研究者詳細 - 蛭田　勇樹

論文 - 蛭田　勇樹

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Mesoporous CaCO3-based alkaline stable reversed-phase HPLC packing material

Mochida Mai, Nagai Yuta, Kumagai Hiroto, Imai Hiroaki, Citterio Daniel, Hiruta Yuki

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 2019年08月

ISSN 0065-7727

Structure-luminescence relationship of near-infrared firefly luciferin analogues

Ikeda Yuma, Hiruta Yuki, Citterio Daniel

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 2019年08月

ISSN 0065-7727

pH-Response tunable mixed-charged polymers for reversible adsorption and desorption of proteins

Hiruta Yuki, Sawada Kazuki, Kaku Taisei, Tokura Yuki, Shiratori Seimei, Citterio Daniel

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 2019年08月

ISSN 0065-7727

Microfluidic paper-based analytical devices (microPADs) with DVD player: Centrifugally assisted flow acceleration for rapid distance readout assays

Maejima Kento, Hiruta Yuki, Citterio Daniel

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 258 2019年08月

ISSN 0065-7727

Biothiol-activatable bioluminescent coelenterazine derivative for molecular imaging in vitro and in vivo

Nomura N., Nishihara R., Nakajima T., Kim S., Iwasawa N., Hiruta Y., Nishiyama S., Sato M., Citterio D., Suzuki K.

Analytical Chemistry （Analytical Chemistry） 91 （ 15 ） 9546 - 9553 2019年08月

研究論文（学術雑誌）, 査読有り, ISSN 00032700

© 2019 American Chemical Society. There is a high demand for sensitive biothiol probes targeting cysteine, glutathione, and homocysteine. These biothiols are known as playing essential roles to maintain homeostasis and work as indicators of many diseases. This work presents a bioluminescent probe (named AMCM) to detect biothiols in live mammalian cells and in vivo with a limit of detection of 0.11 μM for cysteine in solution and high selectivity for biothiols, making it suitable for real-time biothiol detection in biological systems. Upon application to live cells, AMCM showed low cytotoxicity and sensitively reported bioluminescence in response to changes of biothiol levels. Furthermore, a bioluminescence resonance energy transfer system consisting of AMCM combined with the near-infrared fluorescent protein iRFP713 was applied to in vivo imaging, with emitted tissue-permeable luminescence in living mice. In summary, this work demonstrates that AMCM is of high practical value for the detection of biothiols in living cells and for deep tissue imaging in living animals.

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Disposable electrochemical biosensor based on surface-modified screen-printed electrodes for organophosphorus pesticide analysis

Hua Q., Ruecha N., Hiruta Y., Citterio D.

Analytical Methods （Analytical Methods） 11 （ 27 ） 3439 - 3445 2019年07月

研究論文（学術雑誌）, 査読有り, ISSN 17599660

© 2019 The Royal Society of Chemistry. A disposable enzymatic electrochemical biosensor for amperometric organophosphorus pesticide determination based on a screen-printed carbon electrode (SPCE) platform was successfully developed. The working electrode consists of acetylcholine esterase (AChE) immobilized onto the surface of multi-walled carbon nanotube (MWCNT), chitosan (CS) and gold nanoparticle (AuNP)-modified SPCE. Ferricyanide was used as a redox mediator in solution. Two linear dynamic response ranges from 0.01 to 10 μg L-1 and from 10 to 100 μg L-1 were found for paraoxon-ethyl, with a detection limit of 0.03 μg L-1 (calculated as the amount of pesticide resulting in 10% of enzyme inhibition). High sensitivity was achieved due to a synergistic effect of AuNPs and MWCNTs deposited on the SPCE surface. Moreover, 83% of enzyme activity was retained after a dry storage period of 49 days (4 °C). Finally, the results of the electrochemical biosensor for paraoxon-ethyl-spiked spinach sample analysis showed a good agreement with those obtained from conventional HPLC.

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Adsorption-Desorption Control of Fibronectin in Real Time at the Liquid/Polymer Interface on a Quartz Crystal Microbalance by Thermoresponsivity

Li J., Kaku T., Tokura Y., Matsukawa K., Homma K., Nishimoto T., Hiruta Y., Akimoto A., Nagase K., Kanazawa H., Shiratori S.

