論文 - 蛭田　勇樹

Current advances in separation chemistry for antibody purification and analysis

Deol S., Matsuda Y., Hiruta Y.

Analytical Sciences 41 （ 5 ） 653 - 666 2025年05月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り,  ISSN  09106340

　概要を見る

Monoclonal antibodies (mAbs) have become essential in modern therapeutics, offering high specificity and efficacy in treating diseases such as cancer, autoimmune disorders, and infectious diseases. With the increasing demand for mAbs, robust analytical and purification technologies are critical to ensuring their safety, efficacy, and consistency. This review highlights recent advances in the application of HPLC for both mAb analysis and purification. In the analytical domain, techniques such as size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEX), and reversed-phase chromatography (RPLC) are explored for their role in evaluating mAb structural attributes and post-translational modifications (PTMs). These methods are indispensable for ensuring stability and functionality while meeting stringent regulatory requirements. In the context of purification, Protein A affinity chromatography remains the gold standard for initial capture of mAb; however, challenges such as high cost and harsh elution conditions have prompted the development of alternative purification technologies. IEX, HIC, multimodal, and membrane chromatography are also critical in achieving high-purity mAb products through multistep workflows, including intermediate purification and polishing stages. This review emphasizes the central role of HPLC in addressing the complex challenges of mAb manufacturing and characterization. By integrating established and emerging chromatographic techniques, it provides insights into the future directions of therapeutic antibody development.

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Bioluminescence readout lateral flow immunoassay using nanobody targeting aflatoxin B1

Takahashi, S; Hiruta, Y; Citterio, D

ANALYST 150 （ 8 ） 1563 - 1570 2025年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0003-2654

　概要を見る

Multiple signal detection methods are known for lateral flow immunoassays (LFIAs), with colorimetric approaches dominating the field. However, their limited sensitivity is a remaining challenge. Fluorescence-based signaling is regarded as a more sensitive method, but it comes at the cost of partial sacrifice of the user-friendliness of LFIAs due to the requirement of an excitation light source. In this context, bioluminescence providing an inherently high signal to noise ratio without the need of excitation light could be an attractive alternative. But only a few studies have demonstrated the application of bioluminescence signaling in LFIAs. This work aimed at the development of a simple bioluminescence-based LFIA for the detection of aflatoxin B1 (AFB1), used as a model target in a competitive LFIA format. Signal transduction was achieved by nanobody-nanoluciferase (Nluc) fusion proteins. These small-sized recombinant heavy-chain-only antibodies derived from the camelidae family directly linked with the Nluc enzyme produce high intensity glow-type bioluminescence in combination with the furimazine substrate. LFIA devices consisting of a sample pad, nitrocellulose membrane and absorbent pad with AFB1-BSA conjugate deposited at the test line on the nitrocellulose membrane, achieved an LOD of 0.26 ng mL⁻¹ for aqueous AFB1 solutions pre-mixed with Nanobody-Nluc and bioluminescence emission observed on an imaging system. More user-friendly LFIA devices with integrated conjugate pad and pre-deposited Nanobody-Nluc provided clear AFB1 concentration-dependent bioluminescence signals with low background and enabled readout with a standard digital camera, resulting in an LOD of 1.12 ng mL⁻¹. Finally, the LFIA strips have been applied in AFB1-spiked oat milk samples. The LOD of 4.09 ng mL⁻¹ achieved in the real sample matrix is well below the maximum allowable residual concentration of AFB1 in the U.S. (20 ng mL⁻¹).

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Origami Paper-Based Immunoassay Device with CRISPR/Cas12a Signal Amplification

Suzuki, H; Tong, GD; Nath, P; Hiruta, Y; Citterio, D

ACS SENSORS 10 （ 3 ） 1811 - 1821 2025年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2379-3694

　概要を見る

In clinical diagnosis, the determination of target proteins at low concentration levels is generally performed by immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), which is a time-consuming process. To date, paper-based ELISA platforms enabling faster and less expensive analysis have been developed, but their important issue for clinical applications is the limited sensitivity compared to conventional ELISA. To address this challenge, this paper introduces a simple, rapid, and highly sensitive detection method for non-nucleic acid targets achieved by integrating the CRISPR/Cas12a system into paper-based ELISA. An origami-type paper-based device enabling simple assay operation has been designed, and the detection of targets on the paper substrates is based on observing the fluorescence signal induced by the CRISPR/Cas12a enzyme cleaving a probe single-stranded DNA (ssDNA) labeled with fluorophore and quencher (FQ reporter). To enhance sensitivity, antibodies labeled with a network of multiple DNA activating the CRISPR/Cas12a enzyme have been utilized as detection antibodies. As a result, the developed device successfully boosted the detection sensitivity for both human IgG and the hepatitis B virus surface antigen (HBsAg). In particular, the limit of detection (LOD) for HBsAg was estimated to be 12 pg/mL, representing over 10-fold higher sensitivity compared with commercially available HBsAg ELISA kits (LOD: 200 pg/mL). In addition, the fluorescence response toward porcine whole blood samples containing different HBsAg concentrations was also confirmed by capturing images with a smartphone, followed by quantitative data analysis. These results demonstrate the potential applicability of the proposed platform for clinical tests at the point of care.

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The latest developments of near-infrared fluorescent probes from NIR-I to NIR-II for bioimaging

Kuronuma, Y; Watanabe, R; Hiruta, Y

ANALYTICAL SCIENCES 41 （ 6 ） 737 - 757 2025年02月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り,  ISSN  0910-6340

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Abstract: Near-infrared I (NIR-I: 650–950 nm) fluorescence imaging is a powerful tool for deep-tissue biological imaging, addressing the limitations of photon penetration depth in the visible-light region. Over the past decade, NIR imaging has extended to the near-infrared II (NIR-II: 1000–1700 nm) region, offering high-resolution and low background imaging by suppressing light scattering, and autofluorescence of tissues. Near-infrared fluorescent probes from NIR-I to NIR-II, with diverse functionalities, are increasingly utilized across biological fields to meet various detection needs and to explore physiological events in real time and spatial dimensions. This review discusses recent advancements in small-molecule NIR fluorescent dyes and probes, particularly those based on cyanine and rhodamine scaffolds, highlighting examples of their applications in bioimaging.

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Ratiometric Imaging for Quantification of Elevated Ca²⁺ in Neurons Using Synthetic Low-Affinity Fluorescent Probe

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

ACS Chemical Neuroscience 16 （ 4 ） 649 - 658 2025年02月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り,  ISSN  1948-7193

　概要を見る

The availability of various calcium ion (Ca2+) fluorescent probes has contributed to revealing physiological events related to intracellular Ca2+. However, conventional probes face challenges for quantitatively and selectively visualizing high Ca2+ concentrations in cells induced by any stimuli, including biomolecules or electrical signal that disrupt Ca2+ homeostasis. In this report, we designed and synthesized a low-affinity ratiometric Ca2+ probe, KLCA-Fura, utilizing o-aminophenol-N,N-diacetate-O-methylene-methylphosphinate (APDAP) as a ligand, for which we recently demonstrated the suitability as a new low-affinity ligand for Ca2+. KLCA-Fura showed a blue shift in excitation wavelength with increasing Ca2+ concentration based on the intramolecular charge transfer (ICT). Its affinity for Ca2+ is lower than commercially available conventional Ca2+ probes. Furthermore, the selectivity for Ca2+ and the fluorescence intensity were considered sufficient to accurately detect Ca2+. The corresponding acetoxymethyl ester, KLCA-FuraAM, was synthesized for intracellular imaging and applied to Ca2+ quantification in neurons. KLCA-FuraAM enabled quantitative ratiometric monitoring of the two-step Ca2+ concentration increase induced by glutamate stimulation. While this two-step response was not clearly observed with a commercially available low-affinity ratiometric Ca2+ probe, Fura-FF, KLCA-FuraAM has demonstrated the potential to quantitatively visualize the behavior of high Ca2+ concentrations. The ratiometric low-affinity Ca2+ probe, KLCA-Fura, is expected to be a powerful tool for discovering new functions of Ca2+ in neurons.

