Development of Polymer-Lipid Hybrid Nanoparticles for Large-Sized Plasmid DNA Transfection.
Masatoshi Maeki, Shuya Uno, Kaisei Sugiura, Yusuke Sato, Yoichiro Fujioka, Akihiko Ishida, Yusuke Ohba, Hideyoshi Harashima, Manabu Tokeshi
ACS applied materials & interfaces, 16, 2, 2110, 2119, 17 Jan. 2024, [International Magazine]
English, Scientific journal, RNA and DNA delivery technologies using lipid nanoparticles (LNPs) have advanced significantly, as demonstrated by their successful application in mRNA vaccines. To date, commercially available RNA therapeutics include Onpattro, a 21 bp siRNA, and mRNA vaccines comprising 4300 nucleotides for COVID-19. However, a significant challenge remains in achieving efficient transfection, as the size of the delivered RNA and DNA increases. In contrast to RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and their cellular functionalization. Here, we introduce polymer-lipid hybrid nanoparticles designed for efficient, large-sized pDNA transfection. We demonstrated that LNPs loaded with positively charged pDNA-polycation core nanoparticles exhibited a 4-fold increase in transfection efficiency for 15 kbp pDNA compared with conventional LNPs, which encapsulate a negatively charged pDNA-polycation core. Based on assessments of the size and internal structure of the polymer-lipid nanoparticles as well as hemolysis and cellular uptake analysis, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This approach holds promise for accelerating the in vivo delivery of large-sized pDNA and advancing the development of artificial cells.

Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations
Masatoshi Maeki, Niko Kimura, Yuto Okada, Kazuki Shimizu, Kana Shibata, Yusuke Miyazaki, Akihiko Ishida, Kento Yonezawa, Nobutaka Shimizu, Wataru Shinoda, Manabu Tokeshi
Nanoscale Advances, Royal Society of Chemistry (RSC), 2024
Scientific journal, We investigated ethanol-induced structural changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations.

A portable liquid chromatography system based on a separation/detection chip module consisting of a replaceable ultraviolet-visible absorbance or contactless conductivity detection unit
Akihiko Ishida, Takuma Nishimura, Kaito Koyama, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
Journal of Chromatography A, 1706, 464272, 464272, Elsevier BV, Sep. 2023, [Peer-reviewed], [Invited], [Lead author, Corresponding author]
English, 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

Microfluidic paper-based analytical devices for cancer diagnosis
Ahmed A. Shalaby, Chia Wen Tsao, Akihiko Ishida, Masatoshi Maeki, Manabu Tokeshi
Sensors and Actuators B: Chemical, 379, 15 Mar. 2023, [Peer-reviewed]
Scientific journal, Cancer is a leading cause of death worldwide. Early diagnosis of cancer is crucial for successful treatment which, in turn, will decrease mortality. The development of low-cost, accurate, and easy to operate point-of-need (PON) devices to be used for cancer diagnosis and treatment follow-up is a worldwide need, especially in developing countries. Paper-based analytical devices (PADs) are considered a key solution, as they provide a low-cost platform for developing PON biosensors for cancer biomarker detections. There are various types of 2D and 3D PADs according to the type of paper substrate (filter paper, chromatographic paper, nitrocellulose membranes, etc.), fabrication method (wax printing, screen printing, cutting, etc.), detection technique used (colorimetry, fluorescence, chemiluminescence, electrochemiluminescence, electrochemical, etc.), the assay principle and recognition element used (antibodies, aptamers, DNA, nanoparticles, enzymes, etc.). Controlling all these factors determines the performance, accuracy, and sensitivity of the developed devices. This review discusses all these factors in the different PADs used for detection of cancer biomarkers and summarizes the advantages and disadvantages of each one.

Effects of Addition of Blocking Agents on Fluorescence Polarization Immunoassay of Okadaic Acid Using a Microfluidic Device
Shunsuke CHIDA, Kazuki TAKAHASHI, Mao FUKUYAMA, Motohiro KASUYA, Masatoshi MAEKI, Akihiko ISHIDA, Hirofumi TANI, Koji SHIGEMURA, Anatoly V. ZHERDEV, Sergei A. EREMIN, Akihide HIBARA, Manabu TOKESHI
BUNSEKI KAGAKU, 72, 3, 133, 138, Japan Society for Analytical Chemistry, 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, [Peer-reviewed], [Corresponding author]
Japanese, Scientific journal

A fabrication technique for paper-based analytical devices via two-sided patterning with thermal-transfer printer and laminator
Takuya Monju, Manabu Hirakawa, Satoshi Kuboyama, Rikuro Saiki, Akihiko Ishida
Sensors and Actuators B: Chemical, 375, 132886, 132886, Elsevier BV, Jan. 2023, [Peer-reviewed], [Corresponding author]
English, 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, [Peer-reviewed], [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}, Feb. 2022, [Peer-reviewed], [Corresponding author]
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 & bioelectronics, 190, 113414, 113414, 15 Oct. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, Antibody detection methods for viral infections have received broad attention due to the COVID-19 pandemic. In addition, there remains an ever-increasing need to quantitatively evaluate the immune response to develop vaccines and treatments for COVID-19. Here, we report an analytical method for the rapid and quantitative detection of SARS-CoV-2 antibody in human serum by fluorescence polarization immunoassay (FPIA). A recombinant SARS-CoV-2 receptor binding domain (RBD) protein labeled with HiLyte Fluor 647 (F-RBD) was prepared and used for FPIA. When the anti-RBD antibody in human serum binds to F-RBD, the degree of polarization (P) increases by suppressing the rotational diffusion of F-RBD. The measurement procedure required only mixing a reagent containing F-RBD with serum sample and measuring the P value with a portable fluorescence polarization analyzer after 15 min incubation. We evaluated analytical performance of the developed FPIA system using 30 samples: 20 COVID-19 positive sera and 10 negative sera. The receiver operating characteristic curve drawn with the obtained results showed that this FPIA system had high accuracy for discriminating COVID-19 positive or negative serum (AUC = 0.965). The total measurement time was about 20 min, and the serum volume required for measurement was 0.25 μL. Therefore, we successfully developed the FPIA system that enables rapid and easy quantification of SARS-CoV-2 antibody. It is believed that our FPIA system will facilitate rapid on-site identification of infected persons and deepen understanding of the immune response to COVID-19.

