Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Takayuki Kawai Last modified date:2022.07.14

Associate Professor / Inorganic and Analytical Chemistry / Department of Chemistry / Faculty of Sciences

1. Emiko Okubo-Kurihara, Ahmed Ali, Mika Hiramoto, Yukio Kurihara, Yasmine Abouleila, Eman Muhammad Abdelazem, Takayuki Kawai, Yuko Makita, Mika Kawashima, Tsuyoshi Esaki, Hiroaki Shimada, Tetsuya Mori, Masami Yokota Hirai, Takumi Higaki, Seiichiro Hasezawa, Yoshihiro Shimizu, Tsutomu Masujima, Minami Matsui, Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis., Plant physiology, 10.1093/plphys/kiac093, 2022.03.
2. Takayuki Kawai, Yasuhiro Mihara, Makiko Morita, Masahiko Ohkubo, Taiji Asami, Tomonobu M. Watanabe, Quantitation of Cell Membrane Permeability of Cyclic Peptides by Single-Cell Cytoplasm Mass Spectrometry, Analytical Chemistry, 10.1021/acs.analchem.0c03901, 93, 7, 3370-3377, 2021.02.
3. Takayuki Kawai, Recent Advances in Trace Bioanalysis by Capillary Electrophoresis, Analytical Sciences, 10.2116/analsci.20sar12, 37, 1, 27-36, 2021.01.
4. Development of Ultra-sensitive CE/MS and Its Application to Single Cell Metabolome Analysis.
5. Trace omics analysis by ultra-sensitive capillary electrophoresis.
6. Takayuki Kawai, Nobutoshi Ota, Kaori Okada, Akiko Imasato, Yuri Owa, Makiko Morita, Misa Tada, Yo Tanaka, Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration, Analytical Chemistry, 10.1021/acs.analchem.9b01578, 91, 16, 10564-10572, 2019.08, Copyright © 2019 American Chemical Society. Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis-mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (∼10 μm) and tapered (5-10 μm) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triple-quadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation..
7. Yasmine Abouleila, Kaoru Onidani, Ahmed Ali, Hirokazu Shoji, Takayuki Kawai, Chwee Teck Lim, Vipin Kumar, Shinobu Okaya, Ken Kato, Eiso Hiyama, Toshio Yanagida, Tsutomu Masujima, Yoshihiro Shimizu, Kazufumi Honda, Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells, Cancer Science, 10.1111/cas.13915, 110, 2, 697-706, 2019.02, © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics-based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type..
8. Takayuki Kawai, Nobutoshi Ota, Akiko Imasato, Yoko Shirasaki, Koji Otsuka, Yo Tanaka, Profiling of N-linked glycans from 100 cells by capillary electrophoresis with large-volume dual preconcentration by isotachophoresis and stacking, Journal of Chromatography A, 10.1016/j.chroma.2018.06.034, 1565, 138-144, 2018.08, © 2018 Elsevier B.V. Glycan structure is changed in response with pathogenesis like cancer. Profiling of glycans from limited number of pathogenetic cells in an early-stage tissue is essential for discovering effective drugs. For analyzing tiny biological samples, we developed sensitive, high-resolution, and salt-tolerant method for analyzing trace level of N-linked glycans by coupling capillary electrophoresis (CE), laser-induced fluorescence (LIF) detection, and a new online sample preconcentration (OSP) method named “large-volume dual preconcentration by isotachophoresis and stacking (LDIS)” which is composed of two OSP methods, large-volume sample stacking (LVSS) and transient isotachophoresis (tITP). A typical LDIS-CE-LIF protocol was simple: a short-plug of leading electrolyte (LE) and large-volume sample solution were introduced to a capillary, followed by application of constant voltage. In the analysis of glucose ladder labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 10 mM sodium chloride as LE, up to 2300-fold sensitivity increase was achieved with higher resolution than those in normal CE. By applying pressure assist during preconcentration, both viscous gel electrolyte and salty matrix of up to 10 mM NaCl were acceptable. Finally, N-glycans from approximately 100 cells (HeLa, MCF7, and HepG2) were analyzed as the model of localized tumor cells. From 30 to 40 glycans were successfully detected with almost same profile of large-scale sample. N-glycan structure could be predicted by searching glucose-unit value via Glycobase database, indicating that HepG2 expressed more sialylated glycans and MCF-7 expressed less glycans respectively, comparing with HeLa cells. It suggests the potential of LDIS-CE-LIF for discovery of disease-specific N-linked glycans in microscale environment..