Biomacromolecules （Biomacromolecules） 20 （ 4 ） 1748 - 1755 2019年04月

研究論文（学術雑誌）, 査読有り, ISSN 15257797

Copyright © 2019 American Chemical Society. The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

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Liposomes with temperature-responsive reversible surface properties

Nemoto R., Fujieda K., Hiruta Y., Hishida M., Ayano E., Maitani Y., Nagase K., Kanazawa H.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 176 309 - 316 2019年04月

研究論文（学術雑誌）, 査読有り, ISSN 09277765

© 2019 Elsevier B.V. Liposomes composed of egg phosphatidylcholine and cholesterol were modified with the temperature-responsive polymer poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)), and exhibited reversible surface properties with temperature. Completely reversible liposome aggregation due to P(NIPAAm-co-DMAAm) hydration/dehydration was demonstrated over four successive cycles of heating and cooling. The P(NIPAAm-co-DMAAm) polymer was hydrated during cooling, which dispersed the liposomes. The rigidity of the liposomal membrane was one of the factors in the reversible aggregation, as was the modification density of the polymer on the liposomes. A low density on relatively rigid liposomes could maintain the polymer property of reversible hydrated layers below critical solution temperature (LCST) boundary. Above the LCST, temperature-responsive polymers could also transport negatively charged liposomes into cells. The reversible behavior of the temperature-responsive polymer-modified liposomes has not been reported previously and could enable new applications for switching deposit forms as alternative drug carriers.

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In situ monitoring of proteins adsorption and desorption on smart surface using quartz crystal microbalance

Kaku Taisei, Li Jiatu, Nishimoto Taihei, Homma Kenta, Sawada Kazuki, Tokura Yuki, Hiruta Yuki, Akimoto Aya, Nagase Kenichi, Kanazawa Hideko, Shiratori Seimei

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 257 2019年03月

ISSN 0065-7727

Fully inkjet-printed distance-based paper microfluidic devices for colorimetric calcium determination using ion-selective optodes

Shibata H., Hiruta Y., Citterio D.

Analyst （Analyst） 144 （ 4 ） 1178 - 1186 2019年02月

研究論文（学術雑誌）, 査読有り, ISSN 00032654

© 2019 The Royal Society of Chemistry. Although the determination of calcium ions (Ca 2+ ) is of high importance to monitor water hardness, currently available devices for on-site analysis suffer from a lack of user-friendliness and sensitivity. This work demonstrates fully inkjet-printed and low-cost microfluidic paper-based analytical devices (μPADs) for the simple naked-eye colorimetric determination of calcium ions (Ca 2+ ) in drinking and tap water samples. The quantification of Ca 2+ relies on visual readout of the length of a colour-changed detection channel modified with ionophore-doped ion-selective optode nanospheres (nano-optodes), eliminating the requirement of a scanner or a camera. All fabrication steps for deposition of assay reagents have been performed by means of a simple desktop thermal inkjet printer, which is expected to contribute to highly batch-to-batch reproducible device preparation. The detectable Ca 2+ concentrations between 0.05 mmol L -1 and 5 mmol L -1 cover the range recommended by the International Organization for Standardization (0.05-2.5 mmol L -1 ) and the World Health Organization (WHO) guideline for Ca 2+ quantification in drinking water (less than 5 mmol L -1 ). The lowest concentration of Ca 2+ detectable by the naked eye was found to be 0.05 mmol L -1 , which is below the value achieved with previously reported paper-based devices. μPAD quantified Ca 2+ concentrations in tap or drinking waters were within 15% error of the results obtained with a classical complexometric titration. Hence, distance-based μPADs relying on nano-optodes are sensitive and reproducible tools for equipment-free on-site assaying of Ca 2+ in real samples.

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Paper-Based Assay for Ascorbic Acid Based on the Formation of Ag Nanoparticles in Layer-by-Layer Multilayers

Tokura Y., Moriyama Y., Hiruta Y., Shiratori S.