Determination of Deoxynivalenol in Wheat, Barley, Corn Meal, and Wheat-Based Products by Simultaneous Multisample Fluorescence Polarization Immunoassay Using a Portable Analyzer
Ayano Nakamura, Mitsutoshi Aoyagi, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
ACS Food Science & Technology, 1, 9, 1623, 1628, American Chemical Society ({ACS}), 15 Oct. 2021, [Peer-reviewed]
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, [Peer-reviewed], [Corresponding author]
English, Scientific journal

Paper-Based Analytical Device for the On-Site Detection of Nerve Agents
Akinori Yamaguchi, Hajime Miyaguchi, Akihiko Ishida, Manabu Tokeshi
ACS Applied Bio Materials, 4, 8, 6512, 6518, American Chemical Society ({ACS}), 16 Aug. 2021, [Peer-reviewed]
Scientific journal

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, [Peer-reviewed], [International Magazine]
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, [Peer-reviewed], [Corresponding author], [International Magazine]
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 μg/mL, respectively. The simultaneous determination of AA and pH in various beverages was performed with no significant difference compared to results of the conventional method.

One-Step Production Using a Microfluidic Device of Highly Biocompatible Size-Controlled Noncationic Exosome-like Nanoparticles for RNA Delivery
Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
ACS Applied Bio Materials, 4, 2, 1783, 1793, American Chemical Society ({ACS}), 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.

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, 05 Feb. 2021, [Peer-reviewed], [International Magazine]
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 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.

Rapid, sensitive universal paper-based device enhances competitive immunoassays of small molecules
Takeshi Komatsu, Yuki Sato, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
Analytica Chimica Acta, 1144, 85, 95, Elsevier BV, Feb. 2021, [Peer-reviewed], [Corresponding author], [International Magazine]
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.

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.

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}, Jan. 2021
Scientific journal

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.

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}), 25 Aug. 2020, [International Magazine]
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 fragment-based (FBDD) drug design. However, RT crystallography and RT crystallography-based protein-ligand complex analyses require the preparation and measurement of numerous crystals to avoid the X-ray radiation damage. Thus, for the application of RT crystallography to protein-ligand complex analysis, the simultaneous preparation of protein-ligand complex crystals and sequential X-ray diffraction measurement remain challenging. Here, we report an RT crystallography technique using a microfluidic protein crystal array device for protein-ligand complex structure analysis. We demonstrate the microfluidic sorting of protein crystals into microwells without any complicated procedures and apparatus, whereby the sorted protein crystals are fixed into microwells and sequentially measured to collect X-ray diffraction data. This is followed by automatic data processing to calculate the 3D protein structure. The microfluidic device allows the high-throughput preparation of the protein-ligand complex solely by the replacement of the microchannel content with the required ligand solution. We determined eight trypsin-ligand complex structures for the proof of concept experiment and found differences in the ligand coordination of the corresponding RT and conventional cryogenic structures. This methodology can be applied to easily obtain more natural structures. Moreover, drug development by FBDD could be more effective using the proposed methodology.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer
Keine Nishiyama, Yohei Takeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Yutaka Yonezawa, Kunitoshi Imai, Haruko Ogawa, Manabu Tokeshi
Sensors and Actuators B: Chemical, 316, 128160, 128160, Elsevier {BV}, Aug. 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.

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, [Peer-reviewed], [International Magazine]
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, [Peer-reviewed], [International Magazine]
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 μm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 μm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.

Paper-Based Device for the Facile Colorimetric Determination of Lithium Ions in Human Whole Blood
Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
ACS Sensors, 5, 5, 1287, 1294, American Chemical Society ({ACS}), 22 May 2020, [Peer-reviewed], [International Magazine]
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%).

Development of a Paper-based Analytical Chip for the Detection of Bacterial 16S rRNA in Wastewater Samples
Meri Nakajima, Akihiko Ishida, Manabu Tokeshi, Hisashi Satoh
BUNSEKI KAGAKU, 69, 12, 715, 722, JAPAN SOC ANALYTICAL CHEMISTRY, 2020
Japanese, Scientific journal, Analysis of bacteria in the sewage wastewater treatment process is essential for process stabilization and upgrading. Although bacteria are currently being analyzed by molecular biology techniques targeting the 16S rRNA gene, there is a problem that they are time-consuming and labor-intensive. In this study, we developed a paper-based analytical chip by using two types of DNA molecules that specifically bind to bacterial 16S rRNA. We optimized the fabrication method of the detection probe and the paper-based analytical chip, and then detected synthetic DNA having a nucleotide sequence that hybridizes with the designed DNA molecules and bacterial 16S rRNA extracted from an activated sludge sample. We evaluated the amount of nucleic acids quantitatively by taking images of the detection line on the paper-based analytical chip with a smartphone and analyzing its brightness with an open-source image processing program, ImageJ. Our method was able to detect 85 nM of bacterial 16S rRNA concentration in the extract. Nucleic acids that did not hybridize with either of the designed DNA molecules were not detected, demonstrating high selectivity of our method.

An aprotic polar solvent assisted size-tuning method for microfluidic production of lipid-based drug nanocarriers with various sizes
Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1021, 1022, 2020
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.

An Electrochemical Sensor Based on Structure Switching of Dithiol-modified Aptamer for Simple Detection of Ochratoxin A.
Mazaafrianto DN, Ishida A, Maeki M, Tani H, Tokeshi M
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry, 35, 11, 1221, 1226, JAPAN SOC ANALYTICAL CHEMISTRY, Nov. 2019, [Peer-reviewed], [Corresponding author]
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.

Rapid, Sensitive, and Selective Detection of H5 Hemagglutinin from Avian Influenza Virus Using an Immunowall Device
Kenia Chávez Ramos, Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Toshihiro Kasama, Yoshinobu Baba, Manabu Tokeshi
ACS Omega, 4, 15, 16683, 16688, American Chemical Society ({ACS}), 08 Oct. 2019, [Peer-reviewed]
Scientific journal, © 2019 American Chemical Society. Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23-100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.

Cover Picture: A Concentric Ring Electrode for a Wall‐jet Cell in a Microfluidic Device (Electroanalysis 9/2019)
Keine Nishiyama, Koki Hoshikawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
Electroanalysis, 31, 9, 1736, 1743, Wiley, 06 Sep. 2019, [Peer-reviewed], [Corresponding author]
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, 07 Aug. 2019, [Peer-reviewed]
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.

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.

High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer
Osamu Wakao, Ken Satou, Ayano Nakamura, Polina A. Galkina, Keine Nishiyama, Ken Sumiyoshi, Fumio Kurosawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Mikhail A. Proskurnin, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
Lab on a Chip, 19, 15, 2581, 2588, Royal Society of Chemistry ({RSC}), 2019, [Peer-reviewed]
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.

High-throughput fluorescence polarization immunoassay by using a portable fluorescence polarization imaging analyzer
Osamu Wakao, Ken Satou, Ayano Nakamura, Polina A. Galkina, Keine Nishiyama, Ken Sumiyoshi, Fumio Kurosawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Mikhail A. Proskurnin, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
Lab on a Chip, 19, 15, 2581, 2588, Royal Society of Chemistry ({RSC}), 2019, [Peer-reviewed]
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.