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Creating coveted bioluminescence colors for simultaneous multi-color bioimaging

Hattori, M; Wazawa, T; Orioka, M; Hiruta, Y; Nagai, T

Science Advances 11 （ 4 ） eadp4750 2025年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2375-2548

　概要を見る

Bioluminescence, an optical marker that does not require excitation by light, allows researchers to simultaneously observe multiple targets, each exhibiting a different color. Notably, the colors of the bioluminescent proteins must sufficiently vary to enable simultaneous detection. Here, we aimed to introduce a method that can be used to expand the color variation by tuning dual-acceptor bioluminescence resonance energy transfer. Using this approach, we could visualize multiple targets with up to 20 colors through single-shot acquisition using a color complementary metal-oxide semiconductor camera. Overall, this method enables simple and simultaneous observation of multiple biological targets and phenomena.

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3D-printed devices for multiplexed semi-quantitative competitive lateral flow immunoassays

Tong, GD; Misawa, K; Jarujamrus, P; Hiruta, Y; Citterio, D

ANALYTICAL METHODS 17 （ 6 ） 1206 - 1213 2025年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1759-9660

　概要を見る

Lateral flow immunoassays (LFIAs) are widely used for the simple and rapid detection of various targets at the point of need. However, LFIAs enabling the simultaneous detection of multiple analytes and the possibility for naked-eye semi-quantitative analysis are facing various challenges, including the requirement of large sample volumes, low efficiency, and accuracy. This is particularly the case for the competitive immunoassay format targeting the detection of low molecular weight compounds, such as, for example, drugs. Due to limited space for multiple reaction zones on a single planar nitrocellulose membrane, conducting multiplexed tests requires the addition of more test strips, which consequently increases the size of the whole device. To overcome these spatial constraints, two 3D-printed devices fitting eight assay lanes of both backed and unbacked nitrocellulose membranes have been designed. For proof of concept, 8-OHdG, caffeine, and acetaminophen were used as model analytes. Inkjet printing was applied to deposit capture reagents in the form of text symbols while controlling the concentration thresholds for text readability to achieve an intuitive result expression. A comparably small sample volume of 350 mL was sufficient to simultaneously visually distinguish 4 concentration levels of caffeine (0, 4, 10, 175 ng mL-1) and acetaminophen (0, 4, 8, 12 ng mL-1) in mixed solutions without crosstalk. This study demonstrates the potential of 3D-printed LFIA devices for multiplex and semiquantitative analyte detection.

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Development of a Semiquantitative Barcode Readout Approach for Paper-Based Analytical Devices (PADs) for Enzymatic H₂O₂ and Glucose Detection

Manmana, Y; Kinugasa, S; Hiruta, Y; Citterio, D

ANALYTICAL CHEMISTRY 97 （ 3 ） 1500 - 1506 2025年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0003-2700

　概要を見る

The integration of barcode technology with smartphones on paper-based analytical devices (PADs) presents a promising approach to bridging manual detection with digital interpretation and data storage. However, previous studies of 1D barcode approaches have been limited to providing only a “yes/no” response for analyte detection. Herein, a method of using barcode readout for semiquantitative signal detection on PADs has been achieved through the integration of barcode technology with a distance-based measurement concept on PADs. To demonstrate the feasibility of this concept, a PAD fabrication strategy incorporating barcodes was explored, using the enzymatic reaction between horseradish peroxidase (HRP), 3,3′-diaminobenzidine (DAB), and H2O2 as a model system. The enzyme-catalyzed polymerization of DAB to polyDAB in the presence of hydrogen peroxide results in the appearance of color observable by the naked eye inside a paperfluidic channel, with the color-changed length depending on the H2O2 concentration. At the same time, the barcode pattern displayed as a result of this distance-based color evolution overlaid with a paper-based barcode layer can be read using a smartphone application. Parameters affecting the signal readout performance were studied. The developed device can be used to detect H2O2 concentrations in the range of 0.25 to 10 mM within 90 min with 79.6% of barcode signals correctly readable. Additionally, results from different smartphone models showed a consistent reading performance (78.4-79.6%). Finally, the quantification of glucose levels in artificial urine samples was demonstrated. This developed PAD signaling strategy offers end-users more simplicity and can be used as a standalone device or in conjunction with other digital devices.

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Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device

Tong, GD; Nath, P; Hiruta, Y; Citterio, D

Lab on a Chip 25 （ 4 ） 536 - 545 2025年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1473-0197

　概要を見る

Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM – among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.

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Development of Column Packing Materials Using Functional Polymers and Application to Pharmaceutical Analysis

Hiruta, Y

CHROMATOGRAPHY 46 （ 1 ） 11 - 18 2025年

研究論文（学術雑誌）, 単著, 筆頭著者, 最終著者, 責任著者, 査読有り,  ISSN  1342-8284

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IgG-Binding Peptidomimetic Mixed-Charge Polymer-Modified Resins for Chromatographic Purification of Antibodies

Deura, K; Sakama, A; Moriwaki, Y; Citterio, D; Hiruta, Y

ACS APPLIED MATERIALS & INTERFACES 16 （ 49 ） 67468 - 67476 2024年11月

研究論文（学術雑誌）, 共著, 最終著者, 責任著者, 査読有り,  ISSN  1944-8244

　概要を見る

The process of antibody purification using Fc affinity ligands such as protein A, G, and L faces several challenges including high cost, low stability, and loss of antibody activity due to harsh elution conditions. Here, we describe a chromatographic purification of antibodies utilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding peptide (Z34C) derived from the B domain of protein A. The protein A mimetic resins were prepared by modifying the surface of a TOYOPEARL, methacrylate resin with a polymer that mimics the amino acid sequence of Z34C and the functions of histidine and acidic and neutral amino acids using histamine methacrylamide (HisMA), methacrylic acid, and neutral monomers. The therapeutic monoclonal antibody (mAb), rituximab, was retained on the column at pH 7 and eluted under mildly acidic conditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA) optimized for antibody interaction. The injected antibodies were selectively captured on the column by hydrophobic and electrostatic interactions with the protein A mimetic polymer under neutral conditions and eluted by electrostatic repulsion under acidic conditions. The HisMA20-EEMA column successfully purified mAbs from mixtures with BSA, mouse ascites fluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA column consistently achieved 90% antibody recovery in 100 consecutive purifications from cell culture supernatant. The antibody purification method presented in this study is low cost, highly durable, easy to synthesize, and allows for mild elution conditions. The results demonstrate that the approach of mimicking IgG-binding peptides with mixed-charge polymers is useful for the development of column packing materials for antibody purification.

