研究者詳細 - 蛭田　勇樹

論文 - 蛭田　勇樹

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Development of Phosphinate Ligand-Based Low-Affinity Ca<sup>2+</sup> Fluorescent Probes and Application to Intracellular Ca<sup>2+</sup> Imaging

Kumada R., Sakama A., Shindo Y., Kuronuma Y., Iwasawa N., Citterio D., Oka K., Hiruta Y.

Analytical Chemistry （Analytical Chemistry） 95 （ 45 ） 16683 - 16691 2023年11月

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

Divalent metal cations such as calcium ion (Ca2+) and magnesium ion (Mg2+) are indispensable to the regulation of various cellular activities. In this research, we developed the KLCA series utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a target binding site, which was reported recently as a highly free Mg2+-selective ligand. KLCA-301 with orange fluorescence based on a rhodamine fluorophore and KLCA-501 with near-infrared (NIR) fluorescence based on a Si-rhodamine fluorophore were synthesized, intended for application to multicolor imaging. The evaluation of the fluorescence response to Ca2+ and Mg2+ of the KLCA series indicated the applicability as low-affinity Ca2+ probes. While KLCA-301 mainly localized in the cytosol in cultured rat hippocampal neurons, KLCA-501 localized to the cytosol and granular organelles in neurons. Comparison of the fluorescence response of KLCA-301 and the high-affinity Ca2+ probe Fluo-4 upon stimulation by glutamate in stained neurons revealed that KLCA-301 could reflect the secondary large rise of intracellular Ca2+, which Fluo-4 could not detect. In addition, KLCA-501 showed a fluorescence response similar to the low-affinity Ca2+ probe Fluo-5N upon stimulation by glutamate in stained neurons, concluding that KLCA-301 and KLCA-501 could be used as low-affinity Ca2+ probes. The KLCA series offers new options for low-affinity Ca2+ probes. Moreover, KLCA-501 achieved simultaneous visualization of the change in Ca2+ and ATP concentrations and also in mitochondrial inner membrane potential in neurons. KLCA-501 is expected to be a strong tool that enables simultaneous multicolor imaging of multiple targets and elucidation of their relationship in cells.

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Cation/anion-exchange mode switching chromatography utilizing pH-responsive mixed charge polymer-modified silica beads

Kaku T., Deura K., Yoshii T., Citterio D., Hiruta Y.

Molecular Systems Design and Engineering （Molecular Systems Design and Engineering） 9 （ 1 ） 56 - 62 2023年11月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 2058-9689

The separation capacity of a column typically remains constant. By applying stimuli-responsive materials to the stationary phase, the separation capacity in a single column can be tuned; however, the separation mode is not completely switched. In this study, we aimed to develop a cation/anion-exchange mode switching chromatography approach, in which the monomer ratio is adjusted, enabling the surface charge to become either negative or positive in response to mobile phase pH. Three types of beads were prepared, each modified with a pH-responsive mixed-charge polymer combining a cationic monomer, a pH-responsive carboxylic acid monomer, a neutral monomer, and a cross-linking monomer. The composition ratio of the cationic monomer to the pH-responsive carboxylic acid monomer was set at 1 : 2 so that the cation-exchange mode occurs at a pH above the pKa and the anion-exchange mode occurs below the pKa. At a pH below the pKa, the retention factor of the negatively charged compound increased. In contrast, at a pH above the pKa, the retention factor of the positively charged compound increased, confirming the charge switching on the bead surface. Switching to the cation- and anion-exchange mode enabled the separation of five basic antidepressants and acidic non-steroidal anti-inflammatory drugs, respectively. Utilizing a pH-responsive mixed-charge polymer, we attributed a cation/anion-exchange mode to a single column.

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Semi-quantitative microfluidic paper-based analytical device for ionic silica detection

Ogawa M., Katoh A., Matsubara R., Kondo H., Otsuka M., Sawatsubashi T., Hiruta Y., Citterio D.

