal, 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.

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}), 25 Sep. 2020
English, Scientific journal, 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.

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, Sep. 2020
English, Scientific journal, A non-competitive fluorescence polarization immunoassay (FPIA) using a Fab fragment was developed for large molecule quantification. Most conventional FPIAs are homogeneous and competitive immunoassays. With competitive FPIAs, it has been difficult to obtain sufficient detection sensitivity when targeting large molecules like proteins. To overcome this fundamental drawback, we report a non-competitive FPIA using a fluorescence-labeled Fab fragment. C-reactive protein (CRP) was used as model substance for validation of the Fab-based non-competitive FPIA. Quantitative analysis of CRP in phosphate buffered saline (PBS) was successfully achieved and the limit of detection (LOD) of CRP in PBS was 207 ng/mL. Moreover, using far-red emitting fluorescent dye (HiLyte Fluor (TM) 647) as a fluorescent labeling substance of Fab fragment, we measured CRP in human serum without pretreatment of a sample in 10 min around the cut-off value of CRP (10 mu g/mL). The LOD of CRP in human serum was 1.58 mu g/mL. In our proposed method, the reaction was completed in a simple one-step mixing with only one type of fluorescence-labeled Fab fragment reagent, and no washing operation was required. Therefore, rapid protein quantification was achieved with a greatly simplified procedure.

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}), 29 Jul. 2020
English, Scientific journal, 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}), 21 Jul. 2020
English, Scientific journal, 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 mu m-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 mu m-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, AMER CHEMICAL SOC, May 2020
English, Scientific journal, 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%).

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 SCIENCE SA, 21 Apr. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

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
International conference proceedings, 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
International conference proceedings, 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
International conference proceedings, 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.

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
English, Scientific journal, 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 fragmentbased (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, Sensitive, and Selective Detection of H5 Hemagglutinin from Avian Influenza Virus Using an Immunowall Device
Chavez Ramos Kenia, Nishiyama Keine, Maeki Masatoshi, Ishida Akihiko, Tani Hirofumi, Kasama Toshihiro, Baba Yoshinobu, Tokeshi Manabu
ACS OMEGA, 4, 15, 16683, 16688, AMER CHEMICAL SOC, 08 Oct. 2019, [Peer-reviewed]
English, Scientific journal, 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-V C H VERLAG GMBH, Sep. 2019, [Peer-reviewed]
English, Scientific journal, 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, ROYAL SOC CHEMISTRY, 07 Aug. 2019, [Peer-reviewed]
English, Scientific journal, © 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.

High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer
Wakao Osamu, Satou Ken, Nakamura Ayano, Galkina Polina A, Nishiyama Keine, Sumiyoshi Ken, Kurosawa Fumio, Maeki Masatoshi, Ishida Akihiko, Tani Hirofumi, Proskurnin Mikhail A, Shigemura Koji, Hibara Akihide, Tokeshi Manabu
LAB ON A CHIP, 19, 15, 2581, 2588, ROYAL SOC CHEMISTRY, 07 Aug. 2019, [Peer-reviewed]
English, Scientific journal, © 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.

An Electrochemical Sensor Based on Structure Switching of Dithiol-Modified Aptamer for Simple Detection of Ochratoxin A
Donny Nugraha Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
Analytical Sciences, 35, 11, 1221, 1226, JAPAN SOC ANALYTICAL CHEMISTRY, Jul. 2019, [Peer-reviewed]
English, Scientific journal, 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.

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, 15 Apr. 2019, [Peer-reviewed]
English, Scientific journal, 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.

Label-Free Electrochemical Sensor for Ochratoxin A Using a Microfabricated Electrode with Immobilized Aptamer
Mazaafrianto Donny N, Ishida Akihiko, Maeki Masatoshi, Tani Hirofumi, Tokeshi Manabu
ACS OMEGA, 3, 12, 16823, 16830, AMER CHEMICAL SOC, Dec. 2018, [Peer-reviewed]
English, Scientific journal, 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, Aug. 2018
Japanese

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, 09 May 2018, [Peer-reviewed]
English, Scientific journal, 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, 25 Apr. 2018, [Peer-reviewed]
English, Scientific journal, 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, American Institute of Physics Inc., 01 Feb. 2018, [Peer-reviewed]
English, Scientific journal, 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
International conference proceedings, 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, [Peer-reviewed]
English, Scientific journal, 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, Nov. 2017, [Peer-reviewed]
English, Scientific journal, 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, Oct. 2017, [Peer-reviewed]
English, Scientific journal, 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, Oct. 2017, [Peer-reviewed]
English, Scientific journal, 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, Aug. 2017
Japanese

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, Aug. 2017
Japanese

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, Aug. 2017
Japanese

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, Dec. 2016, [Peer-reviewed]
English, Scientific journal, 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, Nov. 2016, [Peer-reviewed]
English, Scientific journal, 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, Nov. 2016, [Peer-reviewed]
English, Scientific journal, 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, Aug. 2016
Japanese

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, Aug. 2016, [Peer-reviewed]
English, Scientific journal, 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, May 2016, [Peer-reviewed]
English, Scientific journal, 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, [Peer-reviewed]
English, Scientific journal, 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, [Peer-reviewed]
English, Scientific journal, 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, [Peer-reviewed]
English, Scientific journal, 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, Nov. 2015, [Peer-reviewed]
English, Scientific journal, 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, Oct. 2015, [Peer-reviewed]
English, Scientific journal, 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.

Development of High-performance Immuno-pillar Devices: Improvement of Antibody-immobilized Solid Support
Nanako Nishiwaki, Toshihiro Kasama, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
BUNSEKI KAGAKU, 64, 5, 329, 335, JAPAN SOC ANALYTICAL CHEMISTRY, May 2015, [Peer-reviewed]
Japanese, Scientific journal, 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.

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, [Peer-reviewed]
English, Scientific journal, 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.

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, [Peer-reviewed]
English, Scientific journal, 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.

Bioluminescence Assay Based on Controlled Inhibitory Effect on Firefly Luciferase
Ryoko Kurishiba, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
LUMINESCENCE, 29, 78, 78, WILEY-BLACKWELL, Aug. 2014, [Peer-reviewed]
English

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, Aug. 2014, [Peer-reviewed]
English

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, Aug. 2014, [Peer-reviewed]
English

A phosphor sheet providing wider color gamut for LCDs　　　　　　　　　　　　　　　
Yasushi Ito, Tomomitsu Hori, Hirofumi Tani, Tsuneo Kusunoki, Hirofumi Kondo
Proceedings of the International Display Workshops, 2, 750, 752, 2012
English, International conference proceedings, A phosphor sheet in which thiogallate green and sulfide red phosphor are contained has been developed. Good color gamut performance of 90% NTSC-xy can be realized by a LCD with that. It has moisture barrier layers on the surface, which can reduce the reaction between these phosphors and moisture.

A phosphor sheet providing wider color gamut for LCDs
Yasushi Ito, Tomomitsu Hori, Hirofumi Tani, Tsuneo Kusunoki, Hirofumi Kondo
IDW/AD '12: PROCEEDINGS OF THE INTERNATIONAL DISPLAY WORKSHOPS, PT 3, 19, 1663, 1665, INST IMAGE INFORMATION & TELEVISION ENGINEERS, 2012
English, International conference proceedings, A phosphor sheet in which thiogallate green and sulfide red phosphor are contained has been developed. Good color gamut performance of 90% NTSC-xy can be realized by a LCD with that. It has moisture barrier layers on the surface, which can reduce the reaction between these phosphors and moisture.

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, Sep. 2009
English, Scientific journal, 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.

Effect of cationic surfactants on enhancement of firefly bioluminescence in the presence of liposomes
Hirofumi Tani, Mizuyo Notani, Tamio Kamidate
ANALYTICAL SCIENCES, 24, 9, 1111, 1115, JAPAN SOC ANALYTICAL CHEMISTRY, Sep. 2008
English, Scientific journal, Firefly bioluminescence (BL) was greatly affected by cationic surfactants coexisting with liposomes containing phosphatidylcholine and cholesterol. In this study, the effects of the type and concentration of cationic surfactants on BL were studied in the presence of the liposomes. Three types of cationic surfactant: benzalkonium chloride (BAC), n-dodecyltrimethylammonium bromide (DTAB), and benzethonium chloride (BZC), were used. As a common effect in these surfactants, BL intensity was increased and then drastically decreased with increasing surfactant concentration. This can be explained by the formation of cationic liposomes as BL enhancers at low concentration of the surfactant, and by the transformation into cationic (mixed) micelles as inhibitors at high concentration. The maximal BL intensity and the concentration for the maximal BL were dependent on the type of the surfactants. To explain the differences in these parameters in the enhanced BL, we determined the distribution coefficient, K, of the surfactants to the liposomal membrane. The result indicated that the surfactant with higher K value gives the maximal BL intensity at lower concentration.

Direct determination of horseradish peroxidase encapsulated in liposomes by using luminol chemiluminescence
Tamio Kamidate, Kanako Komatsu, Hirofumi Tani, Akihiko Ishida
ANALYTICAL SCIENCES, 24, 4, 477, 481, JAPAN SOC ANALYTICAL CHEMISTRY, Apr. 2008
English, Scientific journal, 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.

A unimolecular nanocapsule: Encapsulation property of amphiphilic polymer based on hyperbranched polythreitol
Yoshikazu Kitajyo, Yumiko Nawa, Masaki Tamaki, Hirofumi Tani, Kenji Takahashi, Harumi Kaga, Toshifumi Satoh, Toyoji Kakuchi
POLYMER, 48, 16, 4683, 4690, ELSEVIER SCI LTD, Jul. 2007
English, Scientific journal, Hyperbranched polythreitol (1) with different molecular weights (M-w,M-SLS: 1.18 x 10(4) and 4.79 x 10(4)) was reacted with trityl chloride in DMF to afford a novel amphiphilic polymer (2) consisting of 1 as the hydrophilic core and the trityl groups as the hydrophobic shell. Compound 2 was tested for its ability to act as a unimolecular nanocapsule toward the water-soluble dye, rose bengal (RB). Their encapsulation and release properties were also evaluated by comparison with the degree of substitution (DS) of the trityl groups, i.e., the hydrophobic shell density. The polymers were found to have very good unimolecular nanocapsule characteristics even at extremely low concentrations. The average number of RBs per polymer molecule depended on the hydrophilic core size and the hydrophobic shell density. The increasing DS value led to a decrease in the encapsulated amount due to the decrease in the hydrophilic core space, while the low DS value (less than ca. 20 rnol%) led to a destabilization as a unimolecular nanocapsule and a lower encapsulation ability. In particular, 2 with ca. 23% DS value showed an efficient encapsulation. Based on a release test of the RB-loaded unimolecular nanocapsules, the polymers showed a high RB-holding ability in water. (c) 2007 Elsevier Ltd. All rights reserved.

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, May 2007
English, Scientific journal, 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.