9. Amit V. Patel, Takayuki Kawai, Liping Wang, Stanislav S. Rubakhin, Jonathan V. Sweedler, Chiral Measurement of Aspartate and Glutamate in Single Neurons by Large-Volume Sample Stacking Capillary Electrophoresis, Analytical Chemistry, 10.1021/acs.analchem.7b03435, 89, 22, 12375-12382, 2017.11, © 2017 American Chemical Society. d-Amino acids (d-AAs) are endogenous molecules found throughout the metazoan, the functions of which remain poorly understood. Measurements of low abundance and heterogeneously distributed d-AAs in complex biological samples, such as cells and multicellular structures of the central nervous system (CNS), require the implementation of sensitive and selective analytical approaches. In order to measure the d- and l-forms of aspartate and glutamate, we developed and applied a stacking chiral capillary electrophoresis (CE) with laser-induced fluorescence detection method. The achieved online analyte preconcentration led to a 480-fold enhancement of detection sensitivity relative to capillary zone electrophoresis, without impacting separation resolution or analysis time. Additionally, the effects of inorganic ions on sample preconcentration and CE separation were evaluated. The approach enabled the relative quantification of d-aspartate and d-glutamate in individual neurons mechanically isolated from the CNS of the sea slug Aplysia californica, a well characterized neurobiological model. Levels of these structurally similar d-AAs were significantly different in subpopulations of cells collected from the investigated neuronal clusters..
10. Fumihiko Kitagawa, Tatsuya Ishiguro, Misaki Tateyama, Isoshi Nukatsuka, Kenji Sueyoshi, Takayuki Kawai, Koji Otsuka, Combination of large-volume sample stacking with an electroosmotic flow pump with field-amplified sample injection on cross-channel chips, Electrophoresis, 10.1002/elps.201700155, 38, 16, 2075-2080, 2017.08, © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed-polarity voltages on a cross-channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP-FASI. The applied pressure generated a counter-flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520-fold sensitivity increase was obtained in the LVSEP-FASI analysis of a standard dye, which was 33-times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100-fold sensitivity increase was achieved with the 75-μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d-leucine..
11. Fumihiko Kitagawa, Saeko Kinami, Yuuki Takegawa, Isoshi Nukatsuka, Kenji Sueyoshi, Takayuki Kawai, Koji Otsuka, On-line coupling of sample preconcentration by LVSEP with gel electrophoretic separation on T-channel chips, Electrophoresis, 10.1002/elps.201600184, 38, 2, 380-386, 2017.01, © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim To achieve an on-line coupling of the sample preconcentration by a large-volume sample stacking with an electroosmotic flow pump (LVSEP) with microchip gel electrophoresis (MCGE), a sample solution, a background solution for LVSEP and a sieving solution for MCGE were loaded in a T-form channel and three reservoirs on PDMS microchips. By utilizing the difference in the flow resistance of the two channels, a low-viscosity sample and a viscous polymer solution were easily introduced into the LVSEP and MCGE channels, respectively. Fluorescence imaging of the sequential LVSEP-MCGE processes clearly demonstrated that a faster stacking of anionic fluorescein and successive introduction into the MCGE channel can be carried out on the T-channel chip. To evaluate the preconcentration performance, a conventional MCZE analysis of fluorescein on the cross-channel chip was compared with LVSEP-MCGE on the short T-channel chip, and as a result that the value of sensitive enhancement factor (SEF) was estimated to be 370. The repeatability of the peak height was good with the RSD value of 3.2%, indicating the robustness of the enrichment performance. In the successive LVSEP-MCGE analysis of φX174/HaeIII digest, the DNA fragments were well enriched to a sharp peak in the LVSEP channel, and they were separated in the MCGE channel, whose electropherogram was well-resembled with that in the conventional MCGE. The values of SEF for the DNA fragments were calculated to be ranging from 74 to 108. Thus, the successive LVSEP-MCGE analysis was effective for both preconcentrating and separating DNA fragments..