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Sensor Arrays for Electrochemical Detection of PCR-Amplified Genes Extracted from Cells Suspended in Environmental Waters

Aoki, H; Kawaguchi, M; Kumakura, Y; Kamo, H; Miura, K; Hiruta, Y; Simizu, S; Citterio, D

SENSORS 24 （ 22 ）  2024年11月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Ecological surveys of living things based on DNAs from environmental samples are attractive. However, despite simplicity of water sampling from the target environment, it is still necessary to transport the samples to the laboratory for DNA analysis based on skillful next-generation sequencers. To perform DNA-oriented surveys based on a simple protocol without any special training, we demonstrated, in this study, the detection of genes from cell-containing environmental waters using gene sensor arrays that require no DNA labeling and no external indicators. Cell-suspended PBS or river water were used as models of environmental waters containing living things, and DNA samples were prepared by PCR amplification. Ferrocene-terminated probes were synthesized and immobilized on an electrode array to develop a sensor array. The sensor array showed a large response to a target DNA complementary to the probe and no response to a mismatched DNA, indicating sequence-specific detection. For DNA samples prepared from the cells in PBS, they showed good responses similar to those for the target DNA. They also significantly detected DNA samples from the cells in river water at a general environmental concentration (38 cells mL−1) with 28-fold larger responses than those for 0 cells mL−1.

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Paper-based analytical device for point-of-care nucleic acid quantification combining CRISPR/Cas12a and a personal glucose meter

Tanifuji, Y; Tong, GD; Hiruta, Y; Citterio, D

ANALYST 149 （ 19 ） 4932 - 4939 2024年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0003-2654

　概要を見る

Although CRISPR-based nucleic acid detection has great potential in point-of-care testing due to its simplicity, it has been rarely integrated into paper-based analytical devices (PADs), which are attractive platforms to simplify assays. This work introduces a CRISPR-assisted nucleic acid quantification approach integrated into a PAD with signal readout by a personal glucose meter (PGM). Retention of magnetic beads by filter paper and pre-deposition of all required reagents by freeze-drying stabilized with trehalose enabled the indirect quantification of human papilloma virus (HPV) DNA through a PGM readout without complicated user intervention and complex reagent handling. The calculated limit of detection was 57 pM, which is comparable with other amplification-free CRISPR-based assays detecting nucleic acids. The fully integrated device exhibited good storage stability for up to 4 weeks, suggesting its applicability toward practical point-of-care nucleic acid quantification.

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Lateral flow immunoassay device to detect staphylococcal enterotoxin B (SEB) in durian candy

Relox, PE Jr; Israel, KAC; Hiruta, Y; Citterio, D

ANALYTICAL SCIENCES 41 （ 1 ） 3 - 11 2024年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0910-6340

　概要を見る

Staphylococcal enterotoxin B (SEB), a potent enterotoxin produced by Staphylococcus aureus, has been implicated in incidences of Staphylococcal food poisoning in the Philippines. The use of lateral flow immunoassay devices to detect this toxin in solid food samples, like durian candy, at the point of sampling is constrained by the requirement for sample purification (e.g. centrifugation). This problem is also true with the other applications of LFIA devices on food samples. To overcome this challenge, a lateral flow immunoassay (LFIA) device capable of detecting SEB in unpurified durian candy sample was developed in this study. A modified LFIA device was assembled with three layers of glass fiber pads functioning as sample pads instead of a conventional cellulose fiber pad. Unlike with the cellulose fiber pad, the glass fiber sample pads acted as filter and allowed the flow of a 1:5 dilution of durian candy. The LFIA device applied to spiked 1:5 diluted durian candy samples achieved a visual limit of detection of 5 ng/mL for SEB, which is twofold lower than reported for previous LFIA devices designed to detect SEB in food samples.

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Rational design of pH-responsive near-infrared spirocyclic cyanines: the effects of substituents and the external environment

Sakama, A; Seo, H; Hara, J; Shindo, Y; Ikeda, Y; Oka, K; Citterio, D; Hiruta, Y

CHEMICAL COMMUNICATIONS 60 （ 46 ） 5984 - 5987 2024年06月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り,  ISSN  1359-7345

　概要を見る

pH-responsive spirocyclic cyanine dyes were designed and synthesized. The equilibrium constant for cyclization (pKcycl) could be rationally controlled by changing the nucleophilic moiety and the side chains. Encapsulation in polymeric micelles inhibited the H-aggregation of the dye, and the pKcycl could be shifted according to the amphiphilic polymer employed.

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Basic evaluation of the CRISPR/Cas system stability for application to paper-based analytical devices

Tanifuji Y., Suzuki H., Tong G., Hiruta Y., Citterio D.

Analytical Methods 16 （ 25 ） 4143 - 4149 2024年05月

研究論文（学術雑誌）, 査読有り,  ISSN  17599660

　概要を見る

Despite the promising features of the CRISPR/Cas system for application to point-of-care nucleic acid tests, there are only a few reports on its integration into paper-based analytical devices (PADs) for the purpose of assay simplification. In most cases, paper platforms have only been used for the final signal readout in an assay otherwise performed in a test tube. Therefore, there is very limited information on the suitability of the CRISPR/Cas system for on-device reagent storage. To fill this gap, the current work primarily investigated the influence of various factors, including the type of paper, reagent drying method, effect of stabilizers, and storage condition on the storage stability of reagents necessary for CRISPR-based assays on paper substrates, by comparing the fluorescence signal emitted by the trans-cleavage of the dsDNA-activated Cas12a complex. The results obtained in the form of fluorescence signals emitted after trans-cleavage of a ssDNA probe through a dsDNA-activated Cas12a complex on paper substrates showed that CRISPR-related reagents spontaneously dried at room temperature on BSA blocked paper retained over 70% of their initial activity when stored at −20 °C for 28 days, independent of the type of paper substrates, which was improved by the addition of sucrose as a stabilizer. In addition, reagents dried on paper substrates under the optimized conditions exhibited stronger heat tolerance at temperatures above 65 °C compared to their corresponding solutions. This work is expected to contribute to the future development of fully integrated PADs relying on CRISPR/Cas systems for point-of-care applications requiring no additional reagent handling.

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Simplified capture, extraction, and amplification of cellular DNA from water samples

Kawaguchi M., Aoki H., Kamo H., Miura K., Hiruta Y., Simizu S., Citterio D.

Analytical Sciences （Analytical Sciences）  40 （ 3 ） 501 - 510 2024年03月

研究論文（学術雑誌）, 査読有り,  ISSN  09106340

　概要を見る

DNA analysis in water samples is attracting attention in various fields. However, conventional methods for DNA analysis require a work-intensive and time-consuming sample pre-treatment. In this study, a simplified pre-treatment method for analyzing DNA in water samples was evaluated. The process consists of filtration, DNA extraction, and amplification, which can be achieved within a short time. In the filtration process, two types of filters, firstly a tissue paper (Kimwipe) and then a glass filter (GF/F), were used in sequence. The first large pore size filter enabled a reduction in filtration time by removing large particulate matter impurities present in river water matrix. Cells spiked into 1 L of river water were recovered at more than 90% within approximately 5 min filtration time. Also, DNA was extracted from the captured cells directly on the surface of the filter in only 5 min. Thus, DNA collection and extraction from a water sample can be completed within about 10 min. Furthermore, PCR amplification was performed directly from DNA-attached filter sections, which greatly reduced the number of required pre-treatment steps. Finally, we succeeded in establishing a simple and fast on-site pre-treatment system by using a hand-driven syringe filtration method. This pre-treatment system is expected to offer the possibility for the future establishment of a rapid and easy DNA analysis method applicable to various types of water samples. Graphical abstract: (Figure presented.)

