論文 - 蛭田　勇樹

Single-step bioluminescence lateral flow immunoassays for diagnostics

Shimazu R., Kawahara J., Tomimuro K., Misawa K., Ni Y., Hiruta Y., Merkx M., Citterio D.

23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019 （23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019）     703 - 704 2019年

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© 2019 CBMS-0001. We present two diagnostic platforms, a microfluidic thread-based and a conventional immunochromatographic strip approach, for single-step lateral flow immunoassays (LFIAs) with bioluminescence (BL) signaling requiring only sample application and detection of emission color changes by a digital camera. Both rely on bioluminescence resonance energy transfer (BRET)-switching proteins. The detection of three clinically relevant targets is demonstrated: Cetuximab as example of an antibody-based anticancer drug requiring monitoring of blood drug levels in patients, and HIV1-p17 antibodies and antigens as examples of infectious disease markers.

Paper-Based Antibody Detection Devices Using Bioluminescent BRET-Switching Sensor Proteins

Tenda K., van Gerven B., Arts R., Hiruta Y., Merkx M., Citterio D.

Angewandte Chemie - International Edition （Angewandte Chemie - International Edition）  57 （ 47 ） 15369 - 15373 2018年11月

研究論文（学術雑誌）, 査読有り,  ISSN  14337851

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© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This work reports on fully integrated “sample-in-signal-out” microfluidic paper-based analytical devices (μPADs) relying on bioluminescence resonance energy transfer (BRET) switches for analyte recognition and colorimetric signal generation. The devices use BRET-based antibody sensing proteins integrated into vertically assembled layers of functionalized paper, and their design enables sample volume-independent and fully reagent-free operation, including on-device blood plasma separation. User operation is limited to the application of a single drop (20–30 μL) of sample (serum, whole blood) and the acquisition of a photograph 20 min after sample introduction, with no requirement for precise pipetting, liquid handling, or analytical equipment except for a camera. Simultaneous detection of three different antibodies (anti-HIV1, anti-HA, and anti-DEN1) in whole blood was achieved. Given its simplicity, this type of device is ideally suited for user-friendly point-of-care testing in low-resource environments.

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Protein purification using solid-phase extraction on temperature-responsive hydrogel-modified silica beads

Okubo K., Ikeda K., Oaku A., Hiruta Y., Nagase K., Kanazawa H.

Journal of Chromatography A （Journal of Chromatography A）  1568   38 - 48 2018年09月

研究論文（学術雑誌）, 査読有り,  ISSN  00219673

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© 2018 Elsevier B.V. Recently, the importance of biopharmaceuticals in medical treatments has been increasing, and effective protein purification methods are strongly required for their production. In the present study, a temperature-responsive solid-phase extraction (SPE) column was developed for the purification of proteins without affecting their bioactivity. A temperature-responsive polymer hydrogel-modified stationary phase was prepared by coating aminopropyl silica beads (average diameter, 40–64 μm) with poly(N-isopropylacrylamide) (PNIPAAm)-based thermoresponsive hydrogels. n-Butyl methacrylate and acrylic acid were copolymerized with PNIPAAm as hydrophobic and anionic monomers, respectively. Using these temperature-responsive SPE columns, targeted proteins were retained on the thermoresponsive hydrogel at 40 °C through hydrophobic and electrostatic interactions. After the temperature was reduced from 40 °C to 4 °C, the retained proteins were successfully eluted from the column. Using the temperature-responsive SPE system, lysozyme was successfully separated from ovalbumin without any loss in bioactivity (99.7 ± 0.1%). Rituximab, a monoclonal antibody, was also purified from BSA or hybridoma cell culture medium using the prepared SPE column. Denaturation of rituximab was not observed in the rituximab fraction eluted from the SPE column. These results demonstrate that temperature-responsive polymer-based SPE can be applied in biomedical purifications, while maintaining the biological activity of the proteins.