12. Nobutoshi Ota, Yuri Owa, Takayuki Kawai, Yo Tanaka, Micro/nanoparticle separation via curved nano-gap device with enhanced size resolution, Journal of Chromatography A, 10.1016/j.chroma.2016.05.064, 1455, 172-177, 2016.07, © 2016 Elsevier B.V. Micro/nanoparticles are widely found in industry and biological field to play important roles and particle size distribution is an important factor to evaluate these particles. Nano-gap device has advantages in size determination for particles in diverse size and/or shape, but it has difficulty in practical use due to severe requirement on instrumental alignment to reproduce the gap profile and non-quantitative sample injection based on capillary action. To solve these problems, curved nano-gap device (CGD) was fabricated from two flat glass plates via a simple microfabrication process to gain enhanced size resolution, and pressure-driven liquid delivery system was coupled to CGD. The gap was precisely controlled by wet etching with hydrofluoric acid on a glass plate to obtain the depth of 35.5 ± 15.0 nm on average. CGD utilized glass deflection with 18.1 nm elevation/μm lateral distance that achieved practical size resolutions of 14.5 nm, which was 15.7% smaller than that of conventional linear nano-gap device. Using CGD, particles from 0.5 to 10 μm diameter were trapped and separated. The estimated sizes of the trapped particles matched the suggested values well. Cell sizes were also measured by CGD and the measured values matched with the values found by microscope observation. CGD acquired reproducible instrumental setup that resulted in robust analysis on size of micro/nanoparticles..
13. Amit V. Patel, Takayuki Kawai, Stanislav S. Rubakhin, Jonathan V. Sweedler, Free D-aspartate in nonmammalian animals: Detection, localization, metabolism, and function, D-Amino Acids: Physiology, Metabolism, and Application, 10.1007/978-4-431-56077-7_12, 173-197, 2016.01, © Springer Japan 2016. Many functions of amino acids, including protein synthesis, require that they be in the l-form. As a result, in most biological systems, the levels of free D-amino acids (DAAs) are enzymatically suppressed. However, the site-specific synthesis, accumulation, and release of DAAs do occur. In fact, the accumulation of DAAs in the nervous, exocrine, and endocrine systems suggests that they perform specific functions. The focus here is on the well-studied DAA, D-aspartate; we review the advancements in the analytical approaches used for its detection and characterization and discuss the role it plays in the structural and functional organization of numerous biological systems of nonmammalian animals. The view that D-Asp has specific functions is supported by a large body of experimental data showing its endogenous synthesis, accumulation, release, stimulation of follower cells, uptake, and enzymatic catabolism. A variety of biological models, each having distinct anatomies, morphologies, biochemistries, and behaviors, have been used to investigate the fundamental mechanisms of D-Asp involvement in the normal and pathological functioning of cells and organisms. Many physiological and behavioral effects induced by D-Asp have been documented, demonstrating it has neurotransmitter, hormonal, and neuromodulator roles. Similar to many classical neurotransmitters, D-Asp has physiological roles that are conserved throughout the evolutionary tree, with nearly all studied animals shown to possess and use D-Asp..
14. CE Analysis of Trace Level Biological Sample Using Online Sample Preconcentration Method.
15. T. Kawai, H. Moriguchi, Y. Tanaka, Simple valves on a pdms microchip bonded via patterned oxygen plasma, IEEE Transducers - 18th International Conference on Solid-State Sensors, Actuators and Microsystems, 10.1109/TRANSDUCERS.2015.7181292, 18, 1782-1785, 2015.08, © 2015 IEEE. The simplest form of microfluidic valve is proposed with detailed working mechanism. Conventional on-chip valves are requiring a multiple layers of microchip with complicated fluidic pattern and/or complicated external control system. In contrast, our developed valve can be fabricated in a bilayer microchip with a simple pattern, two channels and a wall separating them. Our valve fabrication was based on a plasma patterning method with a sacrificial aluminum layer, which shield the oxygen plasma activation only in the aimed position of the substrate. This approach is more robust than the conventional «plasma deactivation» approach based on the chemical patterning via micro-contact printing. For the valve regulation, no external control line is necessary but a weak pressure to the injection channel. Although our valve has no simpler structure than that of the hydrophobic valve which has ever been the simplest, our valve has a significant advantage in the repeatable utility. Due to its simplicity, moreover, long term injection more than 120 min was easily and precisely carried out. These highest simplicity and performance are quite suitable for the large integration and mass production of complicated lab-on-a-chip system for the application to biological research..