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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 (vol 15, pg 1790, 2023)

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

ANALYTICAL METHODS 16 （ 9 ） 1416 - 1416 2024年02月

研究論文（学術雑誌）, 最終著者, 責任著者,  ISSN  1759-9660

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Current advances in the development of bioluminescent probes toward spatiotemporal trans-scale imaging

Sakama A., Orioka M., Hiruta Y.

Biophysics and physicobiology 21 2024年

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り

　概要を見る

Bioluminescence imaging has recently attracted great attention as a highly sensitive and non-invasive analytical method. However, weak signal and low chemical stability of the luciferin are conventional drawbacks of bioluminescence imaging. In this review article, we describe the recent progress on the development and applications of bioluminescent probes for overcoming the aforementioned limitations, thereby enabling spatiotemporal trans-scale imaging. The detailed molecular design for manipulation of their luminescent properties and functions enabled a variety of applications, including in vivo deep tissue imaging, long-term imaging, and chemical sensor.

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Nonsequential and Sequential Dual Detection Probe Lateral Flow Immunoassay Device for Staphylococcal Enterotoxin B Detection

Relox, PE; Israel, KAC; Hiruta, Y; Citterio, D

MINDANAO JOURNAL OF SCIENCE AND TECHNOLOGY 22   235 - 259 2024年

ISSN  2244-0410

　概要を見る

Staphylococcal enterotoxins (SEs) are among the leading causes of foodborne illnesses in the Philippines. Rapid and sensitive detection of these toxins in food, particularly staphylococcal enterotoxin B (SEB) - the most potent type of SE - is imperative. Rapid toxin detection can be achieved using a lateral flow immunoassay (LFIA) device. However, low sensitivity has been a persistent issue with LFIA devices. To develop an LFIA device with improved sensitivity, this study investigated the exclusive use of antibodies as biorecognition molecules in a dual-detection probe LFIA device format and the configuration of the LFIA device to release the probes nonsequentially and sequentially. The LFIA device was fitted with two layers of glass fiber sample pads, loaded separately with the two detection probes, and partially separated by a Scotch tape film. For the nonsequential LFIA device, the two sample pads were pretreated with blocking buffers containing the same sugar concentration, whereas for the sequential LFIA device, the two sample pads were pretreated with blocking buffers containing different sugar concentrations. When tested against a filtered, homogenized durian candy sample, the nonsequential LFIA device generated higher signal intensity than the sequential LFIA device. This result suggests that aggregation of the detection probes was superior to sequential binding in enhancing the signal of the LFIA device for SEB detection. The sensitivity of the nonsequential LFIA device at 5 ng/mL was two-fold higher compared to previous POCT LFIA devices for SEB detection in food samples.

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Development of Phosphinate Ligand-Based Low-Affinity Ca²⁺ Fluorescent Probes and Application to Intracellular Ca²⁺ 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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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

　概要を見る

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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Small molecule-based alkaline-earth metal ion fluorescent probes for imaging intracellular and intercellular multiple signals

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

Chemistry Letters （Chemistry Letters）  50 （ 5 ） 870 - 887 2021年05月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り,  ISSN  03667022

　概要を見る

This highlight review overviews recent progress in fluorescence imaging technology for the alkaline-earth metal ions calcium, and magnesium, by small molecule-based fluorescent probes. Fluorescent probes for Ca2+ using 1,2-bis-(o-aminophenoxy)- ethane-N,N,N′,N′-tetraacetic acid (BAPTA) as a recognition unit are introduced. Focus is set on the fluorescence characteristics and intracellular dynamics of Ca2+ fluorescent probes depending on the differences in fluorophore units. In addition, this review comprehensively covers Mg2+ fluorescent probes from Mg2+ selective ligands and fluorophore units to their application. In particular, the influence of the molecular structure of the fluorescent probes on organelle localization and fluorescence wavelength for imaging intracellular signals in organelles and intercellular multiple signal interactions are summarized, providing a strategy for probe design for chemists, and for selecting the appropriate imaging technique for biologists.

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Long-term single cell bioluminescence imaging with C-3 position protected coelenterazine analogues

Mizui Y., Eguchi M., Tanaka M., Ikeda Y., Yoshimura H., Ozawa T., Citterio D., Hiruta Y.

Organic and Biomolecular Chemistry （Organic and Biomolecular Chemistry）  19 （ 3 ） 579 - 586 2021年01月

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り,  ISSN  14770520

　概要を見る

© The Royal Society of Chemistry 2020. Bioluminescence is a powerful imaging modality for monitoring biological phenomena bothin vitroandin vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.

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Anionic fluorophore-assisted fabrication of gold microstructures inside a hydrogel by multi-photon photoreduction

Machida Manan, Shen Weilu, Onoe Hiroaki, Hiruta Yuki, Heisterkamp Alexander, Mazur Eric, Terakawa Mitsuhiro

OPTICAL MATERIALS EXPRESS （Optical Materials Express）  11 （ 1 ） 48 - 58 2021年01月

研究論文（学術雑誌）, 査読有り,  ISSN  2159-3930

　概要を見る

The fabrication of accentuated gold microstructures is demonstrated by multi-photon photoreduction inside an anionic fluorophore-containing hydrogel. We attempted to facilitate gold-ion photoreduction near the focal point of laser pulses, expecting the donation of electrons by the oxidation of fluorophores in the vicinity of gold ions. The presence of anionic FITCdextran also inhibited the spontaneous reduction in untargeted zones, which is attributed to the coordination of gold ions and the anionic FITC-dextran. Simultaneous facilitation and inhibition are promising for the fabrication of dense metal microstructures in the targeted zone while maintaining the hydrogel's light permeability.