ACS Applied Nano Materials （ACS Applied Nano Materials） 2 （ 1 ） 241 - 249 2019年01月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 2574-0970

© 2018 American Chemical Society. A simple, portable, and cost-effective paper-based colorimetric assay is developed for ascorbic acid (AA) with excellent properties, such as fast response, high sensitivity, high selectivity, and good stability. AA is an important nutrient for human life, and adequate quantification of AA is required for controlling the intake of AA for health management. Moreover, point-of-care analysis, which is a simple, easy-to-use, and cost-effective analysis method for unskilled users, has been a focus in the medical and health care fields. We fabricate a colorimetric paper-based assay with poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) layer-by-layer self-assembly containing silver ions for the point-of-care analysis of AA. The color of the assay is changed from white to yellow and then to dark-brown after immersion in AA solution because of the reduction of silver ions to silver nanoparticles by AA and the localized surface plasmon resonance of the generated silver nanoparticles. The assay shows a fast response to AA of only 60 s and a sensitivity to AA (limit of detection is 0.4 ppm). Furthermore, the colorimetric assay exhibits a high selectivity for AA compared with other compounds contained in human fluid and various drinks. The concentration of AA in various commercial products is quantified by the assay with the same accuracy as high-performance liquid chromatography. The design of the PAH/PAA multilayers containing silver ions for a fast response, highly sensitive, and selective colorimetric assay will contribute to the development of point-of-care analysis for the early detection of diseases as well as controlling the intake of nutrients.

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Flow control-based 3D μPADs for organophosphate pesticide detection

Hua Q., Shibata H., Hiruta Y., Citterio D.

Analytical Sciences （Analytical Sciences） 35 （ 4 ） 393 - 399 2019年

査読有り, ISSN 09106340

© 2019 The Japan Society for Analytical Chemistry. Flow control-based paper devices have recently shown great potential for point-of-need analysis, since they allow for the easy operation of multi-step assays by minimizing user operation. In this work, a wax printing method was evaluated as a means to control liquid flow in 3D microfluidic paper-based analytical devices (μPADs). The resulting flow controlbased 3D μPADs were applied to determine paraoxon-ethyl as a typical organophosphate pesticide model system. The analytical procedure is as simple as applying a 200-μL sample solution, resulting in reproducible (relative standard deviation of colorimetric signals from 6 independently fabricated devices, 2.63%) colorimetric signals within 1 h of the assay time with the limit of detection (LOD) reaching 25.0 μg/L. Finally, results obtained for pesticide-spiked water samples analyzed by flow control-based 3D μPADs showed good agreement with those from a conventional HPLC analysis with UV detection.

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Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo

Nishihara R., Paulmurugan R., Nakajima T., Yamamoto E., Natarajan A., Afjei R., Hiruta Y., Iwasawa N., Nishiyama S., Citterio D., Sato M., Kim S., Suzuki K.

Theranostics （Theranostics） 9 （ 9 ） 2646 - 2661 2019年

研究論文（学術雑誌）, 査読有り, ISSN 1838-7640

© Ivyspring International Publisher. Background: Bioluminescence imaging (BLI) is one of the most widely used optical platforms in molecular imaging, but it suffers from severe tissue attenuation and autoluminescence in vivo. Methods: Here, we developed a novel BLI platform on the basis of bioluminescence resonance energy transfer (BRET) for achieving a ∼300 nm blue-to-near infrared shift of the emission (NIR-BRET) by synthesizing an array of 18 novel coelenterazine (CTZ) derivatives, named “Bottle Blue (BBlue)” and a unique iRFP-linked RLuc8.6-535SG fusion protein as a probe. Results: The best NIR-BRET was achieved by tuning the emission peaks of the CTZ derivatives to a Soret band of the iRFP. In mammalian cells, BBlue2.3, one of the CTZ derivatives, emits light that is ∼50-fold brighter than DBlueC when combined with RLuc8.6-535SG, which shows stable BL kinetics. When we used a caged version of BBLue2.3, it showed a BL half decay time of over 60 minutes while maintaining the higher signal sensitivity. This NIR BL is sufficiently brighter to be used for imaging live mammalian cells at single cell level, and also for imaging metastases in deep tissues in live mice without generating considerable autoluminescence. A single-chain probe developed based on this BLI platform allowed us to sensitively image ligand antagonist-specific activation of estrogen receptor in the NIR region. Conclusion: This unique optical platform provides the brightest NIR BLI template that can be used for imaging a diverse group of cellular events in living subjects including protein‒protein interactions and cancer metastasis.

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A biomimetic hybrid material consisting of CaCO<inf>3</inf> mesoporous microspheres and an alternating copolymer for reversed-phase HPLC

Mochida M., Nagai Y., Kumagai H., Imai H., Citterio D., Hiruta Y.