16. Takuya Kubo, Koichi Kanemori, Risa Kusumoto, Takayuki Kawai, Kenji Sueyoshi, Toyohiro Naito, Koji Otsuka, Simple and effective label-free capillary electrophoretic analysis of sugars by complexation using quinoline boronic acids, Analytical Chemistry, 10.1021/acs.analchem.5b00998, 87, 10, 5068-5073, 2015.05, © 2015 American Chemical Society. An effective separation and detection procedure for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at a wide range of pH conditions and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH conditions. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of the 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limits of detection for sorbitol and xylitol by the CE method were estimated at 15 and 27 μM, respectively..
17. Takayuki Kawai, Masato Watanabe, Kojiro Uetani, Yudai Fukushima, Kenji Sueyoshi, Takuya Kubo, Fumihiko Kitagawa, Hiroyuki Yano, Koji Otsuka, Hydrophilic interaction electrokinetic chromatography using bio-based nanofillers, Electrophoresis, 10.1002/elps.201300558, 35, 15, 2229-2236, 2014.08, Hydrophilic interaction (HI)-based separation like HILIC is effective for analyzing hydrophilic biological samples such as carbohydrates, peptides, and metabolites. To overcome the drawbacks of conventional HILIC such as large consumption of organic solvents and easy deterioration of the separation column, we developed HI electrokinetic chromatography (EKC) by employing bio-based nanomaterials as the hydrophilic pseudostationary phase. By mechanical/chemical treatments, cellulose, chitin, and chitosan were processed to 10-nm wide nanofibers/nanowhiskers (NFs/NWs), which are longer/shorter than 1000/200 nm, respectively. In HI-EKC of oligosaccharides using 0.001% uncharged cellulose NFs, strong interaction was observed for the large-size oligosaccharides with the retention factors (k) of up to 1.56, indicating a HILIC-mode interaction. In HI-EKC with 0.1% positively charged chitosan NFs, benzenedisulfonic acid, benzenesulfonic acid (BS), and p-hydroxy BS (HBS) had k values of 0.036, 0.018, and 0.018, respectively, suggesting that the ion-exchange interaction mainly occurred via sulfonate groups. Finally, HI-EKC was demonstrated using 0.05% chitin or chitosan NWs. In both cases using chitin and chitosan NWs, HBS showed much stronger interaction with k > 0.192 compared with BS with k < 0.070. It indicated structural difference between NFs and NWs affected the HI behavior in terms of both the ion-exchange and HILIC modes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim..
18. Takayuki Kawai, Masumi Ueda, Yudai Fukushima, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka, Toward 10000-fold sensitivity improvement of oligosaccharides in capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump combined with field-amplified sample injection, Electrophoresis, 10.1002/elps.201200615, 34, 16, 2303-2310, 2013.08, A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow pump (LVSEP) and field-amplified sample injection (FASI), was investigated in CE to achieve highly sensitive oligosaccharide analysis. In CE with LVSEP-FASI, analytes injected throughout the capillary were concentrated on the basis of LVSEP, followed by an electrokinetic introduction of concentrated analytes from the inlet vial by the FASI mechanism. After switching the inlet vial solution from the sample to running buffer, the concentrated analytes were then separated by CZE. In the present LVSEP-FASI-CZE, pressure was applied to the capillary inlet until the inlet vial solution was exchanged. The applied pressure generated a counterflow against the EOF. It kept the stacked sample zone within the capillary, minimizing loss of concentrated analytes. Fluorescein was first analyzed by LVSEP-FASI-CZE to optimize preconcentration condition. Up to 110000-fold sensitivity increase was obtained with 200 μL of sample, compared to normal CZE with sample injection of 0.3 psi for 3 s (ca. 1.7 nL). From the results, the pressure application improved the efficiency of the FASI-mode concentration significantly at total concentration time longer than 10 min. In the analysis of maltoheptaose, a 10000-fold sensitivity increase was achieved, which is the highest concentration efficiency ever reported in CE of oligosaccharides. The relative standard deviations of the detection time and peak height were 2.4 and 11%, respectively. In the analysis of glucose oligomer, up to 8600-fold sensitivity increases were achieved without reducing the separation performance of conventional CZE. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim..