Encapsulation-release property of amphiphilic hyperbranched D-glucan as a unimolecular reverse micelle
Yoshikazu Kitajyo, Tomoko Imai, Yoko Sakai, Masaki Tamaki, Hirofumi Tani, Kenji Takahashi, Atsushi Narumi, Harumi Kaga, Noriaki Kaneko, Toshifumi Satoh, Toyoji Kakuchi
POLYMER, 48, 5, 1237, 1244, ELSEVIER SCI LTD, Feb. 2007
English, Scientific journal, The synthesis of a novel unimolecular reverse micelle, the hyperbranched D-glucan carbamate (3), was accomplished through the carbamation reaction of the hyperbranched D-glucan (1) with the N-carbonyl L-leucine ethyl ester (2) in pyridine at 100 degrees C. Polymer 3 was soluble in a large variety of organic solvents, such as methanol, acetone, chloroform, and ethyl acetate, and insoluble in water, which remarkably differed from the solubility of 1. The degree of carbamate substitution (DS) for 3 was controlled by the feed rate of 2, and the DS values were in the range of 46.0-93.7%. Polymer 3 possessed the encapsulation ability for water-soluble molecules, such as rose bengal, thymol blue, and alizarin yellow in chloroform, and the encapsulation ability depended on the hydrophilicity of 3 and the molecular size of the dye. The rose bengal (RB) encapsulated polymer (RB/3) showed a slow release from the RB/3 system into water at neutral pH, while the release rate was significantly accelerated by the hydrolysis of the hydrophobic polymer shell under basic conditions. (c) 2007 Elsevier Ltd. All rights reserved.

On-chip bioassay using immobilized sensing bacteria in three-dimensional microfluidic network.
Tani, H., Maehana, K., Kamidate, T.
Methods in molecular biology (Clifton, N.J.), 385, 37, 52, 2007
Scientific journal

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, Jan. 2007
English, Scientific journal, 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.

On-chip genotoxic bioassay based on bioluminescence reporter system using three-dimensional microfluidic network
K Maehana, H Tani, T Kamidate
ANALYTICA CHIMICA ACTA, 560, 1-2, 24, 29, ELSEVIER SCIENCE BV, Feb. 2006
English, Scientific journal, Microchip-based genotoxic bioassay using sensing Escherichia coli strains has been performed. in this method, the assay was conducted in three-dimensional microfluidic network constructed by a silicon perforated microwell array chip and two poly(dimethylsiloxane) (PDMS) multi-microchannel chips. The sensing strains having firefly luciferase reporter gene under transcriptional control of umuD as an SOS promoter were put into the channels on one of the PDMS chips and immobilized in the silicon microwells. Samples containing genotoxic substances and substrates for luciferase were into the channels on the other PDMS chip. The optimum conditions of the assay in the on-chip format have been investigated using mitomycin C(MMC) as a genotoxic substance. As a result, the dose-dependence of bioluminescence intensity was obtained at once on the chip. Additionally, the response ratios of the bioluminescence between mutagen-and non-induced strains were successfully enhanced by improving the on-chip assay methods and conditions. Several well-known genotoxic substances were subjected to the on-chip assay, and were detected with the detection limits comparable to those in the conventional method with reduced time. (c) 2005 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, Jan. 2006
English, Scientific journal, 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, Jul. 2005
English, Scientific journal

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, Jun. 2005
Japanese, Scientific journal, 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.

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, Jun. 2005
English, Scientific journal, 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.

変異原物質のオンチップバイオアッセイ　　　　　　　　　　　　　　　
Electrochemistry, 73, 3, 215, 219, 2005

Chip-based bioassay using bacterial sensor strains immobilized in three-dimensional microfluidic network
H Tani, K Maehana, T Kamidate
ANALYTICAL CHEMISTRY, 76, 22, 6693, 6697, AMER CHEMICAL SOC, Nov. 2004
English, Scientific journal, A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays.

Effects of using a low-copy plasmid and controlling membrane permeability in SOS-based genotoxic bioassay
K Maehana, H Tani, T Shiba, T Kamidate
ANALYTICA CHIMICA ACTA, 522, 2, 189, 195, ELSEVIER SCIENCE BV, Sep. 2004
English, Scientific journal, A whole-cell assay based on bacterial SOS response has been used for genotoxicity evaluation of chemical substances. In this study, for improvement of induction ratio and detection limit in the genotoxic assay, three modifications to an original tester strain, Escherichia coli harboring a multi-copy plasmid-borne fusion of firefly luciferase and the umuD promoter, were examined singly or in combinations. First, the effect of replacement of the multi-copy plasmid with a low-copy one was examined. Bioluminescence (BL) emission from the E. coli strain having the low-copy plasmid induced by mitomycin C (MMC) as a genotoxic substance was decreased compared to that from the original strain, but the BL emission ratio of the induced strain to the non-induced one was increased by two-fold at maximum due to reduced blank BL emission from the non-induced strain. Next, two modifications were made in view of control of the membrane permeability for substances by using toluene or a tolC mutant. Toluene promoted the membrane permeation of substances, while tolC mutation inhibited the efflux of them via cell membranes. The addition of toluene to the strain resulted in an increase in the BL ratio by increasing BL emission from the induced strain. On the other hand, by the use of tolC mutation, the dose-dependency curve of the BL ratio shifted to a lower concentration range of MMC, and the detection limit of MMC was therefore greatly improved. Compared to the original tester strain, in combination with the use of the low-copy plasmid. the maximum induction ratio was increased about four-fold to above 400 by the addition of toluene, whereas the detection limit was improved 1000-fold to below 10 pg ml(-1) by the use of a tolC mutant. The effects of each modification and their combination on the induction ratio and detection limit were examined with various substances. (C) 2004 Elsevier B.V. All rights reserved.

Effects of cytochrome b(5) on drug oxidation activities of human cytochrome P450 (CYP) 3As: similarity of CYP3A5 with CYP3A4 but not CYP3A7
S Yamaori, H Yamazaki, A Suzuki, A Yamada, H Tani, T Kamidate, K Fujita, T Kamataki
BIOCHEMICAL PHARMACOLOGY, 66, 12, 2333, 2340, PERGAMON-ELSEVIER SCIENCE LTD, Dec. 2003
English, Scientific journal, Effects of cytochrome b(5) (b(5)) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b(5) in approximately one third of the substrates employed in this study. Such enhancement by b(5) was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b(5) with any substrates employed. V-max values for midazolam 1'-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b(5), which was also seen with CYP3A4, although the extent of the effects of b(5) on S-50 (K-m) and Hill coefficient differed dependent on substrates used. In contrast, b(5) did not alter any of these kinetic parameters of CYP3A7. The effects of b(5) on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b(5) in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7. (C) 2003 Elsevier Inc. All rights reserved.

Use of cholate derivatives with submicellar concentration for controlling selectivity of proteins in hydrophobic interaction chromatography
H Tani, T Matsubara, T Kamidate
JOURNAL OF CHROMATOGRAPHY A, 1016, 1, 51, 60, ELSEVIER SCIENCE BV, Oct. 2003
English, Scientific journal, Hydrophobic interaction chromatography (HIC) of proteins using a phenyl column has been performed in the presence of various surfactants with micellar and submicellar concentration ranges. Most surfactants were effective for a decrease in the retention of proteins in both concentration ranges. However, the use of anionic cholate derivatives increased the retention of the proteins with high isoelectric point, such as lysozyme, cytochrome c, and trypsin, in submicellar concentration range, and then decreased it above the critical micellar concentration, while the retention of the other proteins was monotonously decreased. The results of frontal chromatographic analysis of the surfactant and capillary electrophoresis for the proteins in the presence of surfactant show that in the submicellar concentration range, cholate derivatives allowed to be adsorbed on the stationary phase. while they exhibited no interactions with the proteins. Thus, it appeared that the increase in the retention of basic proteins was due to the electrostatic attraction between the proteins and cholate-modified stationary phase. We have applied the unique property of cholate to the separation of ovalbumin and lysozyine in egg white sample using hydrophobic chromatography. (C) 2003 dElsevier B.V. All rights reserved.

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, Aug. 2003
English, Scientific journal, 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.

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, Apr. 2003
English, Scientific journal, 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.

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, Mar. 2002
English, Scientific journal, 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+.

Affinity partitioning of histidine-tagged cytochrome b(5) and an oligopeptide in an aqueous micellar two-phase system containing a metal-chelating polymer
K Maehana, H Tani, T Kamataki, T Kamidate
BUNSEKI KAGAKU, 51, 1, 13, 19, JAPAN SOC ANALYTICAL CHEMISTRY, Jan. 2002, [Peer-reviewed]
Japanese, Scientific journal, A metal affinity interaction between metals and proteins was exploited for the selective partitioning of histidine-tagged membrane proteins in an aqueous micellar two-phase system. The system used was a mixture of non-ionic surfactant micelle, Triton X-100, and a metal-chelating polymer, Ni(II)-imminodiacetic acid-poly (ethylene glycol) (NHDA-PEG). The mixture separated into two phases, a polymer-enriched upper phase and a micelle-enriched lower phase, under an appropriate condition. The effect of the Ni-IDA-PEG concentration on the partitioning of cytochrome b(5) with and without a histidine-tag, and a fluorescein isothiocyanate-labeled oligopeptide containing six histidines was examined in two-phase systems containing various pH buffers. Cytochrome b(5) without a histidine-tag was partitioned into a micelle-rich phase, independent of the Ni-IDA-PEG concentration, due to a hydrophobic interaction. On the other hand, with increasing the concentration of Ni-IDA-PEG, the partitioning of histidine-tagged cytochrome b(5) was shifted to the polymer-rich phase in a system containing a phosphate or borate buffer. Upon the addition of imidazole to, or the removal of Ni(II) from the two-phase systems, the histidine-tagged protein showed the same partition behavior as that of the protein without a histidine-tag. These results indicate that the metal-chelating polymer plays the role of an affinity ligand, which enables histidine-tagged membrane proteins to move out from the micelle-rich phase to the polymer-rich phase. When Tris was used as a pH buffer, no affinity partitioning of the protein was observed. Although the oligopeptide also partitioned into the polymer phase in the presence of Ni-IDA-PEG, the partitioning was not dependent on the buffer type. Thus, the partitioning seems to be largely affected by the buffer type as well as the protein structure. The present system has a potential for a simple and rapid purification method for the histidine-tagged membrane proteins.