Ultrasensitive detection of disease biomarkers using an immuno-wall device with enzymatic amplification
Keine Nishiyama, Toshihiro Kasama, Seiya Nakamata, Koya Ishikawa, Daisuke Onoshima, Hiroshi Yukawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
The Analyst, 144, 15, 4589, 4595, Royal Society of Chemistry ({RSC}), 2019, [Peer-reviewed]
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.

Label-Free Electrochemical Sensor for Ochratoxin A Using a Microfabricated Electrode with Immobilized Aptamer
Donny N. Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
ACS Omega, 3, 12, 16823, 16830, American Chemical Society (ACS), 31 Dec. 2018, [Peer-reviewed], [Corresponding author]
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, Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed, aptamers have made significant contributions as bio-recognition sensors. Microdevice systems allow for low reagent consumption, high-throughput of samples, and disposability. Due to these advantages, there has been an increasing demand to develop microfluidic-based aptasensors for analytical technique applications. This review introduces the principal concepts of aptasensors and then presents some advanced applications of microdevice-based aptasensors on several platforms. Highly sensitive detection techniques, such as electrochemical and optical detection, have been integrated into lab-on-a-chip devices and researchers have moved towards the goal of establishing point-of-care diagnoses for target analyses.

A compact fluorescence polarization analyzer with high-transmittance liquid crystal layer
Osamu Wakao, Ken Satou, Ayano Nakamura, Ken Sumiyoshi, Masanori Shirokawa, Chikaaki Mizokuchi, Kunihiro Shiota, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
Review of Scientific Instruments, 89, 2, 024103, American Institute of Physics Inc., 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, 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], [Corresponding author]
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], [Lead author, Corresponding author]
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.

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.

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.

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

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, [Peer-reviewed]
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.

Microchip reversed-phase liquid chromatography with packed column and electrochemical flow cell using polystyrene/poly(dimethylsiloxane)
Akihiko Ishida, Masamichi Natsume, Tamio Kamidate
JOURNAL OF CHROMATOGRAPHY A, 1213, 2, 209, 217, ELSEVIER SCIENCE BV, Dec. 2008, [Peer-reviewed]
English, Scientific journal, A microchip pressure-driven liquid chromatography (LC) with a packed column and an electrochemical flow cell has been developed by using polystyrene (PS) and poly(dimethylsiloxane) (PDMS). The cylindrical separation column with packed octadecyl silica particles was fabricated in the PS substrate. The three electrode system (working, reference, and counter electrode) for amperometric detection was fabricated onto the PS substrate, using the Au deposition, photolithography, and chemical etching. The detector flow cell was formed by sealing the electrode system with a PDMS chip containing a channel. In this flow cell, the effect of working electrode width (in the direction of flow) on chromatographic parameters, such as peak width and peak resolution were studied in electrode width ranging 50-5000 mu m. The effect of electrode width on sensitivity (current intensity. current density, and S/N ratio) was also examined. The sensitivity was discussed by simulating the concentration profile generated around the working electrode. The effects of the column packing size and the column size on the separation efficiency were examined. In this study, a good separation of three catechins was successfully achieved and the detection limits for (+)-catechin, epicarechin, and epigallocatechin gallate were 350, 450, and 160 nM, respectively. (c) 2008 Elsevier B.V. All rights reserved.

Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation
Akihiko Ishida, Yasuko Yamada, Tamio Kamidate
ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 392, 5, 987, 994, SPRINGER HEIDELBERG, Nov. 2008, [Peer-reviewed]
English, Scientific journal, In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

Direct determination of horseradish peroxidase encapsulated in liposomes by using luminol chemiluminescence
Tamio Kamidate, Kanako Komatsu, Hirofumi Tant, Akihiko Ishida
ANALYTICAL SCIENCES, 24, 4, 477, 481, JAPAN SOC ANALYTICAL CHEMISTRY, Apr. 2008, [Peer-reviewed]
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.

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, [Peer-reviewed]
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.

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, [Peer-reviewed]
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.

Reversed-phase liquid chromatography on a microchip with sample injector and monolithic silica column
Akihiko Ishida, Takahiro Yoshikawa, Masamichi Natsume, Tamio Kamidate
JOURNAL OF CHROMATOGRAPHY A, 1132, 1-2, 90, 98, ELSEVIER SCIENCE BV, Nov. 2006, [Peer-reviewed]
English, Scientific journal, In micro total analysis systems, liquid chromatography (LC) works under pressure-driven flow is the essential analysis component. There were not, however, much works on microchip LC. Here we developed a microchip for reversed-phase LC using porous monolithic silica. The chip consisted of a double T-shaped injector and a similar to 40-cm serpentine separation channel. The octadecyl-modified monolithic silica was prepared in the specified part of the channel on the microchip using sol-gel process. Furthermore, the effect of geometry of turn sections on band dispersion at turns was examined under pressure-driven flow. High separation efficiencies of 15,000-18,000 plates/m for catechins were obtained using the LC chip. (c) 2006 Elsevier B.V. All rights reserved.

Firefly bioluminescent assay of ATP in the presence of ATP extractant by using liposomes
T Kamidate, K Yanashita, H Tani, A Ishida, M Notani
ANALYTICAL CHEMISTRY, 78, 1, 337, 342, AMER CHEMICAL SOC, Jan. 2006, [Peer-reviewed]
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, [Peer-reviewed], [Lead author, Corresponding author]
English

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, [Peer-reviewed]
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.

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, [Peer-reviewed]
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.

Determination of hydrogen peroxide by fluorescein chemiluminescence catalyzed with horseradish peroxidase encapsulated in liposome
Tamio Kamidate, Yoshiki Ishida, Hirofumi Tani, Akihiko Ishida
Bunseki Kagaku, 54, 6, 569, 572, 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 × 10-8 M up to 1.0 × 10-5 M. When HRP trapped in liposome was used as a catalyst, the CL intensity was greater than that observed by using HRP dissolved in the bulk solution in the range of 4.0 × 10-7 M up to 1.0 × 10-5 M of H2O2. © 2005 The Japan Society for Analytical Chemistry.

Cationic liposomes enhanced firefly bioluminescent assay of bacterial ATP in the presence of an ATP extractant
N Nakata, A Ishida, H Tani, T Kamidate
ANALYTICAL SCIENCES, 19, 8, 1183, 1185, JAPAN SOC ANALYTICAL CHEMISTRY, Aug. 2003, [Peer-reviewed]
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.