Analytical Sciences （Analytical Sciences） 39 （ 8 ） 1361 - 1370 2023年08月

研究論文（学術雑誌）, 査読有り, ISSN 09106340

Silicate ions (SiO32−), or ionic silica, are known to cause silica scaling in industrial water applications when excess amounts are present; hence, concentrations must be monitored and kept at a constant low level. Ionic silica is conventionally measured by spectrophotometry in the form of its silicomolybdic complex based on the molybdenum blue reaction, but the operation process is complicated and not suitable for on-site detection. To solve these issues, microfluidic paper-based analytical devices (µPADs) have been gaining attention as portable, low-cost analytical devices suitable for on-site detection. The foldable origami type device described in this work enabled silica detection based on the molybdenum blue reaction, in the range of 50–1000 mg/L, with a practically detectable lowest concentration of 50 mg/L. The device showed selectivity for silicate ions and stability over 21 days when stored at 4 °C. The semi-quantitative analytical performance makes the proposed paper-based device attractive for on-site industrial monitoring. Graphical abstract: [Figure not available: see fulltext.]

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Development of prediction model for cloud point of thermo-responsive polymers by experiment-oriented materials informatics

Hayakawa M., Sakano K., Kumada R., Tobita H., Igarashi Y., Citterio D., Oaki Y., Hiruta Y.

Polymer Chemistry （Polymer Chemistry） 14 （ 19 ） 2383 - 2389 2023年04月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 17599954

Thermo-responsive polymers having a lower critical solution temperature (LCST) have attracted attention for biological applications such as drug delivery, diagnosis, and coating materials. In recent years, research on predicting LCST by utilizing machine learning has been conducted. However, since these methods targeted only copolymers combining specific monomer structures, they are not versatile, and multiple trials are still required to obtain new thermo-responsive polymers with the desired LCST. In this study, a prediction model for cloud point temperature (TCP) was built by a combination of materials informatics and chemical insight, named sparse modeling for small data (SpM-S) using a small dataset of polymers collected from the literature as training data. This approach created a model that is interpretable, easy to calculate, and versatile. The prediction accuracy was validated using data from different literature sources and experimental test data. The model was able to predict the TCP of polymers containing monomers not included in the dataset as well as polymers containing monomers included in the dataset. The predictive model has the potential to guide the design of new thermo-responsive polymers, and to contribute to efficient development of thermo-responsive polymers.

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Evaluation of separation performance for eggshell-based reversed-phase HPLC columns by controlling particle size and application in quantitative therapeutic drug monitoring

Yoshii T., Nakano K., Okuda T., Citterio D., Hiruta Y.

Analytical Methods （Analytical Methods） 15 （ 14 ） 1790 - 1796 2023年03月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 17599660

Eggshell-based reversed-phase packing materials were applied to an analytical column for high-performance liquid chromatography. Commercially available eggshell powder was classified by a cyclone system to obtain three types of particles with different diameters (arithmetic mean ± standard deviation: 4.3 ± 3.8, 5.6 ± 3.3, and 9.5 ± 5.5 μm). Sedimentation separation removed tiny particles from each sample, resulting in particles with arithmetic means of 6.6 ± 5.5, 7.3 ± 4.5, and 10.2 ± 5.0 μm, respectively. The unclassified particles and three particle types treated with sedimentation separation were subsequently packed into analytical columns (150 mm × 4.6 mm I.D.), and their separation efficiencies were evaluated by comparing their height equivalent to a theoretical plate (HETP). The column without sedimentation separation exhibited the highest HETP, whereas the columns with sedimentation separation showed better separation efficiency and lower back pressure. The column with the best separation efficiency was applied for the separation of 10 alkylbenzenes and 5 steroids, and all peaks were observed with complete separation (peak resolution: RS > 1.5). Finally, the column was used for quantitative analysis of voriconazole, an azole antifungal agent, and imatinib, a first-generation molecularly targeted drug for cancer treatment, in spiked whole blood. Excellent accuracy (99.1-102.8%) and precision (0.6-1.9%) were observed for the spiked drugs and long-term stability (>3000 column volumes of mobile phase flow) indicated good applicability of the developed eggshell-based column as an analytical column for routine analyses of therapeutic drugs in blood.