19. Takayuki Kawai, Nahoko Naruishi, Hidenori Nagai, Yoshihide Tanaka, Yoshihisa Hagihara, Yasukazu Yoshida, Rotatable reagent cartridge for high-performance microvalve system on a centrifugal microfluidic device, Analytical Chemistry, 10.1021/ac400667e, 85, 14, 6587-6592, 2013.07, Recently, microfluidic lab-on-a-CD (LabCD) has attracted attentions of researchers for its potential for pumpless, compact, and chip-inclusive on-site bioassay. To control the fluids in the LabCD, microvalves such as capillary, hydrophobic, siphon, and sacrificial valves have been employed. However, no microvalve can regulate more than one channel. In a complicated bioassay with many sequential mixing, washing, and wasting steps, thus, an intricate fluidic network with many microchannels, microvalves, and reservoirs is required, which increases assay costs in terms of both system development and chip preparation. To address this issue, we developed a rotatable reagent cartridge (RRC), which was a column-shaped tank and has several rooms to store different reagents. By embedding and rotating the RRC in the LabCD with a simple mechanical force, only the reagent in the room connected to the following channel was injected. By regulating the angle of the RRC to the LabCD, conservation and ejection of each reagent could be switched. Our developed RRC had no air vent hole, which was achieved by the gas-permeable gap between the bottle and cap parts of the RRC. The RRC could inject 230 nL-10 μL of reagents with good recoveries more than 96%. Finally, an enzymatic assay of l-lactate was demonstrated, where the number of valves and reservoirs were well minimized, significantly simplifying the fluidic system and increasing the channel integratability. Well quantitative analyses of 0-100 μM l-lactate could easily be carried out with R2 > 0.999, indicating the practical utility of the RRC for microfluidic bioanalysis. © 2013 American Chemical Society..
20. Takayuki Kawai, Jun Ito, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka, Electrophoretic analysis of cations using large-volume sample stacking with an electroosmotic flow pump using capillaries coated with neutral and cationic polymers, Journal of Chromatography A, 10.1016/j.chroma.2012.09.077, 1267, 65-73, 2012.12, To realize the high-performance and simple-operation analysis of cationic compounds in capillary electrophoresis, we investigated large-volume sample stacking with an electroosmotic flow pump (LVSEP) using capillaries with hydrophilic and weakly cationic inner surface. Three capillary modification methods were employed: thermally passivated physical coating with polymer mixture of poly(vinyl alcohol) and poly(allylamine); covalent modification with random copolymer of acryl amide and 3-(methacryloylamino)propyltrimethylammonium chloride; easily preparable physical coating with dimethyldioctadecylammonium bromide and polyoxyethylene stearate. In these capillaries, the electroosmotic flow (EOF) was well suppressed in the high ionic strength (I) electrolyte under the acidic and basic pH, whereas the EOF was enhanced in the low I electrolyte, indicating a suitable EOF property for the rapid LVSEP and following separation. In the LVSEP-capillary zone electrophoresis (CZE) analyses of benzylamine and 1-naphthylethylamine, up to 550-fold sensitivity increases were successfully obtained in the three capillaries without significantly reducing the repeatability and resolution. LVSEP-cyclodextrin-modified CZE of chlorpheniramine and brompheniramine was also carried out, resulting in up to 380-fold sensitivity enhancement with keeping the baseline separation for the enantiomers. Finally, we performed the LVSEP-CZE analysis of basic proteins, where up to 100-fold sensitivity increases were achieved, but a peak broadening was observed due to the sample adsorption in the low I sample matrix. © 2012 Elsevier B.V..