Affinity partitioning of histidine-tagged cytochrome b5 and an oligopeptide in an aqueous micellar two-phase system containing a metal-chelating polymer
Koji Maehana, Hirofumi Tani, Tetsuya Kamataki, Tamio Kamidate
Bunseki Kagaku, 51, 1, 13, 19, Japan Society for Analytical Chemistry, 2002
Japanese, Scientific journal, A metal affinity interaction between metals and proteins was exploited for the selective partitioning of histidine-tagged membrane proteins in an aqueous micellar two-phase system. The system used was a mixture of non-ionic surfactant micelle, Triton X-100, and a metal-chelating polymer, Ni(II)-imminodiacetic acid-poly (ethylene glycol) (Ni-IDA-PEG). The mixture separated into two phases, a polymer-enriched upper phase and a micelle-enriched lower phase, under an appropriate condition. The effect of the Ni-IDA-PEG concentration on the partitioning of cytochrome b5 with and without a histidine-tag, and a fluorescein isothiocyanate-labeled oligopeptide containing six histidines was examined in two-phase systems containing various pH buffers. Cytochrome b 5 without a histidine-tag was partitioned into a micelle-rich phase, independent of the Ni-IDA-PEG concentration, due to a hydrophobic interaction. On the other hand, with increasing the concentration of Ni-IDA-PEG, the partitioning of histidine-tagged cytochrome b5 was shifted to the polymer-rich phase in a system containing a phosphate or borate buffer. Upon the addition of imidazole to, or the removal of Ni(II) from the two-phase systems, the histidine-tagged protein showed the same partition behavior as that of the protein without a histidine-tag. These results indicate that the metal-chelating polymer plays the role of an affinity ligand, which enables histidine-tagged membrane proteins to move out from the micelle-rich phase to the polymer-rich phase. When Tris was used as a pH buffer, no affinity partitioning of the protein was observed. Although the oligopeptide also partitioned into the polymer phase in the presence of Ni-IDA-PEG, the partitioning was not dependent on the buffer type. Thus, the partitioning seems to be largely affected by the buffer type as well as the protein structure. The present system has a potential for a simple and rapid purification method for the histidine-tagged membrane proteins.

Effect of the geometry of microfabricated flow reactors on chemiluminescent detection of epinephrine with lucigenin
T Kamidate, T Kaide, H Tani, E Makino, T Shibata
LUMINESCENCE, 16, 5, 337, 342, JOHN WILEY & SONS LTD, Sep. 2001
English, Scientific journal, Three types of flow reactors with different lengths (12-126 mm) and widths (0.4-2.0 nun) of channel were made on the silicon chip by microfabrication techniques for the chemiluminescent (CL) detection of epinephrine (EP) with lucigenin (Luc). The volume of each CL reactor was about 10 muL. A solution containing EP and Luc and a solution containing NaOH and periodate were injected successively into each inlet of the CL reactor in the range 20-100 muL/min with a pressure-driven flow system. The intensity of light emission was dependent on the geometry of the flow reactors. These results could be explained in terms of the differences in the diffusion length of the reactants in the flow reactors. The maximum light emission were linearly correlated, with the concentrations of EP over the range from the detection lit-nit of 5.0 x 10(-8) mol/L up to 5.0 x 10(-6) mol/L on the use of the CL reactor with the most promising geometry. Copyright (C) 2001 John Wiley & Sons, Ltd.

Effect of mixing modes on chemiluminescent detection of epinephrine with lucigenin by an FIA system fabricated on a microchip
T Kamidate, T Kaide, H Tani, E Makino, T Shibata
ANALYTICAL SCIENCES, 17, 8, 951, 955, JAPAN SOC ANALYTICAL CHEMISTRY, Aug. 2001
English, Scientific journal, The chemiluminescent (CL) detection of epinephrine (EP) with lucigenin (Luc) was performed using a micro flow cell fabricated on a silicon chip. A solution of EP was injected into the Luc carrier stream. The Luc solution containing EP and an alkaline solution were successively poured into the flow cell by a pressure-driven flow system. Two types of flow cells were fabricated for estimating the effect of the mixing modes in the flow cells on the intensity of light emission. In flow cell 1, two streams entered through separate inlet ports and merged to flow adjacently. In flow cell 2, a Luc solution containing EP was split up to 36 partial flows by passage through the nozzles, and was injected into the alkaline solution. The intensity of light emission in flow cell 2 increased markedly compared to that in flow cell 1. The detection limit of 8.0 x 10(-7) M for EP in flow cell 2 was a factor of six-times better than that in flow cell 1. The improvement in the sensitivity for EP could be explained in terms of the distortion of laminar flow in flow cell 2.

Enhancement of the excluded-volume effect in protein extraction using triblock copolymer-based aqueous micellar two-phase systems
H Tani, Y Suzuki, A Matsuda, T Kamidate
ANALYTICA CHIMICA ACTA, 429, 2, 301, 309, ELSEVIER SCIENCE BV, Feb. 2001
English, Scientific journal, Triblock copolymer surfactants consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), Pluronic L61 (PEO-PPO-PEO, L61) and Pluronic 25R2 (PPO-PEO-PPO, 25R2) were exploited in aqueous micellar two-phase systems for the protein extraction. The extraction was based on the phase separation into surfactant-depleted and -condensed phases (an aqueous and a surfactant-rich phases, respectively) upon warming aqueous micellar solutions of triblock copolymer. In both systems, hydrophilic proteins such as albumin were not extracted into the surfactant-rich phase. On the other hand, hydrophobic cytochrome b(5) was well extracted in the L61 system due to hydrophobic interaction. However, the extraction of cytochrome b(5) was not observed in the 25R2 system. This abnormal extractability of cytochrome b5 in the 25R2 system was explained by the enhanced excluded-volume interaction between cytochrome b(5) and 25R2 micellar network in the surfactant-rich phase, which overcomes the hydrophobic interaction. Additionally, ionic surfactants were added into the systems for controlling extractability of proteins. In the 25R2 system, cationic tetradecyltrimethylammonium was effective for extracting anionic cytochrome bs against the excluded-volume effect, while not for anionic albumin because of its large molecular weight. In 25R2 system containing ionic surfactant, the partitioning of proteins were found to be governed by the hydrophobic, excluded-volume, and electrostatic interactions. Micellar network formed by 25R2 type of surfactant with a strong excluded-volume interaction could provide new selective extraction systems for the separation of proteins. (C) 2001 Elsevier Science B.V. All rights reserved.

Application of horseradish peroxidase-encapsulated liposomes as labels for immunodotblotting
T Suita, H Tani, T Kamidate
ANALYTICAL SCIENCES, 16, 5, 527, 529, JAPAN SOC ANALYTICAL CHEM, May 2000
English, Scientific journal

A kinetic assay for cytochrome b(5) based on electron transfer to cytochrome c
H Tani, T Akasaka, T Kamidate
CHEMISTRY LETTERS, 6, 471, 472, CHEMICAL SOC JAPAN, Jun. 1999
English, Scientific journal, Cytochrome b(5)-mediated electron-transfer reaction from NADH to cytochrome c was exploited for the determination of cytochrome b(5). A linear relationship between cytochrome b(5) concentration and the reducing rate of cytochrome c was obtained in the range of 15-700 pM.

Effect of charged water-soluble polymers on the extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system
H Tani, T Ooura, T Kamidate
BUNSEKI KAGAKU, 47, 12, 965, 970, JAPAN SOC ANALYTICAL CHEM, Dec. 1998, [Peer-reviewed]
Japanese, Scientific journal, The effect of charged water-soluble polymers on the extractability of membrane proteins in a Triton X-114-based aqueous micellar two-phase system was examined. Cytochrome b(5) (Cyt.b(5)), NADH-cytochrome b(5) reductase (fp1), and NADPH-cytochrome P450 reductase (fp2), used as model proteins, were to be well-extracted into a surfactant-rich phase without polymers. In the presence of non-charged dextran, there were no changes in their extractability. However, by the addition of cationic diethylaminoethyldextran at the concentration range of 0.01 similar to 0.3%, the extractability of Cyt.b(5) and fp2 was greatly reduced. In contrast, the use of anionic dextran sulfate at the same concentration range depressed the extraction of fp1 alone. A further increase in the polymer concentration or salt addition resulted in an increase in the extractability. From a calculation of net charge of the proteins, it was shown that Cyt.b(5) and fp2 are negative and fp1 is positive at pH 7.4, where the extraction was conducted. Thus, the depressed extraction of the proteins in the presence of the charged polymers is attributable to the electrostatic interaction between the proteins and the charged polymers in the aqueous phase. These results indicate that charged polymers could be effective for the selective extraction of membrane proteins in a Triton X-114-based aqueous micellar two-phase system.

Enhancement of firefly bioluminescence using liposomes containing cationic components
T Kamidate, T Fujita, H Tani, H Watanabe
ANALYTICAL SCIENCES, 14, 6, 1115, 1119, JAPAN SOC ANALYTICAL CHEM, Dec. 1998
English, Scientific journal, The effect of cationic components in liposome on the bioluminescence (BL) intensity from the firefly luciferin-luciferase reaction with ATP was investigated by use of vesicles formed by the extrusion technique (VET). Stearyltrimethyl-ammonium chloride (STAC), stearylamine (SA) and tetracaine (TC) were used as cationic components. The VET containing STAC and SA enhanced the maximum BL intensity. In contrast, a lowering of the maximum BL intensity was observed in the presence of the VET containing TC. The sensitivity for ATP in the presence of the VET containing STAC was improved by a factor of 2 and 10 times compared to that in the presence of the VET containing SA and that in water alone, respectively. The differences in the BL enhancement between cationic components could be explained in terms of different membrane surface potentials of the VET containing these cationc components.

Aqueous micellar two-phase systems for protein separation
H Tani, T Kamidate, H Watanabe
ANALYTICAL SCIENCES, 14, 5, 875, 888, JAPAN SOC ANALYTICAL CHEMISTRY, Oct. 1998
English, Scientific journal, The extractive technique for protein purification based on two-phase separation in aqueous micellar solutions (aqueous micellar two-phase system (AMTPS)) is reviewed. The micellar solution of a nonionic surfactant, such as polyoxyethylene alkyl ether, which is most frequently used for protein extraction, separates into two phases upon heating above its cloud point. The two phases consist of a surfactant-depleted phase (aqueous phase) and a surfactant-rich phase. Hydrophilic proteins are partitioned to the aqueous phase and hydrophobic membrane proteins are extracted into the surfactant-rich phase. Because of the methodological simplicity and rapidity, this technique has become an effective means, and thus has been widely used for the purification and characterization of proteins. In contrast to polyoxyethylene alkyl ether, micellar solutions of a zwitterionic surfactant, such as alkylammoniopropyl sulfate, separate below the critical temperature. Alkylglucosides can also separate into two phases upon adding water-soluble polymers. Recently, these two-phase systems have been exploited for protein separation. Additionally, hydrophobic affinity ligands, charged polymers, and ionic surfactants have been successfully used for controlling the extractability of proteins in AMTPS.

Separation of microsomal cytochrome b(5) via phase separation in a mixed solution of Triton X-114 and charged dextran
H Tani, T Ooura, T Kamidate, T Kamataki, H Watanabe
JOURNAL OF CHROMATOGRAPHY B, 708, 1-2, 294, 298, ELSEVIER SCIENCE BV, Apr. 1998
English, Scientific journal, The successful introduction of a charged dextran into the Triton X-114 phase separation system for the selective extraction of cytochrome b(5) (cyt. b(5)) in liver microsomes is described. In the absence of charged dextran, 55% of total microsomal proteins and 84% of cyt. b(5) were extracted into the surfactant-rich phase. In the presence of anionic dextran sulfate, the extractability of total microsomal proteins was greatly reduced while that of cyt. b(5) was increased. After triplicate extraction, cyt. b(5) was purified more than 10-fold from microsomes with a recovery of 91% in the surfactant-rich phase. In view of its operational simplicity, this method provides a good means for the partial purification of cyt. b(5) prior to chromatographic separations. (C) 1998 Elsevier Science B.V.