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Evaluation of cellophane as platform for colorimetric assays on microfluidic analytical devices

Shigemori Hiroki, Maejima Kento, Shibata Hiroyuki, Hiruta Yuki, Citterio Daniel

MICROCHIMICA ACTA （Microchimica Acta） 190 （ 2 ） 2023年02月

研究論文（学術雑誌）, 査読有り, ISSN 0026-3672

Due to their low cost, simplicity, and pump-free liquid transport properties, colorimetric assays on paper spots and microfluidic paper-based analytical devices (µPADs) are regarded as useful tools for point-of-care testing (POCT). However, for certain types of colorimetric assays, the “non-transparent” and “white” characters of paper can be a disadvantage. In this work, the possibilities of using cellophane as an alternative platform for colorimetric assays have been investigated. Cellophane is a low cost and easy-to-handle transparent film made of regenerated cellulose. Owing to its hydrophilic character, cellophane-based microfluidic channels fabricated through a print–cut–laminate approach enabled pump-free liquid transport into multiple detection areas, similar to µPADs. In addition, the water absorption characteristics of cellophane allowed the stable immobilization of water-soluble colorimetric indicators without any surface modification or additional reagents. The transparency of cellophane provides possibilities for simple background coloring of the substrates, increasing the dynamic signal range for hue-based colorimetric assays, as demonstrated for two model assays targeting H2O2 (46-fold increase) and creatinine (3.6-fold increase). Finally, a turbidity detection-based protein assay was realized on black background cellophane spots. The lowest limits of detection achieved with the cellophane-based devices were calculated as 7 µM for H2O2, 2.7 mg dL−1 for creatinine, and 3.5 mg dL−1 for protein (human serum albumin). Graphical Abstract: [Figure not available: see fulltext.]

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Fabrication process development and basic evaluation of eggshell-based column packing material for reversed-phase preparative separation

Yoshii Tomoka, Sakama Akihiro, Kanamori Kazuyoshi, Nakanishi Kazuki, Imai Hiroaki, Citterio Daniel, Hiruta Yuki

JOURNAL OF CHROMATOGRAPHY A （Journal of Chromatography A） 1688 2023年01月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 0021-9673

Purification of basic drugs in reversed-phase mode is often difficult, mainly due to adsorption of positively charged compounds to the silica gel-based stationary phase. Since this adsorption can be suppressed under alkaline condition, columns with alkali-resistance are required. In addition, compounds with acid-sensitive structures are sometimes degraded during separation on silica gel-based columns which exhibit acidity due to their surface structure. We prepared an alkali-resistant reversed-phase packing material, Eggshell-PMAcO based on eggshells modified with an amphiphilic copolymer, poly(maleic acid-alt-1-octadecene) (PMAcO). The height equivalent to a theoretical plate (HETP) of the Eggshell-PMAcO column was improved by surface treatment with ammonium acetate buffer (900 mM, pH = 3.7), which is an inexpensive reagent, and the retention behavior for hydrophobic compounds was compared to a typical ODS column based on silica gel, resulting in sufficient selectivity of the eggshell-based column for hydrophobic compounds, as indicated by the ratio of retention factors of pentylbenzene and butylbenzene (Eggshell-PMAcO column: 1.55, ODS column: 1.65). Column temperature-dependent retention behavior of naphthalene was investigated in the temperature range from 25 °C to 45 °C, followed by the calculation of thermodynamic parameters. There was little difference in the standard molar enthalpy (Eggshell-PMAcO: −19.6 kJ/mol, ODS: −21.7 kJ/mol). The absolute value of the standard free Gibbs energy for the Eggshell-PMAcO column was much smaller than that of the ODS column (Eggshell-PMAcO: −0.284 kJ/mol, ODS: −13.0 kJ/mol), indicating that the Eggshell-PMAcO column had a weaker retention strength for naphthalene than the ODS column mainly due to the large difference in the standard molar entropy (Eggshell-PMAcO: −64.9 J/mol K, ODS column: −29.2 J/mol K). The retention capacities for imipramine under neutral (water/methanol) and alkaline (0.1% triethylamine water/methanol) conditions were 0.2 mg and 5 mg, respectively, based on injection mass-dependent HETP, retention factor and symmetry factor. Finally, the prepared column was applied to the purification of a building block for nucleic acid drugs. This study demonstrated that reversed-phase columns, which can be fabricated from eggshells and an amphiphilic copolymer in an inexpensive and eco-friendly way, have the ability to purify basic compounds and acid-sensitive compounds.

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Effect of the side chain composition of mixed-charge polymers on pH-selective cell–membrane interactions

Nakamura Y., Nasu M., Shindo Y., Oka K., Citterio D., Hiruta Y.