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Ion-selective optodes integrated paper-based device for simultaneous colorimetric quantification of salivary Na+, K+ and Ca2+

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

Bunseki Kagaku （Bunseki Kagaku）  70 （ 3 ） 165 - 173 2021年

研究論文（学術雑誌）, 査読有り,  ISSN  05251931

　概要を見る

The quantification of electrolytes in biological fluids plays an important role for evaluating the human health condition. This work proposes a disposable and simple microfluidic paperbased analytical device (µPAD) for the quantitative colorimetric detection of salivary Na+, K+ and Ca2+, relying on ion-selective optodes (ISOs). Nanoscale ISOs composed of pluronic F-127 surfactant-based micelles were used as a colorimetric indicator system, reducing the assay time compared to the previously reported bulk membrane approach. Devices consist of three functionalized paper layers assembled by lamination, with the optical signal read out from the bottom layer. Saliva was chosen as the biological specimen because it is painlessly collected and easy-to-handle, which makes it suitable for POC assays. Simultaneous Na+, K+ and Ca2+ detection by a single µPAD was successfully confirmed by using aqueous metal ion samples with various concentrations of Na+, K+ and Ca2+ within the biologically relevant range. The simultaneous detection of the three cationic species enabled the correction of the three ion quantities by eliminating the interference from other ions using the calculated selectivity coefficients. For proof-of-concept, Na+ quantification in a human saliva sample was conducted by the developed µPADs. The calibration curve for this assay was prepared by applying artificial saliva to the µPADs. The concentrations of Na+ in spiked human saliva were also measured by the µPADs. The obtained recovery rates were 78-132 %, which indicates the quantification of the added Na+ in the real sample matrix. Finally, this µPAD was successfully integrated into a smartphone-based assay, which enables salivary electrolyte quantification in resource poor settings. The developed µPADs satisfy the criteria of low-cost (< $0.3/device), rapidity (< 5 min) and simple analytical procedure compared to the conventional ion-selective electrode (ISE) method.

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Distance readout-based paper device for multiplexed urinalysis

Sawano R., Maejima K., Shibata H., Hiruta Y., Citerrio D.

Bunseki Kagaku （Bunseki Kagaku）  70 （ 3 ） 175 - 181 2021年

研究論文（学術雑誌）, 査読有り,  ISSN  05251931

　概要を見る

Urinalysis is widely used in routine medical checkups. Conventionally, colorimetric paper strips currently available on the market are applied. Although they allow to rapidly obtain results, their use is often not straightforward, because a timed readout is required, followed by comparison with a color chart. D-μPADs (distance-based microfluidic paper-based analytical devices) have gained attention for enabling highly intuitive signal readout. During liquid flow through a paperfluidic channel, target analytes are sequentially depleted from the sample through reaction with colorimetric indicators and/or adsorption to the paper surface, so that the length of color-changed sections correspond to the analyte content. Therefore, D-μPADs are expected to solve the problems of existing urinalysis tests. The final goal is the development of D-μPADs for the simultaneous detection of urinary protein (albumin), glucose and hemoglobin. Colorimetric distance-based calibration curves were acquired for all analytes in artificial urine matrix containing electrolytes and organic compounds. For quantitative assays, evaluation of color-changed distances was conducted by computer software. The first cases of distance-based albumin, glucose and hemoglobin readouts in relatively complex artificial urine matrix are reported. For albumin detection, the color uniformity of the colorimetric indicator was improved by paper surface modification with poly(4-styrenesulfonic acid), an anionic polymer. For glucose detection, signal visibility was significantly improved by paper surface modification with chitosan, a biopolymer that works as immobilization support for enzymes. Finally, we evaluated the effect of urinary components on the assay results. It was confirmed that D-μPADs for the hemoglobin assay require a treatment to reduce the effect of albumin adsorption on the paper substrate.

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Fluorescent and Bioluminescent Probes based on Precise Molecular Design

Kumada Rei, Orioka Mariko, Citterio Daniel, Hiruta Yuki

BUNSEKI KAGAKU （Bunseki Kagaku）  70 （ 10-11 ） 600 - 616 2021年

研究論文（学術雑誌）, 最終著者, 責任著者, 査読有り,  ISSN  0525-1931

　概要を見る

Fluorescence and bioluminescence have been actively applied to various chemical sensors and biosensors, including to cellular/in vivo imaging. On application to the detection of specific targets, it is necessary to develop fluorescent/bioluminescent probes that selectively respond to the target analytes such as molecules, ions or cells by changing their optical properties. The authors have focused on the development of small molecular probes by means of molecular design based on the desired function. In this paper, we describe the approaches to molecular design of fluorescent/bioluminescent probes and their applications, including the introduction of our recent research works. Finally, a future outlook for further functionalization of small molecular probes is provided.

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Near-infrared pH responsive heptamethine cyanine platforms: Modulating the proton acceptor

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

Dyes and Pigments （Dyes and Pigments）  181 2020年10月

研究論文（学術雑誌）, 査読有り,  ISSN  01437208

　概要を見る

© 2020 Elsevier Ltd In this report, we wish to present the rational design, synthesis, and study of optical characteristics of near-infrared (NIR) molecular probes emitting over 800 nm. The title pH sensing platforms consist of a piperazine moiety serving as a hydronium ion receptor, coupled with a rigidified symmetric heptamethine backbone. The spectrally fine-tuned response of the fluorophores was studied in both organic solvents and aqueous buffer solutions, employing combined UV–Vis and steady state fluorimetric techniques. In parallel, computational approach and optical spectroscopy were employed aiming to ascribe the origin of spectral behavior of the amino-substituted heptamethines. Herein, we also investigated the halochromic effect of the differently substituted piperazines on the spectral properties of the NIR cyanine dyes. Under acidic conditions, the protonation of the piperazine moiety promoted a gradual bathochromic shift in the absorption spectra. The Δλ between the neutral and the protonated forms are indirectly related to the modifications applied to the proton acceptor. The calculated pKa values (5.13–7.06) were found to be dependent on the dye chemical structure. A good linear correlation was established between the pKa of the newly synthesized dyes and that of the reported piperazine moieties. This aspect allows the future expansion of the current facile strategy since sensing chromophores with predicted pH response could readily be designed based on pKa databases for substituted piperazine derivatives. A detailed literature study along with NMR and computational methods were found to be in agreement with our assumption, that both nitrogen atoms of the 1,4-disubstituted amine are involved in the sensing process. The covered pKa range allows us to recommend the title fluorophores as promising potential candidates for qualitative analysis of biological samples. A cytotoxicity study by the MTT assay showed that the NIR dyes are safe and applicable for bioanalytical purposes at dye concentrations up to 10 μM. Fluorescence imaging and cellular applications, including staining and visualization of HeLa cervical cancer cells was successfully demonstrated.

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All-printed semiquantitative paper-based analytical devices relying on QR code array readout

Katoh A., Maejima K., Hiruta Y., Citterio D.

The Analyst （The Analyst）  145 （ 18 ） 6071 - 6078 2020年09月

研究論文（学術雑誌）, 査読有り,  ISSN  0003-2654

　概要を見る

The wide spread of smartphones and QR codes for various end-user applications has had an impact beyond traditional fields of use, recently also reaching point-of-care testing (POCT). This work presents the integration of QR code recognition into paper-based analytical devices (PADs) with "distance-based" colorimetric signalling, resulting in semiquantitative readout fully relying on straightforward barcode reader solutions. PADs consist of an array of QR codes arranged in series inside a paperfluidic channel. A mask dye concept has been developed, which enables utilisation of colour changing indicators by initially hiding QR codes. The colour change of the indicator induced by the presence of an analyte of interest results in gradual unmasking of QR codes, which become recognisable by the smartphone barcode reader app. To reproducibly fabricate devices, all fabrication steps were performed by commercial desktop solid ink and inkjet printing. The QR code masking function was optimised by controlling the amount of printed mask dye through adjusting the opacity of printing patterns during the inkjet deposition process. For proof-of-concept, a model assay in the form of colorimetric copper ion (Cu2+) detection in the concentration range of 0.4 mM to 3.2 mM was evaluated. Consistent results independent of the smartphone model and environmental light condition were achieved with a free barcode reader app. To the best of our knowledge, this work is the first demonstration of a semiquantitative assay approach fully relying on QR code readout without digital colour analysis, customised app or hardware modification.