Triton X-114を用いたミセル水性二相分配法による膜タンパク質の抽出に及ぼす荷電水性ポリマーの添加効果
分析化学, 47, 12, 965, 970, 1998

Extraction of proteins based on phase separation in aqueous triblock copolymer surfactant solutions
H Tani, A Matsuda, T Kamidate, H Watanabe
ANALYTICAL SCIENCES, 13, 6, 925, 929, JAPAN SOC ANALYTICAL CHEM, Dec. 1997, [Peer-reviewed]
English, Scientific journal, Triblock copolymer surfactants, Pluronic L61 (L61) and Pluronic 25R2 (25R2), consisting of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) were utilized for the extraction of proteins. The extraction was based on phase separation into surfactant-depleted and -condensed phases (an aqueous phase and a surfactant-rich phase) upon warming aqueous solutions of L61 and 25R2. Hydrophilic cytochrome c was not extracted into the surfactant-rich phase in either Pluronics. However, hydrophobic cytochrome b(5) was extracted with L61, while not with 25R2. The difference in the extractability of cytochrome b(5) and cytochrome c is probably due to the structural difference in L61 and 25R2.

Polymer-induced phase separation in aqueous micellar solutions of octyl-beta-D-thioglucoside for extraction of membrane proteins
H Tani, T Saitoh, T Kamidate, T Kamataki, H Watanabe
BIOTECHNOLOGY AND BIOENGINEERING, 56, 3, 311, 318, JOHN WILEY & SONS INC, Nov. 1997, [Peer-reviewed]
English, Scientific journal, A water-soluble polymer such as polyethylene glycol (PEG), Dextran T-500 (Dx), or diethylaminoethyl-Dextran (DEAE-Dx) induced aqueous micellar solutions of octyl-beta-D-thioglucoside (OTG) to phase separation at 0 degrees C. One of the two phases thus formed is a surfactant-depleted aqueous solution (aqueous phase) of a water-soluble polymer and the other a concentrated OTG solution (surfactant-rich phase). In a combination of OTG with PEG or Dx, cytochrome P450 (P450) and cytochrome b(5) (b(5)) were well extracted into the surfactant-rich phase. The extraction yield of P450 was slightly greater than that of b(5). In contrast to PEG and Dx, DEAE-Dx markedly reduced the extraction of b(5), while that of P450 remained almost unchanged. DEAE-Dx served the dual functions of inducing the phase separation and preventing the extraction of b(5) into the surfactant-rich phase. This depressed extraction of b(5) was reversed by the addition of potassium phosphate. DEAE-Dx and potassium phosphate proved effective in controlling the extractability of b(5). The polymer-induced phase separation provides a new basis for highly efficient extraction of membrane proteins under mild conditions that should be acceptable for thermolabile membrane proteins under physiological conditions. (C) 1997 John Wiley & Sons, Inc.

Selective solubilization of microsomal electron-transfer proteins with alkylglucoside
H Tani, T Saitoh, T Kamidate, T Kamataki, H Watanabe
ANALYTICAL SCIENCES, 13, 5, 747, 751, JAPAN SOC ANALYTICAL CHEM, Oct. 1997, [Peer-reviewed]
English, Scientific journal, A series of alkylglucosides (AGs) was examined for the solubilization of four microsomal electron-transfer proteins (cytochrome P450 (P450), cytochrome b(5) (b(5)), NADPH-cytochrome P450 reductase (fp2), and NADH-cytochrome b(5) reductase (fp1)) in rat liver microsomes. Among the four proteins, b(5) and fp2 were completely solubilized, and thus, almost recovered in the supernatant fraction after centrifugation, while P450 and fp1 were in the pellet. In particular, the solubilization yield of P450 was only about 10%. With a high ratio of alkylglucoside to membrane, along with a low ionic strength, a greater selectivity for b(5) and fp2 could be obtained. Such high selectivity was not observed by the use of sugar ester, bile salt, and poly(oxyethylene) alkylphenyl ether types of surfactants. After re-solubilization of the pellet with sodium cholate, the supernatant fraction contained P450 free From b(5) and the final recovery of P450 was about 40%. Thus, this two-step solubilization provides a simple method for the partial purification of P450 in microsomes.

Selective uptake of copper(II) and zinc(II) ions using liposomes without ionophores
T Kamidate, J Yamaguchi, T Suita, H Tani, H Watanabe
CHEMISTRY LETTERS, 10, 971, 972, CHEMICAL SOC JAPAN, 1997, [Peer-reviewed]
English, Scientific journal, The multilamellar vesicles (MLVs) without ionophores were applied to the separation media of Cu2+ and Zn2+. Mn2+, Fe2+, Co2+ and Ni2+ were impermeable into the MLVs. The uptake rate and efficiency of Cu2+ were dependent on the charge-type of the MLVs surface, while the effect of copper salts on the uptake rate and efficiency was little observed.

Equilibrium study on interactions between proteins and bile-salt micelles by micellar electrokinetic chromatography
T Saitoh, T Fukuda, H Tani, T Kamidate, H Watanabe
ANALYTICAL SCIENCES, 12, 4, 569, 573, JAPAN SOC ANALYTICAL CHEM, Aug. 1996, [Peer-reviewed]
English, Scientific journal, Interactions between proteins and bile-salt micelles were evaluated on the basis of the binding constants, which were obtained from the migration of proteins in micellar electrokinetic chromatography. The binding constants, K-b=[P-b]/([P-w][B]), where [P-b] is the concentration of a protein binding with a bile-salt, [P-w] free protein concentration, and [B] the concentration of bile salt present in the micelles, were successfully determined from the slope of a linear curve of the capacity factor (k') of the protein against [B]. The binding constants were almost identical to those obtained by a gel-filtration method. The value of K-b increased with increasing the hydrophobicity of the bile salt or that of the protein.

PHASE-SEPARATION IN AQUEOUS MICELLAR SOLUTIONS OF NONIONIC SURFACTANTS FOR PROTEIN SEPARATION
T SAITOH, H TANI, T KAMIDATE, H WATANABE
TRAC-TRENDS IN ANALYTICAL CHEMISTRY, 14, 5, 213, 217, ELSEVIER SCI LTD, May 1995, [Peer-reviewed]
English, Scientific journal, The use of phase separation in aqueous micellar solutions of nonionic surfactants is introduced as a separation method membrane proteins. Recent progress phase separation is also described.

POLYMER-INDUCED PHASE-SEPARATION IN AQUEOUS MICELLAR SOLUTIONS OF ALKYLGLUCOSIDES FOR PROTEIN EXTRACTION
T SAITOH, H TANI, T KAMIDATE, T KAMATAKI, H WATANABE
ANALYTICAL SCIENCES, 10, 2, 299, 303, JAPAN SOC ANALYTICAL CHEM, Apr. 1994, [Peer-reviewed]
English, Scientific journal, Aqueous micellar solutions of alkylglucosides were separated into two phases at 0-degree-C upon addition of polyethylene glycol 6000 (PEG) or Dextran T-500 (Dextran). One was an aqueous phase in which hydrophilic proteins, cytochrome c and peroxidase (horseradish), were retained, and the other was a surfactant-rich phase into which hydrophobic membrane proteins, bacteriorhodopsin and cytochrome b5, were extracted. A combination of octyl-beta-D-thioglucoside (OTG) (or nonyl-beta-D-glucoside (NG)) with PEG (or Dextran) was the best choice for extraction of hydrophobic proteins. Extraction yields (50 - 90%) and concentration factors (7 - 30) of the hydrophobic proteins were dependent on the types of nonionic surfactants and water-soluble polymers. Solubilization and phase separation in processing cell membranes could be made in a single step at 0-degree-C. Hence the present method would be useful for the purification of thermolabile proteins.

AGGREGATION OF N-OCTANOYL-N-PHENYLHYDROXYLAMINE IN CARBON-TETRACHLORIDE
T SAITOH, H TANI, T KAMIDATE, H WATANABE, K HARAGUCHI, S ABE
ANALYTICAL SCIENCES, 9, 3, 345, 349, JAPAN SOC ANALYTICAL CHEM, Jun. 1993, [Peer-reviewed]
English, Scientific journal, The aggregation number of N-octanoyl-N-phenylhydroxylamine (C8-PHA) in carbon tetrachloride was determined on the basis of the extraction equilibria of copper(II) chelate with C8-PHA between aqueous and carbon tetrachloride phases. According to the relation of the distribution ratio of copper(II) with its concentration in the organic phase, the formation of a reversed micelle consisting or approximately 13 C8-PHA molecules was deduced when C8-PHA concentration was greater than 0.1 mol dm-3. The Karl Fisher titration results showed that one water molecule was included in the reversed micelle. The polar parameter (E(T)(1)) of the reversed micelle was determined to be 250 kJ mol-1 by measuring absorption spectra of 1-(4-hydroxyphenyl)-2,4,6-triphenylpyridinium betaine (HPTPP) as a function of C8-PHA concentration in carbon tetrachloride. The E(T)(1) value was different from that in carbon tetrachloride, indicating the incorporation of HPTPP into the polar core region where the hydrophilic chelating groups are oriented inward in the reversed micelle. On the basis of the E(T)(1) values in organic solvents, the polar core region could be regarded as being an alcoholic medium.

Aggregation of TV-Octanoyl-TV-phenylhydroxylamine in Carbon TetrachlorideApplication of Supercritical Fluid Extraction and Chromatography to Assay Fat-Soluble Vitamins in Hydrophobic Ointment
Tohru Saitoh, Mitsuhiro Masuda, Shinichi Koike, Mitsuichi Handa, Kazuhiko Sagara, Taku Mizutani, Hirofumi Tani, Tamio Kamidate, Hiroto Watanabe, Kensaku Haraguchi, Shokichi Abe
analytical sciences, 9, 3, 345, 349, 1993, [Peer-reviewed]
English, Scientific journal, The aggregation number of jV-octanoyl-TV-phenylhydroxylamine (C8-PHA) in carbon tetrachloride was determined on the basis of the extraction equilibria of copper(II) chelate with C8-PHA between aqueous and carbon tetrachloride phases. According to the relation of the distribution ratio of copper(II) with its concentration in the organic phase, the formation of a reversed micelle consisting of approximately 13 C8-PHA molecules was deduced when C8-PHA concentration was greater than 0.1 mol dm-3. The Karl Fisher titration results showed that one water molecule was included in the reversed micelle. The polar parameter (Et(1)) of the reversed micelle was determined to be 250 kJ mol1 by measuring absorption spectra of l-(4-hydroxyphenyl)-2,4,6-triphenylpyridinium betaine (HPTPP) as a function of C8-PHA concentration in carbon tetrachloride. The Mt(1) value was different from that in carbon tetrachloride, indicating the incorporation of HPTPP into the polar core region where the hydrophilic chelating groups are oriented inward in the reversed micelle. On the basis of the Et(1) values in organic solvents, the polar core region could be regarded as being an alcoholic medium. © 1993, The Japan Society for Analytical Chemistry. All rights reserved.