Firefly bioluminescence assay of ATP using diethylaminoethyl-dextran as an enhancer in the presence of sodium chloride and ATP extranctant
A Ishida, T Yoshikawa, T Kamidate
ANALYTICAL BIOCHEMISTRY, 316, 1, 127, 130, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2003, [Peer-reviewed]
English, The firefly bioluminescence (BL)(1) technique for measuring biomass, cell status, and activity of adenosine 5'-triphosphate (ATP)-related enzymes and reporter protein has been widely used because of its rapidity, high sensitivity, and robustness [1-9]. This technique is based on the reaction including the oxidative decarboxylation of luciferin by firefly luciferase in the presence of ATP and Mg2+, resulting in the production of light. Recently this BL assay has received considerable attention in the food industry because this method can be used as a rapid monitoring system for the safety of food products and hygiene of food contact surfaces at critical control points of food processing [10-14]. ATP is present in all living cells and the amount of ATP per cell is fairly constant. The presence of ATP in the food products and manufacturing environment suggests contamination of microbes and food residues.
The firefly BL method is subject to lowering of sensitivity because of the inhibition of luciferase by ATP extractants and various salts. The ATP extractants are essential compounds for release of ATP from living cells, such as dilute acids, surfactants, boiling buffers, and organic solvents [15,16]. Among these extractants, trichloroacetic acid (TCA), which is suitable for a variety of types of cells, is the most widely used. This extractant, however, is a rather potent inhibitor of luciferase [2,17,18]. In addition to the ATP extractant, several anions such as perchlorate, nitrate, and halide ions inhibit the enzyme. Chloride ion is contained as a. form of sodium chloride in a variety of foods. Although bacteria can barely survive at a high concentration of NaCl, the presence of bacteria is often detected in foods containing NaCl. Therefore, there should be a considerable loss in sensitivity of the BL assay for monitoring bacterial contamination of those food products because of the inhibition by NaCl and TCA.
Previously, we have found that diethylaminoethyldextran (DEAE-Dx) enhanced the light emission from the BL reaction [19] and have successively applied it to a highly sensitive BL assay of ATP in the presence of TCA or Triton X-100 [20]. On the other hand, the effect of a variety of ions on the BL reaction was studied and the possible mechanisms of the inhibition by several inorganic anions were presented [21-23]. Furthermore the inhibitory effects of inorganic ions in real samples on the BL assay were investigated [24-26]. However, there was no study on improving the sensitivity of the BL assay in the presence of those inhibitors.
In this report, we describe the highly sensitive BL assay under severe conditions, in which, in addition to TCA, chloride ion inhibits luciferase, based on the enhancement effect of DEAE-Dx on the BL emission.

Firefly bioluminescence assay of ATP using diethylaminoethyl-dextran as an enhancer in the presence of sodium chloride and ATP extranctant
A Ishida, T Yoshikawa, T Kamidate
ANALYTICAL BIOCHEMISTRY, 316, 1, 127, 130, ACADEMIC PRESS INC ELSEVIER SCIENCE, May 2003
English, The firefly bioluminescence (BL)(1) technique for measuring biomass, cell status, and activity of adenosine 5'-triphosphate (ATP)-related enzymes and reporter protein has been widely used because of its rapidity, high sensitivity, and robustness [1-9]. This technique is based on the reaction including the oxidative decarboxylation of luciferin by firefly luciferase in the presence of ATP and Mg2+, resulting in the production of light. Recently this BL assay has received considerable attention in the food industry because this method can be used as a rapid monitoring system for the safety of food products and hygiene of food contact surfaces at critical control points of food processing [10-14]. ATP is present in all living cells and the amount of ATP per cell is fairly constant. The presence of ATP in the food products and manufacturing environment suggests contamination of microbes and food residues.
The firefly BL method is subject to lowering of sensitivity because of the inhibition of luciferase by ATP extractants and various salts. The ATP extractants are essential compounds for release of ATP from living cells, such as dilute acids, surfactants, boiling buffers, and organic solvents [15,16]. Among these extractants, trichloroacetic acid (TCA), which is suitable for a variety of types of cells, is the most widely used. This extractant, however, is a rather potent inhibitor of luciferase [2,17,18]. In addition to the ATP extractant, several anions such as perchlorate, nitrate, and halide ions inhibit the enzyme. Chloride ion is contained as a. form of sodium chloride in a variety of foods. Although bacteria can barely survive at a high concentration of NaCl, the presence of bacteria is often detected in foods containing NaCl. Therefore, there should be a considerable loss in sensitivity of the BL assay for monitoring bacterial contamination of those food products because of the inhibition by NaCl and TCA.
Previously, we have found that diethylaminoethyldextran (DEAE-Dx) enhanced the light emission from the BL reaction [19] and have successively applied it to a highly sensitive BL assay of ATP in the presence of TCA or Triton X-100 [20]. On the other hand, the effect of a variety of ions on the BL reaction was studied and the possible mechanisms of the inhibition by several inorganic anions were presented [21-23]. Furthermore the inhibitory effects of inorganic ions in real samples on the BL assay were investigated [24-26]. However, there was no study on improving the sensitivity of the BL assay in the presence of those inhibitors.
In this report, we describe the highly sensitive BL assay under severe conditions, in which, in addition to TCA, chloride ion inhibits luciferase, based on the enhancement effect of DEAE-Dx on the BL emission.

Direct detection of horseradish peroxidase as a marker molecule encapsulated in liposomes via use of fluorescein chemiluminescence
T Kamidate, Y Ishida, H Tani, A Ishida
CHEMISTRY LETTERS, 32, 4, 402, 403, CHEMICAL SOC JAPAN, 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.

Preconcentration of catecholamines into liposomes with imposed pH gradients
Akihiko Ishida, Manabu Ikemoto, Yoshiki Ishida, Tamio Kamidate
Bulletin of the Chemical Society of Japan, 76, 5, 985, 989, Chemical Society of Japan, 2003, [Peer-reviewed]
English, Scientific journal, The preconcentrations of epinephrine (EP) and norepinephrine (NE) into phosphatidylcholine liposomes with imposed pH gradient across the membrane were investigated by adding the catecholamine (CA) to the external medium of liposomes. To determine the CAs entrapped into liposomes, a simple method was also developed, based on the adsorption of CAs onto alumina and chemiluminescence detection. The uptake of the CAs into the liposomes was examined over a wide ΔpH range of 0-5 units (internal pH 5.0). The maximum uptakes of EP and NE were 70% and 88%, respectively, around an external pH of 9 (ΔpH4), where zwitterionic species of EP and NE are dominant. The final concentrations of EP and NE in internal volumes were 34- and 43-fold greater than the initial concentrations of EP and NE, respectively. The uptake of the CAs was studied by using a mathematical model, which indicates that the uptake is contributed by the molar fraction of the zwitterion species in the external volume as well as the pH gradient. The uptake of EP was found to suffer from a steric hindrance due to a methyl group substituted for the hydrogen of the amino group. This study proves the capability of the liposomes for accumulation media of CAs.