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Text-Displaying Semiquantitative Competitive Lateral Flow Immunoassay Relying on Inkjet-Printed Patterns

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

ACS Sensors （ACS Sensors）  5 （ 7 ） 2076 - 2085 2020年07月

研究論文（学術雑誌）, 査読有り,  ISSN  2379-3694

　概要を見る

Copyright © 2020 American Chemical Society. This work describes a colorimetric signaling approach for competitive lateral flow immunoassays (LFIAs) enabling sensitive and semiquantitative direct visual result readout in the form of "text", demonstrated on the example of 8-hydroxy-2′-deoxyguanosine (8-OHdG) detection. The distinctive feature of the developed text-displaying LFIA (TD-LFIA) is the test zone system consisting of a combination of two types of inkjet-deposited capture molecules referred to as "mask antigen"and "text antibody", allowing for sensitive turn-on signaling as opposed to the inverse response of conventional competitive LFIAs. The user operation is limited to sample application, followed by direct reading of assay results written in text after approximately 10 min. TD-LFIAs enabled the visual detection of 8-OHdG at concentrations down to 3 ng/mL, which is a 2-3 orders of magnitude lower visual detection limit than that achieved with the corresponding conventional design and is comparable to the existing LFIAs relying on external signal readout equipment. Highly reproducible observer-independent assay performance was confirmed, and the result interpretation is not influenced by sample color and readout timing. Making use of customizable threshold settings for text appearance, a device for semiquantitative assays was developed and successfully applied to the detection of 8-OHdG at four concentration levels (trace, low, medium, and high) in 54 human urine samples within the clinically relevant concentration range. The sensitive and intuitive signaling method of the developed system offers great potential for an alternative competitive LFIA platform suitable for real-world point-of-care testing applications.

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Thread-Based Bioluminescent Sensor for Detecting Multiple Antibodies in a Single Drop of Whole Blood

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

ACS Sensors （ACS Sensors）  5 （ 6 ） 1786 - 1794 2020年06月

研究論文（学術雑誌）, 査読有り,  ISSN  2379-3694

　概要を見る

Copyright © 2020 American Chemical Society. Antibodies are important biomarkers in clinical diagnostics in addition to being increasingly used for therapeutic purposes. Although numerous methods for their detection and quantification exist, they predominantly require benchtop instruments operated by specialists. To enable the detection of antibodies at point-of-care (POC), the development of simple and rapid assay methods independent of laboratory equipment is of high relevance. In this study, we demonstrate microfluidic thread-based analytical devices (μTADs) as a new platform for antibody detection by means of bioluminescence resonance energy-transfer (BRET) switching sensor proteins. The devices consist of vertically assembled layers including a blood separation membrane and a plastic film with a sewn-in cotton thread, onto which the BRET sensor proteins together with the substrate furimazine have been predeposited. In contrast to intensity-based signaling, the BRET mechanism enables time-independent, ratiometric readout of bioluminescence signals with a digital camera in a darkroom or a smartphone camera with a 3D-printed lens adapter. The device design allows spatially separated deposition of multiple bioluminescent proteins on a single sewn thread, enabling quantification of multiple antibodies in 5 μL of whole blood within 5 min. The bioluminescence response is independent of the applied sample volume within the range of 5-15 μL. Therefore, μTADs in combination with BRET-based sensor proteins represent user-friendly analytical tools for POC quantification of antibodies without any laboratory equipment in a finger prick (5 μL) of whole blood.

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Metal-Free Colorimetric Detection of Pyrophosphate Ions by Inhibitive Nanozymatic Carbon Dots

Chen C.Y., Tan Y.Z., Hsieh P.H., Wang C.M., Shibata H., Maejima K., Wang T.Y., Hiruta Y., Citterio D., Liao W.S.

ACS sensors （ACS sensors）  5 （ 5 ） 1314 - 1324 2020年05月

研究論文（学術雑誌）, 査読有り,  ISSN  2379-3694

　概要を見る

The pyrophosphate ion (P2O74-, PPi) plays a critical role in various biological processes and acts as an essential indicator for physiological mechanism investigations and disease control monitoring. However, most of the currently available approaches for PPi species detection for practical usage still lack appropriate indicator generation, straightforward detection requirements, and operation convenience. In this study, a highly sensitive and selective PPi detection approach via the use of nanozymatic carbon dots (CDs) is introduced. This strategy eliminates the common need for metal ions in the detection process, where a direct indicator-PPi interaction is adopted to provide straightforward signal reports, and importantly, through a green indicator preparation. The preparation of this nanozymatic CDs' indicator utilizes an aqueous solution refluxing, employing galactose and histidine as the precursor materials. The mild conditions of the solution refluxing produce fluorescent CDs exhibiting peroxidase-mimic properties, which can catalyze the o-phenylenediamine oxidation under the presence of H2O2. The introduction of PPi species, interestingly, inhibits this process very efficiently, the extent of which can be colorimetrically monitored by the generated yellow product 2,3-diaminophenazine. Spectroscopic results point to CD surface functional groups' selective binding toward PPi species, which severely interferes with the electron transfer process in the enzymatic catalysis. Relying on this CD peroxidase-mimetic property inhibition, sensitive and selective recognition of PPi reaches a detection limit of 4.29 nM, enabling practical usage in complex matrixes. Owing to the superior compatibility and high stability of nanozymatic CDs, they can also be inkjet-printed on paper-based devices to create a portable and convenient platform for PPi detection. Both the solution and the paper-device-based selective recognitions confirm this unique and robust metal-free inhibitive PPi detection, which is supported by a convenient green preparation of nanozymatic CDs.

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Quantitative evaluation of luminescence intensity from enzymatic luminescence reaction of coelenterazine and analogues

Ikeda Y., Tanaka M., Nishihara R., Hiruta Y., Citterio D., Suzuki K., Niwa K.

Journal of Photochemistry and Photobiology A: Chemistry （Journal of Photochemistry and Photobiology A: Chemistry）  394 2020年05月

研究論文（学術雑誌）, 査読有り,  ISSN  10106030

　概要を見る

© 2020 Elsevier B.V. Recent advances in bioluminescence technology rely on the discovery of luciferin-luciferase pairs having excellent luminescence activity. Here, we focus on coelenterazine (CTZ) and its analogues and quantitatively investigate reaction factors, including bioluminescence reaction quantum yield (φBL) and enzyme reaction kinetics (turnover rate, kcat), determining the luminescence intensity to design brighter reaction systems. Initially, it was confirmed that the CTZ was kept stable during the storage and the φBL value measuring experiment in terms of comparative study using benzyl-protected CTZ. Using a luminometer whose absolute responsivity is calibrated for each luminescence reaction spectrum, we absolutely measured φBL and other reaction factors of CTZ and 3 types of blue-shifted CTZ analogues, CTZ 400a, 6-pi-H-CTZ and 6-pi-Ph-CTZ. The results suggested that there was no significant difference in reaction kinetics, but a clear difference in φBL (up to 22-fold), which was presumably due to an improvement in fluorescence quantum yield (φFL) of the corresponding coelenteramide (CTMD), oxidized form of CTZ. Our evaluation suggests that the design of CTZ analogues considering φFL is important for developing a bright luminescent reaction system.