ポリカチオン/DNA複合体搭載脂質ナノ粒子による効率的な長鎖DNAトランスフェクション
宇野秀哉, 真栄城正寿, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 日比野光恵, 谷博文, 原島秀吉, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2023, 2023

カリウム・硝酸・リン酸イオンの同時定量のための紙分析デバイスの開発
才木陸朗, 石田晃彦, 日比野光恵, 真栄城正寿, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2023, 2023

紙ベースの分析デバイスを用いた生物発光レポーターアッセイ
古坐あすか, 日比野光恵, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2023, 2023

LEDベースのUV吸光度検出小型ユニットからなるパームトップ液体クロマトグラフ
小山魁人, 石田晃彦, 日比野光恵, 真栄城正寿, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集(Web), 72nd, 2023

酵素と基質をセルロース媒体に固定化した電気化学ATP-乳酸センサー
九鬼静香, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

ルシフェラーゼ発現大腸菌の紙デバイス上での発現誘導
古坐あすか, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

抗SARS-CoV-2抗体およびSARS-CoV-2スパイクタンパク質断片の非競合蛍光偏光免疫分析法
高橋和希, 福山真央, 粕谷素洋, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

薬剤送達ナノ粒子作製用ガラス製マイクロ流体システムの開発
岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

タンパク質分析用ペーパー分析デバイスの開発を目的としたろ紙上でのホタルルシフェラーゼ活性の制御
野中康伸, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

タンパク質-リガンド複合体解析のためのマイクロ流体デバイスの開発
舟久保智瑛, 真栄城正寿, 上野剛, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2022, 2022

ルシフェラーゼ発現大腸菌の紙デバイスへの固定化と発現誘導
古坐あすか, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集(Web), 71st, 2022

カリウムイオン定量のための距離ベース紙分析デバイスの開発
才木陸朗, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 46th, 2022

蛍光ナノダイヤモンド搭載セラノスティクス用脂質ナノ粒子の作製
杉浦魁星, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 45th, 2022

タンパク質含有ハイドロゲルによるX線結晶構造解析法の開発
一町田由貴, 真栄城正寿, 真栄城正寿, 上野剛, 小西真昌, 坂井直樹, 石田晃彦, 谷博文, 山本雅樹, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 45th, 2022

DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR FOR DIAGNOSING SEVERITY USING BLOOD LACTATE / ATP RATIO AS A BIOMARKER
九鬼静香, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, Chemical Sensors, 37, Supplement B, 2021

ポータブル蛍光偏光測定装置を用いたヒト血清中の抗SARS-CoV-2抗体の検出
高橋和希, 西山慶音, 福山真央, 粕谷素洋, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 分析化学討論会講演要旨集(Web), 81st, 2021

Development Of An Electrochemical Sensor For Diagnosing Severity Using Blood Lactate / ATP Ratio As A Biomarker
九鬼静香, 石田晃彦, 真栄城正寿, 谷博文, 渡慶次学, 電気化学秋季大会講演要旨集(CD-ROM), 2021, 2021

マイクロ流体デバイスを用いたプラスミドDNA搭載脂質ナノ粒子の作製
宇野秀哉, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 43rd, 2021

蛍光偏光免疫分析法によるオカダ酸の検出
千田駿亮, 高橋和希, 福山真央, 粕谷素洋, 真栄城正寿, 石田晃彦, 谷博文, ZHERDEV Anatoly V., EREMIN Sergei A., EREMIN Sergei A., 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 44th, 2021

Affibodyを用いた非競合蛍光偏光免疫分析法によるEGFRの検出
高橋和希, 福山真央, 粕谷素洋, 真栄城正寿, 石田晃彦, 谷博文, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集(CD-ROM), 44th, 2021

蛍光偏光免疫測定法によるヒト血清中の抗SARS-CoV-2抗体の検出
高橋和希, 西山慶音, 福山真央, 粕谷素洋, 真栄城正寿, 石田晃彦, 谷博文, 火原彰秀, 渡慶次学, キャピラリー電気泳動シンポジウム講演要旨集, 40th (CD-ROM), 2020

ナノ構造体を用いた脂質ナノ粒子のサイズ分離
清水一樹, 真栄城正寿, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

ペーパーデバイスによる懸濁試料の簡易・迅速分析のための前処理ユニットの開発
前田陵我, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

血中リチウムイオン測定のための全自動分析システムの開発
小松雄士, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

ポータブルHPLCのためのLEDおよびフォトダイオードを用いた小型吸光検出モジュールの開発
西村卓馬, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

ルシフェラーゼの活性制御に基づくオンチップ生物発光アッセイの開発
野中康伸, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

常温X線結晶構造解析のためのマイクロデバイスの開発
舟久保智瑛, 真栄城正寿, 真栄城正寿, 伊藤翔, 伊藤翔, 上野剛, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

生物発光分析のためのペーパーデバイスの開発とキナーゼ分析への応用
高田一生, 渡慶次学, 谷博文, 石田晃彦, 真栄城正寿, 分析化学討論会講演要旨集(Web), 80th, 2020

高感度免疫分析デバイスの開発とH5型鳥インフルエンザウイルス検出への応用
西山慶音, RAMOS Kenia Chavez, 真栄城正寿, 石田晃彦, 谷博文, 笠間敏博, 馬場嘉信, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

乳酸/ATPの血中濃度比による重症度診断のための電気化学酵素センサーの開発
九鬼静香, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集(Web), 80th, 2020

Development of Micro Device for Simple and Rapid Measurement of Progesterone Using Paper Substrates.
小松雄士, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 日本化学会春季年会講演予稿集(CD-ROM), 100th, 2020

紙ベースイムノアッセイのための抗体の簡便な固定化法
前田陵我, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 42nd (Web), 2020

ナノ構造体搭載デバイスによる脂質ナノ粒子のサイズ分離
清水一樹, 真栄城正寿, 真栄城正寿, 石田晃彦, 谷博文, 西井準治, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 42nd (Web), 2020

ガラス製マイクロ流体デバイスを用いたsiRNA搭載脂質ナノ粒子の作製と大量生産用集積化デバイスの開発
真栄城正寿, 真栄城正寿, 岡田悠斗, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 日本DDS学会学術集会プログラム予稿集, 36th, 2020

DDSナノ粒子作製用ガラス製マイクロ流体デバイスの開発
岡田悠斗, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 42nd (Web), 2020

マイクロデバイスを用いたタンパク質結晶の室温複合体構造解析
竹田怜央, 真栄城正寿, 真栄城正寿, 伊藤翔, 伊藤翔, 上野剛, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 42nd (Web), 2020

UNDERSTANDING THE LIPID NANOPARTICLES STRUCTURE DYNAMICS USING A TIME-RESOLVED SAXS MEASUREMENT
Masatoshi Maeki, Niko Kimura, Kazuki Shimizu, Kento Yonezawa, Nobutaka Shimizu, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi, Proc. Micro TAS 2019, 1478, 1479, Oct. 2019, [Peer-reviewed]
This paper reports a time-resolved small angle X-ray scattering (SAXS) for understanding lipid nanoparticles (LNPs) structure dynamics. The structure and its dynamics are the indispensable factors to ensure the activity and therapeutic effect of LNP-based nanomedicines. However, the structure dynamics of LNPs are not well understood because of the limitation of measurement methodology. To overcome the problem, we developed microfluidic-based SAXS measurement technique combined with a synchrotron X-ray source. We confirmed the LNPs decomposition process and LNPs aggregation process by pH change of the solution., English

PORTABLE FLUORESCENCE POLARIZATION ANALYZER FOR ON-SITE MULTISAMPLE IMMUNOASSAY
Ayano Nakamura, Osamu Wakao, Ken Satou, Mitsutoshi Aoyagi, Kazuhiko Nishimura, Chikaaki Mizokuchi, Ken Sumiyoshi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi, Proc. Micro TAS 2019, 1393, 1394, Oct. 2019, [Peer-reviewed]
This paper reports on a portable fluorescence polarization (FP) analyzer capable of on-site multisample immunoassay. The FP analyzer is small-size (W × D × H = 65 cm3), low cost (< $5,000), and high throughput (96 samples simultaneously). Using this analyzer, we demonstrated FP immunoassay (FPIA) for deoxynivalenol (DON), one of several mycotoxins that frequently infect wheat and other grains, spiked into wheat samples. The assay with the compact FP analyzer had sufficient accuracy to quantify DON in wheat in comparison with LC-MS/MS. Furthermore, multisample immunoassay was conducted by using a microdevice with 96 chambers., English

DEVELOPMENT OF A THREE-DIMENSIONAL MICROMIXER DEVICE FOR PRODUCTION OF VARIOUS LIPID-BASED NUCLEIC ACID NANOCARRIERS
Niko Kimura, Masatoshi Maeki, Yusuke Sato, Kosuke Sasaki, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi, Proc. Micro TAS 2019, 368, 369, Oct. 2019, [Peer-reviewed]
This paper describes development of a three-dimensional (3D) baffle mixer device for precise size control of various types of lipid nanoparticles (LNPs) with high encapsulation efficiency of short interfering RNAs (siRNAs). The 3D baffle mixer device achieved more precise size control of various LNPs than that of the conventional micromixer device. In addition, the 3D baffle mixer enabled effective capturing of siRNAs into LNPs without any assistance of electrostatic interaction between lipid molecules and siRNAs. The 3D baffle mixer device is expected to become one of the key platforms for production of novel lipid-based nucleic acid nanocarriers., English

HIGH-THROUGHPUT X-RAY CRYSTALLOGRAPHY BASED ON THE PROTEIN CRYASTAL ARRAY
Reo Takeda, Masatoshi Maeki, Sho Ito, Go Ueno, Kunio Hirata, Akihiko Ishida, Hirofumi Tani, Masaki Yamamoto, Manabu Tokeshi, Proc. Micro TAS 2019, 191, 192, Oct. 2019, [Peer-reviewed]
This paper reports development of a microfluidic-based high-throughput X-ray crystallography for protein-ligand complex structure analysis. Three-dimensional (3D) structure analysis of a protein-ligand complex requires complicated procedures. To improve the throughput performance, we developed a protein crystal array-based microfluidic device. The microfluidic device can effectively capture protein microcrystals into the microarray and continuously prepare protein-ligand complex samples. We determined lysozyme- p-toluenesulfonic acid complex and thaumatin-selenourea complex structures by serial on-chip X-ray diffraction measurement at room temperature., English

FLUORESCENCE SIGNAL AMPLIFICATION FOR SENSITIVE ENZYME IMMUNOASSAY UTILIZING AN IMMUNO-WALL
Keine Nishiyama, Toshihiro Kasama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi, Proc. Micro TAS 2019, 689, 690, Oct. 2019, [Peer-reviewed]
This paper reports a novel method of signal amplification based on the accumulation of enzymatic fluorescent products at a wall-like structure (immuno-wall) in a microchannel for sensitive immunoassay. We found the function of an immuno-wall as a media for the enrichment of fluorescence molecules. We combined this function with the amplification of the fluorescence signal by an enzymatic reaction to produce a synergistic effect. The performance of the device was evaluated with human C-reactive protein (CRP) as a model target. The limit of detection of CRP was 2.5 pg/mL. This value was approximately 3 orders of magnitude lower than that obtained with a fluorescent dye-labeled antibody (1.7 ng/mL)., English