Polymer Journal （Polymer Journal） 55 （ 11 ） 1179 - 1188 2023年

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 00323896

Zwitterionic polymers are characterized by their antifouling ability due to their net-neutral charge and affinity for cancer cell surfaces. We developed six pH-responsive mixed-charge polymers and investigated the effect of their side chain hydrophobicity on their interaction with cancer cell membranes. The polymers were synthesized using [2-(methacryloyloxy)ethyl]trimethylammonium chloride, carboxylic acid monomers, and butyl methacrylate (BMA) as the neutral spacer moiety, and differences in hydrophobicity were created by altering the type of carboxylic acid monomer and ratio of the spacer. The surface charge of the polymers was designed to switch from net-neutral to positive in response to the weakly acidic tumor environment. This tuning occurred in a more cancer-like environment with the usage of a more hydrophobic anionic monomer and a higher ratio of BMA due to the weakening of electrostatic interactions. This trend was further confirmed by evaluation of the interaction between the polymers and heparin, an anionic polysaccharide that is used as a model for cancer cell surfaces. The affinity of the mixed-charge polymers for cells was then evaluated, and both the hydrophobicity and pH-responsive nature of these polymers led to pH-selective toxicity, which was assumed to be caused by the disruption of cell membrane integrity.

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Amphiphilic Copolymer-Modified Eggshell-Based Column Packing Materials for the Preparative Separation of Basic Drugs

Yoshii T., Mochida M., Kaizu K., Soda Y., Kanamori K., Nakanishi K., Sato T., Imai H., Citterio D., Hiruta Y.

ACS Applied Polymer Materials （ACS Applied Polymer Materials） 4 （ 10 ） 6949 - 6957 2022年10月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 2637-6105

A way to utilize eggshells is desired because most of the eggshell waste is disposed of in landfills. Despite eggshells having attractive features such as a thermodynamically stable porous crystalline structure, their effective utilization as a functional material has yet to be demonstrated. Herein, we developed an alkali-stable column packing material for reversed-phase preparative high-performance liquid chromatography (HPLC) based on eggshells. Nanocrystals and biopolymers present on eggshell powder were removed by surface treatment with buffered ethylenediaminetetraacetic acid (EDTA) and sodium hypochlorite solutions, resulting in bare calcium carbonate with a uniform porous surface. The obtained particles were modified with poly(maleic acid-alt-1-octadecene) (PMAcO) having hydrophobic alkyl chains and anionic carboxylate groups. The prepared PMAcO-modified eggshell-based material was applied to an HPLC column, and its performance is evaluated. The column succeeded in the separation of alkylbenzenes, steroids, and diastereomers. It was also used for the separation of basic tricyclic antidepressants in an alkaline mobile phase, showing excellent retention capacity and alkali stability. This study demonstrates the potential of eggshell utilization, providing not only a practical stationary phase for HPLC but also a solution for recycling calcium carbonate-based biomineral waste.

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Quantitative evaluation of reversed-phase packing material based on calcium carbonate microspheres modified with an alternating copolymer

Kaizu K., Mochida M., Imai H., Citterio D., Hiruta Y.

Journal of Chromatography A （Journal of Chromatography A） 1677 2022年08月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 00219673

Considering the vulnerability of silica gel to alkaline mobile phases, a highly alkaline stable stationary phase for HPLC is required to separate basic compounds with high separation efficiency. To address this issue, we have developed a high alkaline stable packing material (CaCO3-PMAcO) based on mesoporous calcium carbonate microspheres modified with poly(maleic acid-alt-1-octadecene). In this study, we report further investigation of the separation performance of CaCO3-PMAcO column by systematically evaluating the effects of particle size and chromatographic conditions. Based on the theory of the van Deemter equation, the separation efficiency was related to the size of CaCO3-PMAcO particles (2.9 – 5.7 µm). The evaluation of thermodynamics of retention by changing the column temperature from 20 °C to 45 °C implied that the retention mode was dominated by hydrophobic interaction associated with the exothermic enthalpy changes (-11.1 to -12.5 kJ/mol). The results of column selectivity tests revealed that the CaCO3-PMAcO column had hydrophobic selectivity comparable to C18 silica gel columns (αP/B; CaCO3-PMAcO column: 1.53, C18 column: 1.69), and higher shape/steric selectivity (αTri/Ter; CaCO3-PMAcO column: 1.56, C18 column: 0.955). In practice, the CaCO3-PMAcO column could be applied to the separation of not only alkylbenzenes and polycyclic aromatic hydrocarbons, but also to basic tricyclic antidepressants by using an alkaline mobile phase (pH 12).