Kamidate, T., Ishida, Y., Tani, H., and Ishida, A.:"Direct Detection of Horseradish Peroxidase as a Marker Molecule Encapsulated in Liposomes via Use of Fluorescein Chemiluminescence", Chemistry Letters,32:402-403 (2003)*　　　　　　　　　　　　　　　
2003

Ishida, A., Ikemoto, M., Ishida, Y., Kamidate, T.: "Preconcentration of Catecholamines into Liposomes with Imposed pH Gradients", Bulletin of the Chemical Society of Japan, 76:985-989 (2003).*　　　　　　　　　　　　　　　
2003

Enhanced firefly bioluminescence assay of ATP in the presence of ATP extractants by using diethylaminoethyl-dextran
A Ishida, T Yoshikawa, T Nakazawa, T Kamidate
ANALYTICAL BIOCHEMISTRY, 305, 2, 236, 241, ACADEMIC PRESS INC ELSEVIER SCIENCE, Jun. 2002, [Peer-reviewed]
English, Scientific journal, A highly sensitive ATP biolumineseence assay with diethylaminoethyl-dextran (DEAE-Dx) in the presence of ATP extractants such as trichloroacetic acid (TCA) and Triton X-100 is described. These ATP extractants inhibited the activity of firefly luciferase, resulting in a remarkable decrease in the intensity of light emission. However, DEAE-Dx enhanced the intensity of light emission as long as firefly luciferase was active in the presence of the ATP extractants. When DEAE-Dx was used for the assay, the detection limits for ATP in the presence of TCA and Triton X-100 were 0.3 and 0.5 pM, respectively, in aqueous ATP standard solution. The detection limit in the presence of DEAE-Dx was improved 13- to 20-fold compared to that in the absence of DEAE-Dx. The method was applied to the determination of ATP in Escherichia coli extracts. When a 5% solution of TCA was used for the extraction of ATP from E. coli cells, the detection limit corresponded to 250 cells ml(-1) of E. coli. (C) 2002 Elsevier Science (USA).

Uptake of transition metal ions using liposomes containing dicetylphosphate as a ligand
T Kamidate, Y Hashimoto, H Tani, A Ishida
ANALYTICAL SCIENCES, 18, 3, 273, 276, JAPAN SOC ANALYTICAL CHEMISTRY, Mar. 2002, [Peer-reviewed]
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+.

Development of simple methods for the visual determination of trace metal ions by using characteristic color-forming reaction systems of metal chelates
Akihiko Ishida
Bunseki Kagaku, 49, 1, 71, 72, Japan Society for Analytical Chemistry, 2000, [Peer-reviewed]
Japanese, Scientific journal, The importance of simple analytical methods that require no sophisticated instruments is increasing. For simple measurements, visual methods based on the perception of color change and the recognition of figure are more suitable. The author has developed simple methods for the visual determination of trace metal ions, based on construction of the following chemical systems. First, for the visual sensitive detection of trace aluminium, a simple concentration method was developed. After a drop of solution are evaporated on a hydrophobic substrate, components, such as several pH buffers or poly(vinyl alcohol), form a ring-like solid phase. Aluminium ion was concentrated into the ring as a fluorescent chelate of 2,2′-dihydroxyazobenzene and distinctly detected. This method has been successively applied to the determination of ppb levels of aluminium in tealeaves. Second, a color-forming reaction system has been developed, which makes it possible to visualize the concentration of metal ions by using a color property of the Xylenol Orange (XO)-metal complex system. The Xylenol Orange-Fe(III) system gives yellow, red, and purple blue along with an increase in the iron concentration, owing to a sequential formation of FeIII(xo) and FeIII2(xo). A field test for iron leached from rock samples was developed by using the color reaction, and was applied to the prediction of water quality in aquifers.

Field screening test for iron leached from well bore rock samples by using color development of iron(III)-xylenol orange system
A Ishida, E Kaneko, T Yotsuyanagi
CHEMISTRY LETTERS, 28, 4, 351, 352, CHEMICAL SOC JAPAN, Apr. 1999, [Peer-reviewed]
English, Scientific journal, A field test for iron leached from rocks has been developed by using a purple blue iron(III)-Xylenol Orange (XO) complex formed at the iron(III) concentrations above the molar concentration of XO. The test is performed in an aqueous drop on a Tefron(R) plate. When the iron content exceeds a criterion, a distinct color change from yellow to purple blue appears in the drop containing a 10-mg rock sample, HCl, XO, and (NH4)(2)S2O8. This method was applied to the prediction of water quality in aquifers.

A New Confined Spot Test with Hydrophobic Filter Paper and Its Application to the Visual Fluorometric Determination of Trace Aluminium with 2,2'-Dihydroxyazobenzene
KANEKO Emiko, ISHIDA Akihiko, DEGUCHI Yuji, YOTSUYANAGI Takao
Chemistry Letters, 1994, 9, 1615, 1618, The Chemical Society of Japan, 05 Sep. 1994
English, A new sensitive and simple spot test has been developed for the visual determination of trace element in water, based on the formation of a size confined colored ring on hydrophobic filter paper. When 0.1 cm³ of aqueous solution is spotted on the water-repelling surface and evaporated in an oven, a small fleck of 6 mm diameter edged with a colored analyte is formed. This method has been successfully applied to the visual fluorometric determination of trace aluminium(III) ion with 2,2′-dihydroxyazobenzene (DHAB or H₂L). The 1 : 1 chelate, [AlL]⁺, becomes localized in a concentric ring zone at pH 6.5. The detection limit is 2 × 10⁻⁸ mol dm⁻³ (0.5 ppb) by visual fluorometry with a UV lamp in the dark.

粒径制御型ハイブリッドエクソソーム作製法の開発
大山祥大, 真栄城正寿, 真栄城正寿, 日比野光恵, 石田晃彦, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

タンパク質結晶のサイズ分離デバイスの開発とX線結晶構造解析への応用
平山兼光, 真栄城正寿, 真栄城正寿, 真栄城正寿, 上野剛, 坂井直樹, 日比野光恵, 石田晃彦, 山本雅樹, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

深紫外発光ダイオードを光源とするポータブル吸光度検出液体クロマトグラフの開発
小山魁人, 石田晃彦, 日比野光恵, 真栄城正寿, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

タンパク質X線結晶構造解析の自動化のためのオールインワンデバイスの開発
一町田由貴, 真栄城正寿, 真栄城正寿, 真栄城正寿, 上野剛, 小西真昌, 坂井直樹, 日比野光恵, 石田晃彦, 山本雅樹, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

人工エクソソームのRNA送達における膜タンパク質の機能解明
荒谷響子, 丹羽彩由花, 真栄城正寿, 真栄城正寿, 日比野光恵, 石田晃彦, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

アプタマーを用いた非競合蛍光偏光分析法における検出感度の向上
千田駿亮, 植咲月, 福山真央, 粕谷素洋, 日比野光恵, 石田晃彦, 真栄城正寿, 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), 2024, 2024

マイクロ流体デバイスを用いた核酸搭載ウイルス様粒子作製法の開発
杉浦魁星, 岡田悠斗, 真栄城正寿, 真栄城正寿, 日比野光恵, 石田晃彦, 渡慶次学, 化学系学協会北海道支部冬季研究発表会(Web), 2024, 2024