フローデバイスを用いたフルオレセイン振動化学発光
池田晋吾, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集, 79th, 2019

マイクロデバイスを用いたタンパク質-リガンド複合体構造解析
真栄城正寿, 真栄城正寿, 竹田怜央, 伊藤翔, 伊藤翔, 上野剛, 平田邦生, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学, 分析化学討論会講演要旨集, 79th, 2019

電気浸透流ポンプとオンチップカラムに基づく可搬型イオンクロマトグラフの開発
石田晃彦, 西村卓馬, 真栄城正寿, 谷博文, 渡慶次学, イオンクロマトグラフィー討論会講演要旨集, 36th, 2019

核酸搭載脂質ナノ粒子の動的構造解析
真栄城正寿, 真栄城正寿, 木村笑, 清水一樹, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 40th, 2019

核酸ナノキャリア製造のための新規バッフルデバイスの開発
木村笑, 真栄城正寿, 真栄城正寿, 石田明彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 40th, 2019

蛍光偏光免疫分析法によるH5亜型鳥インフルエンザウイルスの検出
西山慶音, 武田洋平, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 小川晴子, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 40th, 2019

マイクロデバイスを用いたX線結晶構造解析の高速化とリガンドスクリーニングへの応用
竹田怜央, 真栄城正寿, 真栄城正寿, 伊藤翔, 伊藤翔, 上野剛, 平田邦夫, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 39th, 2019

脂質ナノ粒子形成後の二次希釈操作が粒子サイズへ与える影響の解明
木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 39th, 2019

小角X線溶液散乱法による脂質ナノ粒子の動的構造解析法の開発
真栄城正寿, 木村笑, 石田晃彦, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集(Web), 68th, 2019

電気浸透流ポンプとオンチップカラムに基づくオールインワンコンパクトLCシステムの開発
石田晃彦, 西村卓馬, 真栄城正寿, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集(Web), 68th, 2019

MICROSCOPIC REAL-TIME MEASUREMENT OF PROTEIN CRYSTAL GROWTH IN MICROFLUIDIC DEVICES
Masatoshi Maeki, Shohei Yamazaki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi, Proc. Micro TAS 2018, 4, 2192, 2193, Nov. 2018, [Peer-reviewed]
This paper reports a microscopic real-time measurement of protein crystal growth in microfluidic devices to understand the effect of micro space on the protein crystal growth. Lysozyme crystallization behavior was observed using an optical microscope and we found that the orientation percentage of the (1 1 0) phase was depending on the depth of the crystallization chambers. The concentration profile and growth rate of lysozyme crystal in the microfluidic device was determined by in situ Raman spectroscopy and laser confocal microscopy-combined with differential interference contrast microscopy (LCM-DIM). The crystal growth rate in 10 and 20 μm depth crystallization chamber was 10 times slower than that of the other devices., English

Microfluidic stepwise and rapid ethanol dilution for precise size control of lipid nanoparticles　　　　　　　　　　　　　　　
Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Proc. Micro TAS 2018, 1404, 1405, Nov. 2018, [Peer-reviewed]
English

ナノピラーデバイスを用いたDNAのサイズ分離と無標識検出　　　　　　　　　　　　　　　
阿尻 大雅, 安井 隆雄, 笠 晴也, 真栄城 正寿, 石田 晃彦, 谷 博文, 西井 準治, 馬場 嘉信, 渡慶次 学, 日本分析化学会講演要旨集, 67年会, 125, 125, Aug. 2018
(公社)日本分析化学会, Japanese

電気化学検出液体クロマトグラフィーチップの開発とその尿中シュウ酸分析への応用
藤井大地, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2018, 2018

マイクロ流体デバイスにおけるルミノール化学発光の増感効果
菊地優仁, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2018, 2018

マイクロデバイスを用いた小麦中デオキシニバレノールの多サンプル同時蛍光偏光免疫分析
中村彩乃, 若尾摂, 佐藤憲, 住吉研, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 37th, 2018

微小空間内におけるリゾチームの結晶化制御および晶析挙動解析
山崎翔平, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2018, 2018

Simultaneous Multiplex Determination of Deoxynivalenol in Wheat By Fluorescence Polarization Immunoassay Using Microdevice
中村彩乃, 若尾摂, 佐藤憲, 住吉研, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム, 17, 2, 2018

オンチップ液体クロマトグラフィーのためのウォールジェット式電気化学検出器の開発
干川晃生, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集, 67th, 2018

マイクロデバイスを用いた小型蛍光偏光測定装置の開発とその応用
中村彩乃, 若尾摂, 佐藤憲, 溝口親明, 住吉研, 黒澤文夫, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 38th, 2018

ペーパーデバイスによるPOCTのための高感度競合イムノアッセイの実現
前田陵我, 小松雄士, 佐藤優樹, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 38th, 2018

ハイスループット蛍光偏光イムノアッセイデバイスの開発
若尾摂, 中村彩乃, 佐藤憲, 溝口親明, 住吉研, 黒澤文夫, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 38th, 2018

シクロデキストリンと両親媒性物質を用いたホタルルシフェラーゼの活性制御と生物発光分析法への応用
工藤裕貴, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 分析化学討論会講演要旨集, 78th, 2018

酵素免疫測定法を利用した高感度イムノウォールデバイスの開発
西山慶音, 笠間敏博, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 38th, 2018

ペーパーデバイスによるリチウムイオン濃度測定
小松雄士, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2018, 2018

微小空間におけるタンパク質結晶成長のリアルタイム計測
竹田怜央, 山崎翔平, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 38th, 2018

イムノウォールデバイスの開発と遺伝子変異たんぱく質の検出
中股征哉, 笠間敏博, 笠間敏博, 長谷哲成, 與語直之, 與語直之, 小沢直也, 真栄城正寿, 石田晃彦, 佐藤光夫, 加地範匡, 谷博文, 長谷川好規, 馬場嘉信, 馬場嘉信, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2018, 2018

iLiNPデバイスによるpH応答性カチオン性脂質ナノ粒子の粒径制御
木村笑, 真栄城正寿, 岡部奈々, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 渡慶次学, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 37th, 2018

A Small-Sized Lipid Nanoparticles Production Method Using Microfluidic Devices with Baffle Structures
N. Kimura, M. Maeki, Y. Sato, A. Ishida, H. Tani, H. Harashima, M. Tokeshi, Proc. Micro TAS 2017, 965, 966, Oct. 2017
This paper reports a lipid nanoparticles (LNP) production method and its formation behavior using microfluidic devices with baffle structures. The microfluidic devices showed great mixing efficiency at 500 μL/min, and we achieved 20 nm-sized LNPs production that chaotic micromixers were not able to produce at the same flow rate condition. Additionally, we found that the smaller-sized LNPs/siRNA prepared by baffle structures have higher penetration efficiency than that of the larger-sized LNPs, but all of them showed the gene silencing activity. The microfluidic devices with baffle structures are expected to be a practicable apparatus for DDSs application., English

High-Throughput Fluorescence Polarization Measurement System Towards Molecular Interaction Analysis
O. Wakao, M. Maeki, A. Ishida, H. Tani, A. Hibara, M. Tokeshi, Proc. Micro TAS 2017, 557, 558, Oct. 2017, [Peer-reviewed]
This report demonstrated simultaneous fluorescence polarization (FP) measurement for 100 samples. The measurement was based on the synchronization detection between the switching of a liquid crystal (LC) layer and the sampling rate of an image sensor. This offered captured FP signals from multisample as single FP image, and the values of FP signals were obtained separately by image analysis. The results showed the analytical performance of our system was comparable to that of conventional apparatus and which implies our system can be applicable to molecular interaction analysis., English

A Microfluidic-Based Technique for Creating Amyloid Nanostructures and Its Application to Enzyme Reaction　　　　　　　　　　　　　　　
M. Maeki, S. Sato, A. Ishida, H. Tani, M. Tokeshi, Proc. Micro TAS 2017, 1279, 1280, Oct. 2017, [Peer-reviewed]
English

分子間相互作用測定のための多サンプル同時蛍光偏光測定システムの開発
若尾摂, 住吉研, 溝口親明, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, センサ・マイクロマシンと応用システムシンポジウム(CD-ROM), 34th, 2017

小型蛍光偏光イムノアッセイ装置の開発
若尾摂, 住吉研, 溝口親明, 真栄城正寿, 石田晃彦, 谷博文, 重村幸治, 火原彰秀, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 36th, 2017

微小空間によるタンパク質結晶化制御と分光法を用いた晶析挙動解析
山崎翔平, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 36th, 2017

マイクロデバイスを用いたアミロイド構造体作製法の開発と酵素反応への応用
真栄城正寿, 佐藤紗耶, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 36th, 2017

高効率免疫分析を可能とするイムノウォールデバイスの開発
中股征哉, 笠間敏博, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 36th, 2017

DNA分離のためのナノピラーデバイス作製の簡便化とその応用
阿尻大雅, 安井隆雄, 安井隆雄, 笠晴也, 真栄城正寿, 石田晃彦, 谷博文, 西井準治, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, キャピラリー電気泳動シンポジウム講演要旨集, 37th, 2017

バッフル構造を配置した流路デザインによる粒径の精密制御
木村笑, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 35th, 2017

マイクロデバイスを用いた脂質ナノ粒子作製と生体内動態挙動
真栄城正寿, 木村笑, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学工学会秋季大会研究発表講演要旨集(CD-ROM), 49th, 2017

Lipid nanoparticle-size control by microchannel with baffle structures
木村笑, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学とマイクロ・ナノシステム, 16, 2, 2017

Development of a New Technique for Washing Steps in Multistep Assays Using Microfluidic Paper-based Analytical Devices
Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi, Proceedings of MicroTAS 2016, 970, 971, Oct. 2016, [Peer-reviewed]
This paper presents a novel washing technique for microfluidic paper-based analytical devices (μPADs) to remove unbound antigen or antibodies from paper substrates in multistep assays and achieve higher sensitivity and reproducibility relying on spontaneous capillary force of the paper., English, Introduction international proceedings

Fluorescence Polarization-based Multiple Samples Detection Using Microchamber Array Towards High-throughput Molecular Interaction Analysis
Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi, Proceedings of MicroTAS 2016, 1400, 1401, Oct. 2016, [Peer-reviewed]
We report a new high-throughput homogeneous assay format using a microchamber array towards molecular interactions analysis. The assay format is based on the fluorescence polarization (FP) detection using liquid-crystal display (LCD) that can switches a direction of FP by changing its images. In this work, we demonstrate a multiple sample FP immunoassay (FPIA) of prostaglandin E2 in a microchamber array to simultaneously analyze 25 samples. FP signals were imaged simultaneously and their values were acquired separately. This result showed our format with high-throughput had a measurement performance comparable to that of a conventional apparatus designed to single analysis., English, Introduction international proceedings