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Poly(N-isopropylacrylamide)-based temperature- and pH-responsive polymer materials for application in biomedical fields

Hiruta Yuki

POLYMER JOURNAL （Polymer Journal） 54 （ 12 ） 1419 - 1430 2022年08月

研究論文（学術雑誌）, 筆頭著者, 最終著者, 責任著者, 査読有り, ISSN 0032-3896

The synthesis and application of stimuli-responsive polymer materials have been extensively studied. Among stimuli-responsive polymers, thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) is the most widely investigated. PNIPAAm-based polymers undergo a reversible hydrophilic/hydrophobic phase transition in response to temperature. In addition, by introducing sites that are responsive to physical and chemical stimuli into PNIPAAm-based polymers, they also undergo phase transitions in response to stimuli, such as light, pH, oxidation/reduction, and enzyme activity. In this focus review, recent advancements in the applications of stimuli-responsive polymers based on PNIPAAm in biomedical fields are summarized, with an emphasis on our own research. In particular, a summary of the design of polymers for application in the separation and purification of (bio)pharmaceutical products and controlled cellular uptake is provided. First, temperature-responsive chromatography with PNIPAAm-modified silica beads is introduced. Thereafter, temperature- and pH-responsive polymers based on PNIPAAm used in imaging and drug delivery applications are discussed. Finally, the conclusions are presented, and future perspectives for the biomedical applications of stimuli-responsive polymers are discussed.

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Organelle-selective near-infrared fluorescent probes for intracellular microenvironment labeling

Kurutos A., Shindo Y., Hiruta Y., Oka K., Citterio D.

DYES AND PIGMENTS （Dyes and Pigments） 204 2022年08月

研究論文（学術雑誌）, 査読有り, ISSN 0143-7208

The design, synthesis, and bio-applicability of a series of 8 near-infrared (NIR) molecular probes with tunable hydrophilic/hydrophobic properties are described in this paper. The title dyes comprise of symmetric heptamethine cyanines with various N-quaternary heterocyclic moieties including indole, benzo[e]indol, benzothiazole and naphtho[1,2-d]thiazole, along with an extra positive charge in close proximity to the polymethine backbone. Combined UV–Vis and spectrofluorimetric techniques in both organic and aqueous-buffered solutions were employed to investigate the optical properties of the NIR chromophores. Two of the fluorophores emerged remarkable fluorescence quantum yield (Φ = 0.083), while their brightness was found up to 3-fold that of the FDA approved Indocyanine green – ICG (Φ = 0.027). What is more, two of the dyes appear to outperform the later clinical benchmark fluorophore in terms of photostability. While the ICG was completely faded after 1 h of constant irradiation, nearly 70% of the original emission signal could be observed for one of the novel molecular probes. The log Pow values determined by a combination of HPLC and UV–Vis (partition between 1-octanol/water) cover a relatively large range, from −2.14 up to 7.92. Investigation on the cytotoxicity by means of the MTT assay revealed, that present dyes are suitable for bioanalytical purposes and are safe to use. This paper also includes fluorescence imaging and cellular applications, such as labeling of HeLa cervical cancer cells. The results from the confocal microscopy reveal the superior staining capability of current heptamethines since they show significantly brighter signal than the commercial ICG probe. An organelle-selective fluorescent staining was achieved depending on the dye chemical structure. Co-localization within the intracellular microenvironment (linear or granular - cytosol, mitochondria, lysosome, endoplasmatic reticulum or Golgi apparatus) was confirmed without interference, employing several conventional organelle dye trackers. To the best of our knowledge, heptamethine dyes represent the first example of a series NIR emitting probes, that exhibit rational structural modifications aiming to fine-tune the hydrophilic/hydrophobic properties furnishing organelle-targeted staining.

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Feasibility of Using Poly[oligo(ethylene glycol) Methyl Ether Methacrylate] as Tumor-Targeted Carriers of Diagnostic Drugs

Sano K., Umemoto K., Miura H., Ohno S., Iwata K., Kawakami R., Munekane M., Yamasaki T., Citterio D., Hiruta Y., Mukai T.