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Paper-Based Device for Naked Eye Urinary Albumin/Creatinine Ratio Evaluation

Hiraoka R., Kuwahara K., Wen Y.C., Yen T.H., Hiruta Y., Cheng C.M., Citterio D.

ACS Sensors （ACS Sensors）  5 （ 4 ） 1110 - 1118 2020年04月

研究論文（学術雑誌）, 査読有り,  ISSN  2379-3694

　概要を見る

Copyright © 2020 American Chemical Society. This paper introduces the concept of "Drawing-PADs" (Drawing paper-based microfluidic analytical devices) allowing to intuitively evaluate the urinary albumin (Alb) index, a clinically important parameter used for the early detection of renal deficiencies related to diabetes, among others. To enable regular monitoring of the Alb index, a simple examination method suitable for self-diagnosis is highly desirable. The Drawing-PADs rely on the simultaneous naked eye detection of Alb and creatinine (Cre) on a single device according to the distance-based microfluidic PAD (μPAD) approach. The Alb index is visualized by simply drawing a straight line connecting the top of two color-changed assay channel sections (Alb and Cre channels), followed by visually confirming the position of the intercept of the drawn straight line. The semiquantitative Alb index evaluation performed with Drawing-PADs does not require any equipment such as a camera, software, or a color reference chart. The obtained results are independent of the sample volume and are not influenced by changes in the absolute Alb and Cre concentrations caused by urine excretion variations, making spot urine assays possible. Classification of Alb index values according to clinically relevant criteria (normoalbuminuria, microalbuminuria, and macroalbuminuria) is readily achieved within 15 min and has been validated for 15 human urine samples including diabetic patients and healthy volunteers.

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Centrifugal Paperfluidic Platform for Accelerated Distance-Based Colorimetric Signal Readout

Maejima Kento, Hiruta Yuki, Citterio Daniel

ANALYTICAL CHEMISTRY （Analytical Chemistry）  92 （ 7 ） 4749 - 4754 2020年04月

研究論文（学術雑誌）, 査読有り,  ISSN  0003-2700

　概要を見る

Copyright © 2020 American Chemical Society. Distance-based readout is one of the most user-friendly and simple colorimetric signaling methods widely applied for paper-based analytical devices (PADs). This work presents the integration of distance readout PADs into a centrifugal platform enabling affordable, rapid, and sample volume-independent colorimetric assays. Centrifugally assisted distance-based PADs (CD-PADs) eliminate the requirement to use micropipets for sample introduction and reduce the overall time for distance-based assays. All device fabrication steps were performed through computer-controlled print, cut, and laminate (PCL) techniques oriented toward mass production. The inexpensive centrifugal platform was built on a recycled DVD player combined with an open source microcomputer (Arduino). Assay protocols, including rotational velocity and rotation time, were optimized to obtain a maximum dynamic range and reproducible results for sample volume metering (coefficient of variation 3.62%). A colorimetric Ni2+ assay chosen to demonstrate measurements on CD-PADs allowed the detection of nickel ions (Ni2+) with naked-eye interpretation within 1.5 min and a limit of detection (LOD) of 44.1 ng of Ni2+, which to the best of our knowledge is the lowest value reported for a distance-based Ni2+ assay.

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pH-Responsive Tunable Mixed-Charge Polymers for pH-Selective Interaction with Anionic Biological Constituents

Hiruta Yuki, Sawada Kazuki, Mizui Yuki, Citterio Daniel

BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN （Bulletin of the Chemical Society of Japan）  93 （ 4 ） 547 - 552 2020年04月

研究論文（学術雑誌）, 筆頭著者, 責任著者, 査読有り,  ISSN  0009-2673

　概要を見る

A pH-responsive mixed-charge polymer consists of uniformly distributed cationic and anionic units in the polymer chain. Carboxylic acid monomers are generally used as anionic monomers. The pH responsive range is confined to a relatively narrow pH of Â5, which is limiting the biological application of pH-responsive polymers. Therefore, we investigated the effect of a series of carboxylic acid and neutral monomers on the pKa value of pH-responsive mixed-charge polymers. We synthesized the polymers using [2-(methacryloyloxy)ethyl]-trimethylammonium chloride, carboxylic acid monomers, and neutral monomers as the spacer moiety. As the monomer hydrophobicity and the composition ratio of neutral monomers increased, the pKa shifted to higher values. Finally, the polymer pKa values were tuned from 4.6 to 6.1. We confirmed that the pH-dependent interaction between anionic tissue constituent and pH-responsive mixed-charge polymers depends on their pKa.

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Ring-Fused Firefly Luciferins: Expanded Palette of Near-Infrared Emitting Bioluminescent Substrates

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

Analytical chemistry （Analytical chemistry）  92 （ 6 ） 4235 - 4243 2020年03月

研究論文（学術雑誌）, 査読有り,  ISSN  00032700

　概要を見る

Firefly bioluminescence is broadly applied as a noninvasive imaging modality in the biomedical research field. One limitation in firefly bioluminescence imaging is the limited variety of luciferins emitting in the near-infrared (NIR) region (650-900 nm), where tissue penetration is high. Herein, we describe a series of structure-inherent NIR emitting firefly luciferin analogues, NIRLucs, designed through a ring fusion strategy. This strategy resulted in pH-independent structure-inherent NIR emission with a native firefly luciferase, which was theoretically supported by quantum chemical calculations of the oxidized form of each luciferin. When applied to cells, NIRLucs displayed dose-independent improved NIR emission even at low concentrations where the native d-luciferin substrate does not emit. Additionally, excellent blood retention and brighter photon flux (7-fold overall, 16-fold in the NIR spectral range) than in the case of d-luciferin have been observed with one of the NIRLucs in mice bearing subcutaneous tumors. We believe that these synthetic luciferins provide a solution to the longstanding limitation in the variety of NIR emitting luciferins and pave the way to the further development of NIR bioluminescence imaging platforms.

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Traffic-light-type microfluidic paper-based analytical device for hydrogen peroxide detection

Ohta S., Hiraoka R., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences （MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences）     444 - 445 2020年

　概要を見る

© 2020 CBMS-0001 This work reports an intuitive signaling approach enabling naked-eye semi-quantitative result readout in the form of a “traffic-light” for microfluidic paper-based analytical devices (µPADs). The detection of hydrogen peroxide has been demonstrated with the aim of quantifying metabolites relevant as biomarkers of various lifestyle-related diseases. User operation is limited to simple sample application, followed by observing the traffic-light signal after approximately 10 min. Due to its intuitiveness with very low probability of result misinterpretation, this system is expected to offer great potential for home-based self-diagnostic point-of-care testing (POCT) applications.

Ink-jet printed microfluidic paper-based analytical devices relying on QR code detection

Katoh A., Maejima K., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences （MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences）     456 - 457 2020年

　概要を見る

© 2020 CBMS-0001 Microfluidic paper-based analytical devices (µPADs) that display semi-quantitative analysis results by reading a QR code have been developed. The only user operation required is to apply the sample and to read the displayed QR code with a mobile device to obtain the result. Copper ions were detected as a model target and the copper ion concentration-dependent display of QR codes was achieved. This simple assay method can be applied to other targets and has great potential for point-of-care testing (POCT) applications.