Effect of the Grooved Structures and the Ethanol Concentration on the Small-sized Lipid Nanoparticles Formation　　　　　　　　　　　　　　　
Yuka Fujishima, Masatoshi Maeki, Yusuke Sato, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi, Proceedings of MicroTAS 2016, 1412, 1413, Oct. 2016, [Peer-reviewed]
English, Introduction international proceedings

がん診断のためのエクソソーム分析法の開発　　　　　　　　　　　　　　　
阿尻 大雅, 安井 隆雄, 真栄城 正寿, 石田 晃彦, 谷 博文, 馬場 嘉信, 渡慶次 学, 日本分析化学会講演要旨集, 65年会, 165, 165, Aug. 2016
(公社)日本分析化学会, Japanese

分子間相互作用測定のための蛍光偏光イメージングシステムの開発
若尾摂, 真栄城正寿, 石田晃彦, 谷博文, 火原彰秀, 渡慶次学, 分析化学討論会講演要旨集, 76th, 2016

マイクロデバイスを用いたクロラムフェニコールの蛍光偏光免疫測定
青木琴, 若尾摂, 真栄城正寿, 石田晃彦, 谷博文, EREMIN Sergei, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 33rd, 2016

蛍光偏光イメージングによる複数サンプルの同時イムノアッセイ
若尾摂, 真栄城正寿, 石田晃彦, 谷博文, 火原彰秀, 渡慶次学, 化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM), 2016, 2016

ATP測定のための電気化学酵素センサーの開発
西山慶音, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM), 2016, 2016

マイクロチップ電気化学検出LCデバイスの生体試料分析への応用
藤井大地, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 日本分析化学会年会講演要旨集, 65th, 2016

ルシフェラーゼ内封リポソームを用いたオンチップイムノアッセイ
中谷友祐, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM), 2016, 2016

マイクロデバイスによるタンパク質の立体構造解析
真栄城正寿, 真栄城正寿, 山崎翔平, 杉島正一, 渡邉啓一, 石田晃彦, 谷博文, 宮崎真佐也, 渡慶次学, 分析化学討論会講演要旨集, 76th, 2016

PDMS製チップホルダーを用いたペーパーマイクロ分析チップの開発
MOHAMMADI Saeed, BUSA Lori Shayne Alamo, 真栄城正寿, 石田晃彦, 谷博文, 渡慶次学, 渡慶次学, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 34th, 2016

がん診断のためのエクソソームの無標識検出
阿尻大雅, 安井隆雄, 安井隆雄, 石田晃彦, 谷博文, 真栄城正寿, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 化学系学協会北海道支部冬季研究発表会講演要旨集(CD-ROM), 2016, 2016

Label-free detection of extracellular vesicles for cancer diagnosis　　　　　　　　　　　　　　　
Taiga Ajiri, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi, Micro Total Analysis Systems 2015, 1789-1791, 27 Oct. 2015, [Peer-reviewed]
English, Report scientific journal

Fluorescence Polarization Imaging for Multisample Immunoassay
Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manadu Tokeshi, Proceedings of MicroTAS 2015, 1816, 1818, Oct. 2015, [Peer-reviewed]
This paper reports a unique fluorescence polarization (FP) imaging system with a microchip applicable to multisample simultaneous FP immunoassay. The measurement principle of the system has already been reported [1]. In this work, we present a FP imaging immunoassay for multisample of physiologically active substances (prostaglandin E2, PGE2). FP signals of all samples in the captured by an image sensor were imaged simultaneously, and their values were acquired separately from the image analysis. The analytical performance of this assay system was comparable to that of conventional apparatus. The result is the first demonstration of the multisample FP imaging immunoassay., English, Introduction international proceedings

Microfluidic Approach for Production of Lipid Nanoparticles-Based Nano Medicine
Masatoshi Maeki, Proceedings of MicroTAS 2015, 838, 840, Oct. 2015, [Peer-reviewed]
This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm., English, Introduction international proceedings

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 特集号「生(bio)」, 64, 5, 329-335, 09 Jun. 2015, [Peer-reviewed]
Japanese, Report scientific journal

脂質ナノ粒子の粒径制御のためのマイクロ流体デバイス設計
真栄城正寿, 藤島由佳, 佐藤悠介, 石田晃彦, 谷博文, 原島秀吉, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 32nd, 2015

LCD-CCD Synchronization Detection for Fluorescece Polarization Immunoassay　　　　　　　　　　　　　　　
Osamu Wakao, Yusaku Fujii, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi, Proceedings of the Micro TAS 2014 Symposium, 2271, 2273, Oct. 2014, [Peer-reviewed]
English, Introduction international proceedings

ナノ構造体を用いた無標識DNAの高感度検出
阿尻大雅, 安井隆雄, 安井隆雄, 石田晃彦, 谷博文, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 29th, 2014

マイクロ流路内におけるリポソーム形成メカニズムの考察
齋藤竜亮, 真栄城正寿, 安井隆雄, 安井隆雄, 石田晃彦, 谷博文, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 30th, 2014

ナノ構造体の光回折を利用した高感度無標識検出法の開発
阿尻大雅, 安井隆雄, 安井隆雄, 石田晃彦, 谷博文, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 30th, 2014

イムノウォールデバイスによる疾病マーカーのマルチプレックスアッセイ
西脇奈菜子, 笠間敏博, 石田晃彦, 谷博文, 馬場嘉信, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 30th, 2014

DEVELOPMENT OF 3RD GENERATION IMMUNO-PILLAR DEVICE FOR HIGH SENSITIVE DETECTION OF DISEASE MARKERS　　　　　　　　　　　　　　　
N. Nishiwaki, T. Kasama, A. Ishida, H. Tani, Y. Baba, M. Tokeshi, Micro Total Analysis Systems 2013, 1164-1166, 28 Oct. 2013, [Peer-reviewed]
English, Report scientific journal

マイクロ流体デバイスを用いるDDSナノキャリアの作製
齋藤竜亮, 安井隆雄, 安井隆雄, 石田晃彦, 谷博文, 加地範匡, 加地範匡, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 28th, 2013

イムノピラーチップの高性能化
西脇奈菜子, 石田晃彦, 谷博文, 笠間敏博, 笠間敏博, 馬場嘉信, 馬場嘉信, 馬場嘉信, 渡慶次学, 渡慶次学, 渡慶次学, 化学とマイクロ・ナノシステム学会研究会講演要旨集, 27th, 2013

Portable Liquid Chromatography System Based on Battery-Powered Electroosmotic Pump and Microchip with Packed Column and Electrochemical Detector　　　　　　　　　　　　　　　
Akihiko Ishida, Takehiro Fujimoto, Satoshi Yokokawa, Hirofumi Tani, Manabu Tokeshi, Ichiro Yanagisawa, Proceedings of the MicroTAS 2012 Symposium, 1183, 1185, Oct. 2012, [Peer-reviewed]
English, Summary international conference

On-Chip Bioluminescence Assay of ATP and Kinases Using Immobilized Firefly Luciferase in Three-Dimensional Microfluidic Chip　　　　　　　　　　　　　　　
Hirofumi Tani, Atsuki Morisaki, Akihiko Ishida, Manabu Tokeshi, Proceedings of the MicroTAS 2012 Symposium, 1618, 1620, Oct. 2012, [Peer-reviewed]
English, Summary international conference

分析化学用語辞典　　　　　　　　　　　　　　　
オーム社, 2011

改訂六版 分析化学便覧　　　　　　　　　　　　　　　
丸善, 2011

マイクロチップ液体クロマトグラフィーのためのオンチップ電気化学フローセルの開発
石田 晃彦, 夏目 大道, 谷 博文, Proceedings of the Chemical Sensor Symposium, 42, 97, 99, Sep. 2006
電気化学会化学センサ研究会, Japanese

O-13 On-chip bioluminescence reporter assay for genotoxic substances using three-dimensional microfluidic network(Oral Session(2)-DNA damage, Anti-mutagens, micronuclei test, Development of tests-)
Tani H, Maehana K, Kamidate T, 日本環境変異原学会大会プログラム・要旨集, 34, 101, 101, 2005
日本環境変異原学会, English

P-50 On-chip bioluminescence reporter assay for genotoxic substances using three-dimensional microfluidic network(Poster Presentations(1))
Tani H, Maehana K, Kamidate T, 日本環境変異原学会大会プログラム・要旨集, 34, 129, 129, 2005
日本環境変異原学会, English

On-Chip Bioassay for Mutagens　　　　　　　　　　　　　　　
73, 3, 215, 219, 2005

Micelle-mediated extraction
H Tani, T Kamidate, H Watanabe, JOURNAL OF CHROMATOGRAPHY A, 780, 1-2, 229, 241, Sep. 1997
The extraction technique based on phase separation in aqueous micellar solutions is reviewed. The technique has now been utilized for separation and preconcentration of metal chelates, organic compounds, and proteins. Additionally, the phase behavior of the micellar solutions and recent advances in the phase separation technique are also described. In the extraction of metal chelates, distribution equilibria are considered. In contrast to conventional solvent extraction, the distribution of metal chelates into a condensed surfactant phase (surfactant-rich phase) was dependent on metal ions. Proteins were extractable into the surfactant-rich phase according to their hydrophobicity. The recent use of affinity ligands and water-soluble polymers for controlling extractability of proteins are also introduced. (C) 1997 Elsevier Science B.V., ELSEVIER SCIENCE BV, English

バイオ医薬の開発技術とシーズ　　　　　　　　　　　　　　　
シーエムシー出版, 2009

Microchip-based Assay Systems, Methods and Applications　　　　　　　　　　　　　　　
Humana Press, 2007

水の分析(第5版)　　　　　　　　　　　　　　　
化学同人, 2005

リポソーム応用の新展開～人工細胞の開発に向けて～　　　　　　　　　　　　　　　
エヌ・ティー・エス, 2005

演習で学ぶ環境　　　　　　　　　　　　　　　
三共出版, 2002

環境の化学分析　　　　　　　　　　　　　　　
三共出版, 1998

Development of high performance paper devices and its application development
Grants-in-Aid for Scientific Research
01 Apr. 2020 - 31 Mar. 2023
渡慶次 学, 谷 博文
本研究では、①分析性能の高い紙デバイスの研究、②免疫分析デバイスの開発、③細胞アッセイ及び細胞分離デバイスの開発、④教育ツール用デバイスの開発に取り組む。本年度は、この中の①、②、④に取り組んだ。
①双極性障害の治療薬であるリチウム製剤の血中濃度測定をモデル系として、高性能紙デバイスシステムを構築した。血漿分離と血漿中のリチウムイオンの検出を性質の異なる紙を利用することで、わずか1滴（20マイクロリットル）の試料量で1分以内に測定することに成功した。検出性能は、遠心分離と吸光光度計による従来法と同等であった。
②競合免疫分析が可能な3次元紙デバイスを開発した。流路を3次元化することで流路長を短くすることが可能となり、アッセイ時間を短縮することができた。デバイスは3層構造になっており、反応領域と検出領域を別の層にすることで、分析性能を向上させることができた。さらに、非競合免疫分析の酵素免疫分析法（ELISA）の基礎検討も実施した。
③紙デバイスは、安価で簡便ということから、教育ツールとして適している。しかし、濃度定量するためには、マイクロピペットによる試料導入が必要である。そこでマイクロピペット等を用いずに、サンプルに浸漬するだけで、サンプル中の測定対象の濃度を定量できる紙デバイスを開発した。アスコルビン酸とpHの同時測定が可能なデバイスを開発し、測定結果の浸漬時間依存性を評価したところ、測定結果は浸漬時間（3秒から1分）に依存しないことも確認した。測定性能も従来法を同等であることを確認した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20H02760