Development of a SARS-CoV-2-like particles preparation method using microfluidic devices and its application to nucleic acid delivery
杉浦魁星, 岡田悠斗, 真栄城正寿, 真栄城正寿, 石田晃彦, 渡慶次学, 化学とマイクロ・ナノシステム, 23, 1, 2024

マイクロ流体デバイスを用いた人工エクソソームの開発
丹羽彩由花, 大山祥大, 真栄城正寿, 真栄城正寿, 日比野光恵, 石田晃彦, 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

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

小型測定デバイスからなるATPの簡便かつ迅速な比色定量システム
菊知龍哉, 石田晃彦, 日比野光恵, 真栄城正寿, 渡慶次学, 日本分析化学会年会講演要旨集(Web), 72nd, 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

非競合蛍光偏光分析法の検出感度に影響する因子:トレーサーの蛍光位置および蛍光色素の寿命
千田駿亮, 植咲月, 福山真央, 粕谷素洋, 日比野光恵, 石田晃彦, 真栄城正寿, 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), 48th, 2023

タンパク質のX線結晶構造解析の自動化に向けたマイクロ流体デバイスの開発
一町田由貴, 真栄城正寿, 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, 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), 48th, 2023

マイクロ流体デバイスを用いたSARS-CoV-2様粒子作製法の開発と薬剤送達への応用
杉浦魁星, 岡田悠人, 真栄城正寿, 真栄城正寿, 石田晃彦, 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), 48th, 2023

マイクロ流体デバイスを用いたハイブリッドエクソソームの作製
大山祥大, 真栄城正寿, 日比野光恵, 石田晃彦, 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), 48th, 2023

Title Development of a portable liquid chromatography system with an electroosmotic pump and chip-based column
石田晃彦, 小山魁人, 日比野光恵, 真栄城正寿, 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, 43rd (CD-ROM), 2023

Improved Sensitivity of Bioluminescence Assay in a Paper-Based Analytical Device as a Platform
田中楓子, 大山祥大, 石田晃彦, 真栄城正寿, 渡慶次学, 藤原政司, 田島健次, 谷博文, セルロース学会年次大会講演要旨集, 30th, 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

エクソソームによるリキッドバイオプシーのための標準脂質ナノ粒子の開発
大山祥大, 丹羽彩由花, 真栄城正寿, 真栄城正寿, 真栄城正寿, 石田晃彦, 谷口正輝, 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

マイクロ流体デバイスを用いた人工エクソソーム作製法の開発
丹羽彩由花, 大山祥大, 真栄城正寿, 真栄城正寿, 石田晃彦, 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

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

マイクロ流体デバイスを用いた長鎖プラスミドDNA搭載ナノ粒子の作製
宇野秀哉, 真栄城正寿, 真栄城正寿, 佐藤悠介, 石田晃彦, 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), 46th, 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

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]
This paper reports a bottom-up fabrication technique of amyloid nanostructures and its application to an enzyme immobilized microfluidic device. The process consists 3 steps: (1) immobilization of lysozymes, (2) denaturation and growth of amyloid fibrils, and (3) immobilization of enzymes on the amyloid fibrils. The growth of amyloid fibrils was able to control by the concentration of lysozyme solution and incubation time. We evaluated the performance of enzyme immobilized amyloid fibrils by hydrolysis reaction. As a results, synthetic peptides and proteins were effectively digested compared with conventional method., 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]
This paper describes lipid nanoparticles (LNPs) formation behavior in microfluidic devices equipped with the staggered herringbone micromixers (SHM) in order to control the LNPs size precisely. Three types of microfluidic devices with different heights of mixer structures were fabricated to examine the effect of mixer structure on the LNPs formation behavior. We found the height of the grooved structures, the lipid concentration, and the ethanol concentration were significant factors for controlling LNPs size and its distribution., 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, 1791, 27 Oct. 2015, [Peer-reviewed]
We have developed a label-free detection method of biomolecules using nanostructures [1]. The principle of this method is based on intensity changes of diffracted light derived from the nanostructures. This method is very simple and useful for label-free detection of biomolecules, but further improvement in sensitivity was necessary to apply it to clinical applications. In this paper, we optimized optical system and device design to improve the sensitivity, and applied it to measure extracellular vesicles for cancer diagnosis. These results showed that our method had a potential to be a first screening method for cancer diagnosis., 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

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, 2, 1164-1166, 1166, 28 Oct. 2013, [Peer-reviewed]
We previously developed immuno-pillar devices for rapid and easy-to-use immunoassay [1-3]. However, improvement in the detection sensitivity (nM-pM) has still remained as a problem toward the screening test for disease markers with low cut-off values. We report here a third-generation immuno-pillar device in which the primary antibodies are covalently bounded to polymers on microbeads. By using this device we achieved pM-fM detection sensitivity of C-reactive protein (CRP) spiked in human serum. Moreover, we demonstrated that the device has long-term stability. From these results, it was proved that the third-generation device has capability required for practical use., 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

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

日本分析化学会北海道支部編「水の分析(第5版)」<共著>　　　　　　　　　　　　　　　
化学同人, 2005

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

Highly sensitive visual fluorometry of aluminium at ppb level with ring-like solid phase of poly(vinyl alcohol)
A Ishida, E Kaneko, T Yotsuyanagi, CHEMISTRY LETTERS, 3, 217, 218, Mar. 1999
By constructing a self-ordered nearly perfect ring system, a new type of visual test method has been developed. Poly(vinyl alcohol) forms a sharp ring-like solid phase (10.6 mm diameter) when a 100-mu l drop of its aqueous solution was evaporated on a poly(vinyl chloride) plate. Aluminium ion of 1-30 ppb has been determined visually as a fluorescent 1:1 chelate of 2,2'-dihydroxyazobenzene concentrated into the ring., CHEMICAL SOC JAPAN, English

マイクロ流体分析　　　　　　　　　　　　　　　
渡慶次 学, 真栄城 正寿, 佐藤 記一, 佐藤 香枝, 火原 彰秀, 石田 晃彦, Chapter5 マイクロチップクロマトグラフィー
Oct. 2020, [Joint work]

農産物・食品検査法の新展開 《普及版》
山本, 重夫
シーエムシー出版, Aug. 2016, 9784781311166, x, 265p, Japanese

農産物・食品検査法の新展開 = Advanced inspection technology of agricultural products and foods
山本, 重夫, 第IV編第1章色で見分ける細菌汚染スクリーニング法～新しいキシレノールオレンジ-鉄錯体法の技術～
シーエムシー出版, Jul. 2010, 9784781302478, x, 265p, Japanese, [Contributor]

水の分析
日本分析化学会北海道支部, 厚谷, 郁夫, 田中, 俊逸, 4.7 フローインジェクション分析（FIA）
化学同人, Jun. 2005, 9784759809916, x, 472p, Japanese, [Contributor]