Journal of Materials Chemistry B （Journal of Materials Chemistry B） 7 （ 31 ） 4771 - 4777 2019年

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 20507518

This journal is © The Royal Society of Chemistry. We developed a biomineral-inspired hybrid material composed of CaCO3 and an organic polymer as a column packing material for HPLC. This material combines a hierarchical mesoporous structure and the functionality of the polymer. The surface of monodispersed mesoporous CaCO3 microspheres was modified with poly(maleic acid-alt-1-octadecene) (PMAcO) comprising hydrophobic alkyl chains and anionic carboxylate groups. PMAcO adsorbed onto the surface of CaCO3 through electrostatic interaction between Ca2+ sites and carboxylate groups, resulting in an octadecene coated microsphere interface. These microspheres were applied as a HPLC column and exhibited reversed-phase retention behavior in the separation of alkylbenzenes. This column showed high alkaline mobile phase resistance compared with the conventionally applied ODS column packing material. Quantitative analysis of the basic antidepressants clomipramine and imipramine spiked into whole blood was achieved with an alkaline mobile phase, demonstrating the potential of the biomineral-inspired material as a HPLC stationary phase for practical applications in routine analyses of basic drugs requiring alkaline mobile phases.

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Crosslinked Poly(N-Isopropylacrylamide)-Based Microfibers as Cell Manipulation Materials with Prompt Cell Detachment

Konishi T., Mizutani Akimoto A., Nishimoto T., Tokura Y., Tenjimbayashi M., Homma K., Matsukawa K., Kaku T., Hiruta Y., Nagase K., Kanazawa H., Shiratori S.

Macromolecular Rapid Communications （Macromolecular Rapid Communications） 40 （ 23 ） 2019年

研究論文（学術雑誌）, 査読有り, ISSN 10221336

© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Stimuli-responsive smart materials are a key to the realization of next-generation medical technologies. Among them, the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) is attracting particular attention because it is easy to use in physiological conditions. PNIPAAm-grafted surfaces can undergo temperature-modulated cell adhesion and detachment without proteolytic enzymes, and can be used as cell-separating materials through selective cell adhesion/detachment. However, cell detachment at reduced temperatures is problematic because it takes several hours. A novel thermoresponsive crosslinked microfiber system that can greatly reduce the cell detachment time is introduced in this study. The crosslinked fibers provide temperature-dependent volume change, and enable cell detachment within 10 min of reducing the temperature, which is one-sixth of the time required in previous studies. The prompt cell detachment is thought to arise from a completely new mechanism derived from fiber swelling. This system will make a significant contribution as a novel cell manipulating system for next-generation medical technology.

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Microfluidic Paper-Based Analytical Devices for Colorimetric Detection of Lactoferrin

Kudo H., Maejima K., Hiruta Y., Citterio D.

SLAS Technology （SLAS Technology） 25 （ 1 ） 47 - 57 2019年

研究論文（学術雑誌）, 査読有り, ISSN 24726303

© 2019 Society for Laboratory Automation and Screening. Lactoferrin is an abundant glycoprotein in human body fluids and is known as a biomarker for various diseases. Therefore, point-of-care testing (POCT) for lactoferrin is of interest. Microfluidic paper-based analytical devices (µPADs) have gained a lot of attention as next-generation POCT device candidates, due to their inexpensiveness, operational simplicity, and being safely disposable. This work presents a colorimetric sensing approach for quantitative lactoferrin analysis. The detection mechanism takes advantage of the high affinity of lactoferrin to ferric ions (Fe3+). Lactoferrin is able to displace an indicator from a colorimetric 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-Fe3+ complex, resulting in a color change. A 5-Br-PADAP-Fe3+ complex was encapsulated into water-dispersible poly(styrene-block-vinylpyrrolidone) particles, whose physical entrapment in the cellulosic fiber network results in the immobilization of the complex to the paper matrix. The complex-encapsulating particles showed a color change response in accordance with lactoferrin concentration. Both color intensity-based paper well plates and distance readout-based µPADs are demonstrated. Color intensity-based devices allowed quantitative analysis of lactoferrin concentrations with a limit of detection of 110 µg/mL, using a smartphone and a color readout app. On the other hand, distance readout-based µPADs showed changes of the length of colored sections in accordance with lactoferrin concentration. In summary, we successfully developed both colorimetric intensity-based paper wells and distance-based µPADs for lactoferrin detection. This work demonstrates a user-friendly colorimetric analysis platform for lactoferrin without requiring lab equipment and expensive antibodies.