ACS APPLIED POLYMER MATERIALS （ACS Applied Polymer Materials） 4 （ 7 ） 4734 - 4740 2022年06月

研究論文（学術雑誌）, 責任著者, 査読有り, ISSN 2637-6105

Poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) is a water-soluble polymer in which multiple short ethylene glycol (EG) chains are grafted along the hydrophobic methyl methacrylate backbone. We investigated whether POEGMA could be used as a drug carrier for tumor diagnosis. For this purpose, we synthesized POEGMA derivatives with different molecular weights (11, 21, and 30 kDa) and EG side chain lengths, and labeled them with radioisotopes (indium-111; 111In) or fluorescence dyes (indocyanine green; ICG) as signal emitters. Fluorescence microscopy studies using colon26 tumor cells treated with ICG-labeled POEGMA derivatives revealed that the POEGMA derivatives were efficiently taken up by tumor cells compared to the control, poly(ethylene glycol) (PEG), suggesting the involvement of a POEGMA-specific cellular uptake pathway. The radiolabeled POEGMA derivatives were intravenously injected into colon26 tumor-bearing mice, and their biodistribution was evaluated. As the molecular weight of POEGMA derivatives increased, the circulating time was prolonged, and their accumulation in the liver and spleen increased. In particular, 21 kDa POEGMA showed excellent tumor uptake and tumor-to-normal tissue ratio. Therefore, we investigated the effect of EG chain length on the tumor uptake of POEGMA (21 kDa) and observed high tumor uptake for all POEGMA derivatives; however, relatively higher abdominal uptake was observed for POEGMA derivatives with longer EG chain lengths. Finally, in vivo fluorescence imaging using optimized POEGMA derivatives labeled with ICG clearly visualized the tumor tissues, suggesting that the POEGMA derivatives could be suitable drug carriers for sensitive tumor diagnosis.

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A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

Orioka M., Eguchi M., Mizui Y., Ikeda Y., Sakama A., Li Q., Yoshimura H., Ozawa T., Citterio D., Hiruta Y.

Bioconjugate Chemistry （Bioconjugate Chemistry） 33 （ 3 ） 496 - 504 2022年03月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 10431802

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

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Traffic light type paper-based analytical device for intuitive and semi-quantitative naked-eye signal readout

Ohta Sera, Hiraoka Ryuya, Hiruta Yuki, Citterio Daniel

LAB ON A CHIP （Lab on a Chip） 22 （ 4 ） 717 - 726 2022年02月

研究論文（学術雑誌）, 査読有り, ISSN 1473-0197

Microfluidic paper-based analytical devices (μPADs) have attracted great attention as potential candidates for point-of-care testing (POCT). Nevertheless, only a limited number of μPADs expected to satisfy the standard of Clinical Laboratory Improvement Amendments (CLIA) waived tests as issued by the US Food and Drug Administration (FDA) have been reported. This work introduces a “traffic light type μPAD”, enabling highly intuitive semi-quantitative equipment-free naked-eye readout with no need for calibration, subjective interpretation or calculation. Assay results are displayed as traffic light colours reporting 5 analyte concentration levels (green/green & yellow/yellow/yellow & red/red). The device has been designed to never display all three colours simultaneously, eliminating any risk for misinterpretation. The mechanism relies on the modulation of sample flow through a network of paperfluidic channels modified with a hydrophobic to hydrophilic phase-switching substance responsive to H2O2. User operation is limited to sample application, followed by observing a clear and time-independent traffic light signal after approximately 10-30 min. Multiple factors influencing the H2O2 concentration-dependent appearance of a specific traffic light signal were studied. Making use of the possibilities for customising the concentration threshold levels for traffic light colour appearance, quantification of glucose at 5 levels in a clinically relevant concentration range was demonstrated in artificial urine as a model proof-of-concept. This platform is expected to offer the possibility for the future detection of other important metabolites.

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Microfluidic thread-based analytical devices for point-of-care detection of therapeutic antibody in blood

Shimazu R., Tomimuro K., Ni Y., Malegori C., Hamedpour V., Hiruta Y., Oliveri P., Merkx M., Citterio D.