Cellophane-based analytical devices for pump-free liquid transport and background-tinted colorimetric assays

Shigemori H., Maejima K., Shibata H., Hiruta Y., Citterio D.

MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences （MicroTAS 2020 - 24th International Conference on Miniaturized Systems for Chemistry and Life Sciences）     422 - 423 2020年

　概要を見る

© 2020 CBMS-0001 This report suggests utilizing “cellophane” as a new substrate material for point-of-care testing (POCT) devices. Cellophane-based microfluidic devices were fabricated by a print, cut and lamination (PCL) methodology and sample liquid was transported into four assay areas without external pumping. Furthermore, making use of the transparency of cellophane, background-tinted devices were fabricated. Based on a blue background-tinted cellophane device, the dynamic range of a hue-based H2O2 assay was 46-fold increased compared to performing the same assay on a white background device. Finally, turbidity testing for albumin was implemented on black background-tinted devices with a visual detection limit of 15 mg/dL.

Fabrication and application of microfluidic paper-based analytical devices (μPAD)

Shigemori H., Hiruta Y., Citterio D.

Kinoshi Kenkyu Kaishi/Annals of the High Performance Paper Society, Japan （Kinoshi Kenkyu Kaishi/Annals of the High Performance Paper Society, Japan）  59   17 - 23 2020年

ISSN  02885867

　概要を見る

Bio)chemical sensors based on paper substrates show attractive features such as being low cost, portable and disposable. Therefore, the technology is currently used in urinary dipsticks and immunochromatographic test strip. In recent years, many research groups report studies on microfluidic paper-based analytical devices (μPADs). In spite of the simplicity of paper-based platforms, μPADs enable performing complicated analytical procedures, multiplex target analysis and equipment-free detection. Due to these useful functions, μPADs are paid attention for point-of-care testing (POCT) and for field-deployable analysis. Herein, we introduce the fabrication methodology and clinical applications of μPADs developed by our research group.

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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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

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

　概要を見る

© 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

　概要を見る

© 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 Mg²⁺ and Application to Multi-Color Imaging of Mg²⁺, 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

　概要を見る

© 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

　概要を見る

© 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

　概要を見る

© 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.

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）     697 - 698 2019年

　概要を見る

© 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.

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

　概要を見る

© 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

　概要を見る

© 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

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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

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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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Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging

Yamada, Arisa, Hiruta, Yuki, Wang, Jian, Ayano, Eri, Kanazawa, Hideko

Biomacromolecules 16 （ 8 ） 2356 - 2362 2015年08月

研究論文（学術雑誌）, 査読有り,  ISSN  1525-7797

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The effects of anionic electrolytes and human serum albumin on the LCST of poly(N-isopropylacrylamide)-based temperature-responsive copolymers

Hiruta, Yuki, Nagumo, Yuhei, Suzuki, Yuichi, Funatsu, Takaaki, Ishikawa, Yuki, Kanazawa, Hideko

Colloids and Surfaces B-Biointerfaces 132   299 - 304 2015年08月

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

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Near IR Emitting Red-Shifting Ratiometric Fluorophores Based on Borondipyrromethene

Hiruta, Yuki, Koiso, Hikaru, Ozawa, Hitoshi, Sato, Hiroyasu, Hamada, Kensaku, Yabushita, Satoshi, Citterio, Daniel, Suzuki, Koji

Organic Letters 17 （ 12 ） 3022 - 3025 2015年06月

研究論文（学術雑誌）, 筆頭著者, 査読有り,  ISSN  1523-7060

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pH/temperature-responsive fluorescence polymer probe with pH-controlled cellular uptake

Yuki Hiruta, Takaaki Funatsu, Minami Matsuura, Jian Wang, Eri Ayano, Hideko Kanazawa

Sensors and Actuators B: Chemical 207   724 - 731 2015年02月

研究論文（学術雑誌）, 筆頭著者, 査読有り

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Effects of terminal group and chain length on temperature-responsive chromatography utilizing poly(N-isopropylacrylamide) synthesized via RAFT polymerization

Hiruta, Yuki, Nagumo, Yuhei, Miki, Atsushi, Okano, Teruo, Kanazawa, Hideko

RSC Advances 5 （ 89 ） 73217 - 73224 2015年

研究論文（学術雑誌）, 筆頭著者, 査読有り,  ISSN  2046-2069

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Temperature-Responsive Fluorescence Polymer Probes with Accurate Thermally Controlled Cellular Uptakes.

Yuki Hiruta, Mirai Shimamura, Minami Matsuura, Yutaro Maekawa, Takaaki Funatsu, Yuichi Suzuki, Eri Ayano, Teruo Okano and Hideko Kanazawa.

ACS Macro Letters 3   281-285 2014年03月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り

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Steric Hindrance Effects in Tripodal Ligands for Extraction and Back-extraction of Ag+.

Yuki Hiruta, Takafumi Watanabe, Etsuko Nakamura, Naoko Iwasawa, Hiroyasu Sato, Kensaku Hamada, Daniel Citterio, and Koji Suzuki.

RSC Advances 4   9791-9798 2014年01月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り

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Temperature-Responsive Chromatography Using a Functional Polymer Modified Stationary Phase with Molecular Recognition Sites

Sakata, Kazuki, Ohkubo, Kohei, Hiruta, Yuki, Ayano, Eri, Kanazawa, Hideko

KOBUNSHI RONBUNSHU 71 （ 7 ） 293 - 301 2014年

研究論文（学術雑誌）, 査読有り,  ISSN  0386-2186

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Li+-selective optodes - effect of fluoroionophore distribution in mesoporous silica thin films on Li+ response.

Yuki Hiruta, Chikai Sato, Youhei Takahashi, Kei Kubobuchi, Yushi Shichi, Daniel Citterio and Koji Suzuki.

RSC Advances 3   6499-6506 2013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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Highly Durable Double Sol-Gel Layer Ratiometric Fluorescent pH Optode Based on the Combination of Two Types of Quantum Dots and Absorbing pH Indicators.

Yuki Hiruta, Naoto Yoshizawa, Daniel Citterio, and Koji Suzuki.

Analytical Chemistry 84   10650-10656 2012年12月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り

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A Fast Response pH Optode Based on a Fluoroionophore Immobilized to a Mesoporous Silica Thin Film.

Yuki Hiruta, Yosuke Ando Daniel Citterio, and Koji Suzuki.

Analytical Sciences 26   297-301 2010年03月

研究論文（学術雑誌）, 共著, 筆頭著者, 査読有り

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Structural characteristics and optical properties of a series of solvatochromic fluorescent dyes displaying long-wavelength emission

Ando, Yosuke, Homma, Yuya, Hiruta, Yuki, Citterio, Daniel, Suzuki, Koji

Dyes and Pigments 83 （ 2 ） 198 - 206 2009年11月

研究論文（学術雑誌）, 査読有り,  ISSN  0143-7208

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A highly Li+-selective glass optode based on fluorescence radiometry.

Yosuke Ando, Yuki Hiruta, Daniel Citterio, and Koji Suzuki.

Analyst 134   2314-2319 2009年09月

研究論文（学術雑誌）, 共著, 査読有り

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