Development of next-generation diagnostic chip and its application to clinical diagnosis
Grants-in-Aid for Scientific Research
31 May 2012 - 31 Mar. 2015
TOKESHI Manabu, YUZAWA Yukio, AKIYAMA Shinichi, TANI Hirofumi, ISHIDA Akihiko
We developed a diagnostic chip with a five biomarker panel for diabetic nephropathy. The biomarker panel of MCP-1, L-FABP, Angiotensinogen, CTGF, and Collagen IV was adopted for diabetic nephropathy. With standard and patient samples, we evaluated the performance of the panel diagnostic chip. For the detection of biomarkers, we confirmed the chip provides rapid analysis (total assay time of 12 min) with high sensitivity and it uses small volumes of the sample and reagent (0.5 μL each), and the obtained results correlated with that of conventional ELISA.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Hokkaido University, 24245013

The development of nanor eactor for high sensitive chemi luminescence analysis by using liposomes
Grants-in-Aid for Scientific Research
2006 - 2007
KAMIDATE Tamio, TANI Hirofumi, ISHIDA Akihiko
Horseradish peroxidase (HRP) -encapsulated liposomes were prepared by freeze-thawing method. The number of HRP molecules trapped in liposomes was three times greater than that prepared by extrusion method HRP trapped in liposomes was directly detected by using luminol chemiluminescence (CL) with H_2O_2 without lysis of liposomes. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H_2O_2 by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalysed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalysed luminol CL in a lipid-free bulk solution. In addition, HRP-trapped liposomes were combined to antibody in order to apply HRP-trapped liposomes to the marker in immunoassay. The CL intensity observed in antibody combined HRP-trapped liposomes was greater 125 times that in antibody combined HRP by avidin-biotin bond.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 18350037

3次元微小流体ネットワークを用いたバイオアッセイシステムの構築
科学研究費助成事業
2005 - 2006
谷 博文
本年度は,変異原物質に応答してルシフェラーゼを発現する試験大腸菌株を用いて生物発光オンチップバイオアッセイを試みた。昨年度に確立した分析フォーマットによりマイトマイシンCなどの各種変異原物質に適用したところ,変異原の強さならびに濃度に応じた生物発光をチップ上から一斉に検出することが可能となった。また,大腸菌をチップに固定化した場合の保存安定性について,生物発光強度ならびに応答比の観点から検討を行った。固定化する菌体濃度およびアガロース濃度に関して最適化された条件下で検討した結果,本チップは4℃にて最大16日間保存可能であることが分かった。一方,培地成分をアガロースに添加した場合,試験菌を30日以上保存可能であることが分かった。しかし,本法で利用するホタル生物発光に対して培地成分による強い阻害効果が見られ,発光強度が大きく減少する結果となった。したがって,試験菌株のレポータータンパク質をホタルルシフェラーゼから培地成分による阻害を受けない発光触媒酵素に変換することでより長期問の保存が可能になるものと期待される。生体内での代謝により変異原性を発現する物質(前変異原物質)について本オンチップアッセイにより検出可能か検討した。前変異原物質にはTrp-P-2を用い,代謝活性化には齧歯類の代謝酵素を含むS9画分を使用した。Trp-P-2とS9を予め混合して代謝活性化を行い,試験菌を固定化したチップに導入した結果をFig.に示す。S9で処理したTrp-P-2を導入したチップからのみ発光が見られた。また,発光応答比は2を超え,Trp-P-2濃度に依存する結果が得られた。このことから,本アッセイを前変異原物質に対して適用可能であることが示された。
日本学術振興会, 若手研究(B), 北海道大学, 17750063

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
2005 - 2006
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, 17655028

The investigation for high sensitive chemi luminescence method using nanoreactor as a marker
Grants-in-Aid for Scientific Research
2003 - 2004
KAMIDATE Tamio, TANI Hirofumi, ISHIDA Akihiko
Liposomes encapsulated horseradish peroxidase(HRP) were prepared for applying the inner water phase in liposome to the nanoreactor for HRP-catalyzed chemiluminecence(CL) reaction. Phosphatidylcholine(PC), phosphatidylglycerol dimyristoyl(DMPG) and cholesterol were used as a component for liposome. The mole % of PC : DMPG : Chel was 8:1:1. The HRP-trapped liposome was prepared by extrusion method with polycarbonate filter. Homogentisic acid γ-lactone(HAL) and luminol were used as a CL reagent for 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 ρ-iodophenol (ρ-IP) enhanced luminol CL. Maximal light emission in HAL CL appeared more rapidly compared to that in ρ-IP enhanced CL methods, thus resulting in remarkable reduction of CL measurement time. The detection limit for HRP in HAL CL was the same as that in ρ-IP enhanced lnminol CL. The linear range of calibration curve for HRP in HAL CL was improved by factors of 50 compared with that in ρ-IP enhanced luminol CL. From these results, it was found that HAL CL were superior to ρ-IP enhanced luminol CL for the determination of HRP encapsulated in liposomes.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 15350038

環境応答型分子集合体を導入した化学発光検出用マイクロDNA分析システムの開発
科学研究費助成事業
2001 - 2002
谷 博文
本年度は,DNAのハイブリダイゼーションに及ぼす会合性高分子の影響について検討した.温度感応型会合性高分子の末端にチオール基を導入し,プローブDNAと同時にシリコン基板に蒸着した金表面上に固定化した.この会合性高分子は加温するとコンパクトに折り畳まれ,冷却すると再び伸長する.したがって,プローブDNAとのアニールに必要な温度に冷却したとき立体的々にハイブリダイゼーションを阻害することが期待される.そこで,これまで行ってきた化学発光法によりハイブリダイゼーション効率ならびに選択性について会合性高分子の影響を検討した.しかしながら,会合性高分子の共存によりDNAの固定化量が大きく低下し,完全に相補性のあるDNAとのハイブリダイゼーションを検出することができなかった.そこで,分子量のより小さい高分子を使用したところ,DNAの結合量が増加し,ハイブリダイゼーションの検出が可能となった.諸条件についてさらに検討し,会合性高分子の相転移温度前後でのハイブリダイゼーションの量を比較したが,顕著な違いは見られず,また相補性の違いを識別することはできなかった.これらの結果から,会合性高分子の同時固定化によるDNAのハイブリダイゼーションの制御が困難であることが明らかとなった.今回の場合,DNAと環境応答型の反応制御を別々に使用したが,例えば両者を結合させたプローブDNAを用いることによりハイブリダイゼーションの制御が可能になるものと考える.一方,会合性高分子共存下における化学発光については,フルオレセインやジブロモフルオレセインを発光基質に用いたところ,相転移温度前後での発光量の顕著な変化が見られた.これにより,会合性高分子を用いた化学発光反応の制御が可能であることが明らかとなった.
日本学術振興会, 若手研究(B), 北海道大学, 13750745

The investigation of chemiluminescence reaction in the micro reactor and its application to the detection in micro analytical system
Grants-in-Aid for Scientific Research
1999 - 2000
KAMIDATE Tamio, TANI Hirofumi
Three types of micro flow reactors with different length and width of channel were made by microfabrication techniques for the chemiluminescent (CL) detection of epinephrine (EP) with lucigenin. The volume of each CL reactor was about 10 μl. A solution containing EP and lucigenin and a solution containing NaOH and periodate were injected successively into each inlet of the CL reactor in the range from 20 to 100 μl min^<-1> with a pressure-driven flow system. The intensity of light emission was dependent on geometry of the CL reactors. These results could be explained on the basis of the difference in diffusion and mixing of the reactants in the CL reactors. The maximum light emission were linearly correlated with the concentrations of EP over the range from the detection limit of 5.0x10^<-8> M up to 5.0x10^<-6> M on the use of the CL reactor with the most promising geometry. In addition, we have investigated the enhanced CL reactions for applying to the micro analytical system.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B)., HOKKAIDO UNIVERSITY, 11450319

生物・化学発光反応を用いた高性能分析法の開発とマイクロ分析システムへの応用　　　　　　　　　　　　　　　
1999
Competitive research funding

Development of High-performance bioanalytical systems based on Bio- and Chemiluminescence and Their Application to uTAS　　　　　　　　　　　　　　　
1999
Competitive research funding

ミセル水性二相分配法を用いたタンパク質の高性能分離法の開発
科学研究費助成事業
1997 - 1998
谷 博文
1. エチレンオキシド(EO)とプロピレンオキシド(PO)からなる両親媒性高分子(Pluronic)ミセル水溶液の二相分配系におけるタンパク質の抽出挙動を調べた.また,抽出に及ぼすイオン界面活性剤の添加の影響についても検討した。PluronicにはEOとPOの配列が異なるL61と25R2の二つのタイプを用いた。L61の系では,疎水性タンパク質が抽出された。この系にタンパク質と電荷が同符号のイオン界面活性剤を添加すると抽出は抑制され,逆に異符号のとき抽出は促進された.これは,界面活性剤相においてL61とイオン界面活性剤が荷電混合ミセルを形成し,タンパク質と静電的相互作用をしたためと考えられる。一方,25R2の系においては,サイズの大きな疎水性タンパク質は抽出されなかった。また,タンパク質と電荷が異符号のイオン界面活性剤を添加しても,抽出は促進されなかった。これは,25R2ミセルがネットワークを形成し,タンパク質に対して排除効果が働くためと考えられる.したがって,本二相分配系においては,タンパク質の電荷ならびにサイズが抽出を制御していることを明らかにした.
2. 昨年度までにTritonX-114/硫酸デキストランミセル水性二相分配系が肝ミクロゾーム(Ms)中のチトクロムb_5(b_5)の選択的抽出に優れた系であることを明らかにした。本年度はこの二相分配系を用いてブタ肝Msからb_5の精製を行った。ボリマー濃度,pHならびにイオン強度などの抽出条件を検討した結果,わずか3回の繰り返し抽出を行うことで,SDS-電気泳動において単一のバンドになるまでb_5を精製することができた。従来法では,同程度の精製度を得るのに数日から数週間を要していたのに対して,本法はわずか数時間の作業で精製を行うことが可能であった。したがって,本二相分配法を用いたb_5の迅速な精製法を確立することができた。
日本学術振興会, 奨励研究(A), 北海道大学, 09750884