Sensors and Actuators B: Chemical （Sensors and Actuators B: Chemical） 352 2022年02月

研究論文（学術雑誌）, 査読有り, ISSN 09254005

In therapies involving therapeutic antibodies, therapeutic drug monitoring (TDM) has the potential to substantially increase clinical efficacy and to enable more economical drug use, because the clearance from the bloodstream after administration is patient dependent. Since TDM must be performed continuously and over extended time periods, it should ideally be achieved through an economical and easy to perform point-of-care (POC) assay. Here, we introduce a new analytical method for POC TDM of the clinically important therapeutic antibody cetuximab, using microfluidic thread-based analytical devices (µTADs) as a platform for a bioluminescence resonance energy-transfer (BRET) switching sensor protein for simple quantitative antibody detection. The µTAD is pre-treated with NaCl for blood plasma separation and a BRET switching sensor protein and its substrate for cetuximab detection, integrating all elements required for a reagent-free, single-step assay. A change in bioluminescence emission color depending on cetuximab concentrations occurs after whole blood application. The device design enables the quantification of cetuximab within the therapeutically relevant blood concentration range in 2.5 min using only 2 µL of whole blood. The results obtained are independent of the blood volume. This µTAD can be stored for about one month at − 20 °C, a temperature compatible with commonly available freezers. The application of mathematical morphology recognition (MMR)-integrated image processing techniques for the automatic selection of regions of interest (ROIs) yields results with minimal error. The simplicity of use, rapidity, and cost-effectiveness of this µTAD are expected to offer the possibility of achieving TDM of therapeutic antibodies at POC for individual patients.

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Colorimetric paper-based sarcosine assay with improved sensitivity

Masumoto M., Ohta S., Nakagawa M., Hiruta Y., Citterio D.

Analytical and Bioanalytical Chemistry （Analytical and Bioanalytical Chemistry） 414 （ 1 ） 691 - 701 2022年01月

研究論文（学術雑誌）, 査読有り, ISSN 16182642

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed. Graphical abstract: [Figure not available: see fulltext.]

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Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues

Ikeda Y., Orioka M., Nomoto T., Hiruta Y., Nishiyama N., Citterio D.

ChemBioChem （ChemBioChem） 22 （ 21 ） 3067 - 3074 2021年11月

研究論文（学術雑誌）, 査読有り, ISSN 14394227

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

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Anion species-triggered antibody separation system utilizing a thermo-responsive polymer column under optimized constant temperature

Nomoto D., Nagase K., Nakamura Y., Kanazawa H., Citterio D., Hiruta Y.

Colloids and Surfaces B: Biointerfaces （Colloids and Surfaces B: Biointerfaces） 205 2021年09月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り, ISSN 09277765

Although the field of antibody drugs has grown larger, the antibody production still faces several challenges. Effective antibody purification is required, but the conventional purification method for antibodies is cost intensive and often causes aggregation problems, indicating the need for new alternative antibody purification methods. In the present study, a constant temperature antibody purification system for use with a thermo-responsive polymer column was developed based on switching of anion species in eluents. By adjusting the temperature for each antibody, the developed column enabled separation of the therapeutic monoclonal antibodies, rituximab and trastuzumab, from contaminants without changing salt concentration or pH of the eluents. The thermo-responsive hydrogel-modified column packing material was synthesized by introducing n-butyl methacrylate, acrylic acid, N,N′-methylenebisacrylamide and N-isopropylacrylamide to the surface of silica beads with an initiator by a graft-from approach. Elution behavior of antibodies with three types of anions, such as citrate, phosphate, and chloride were tested under three different temperature conditions. It was demonstrated that the thermo-responsive hydrogel grafted column showed a switchable antibody retention behavior at constant temperature and salt concentration, with antibody adsorption by NaCl eluent and desorption by citric acid buffer eluent.

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Interference-based wide-range dynamic tuning of the plasmonic color of single gold nanoparticles

Nakayama Bokusui, Nakabayashi Takahito, Esashika Keiko, Hiruta Yuki, Saiki Toshiharu

OPTICS EXPRESS （Optics Express） 29 （ 10 ） 15001 - 15012 2021年05月

研究論文（学術雑誌）, 査読有り, ISSN 1094-4087

Dynamic tuning of nanoscale coloration by exploiting localized surface plasmon resonance of gold nanoparticles (AuNPs) combined with an interference coloration mechanism is demonstrated experimentally. When interference between the scattering field from AuNPs and the reflected field from the substrate is observed under back-scattering white-light microscopy, the AuNPs exhibit various colors depending on their distance to the substrate. When the numerical aperture of the microscope objective is optimized, much greater coverage of the color space than was achieved with previously reported plasmon-based approaches is attained. Also, color tunability is examined by exploiting the temperature-induced volume change of a temperature-responsive hydrogel with embedded AuNPs to dynamically modify the distance to the substrate.

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蛭田 勇樹

論文 - 蛭田 勇樹

蛭田　勇樹

論文 - 蛭田　勇樹