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LAT1-targeting thermoresponsive fluorescent polymer probes for cancer cell imaging

Matsuura, Minami, Ohshima, Mariko, Hiruta, Yuki, Nishimura, Tomohiro, Nagase, Kenichi, Kanazawa, Hideko

International Journal of Molecular Sciences （International Journal of Molecular Sciences）  19 （ 6 ） 116 - 127 2018年06月

研究論文（学術雑誌）, 査読有り,  ISSN  1422-0067

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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of L-[3 H]-leucine, one of the substrates for LAT1 uptake, was investigated. L-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of L-[3 H]-leucine, while P(NIPAAm-co-DMAAm) and L-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that L-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that L-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

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Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells

Hemmi M., Ikeda Y., Shindo Y., Nakajima T., Nishiyama S., Oka K., Sato M., Hiruta Y., Citterio D., Suzuki K.

Chemistry - An Asian Journal （Chemistry - An Asian Journal）  13 （ 6 ） 648 - 655 2018年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1861-4728

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© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc-1 (sulfinate ester luciferin), a chemoselective probe exhibiting a ratiometric and turn-on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the “caged” luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc-1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS-7 cells and human breast adenocarcinoma MCF-7 cells.

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Development of nanocarriers functionalized with stimuli-responsive polymer for controlled cellular uptake

Yamanouchi T., Katsuyama N., Hiruta Y., Ayano E., Kanazawa H.

Kobunshi Ronbunshu （Kobunshi Ronbunshu）  75 （ 2 ） 116 - 127 2018年

研究論文（学術雑誌）, 査読有り,  ISSN  0386-2186

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©2018, The Society of Polymer Science, Japan. Drug delivery system (DDS) is a technology that controls sustained release of medicines in the body and localized delivery to diseased tissues. Therefore, it is possible to keep the concentration of medicine at therapeutically effective concentrations for a long time and to reduce side effects. We have achieved controlled intracellular uptake, and release of medicines by nanocarriers modified with stimuli-responsive polymers based on poly(N-isopropylacrylamide), which undergoes a reversible phase transition in response to external temperature stimulation. In this article, we introduce our recent research on the design of temperature- and pH-responsive polymers, which enables selective intracellular uptake with external stimulation, and applications to DDS of an anti-tumor agent and siRNA by micelle and liposomes modified with those polymers.

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Thread-based analytical device for antibody detection in whole blood by using bioluminescent sensor proteins

Tomimuro K., Tenda K., Hiruta Y., Merkx M., Citterio D.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 （22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018）  2   1018 - 1021 2018年

　概要を見る

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Bioluminescent protein integrating microfluidic thread-based analytical devices (µTADs) for point-of-care testing (POCT) of antibodies in whole blood have been developed. Devices consist of a blood separation membrane and a plastic film layer with sewn-in cotton thread, in which proteins and luciferin for signal generation are pre-deposited. Colorimetry-based response curves for three model antibodies spiked into porcine whole blood have been obtained. Stable signals from 6-18 min after sample application and no cross-talk when assaying multiple different antibodies are observed. The µTADs offer quantitative simultaneous detection of multiple antibodies in a small volume of whole blood (8 µL).

Text-displaying competitive immunochromatographic strips enabling naked-eye semi-quantitative analysis

Misawa K., Yamamoto T., Watanabe D., Hiruta Y., Yamazaki H., Citterio D.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 （22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018）  2   1022 - 1025 2018年

　概要を見る

Copyright © (2018) by Chemical and Biological Microsystems Society. All rights reserved. Competitive lateral flow immunochromatographic assays (LFIAs) enabling naked-eye semi-quantitative result readout in the form of “text” have been developed. User operation is limited to sample application, followed by direct reading of assay results written in text representing the target concentration. LFIA strips enabled the detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG) at concentrations down to 5 ng/mL, which is comparable to existing immunochromatography using external readout equipment. The system is readily adaptable to other analytical targets and allows for high flexibility in text design and detectable analyte concentrations. The simple assay handling and intuitive data interpretation offer great potential for point-of-care testing applications.