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Near-Infrared Fluorescent Probes for Imaging of Intracellular Mg<sup>2+</sup> and Application to Multi-Color Imaging of Mg<sup>2+</sup>, ATP, and Mitochondrial Membrane Potential

Murata O., Shindo Y., Ikeda Y., Iwasawa N., Citterio D., Oka K., Hiruta Y.

Analytical Chemistry （Analytical Chemistry） 92 （ 1 ） 966 - 974 2019年

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 00032700

© 2019 American Chemical Society. The magnesium ion (Mg2+) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg2+, there is a great need for the development of Mg2+-selective fluorescent probes. However, conventional Mg2+ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg2+-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged β-diketone as a selective binding site for Mg2+ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg2+. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg2+. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg2+ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg2+ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg2+ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multi-color imaging of intracellular Mg2+.

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Inkjet-printed pH-independent paper-based calcium sensor with fluorescence signal readout relying on a solvatochromic dye

Shibata H., Ikeda Y., Hiruta Y., Citterio D.

Analytical and Bioanalytical Chemistry （Analytical and Bioanalytical Chemistry） 412 （ 14 ） 3489 - 3497 2019年

研究論文（学術雑誌）, 査読有り, ISSN 16182642

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. A challenge for paper-based cation sensors relying on classical carrier-based ion-selective optodes (ISOs) is their pH-cross response caused by the use of H+-sensitive chromoionophores as optical signal transducers. This work demonstrates fully pH-independent fluorescence-based calcium detection with a paper-based plasticizer-free ISO. To achieve a pH-independent assay, a solvatochromic dye (SD) instead of a traditional H+-sensitive chromoionophore has been applied to the paper-based ISO by means of inkjet printing technology. The detection principle depends on an ionophore-driven phase-transfer ion-exchange reaction between target cations and the positively charged SD, which no longer involves H+ in the optical signal transduction process. The developed paper-based ISOs with the SD resulted in Ca2+ concentration-dependent response curves not affected by the sample pH (pH 6.0, 7.0, and 8.0). The dynamic range obtained for Ca2+ detection was from 10−5 to 1 mol L−1 with a detection limit of 19.3 μmol L−1. Additionally, excellent selectivity derived from the used ionophore has been confirmed. As a simple practical application, the determination of Ca2+ in mineral water has been achieved without the pH-buffering process required for conventional cation-exchange ISOs.

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Distance readout-based paper devices for multiplexed urinalysis

Sawano R., Hiroyuki S., Maejima K., Hiruta Y., 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） 716 - 717 2019年

© 2019 CBMS-0001. This work reports a basic study to simplify urinalysis, using distance-based microfluidic paper-based analytical devices (D-µPADs) for multiplexed detection. Intuitive detection of albumin (Alb), glucose (Glu) and hemoglobin (Hb) in artificial urine samples is demonstrated by a colorimetric distance-based readout method. The response ranges for the three analytes (15-1000 mg/dL, 25-2000 mg/dL, 0.25-1.0 mg/dL), fully cover the physiologically relevant domain. User operation is limited to sample application and readout of the color-changed length corresponding to the analyte concentration.

Paper-based device with integrated ion-selective optodes for colorimetric quantification of salivary metal ions

Suenaga Y., Shibata H., Hiruta Y., Citterio D.

© 2019 CBMS-0001. A disposable and simple PPAD (microfluidic paper-based analytical device) for the quantitative colorimetric detection of salivary cation species, relying on ISOs (ion-selective optodes) is introduced. This work demonstrates the applicability of ISO-integrated PPADs to real saliva samples. Calibration enabling accurate salivary cation quantification was achieved by using artificial saliva. Multiple sensing areas on a single device can detect three different types of ions within 3 min by simple application of 20 PL sample. Three kinds of ISOs were optimized for salivary Na+, K+ and Ca2+ quantification. For proof-of-concept, salivary Na+ concentration was accurately quantified by the developed PPAD.

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蛭田　勇樹

論文 - 蛭田　勇樹