リポソーム応用の新展開 : 人工細胞の開発に向けて
秋吉, 一成, 辻井, 薫, 奥, 直人, 久保井, 亮一, 宝谷, 紘一, 第4節リポソームを利用した高感度計測法
エヌ・ティー・エス, Jun. 2005, 4860430859, 4, 10, 683, 10, 12p, 図版14p, Japanese, [Contributor]

北海道地域3大学新技術説明会にて成果発表　　　　　　　　　　　　　　　
2012

第２４回国際計量計測展にて研究成果の発表と試作品の展示　　　　　　　　　　　　　　　
2010

第3回次世代医療システム産業化フォーラム2010にて技術発表　　　　　　　　　　　　　　　
2010

北海道大学・科学技術振興機構主催の北海道大学 新技術説明会にて技術発表　　　　　　　　　　　　　　　
2009

北海道大学工学系イノベーションブリッジ2007にて研究成果発表　　　　　　　　　　　　　　　
2007

育種・染色体操作を用いたサケ科魚類の魚卵アレルゲン性低減化の試み
科学研究費助成事業 基盤研究(C)
01 Apr. 2020 - 31 Mar. 2023
清水 裕, 笹岡 友季穂, 藤本 貴史, 平松 尚志, 石田 晃彦, 渡慶次 学, 佐伯 宏樹
進捗状況を3項目に分けて述べる。
1.不妊魚の作出：ニジマスの卵と凍結保存したブラウントラウトあるいはサクラマスの精子を受精し、第二極体放出阻止処理により作出された雑種三倍体候補（A：ニジマス×ブラウントラウト、B：ニジマス×サクラマス）の倍数性を調査した。その結果、候補Aでは全25個体で、候補Bでは1個体を除く30個体が三倍体であった。これらの個体はPITタグで標識し、現在も継続して飼育している。（藤本）
2.魚卵アレルゲン検知系の構築：引き続き、ニジマス卵アレルゲンであるβ’-component（BC）のペーパー免疫分析デバイスの構築に取り組んだ。デバイスは濾紙にインクを印刷して加熱する常法により作製し、インクで囲まれた領域を分析反応ゾーンとした。分析は、反応ゾーンに新規作製した抗BC抗体を固定化して試料、酵素標識抗BC抗体、発色試薬を順に加えて行う手順とした。本手法では、従来並みの感度（検出感度：約1 ng/mL）の分析が従来の1/100の時間（約20分）で可能となった。（渡慶次、石田）
加えて、交雑種に対応した検知系に使用する抗体の作成のため、サクラマス排卵からBCを精製し、これを家兎に免疫し抗血清を作製した。（平松）
3.不妊化魚の魚卵アレルゲン性の調査：全23個体の不妊化三倍体ニジマスの内臓組織に含まれるBCを測定したが、全個体の筋肉には魚卵アレルギー発症リスクが認められなかった。しかし、5個体において生殖線から少量のBCが検出されたが、その内3個体は目視にて明確な生殖腺の発達が観られた。目視で生殖線の発達が確認できなかった20個体について、生殖腺の組織切片を作製・観察し、その発達状況を確認した。その結果、３個体で発達中の卵母細胞が散在しているのが確認され、生殖腺の外観だけでアレルギー発症リスクの有無を見分けるのは困難であることが判明した。（清水、佐伯、笹岡、平松）
日本学術振興会, 基盤研究(C), 北海道大学, 20K05903

Construction of a hybrid electrochemical micro-cell and its application to the analyses of trace biological substances
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2017 - 31 Mar. 2020
Ishida Akihiko
An electrochemical detector for next-generation miniaturized analytical devices for testing blood and urine has been developed. Miniaturization of devices allowing automatic flow-based measurement is one of the worldwide topics because it enables rapid diagnosis near the patient. These devices also require improving sensitivity. However, increasing the electrode area is counterproductive, and no other approach has been attempted. Therefore, this study focused on not the electrode area, but the geometry of the electrode. It demonstrated that the sensitivity was successfully improved by changing the geometry of the conventional shape of a disc to a concentric array of multiple ring electrodes. The proposed array electrode allowed the detection of a trace amount of dopamine in blood without special pre-concentration of a sample.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 17K05892

2018年度 現場での即時検査が可能な高性能可搬型分析装置の開発 補助事業　　　　　　　　　　　　　　　
研究補助開発研究
Apr. 2018 - Mar. 2019
石田 晃彦
JKA, Principal investigator, Competitive research funding

ポータブル分析装置の高感度なマイクロ検出器の開発　　　　　　　　　　　　　　　
研究成果展開事業（研究成果最適展開支援プログラム（A-STEP））フィージビリティスタディ（ＦＳ）ステージ 探索タイプ
Jan. 2015 - Dec. 2015
石田 晃彦
科学技術振興機構, Principal investigator, Competitive research funding

Development of next-generation diagnostic chip and its application to clinical diagnosis
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
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

Construction of a highly sensitive electrochemical detector on a microchip device with an integrated single comb-like array electrode and its application
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
2011 - 2013
ISHIDA Akihiko
In this study, a single comb-like electrode was studied, which was formed by arranging a number of microelectrodes at a given spacing. A thorough examination of the shape factors of the electrode led to electrochemical detection of more trace amounts of substances. High sensitivity has been achieved by the electrode alone with a characteristic shape. The electrode has good utility because a wide variety of target substances can be detected and the detection can be performed with a relatively cheap measuring apparatus. This electrode is expected to be used as a powerful detector for microfluidic analysis devices possibly demanded in a field of clinical diagnosis in near future.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 23550087

刺激応答性ベシクルを利用する高感度電気化学検出バイオ分析チップの開発
科学研究費助成事業 若手研究(B)
2006 - 2007
石田 晃彦
本研究では,微小流路内においてベシクル内から漏出させた電気化学活性物質をアンペロメトリーにより高感度に検出することが重要な課題である。電流値は電極面内に形成する測定物質の濃度勾配の積算値であるから,感度を上げるには作用電極の面積または濃度勾配を大きくすることが必要である。これまでに,電極面積を大きくすることは,S/N比を低下させるため有効ではないことを微小流体デバイスを用いて示した。これは,物質が電極反応により消費されながら流れる結果,電極の下流ほど電極垂直方向の濃度勾配が小さくなるためである。そこで,与えられた面積の中で濃度勾配を増加させることが必要になる。そのため,本研究では一本の電極を複数に分割して間隔を置いて配置するくし状の電極を検討した。電極がない部分で試料が補充されるので,下流側での濃度勾配の減少を抑えることができるからである。さらに十分な間隔があれば,分割したどの電極上でも同様の濃度勾配が形成されると推察される。くし形の電極については,これまでに2対の電極を用いて酸化還元を繰り返して感度を上げる検討は行われていたが,くし形電極単独での詳細な検討はなかった。そこで,ポリマー基板およびポリジメチルシロキサンを用いて,間隔をあけた2本の作用電極をもつ微小流体デバイスを作製し,このアプローチの妥当性を検証した。その際,微小流体デバイスで用いられる流速範囲で,物質の種類を変えることにより電流応答の拡散係数依存性を調べた。その結果,どの拡散係数およびどの流速でも間隔の増加とともに電流値は増加し,200μm近辺で最大一定となった。また,電流値を様々な拡散係数および流速で数値シミュレーションしたところ実験結果と一致した。以上から,このアプローチが妥当であり,微小流体デバイスで用いられる流速で様々な電気活性物質に適用できることを明らかにした。
日本学術振興会, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 18750056