Intracellular localization and delivery of plasmid DNA by biodegradable microsphere-mediated femtosecond laser optoporation

Ishii, Atsuhiro, Hiruta, Yuki, Heinemann, Dag, Heisterkamp, Alexander, Kanazawa, Hideko, Terakawa, Mitsuhiro

JOURNAL OF BIOPHOTONICS 10 （ 12 ） 1723 - 1731 2017年12月

研究論文（学術雑誌）, 査読有り,  ISSN  1864-063X

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Analysis of Psychoactive Drugs by Temperature-Responsive Chromatography

Mikuma, Toshiyasu, Kuroki, Tatsumi, Yoshikawa, Mitsuki, Uchida, Ryo, Hiruta, Yuki, Kanazawa, Hideko

Chromatography 38 （ 3 ） 115 - 121 2017年09月

研究論文（学術雑誌）, 査読有り

Dual temperature- and pH-responsive polymeric micelle for selective and efficient two-step doxorubicin delivery

Yuki Hiruta, Yuki Kanda, Naoya Katsuyama, Hideko Kanazawa

RSC Advances 47   29540 - 29549 2017年06月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り

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Reversible conformational changes in parallel type G-quadruplex structure inside a thermoresponsive hydrogel

Erica Hasuike, Aya Mizutani Akimoto, Reiko Kuroda, Ko Matsukawa, Yuki Hiruta, Hideko Kanazawa, and Ryo Yoshida

Chemical Communications 53   3142 - 3144 2017年02月

研究論文（学術雑誌）, 共著, 査読有り

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Design and synthesis of temperature-responsive polymer/silica hybrid nanoparticles and application to thermally controlled cellular uptake

Yuki Hiruta, Ryo Nemoto, Hideko Kanazawa

Colloids and Surfaces B: Biointerfaces 153   2 - 9 2017年02月

研究論文（学術雑誌）, 共著, 筆頭著者, 責任著者, 査読有り,  ISSN  0927-7765

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Transcutaneous drug delivery by liposomes using fractional laser technology

Takahiro Fujimoto, Jian Wang, Kazuki Baba, Yuka Oki, Yuki Hiruta, Masayuki Ito, Shinobu Ito, Hideko Kanazawa

Lasers in Surgery & Medicine 49   525 - 532 2016年12月

研究論文（学術雑誌）, 査読有り

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The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography

Toshiyasu Mikuma, Ryo Uchida, Mizuki Kajiya, Yuki Hiruta, Hideko Kanazawa

Analytical and Bioanalytical Chemistry 409 （ 4 ） 1059 - 1065 2016年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1618-2650

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Rapid and Simultaneous Analysis of Psychotropic Drugs by Ultra-High-Speed HPLC

Yoshikawa, Mitsuki, Uchida, Ryo, Kuroki, Tatsumi, Mikuma, Toshiyasu, Hiruta, Yuki, Nagata, Yoshiko, Kanazawa, Hideko

BUNSEKI KAGAKU 65 （ 4 ） 173 - 179 2016年04月

研究論文（学術雑誌）,  ISSN  0525-1931

Temperature-Responsive Molecular Recognition Chromatography Using Phenylalanine and Tryptophan Derived Polymers Modified Silica Beads

Yuki Hiruta, Ryosuke Kanazashi, Eri Ayano, Teruo Okano, Hideko Kanazawa

Analyst 141   910 - 917 2016年02月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り

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有機・無機ハイブリッド材料を用いた傾向検出型オプティカルpHセンサー

蛭田 勇樹

分析化学    99 - 100 2016年02月

研究論文（学術雑誌）, 単著, 筆頭著者, 最終著者, 査読有り

Temperature-responsive Solid-phase Extraction Column for Biological Sample Pretreatment

Akimaru, Michiko, Okubo, Kohei, Hiruta, Yuki, Kanazawa, Hideko

Analytical Sciences 31 （ 9 ） 881 - 886 2015年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0910-6340

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A Fluorous Biphasic Solvent Extraction System for Lanthanides with a Fluorophilic beta-Diketone Type Extractant

Nakamura, Etsuko, Hiruta, Yuki, Watanabe, Takafumi, Iwasawa, Naoko, Citterio, Daniel, Suzumt, Koji

Analytical Sciences 31 （ 9 ） 923 - 928 2015年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0910-6340

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