The development of nanor eactor for high sensitive chemi luminescence analysis by using liposomes
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
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, Coinvestigator not use grants, Competitive research funding, 18350037

化学発光速度を指標とするリポソームの膜透過性の評価法の開発
科学研究費助成事業 萌芽研究
2005 - 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, Coinvestigator not use grants, Competitive research funding, 17655028

液体クロマトグラフィーの高性能マイクロシステムの構築とその生体分子分析への応用
科学研究費助成事業 若手研究(B)
2004 - 2005
石田 晃彦
本課題の目的は,液体クロマトグラフィー(LC)の要素をガラスやポリマーチップ上に集積化したLCチップを作製し,これを生体分子の分離分析に適用してその有用性を実証することである。本年度は検出部である電極の面積が分離性能および感度(S/N比)に与える影響を検討した。その際,LCチップ間で生じる分離カラムの性能誤差が応答に影響しないよう同一の分離カラムで検討するために,分離カラムから出たバンドを分岐させ,それぞれ異なる電極で同時に検出するLCチップを作製した。電極は金をチップ上に蒸着することにより作製した。分岐部でバンドが等分されることについては蛍光顕微鏡法により確認した。このLCチップを用いて検討した結果,分離度は電極面積に依存しないことを確認した。これは分離カラム(2.5cm長,約10000段/m)から出た試料のバンド幅が電極幅よりも十分に大きいためである。バンド幅と理論段の関係を考慮すると,この結果は小さな分離カラムをもつLCチップ全般にあてはまるといえる。一方,S/N比に関しては電極面積が大きいほど減少した。この理由を電極表面上の試料の流体・拡散シミュレーションに基づいて考察した。すなわち,試料の電極反応が電極上流部で終了するため,電極面積が大きいほど下流部で面積過剰となり電極単位面積当たりに得られる電流値が減少するためと考察した。電極幅は50μm幅が最も有効であった。最後に,医学的な効果が報告されているカテキン類((+)-カテキン,エピカテキン,没食子酸エピガロカテキン)の分離を試みた。その結果,ベースライン分離を達成し,それぞれの検出限界は数百nMレベルであった。また,市販の各種茶飲料のカテキン分析に応用したところ,各成分の分離に加えて,茶葉の発酵の程度が各試料のクロマトグラムに反映していることが確認でき,実試料に対しても良好に適用できることを明らかにした。
日本学術振興会, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 16750056

The investigation for high sensitive chemi luminescence method using nanoreactor as a marker
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
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, Coinvestigator not use grants, Competitive research funding, 15350038

高輝度光微細加工による生体内物質分析用微小電気化学反応セル創製に関する研究
科学研究費助成事業 萌芽研究
2003 - 2004
坂入 正敏, 石田 晃彦
本研究課題は、高輝度光(集光したパルスYAGレーザー照射)表面微細加工法と電気化学的手法により、酵素反応によって生体内で生成される過酸化水素などの、生体微量物質分析用微小電気化学セルをアルミニウム上に作製すること、さらに、対極などを取り付け微小電気化学分析システムにして、その性能を評価することを目的としている。
昨年度は、アルミニウムを用いてアノード酸化、レーザー加工、電気めっきを用いて、2電極型システムを作製でき、K_3Fe(CN)_6/K_4Fe(CN)_6の混合溶液(約1.5μl)を用いてCV測定を行ったところ、アノードピーク電流の走査速度依存性や濃度依存性を確認できたが、ピーク形状および液漏れの問題があった。そこで、本年度は、流路などの形状の改良および作製工程の改良を行い、より完成度の高い生体微量物質分析用微小電気化学セルの作製およびその電気化学的特性評価を行った。
微細溝および微細貫通孔には、レーザー照射後、再度アノード酸化することで、保護性の皮膜を形成し、電極部にはめっきにより金を析出させた。これら、再アノード酸化および金めっきにより、各種溶液で安定に動作可能な、理論内容量1.5μlの微小電気化学セルを作製できた。作製したセルを用いて、電気化学測定を行った結果、従来のマクロ電極と同様のサイクリックボルタモグラムが測定できた。すなわち、アノードピーク電流は電位走査速度の平方根に比例して増加し、その傾きから電極面積を見積もった結果、妥当な値を得ることができた。
日本学術振興会, 萌芽研究, 北海道大学, Coinvestigator not use grants, Competitive research funding, 15656177

自己組織化システムを用いる高性能サイズ制御スポットテスト法
科学研究費助成事業 特別研究員奨励費
1998 - 1998
石田 晃彦
日本学術振興会, 特別研究員奨励費, 東北大学, 97J06075

マイクロチップを利用する化学分析システムの開発　　　　　　　　　　　　　　　
Competitive research funding

血液検体のＡＴＰ測定方法及びキット　　　　　　　　　　　　　　　
Patent right, 石田 晃彦
特許6295408
02 Mar. 2018

ポータブルな液体クロマトグラフ及び液体クロマトグラフィー　　　　　　　　　　　　　　　
Patent right, 石田 晃彦
特許5935696
20 May 2016

検体中に含まれるアデノシン三リン酸の分析方法
Patent right, 石田 晃彦, 山田 泰子, 上舘 民夫, 国立大学法人 北海道大学
JP2006308601, 25 Apr. 2006
WO2006-118093, 09 Nov. 2006
特許第4940432号
30 May 2012
200903081314391360

細胞中ATPの定量法
Patent right, 上舘 民夫, 谷 博文, 石田 晃彦, 三菱化学メディエンス株式会社
特願2004-061585, 05 Mar. 2004
特開2005-245342, 15 Sep. 2005
特許第4546115号
09 Jul. 2010
201103016902185272

イオン性検体のスポットテスト法
Patent right, 四ツ柳 隆夫, 金子 恵美子, 宮川 貴行, 石田 晃彦, 株式会社東北テクノアーチ
特願平6-048632, 18 Mar. 1994
特開平7-260693, 13 Oct. 1995
特許第3393701号
31 Jan. 2003
201103034197155383

試料中成分溶出量の簡易判定方法及びその簡易判定用キット
Patent right, 石田 晃彦, 金子 恵美子, 四ツ柳 隆夫, 株式会社東北テクノアーチ
特願平10-177968, 21 May 1998
特開平11-326309, 26 Nov. 1999
200903018923337891