21. Takayuki Kawai, Hiroshi Koino, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka, Highly sensitive chiral analysis in capillary electrophoresis with large-volume sample stacking with an electroosmotic flow pump, Journal of Chromatography A, 10.1016/j.chroma.2012.02.001, 1246, 28-34, 2012.07, To improve the sensitivity in chiral analysis by capillary electrophoresis without loss of optical resolution, application of large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. Effects of the addition of cyclodextrin (CD) into a running solution on the LVSEP preconcentration was theoretically studied, where the preconcentration efficiency and effective separation length would be slightly increased if the effective electrophoretic velocity (v ep,eff,BGS) of the analytes was decreased by interacting with CD. In LVSEP-CD-modified capillary zone electrophoresis (CDCZE) and LVSEP-CD electrokinetic chromatography with reduced v ep,eff,BGS, up to 1000-fold sensitivity increases were achieved with almost no loss of resolution. In LVSEP-CD-modified micellar electrokinetic chromatography of amino acids with increased v ep,eff,BGS, a 1300-fold sensitivity increase was achieved without much loss of resolution, indicating the versatile applicability of LVSEP to many separation modes. An enantio-excess (EE) assay was also carried out in LVSEP-CDCZE, resulting in successful analyses of up to 99.6% EE. Finally, we analyzed ibuprofen in urine by desalting with a C 18 solid-phase extraction column. As a typical result, 250ppb ibuprofen was well concentrated and optically resolved with 84.0-86.6% recovery in LVSEP-CDCZE, indicating the applicability of LVSEP to real samples containing a large amount of unnecessary background salts. © 2012 Elsevier B.V..
22. Takayuki Kawai, Masato Watanabe, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka, Highly sensitive oligosaccharide analysis in capillary electrophoresis using large-volume sample stacking with an electroosmotic flow pump, Journal of Chromatography A, 10.1016/j.chroma.2011.09.032, 1232, 52-58, 2012.04, To obtain high sensitivity in capillary electrophoresis of oligosaccharide without reducing the high resolution with an easy experimental procedure, large-volume sample stacking with an electroosmotic flow pump (LVSEP) was investigated. As a fundamental study, effect of the conductivity of a sample solution in LVSEP was examined. It was revealed that LVSEP was successfully carried out even in using a sample solution with the ionic strength of 150μM and the conductivity ratio of 20, indicating a good applicability of LVSEP to the analysis of real samples containing salts. When glucose oligomer was analyzed as a model sample in LVSEP-capillary zone electrophoresis (CZE), all peaks were well resolved with decreasing only 5% of the peak-to-peak distance, which suggested 95% of the whole capillary could be used for the effective separation. In the analysis of maltoheptaose, a good calibration line with correlation coefficient of 0.9995 was obtained. The limit of detection was estimated as 2pM, which was 500-fold lower than that in the conventional CZE. N-linked glycans released from three glycoproteins, bovine ribonuclease B, bovine fetuin, and human α 1-acid glycoprotein were also analyzed by LVSEP-CZE. By the sample purification with a gel filtration column, further sample dilution to reduce the sample conductivity for LVSEP was not needed. All glycan samples were well concentrated and separated with up to a 770-fold sensitivity increase. The run-to-run repeatabilities of the migration time, peak height, and peak area were good with relative standard deviations of 0.1-1.3%, 1.2-1.7%, and 2.8-4.9%, respectively. © 2011 Elsevier B.V..
23. Kenji Sueyoshi, Kota Hashiba, Takayuki Kawai, Fumihiko Kitagawa, Koji Otsuka, Hydrophobic labeling of amino acids: Transient trapping-capillary/microchip electrophoresis, Electrophoresis, 10.1002/elps.201000567, 32, 10, 1233-1240, 2011.05, Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim..
24. Takayuki Kawai, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka, Microchip electrophoresis of oligosaccharides using large-volume sample stacking with an electroosmotic flow pump in a single channel, Analytical Chemistry, 10.1021/ac1008145, 82, 15, 6504-6511, 2010.08, The applicability of an online preconcentration technique, large-volume sample stacking with an electroosmotic flow pump (LVSEP), to microchip zone electrophoresis (MCZE) for the analysis of oligosaccharides was investigated. Since the sample stacking and separation proceeded continuously without polarity switching in LVSEP, a single "straight" channel microchip could be employed. In the MCZE analysis of oligosaccharides, sample adsorption onto the channel surface should be suppressed, so the straight microchannel was modified with poly(vinyl alcohol) (PVA). So far, the mechanism of LVSEP in the polymer-coated capillary or microchannel has not been reported, and thus, the LVSEP process in the PVA-coated channel was investigated by fluorescence imaging. Although it is well-known that the PVA coating can suppress the electroosmotic flow (EOF), an enhanced EOF with a mobility of 4.4×10 -4 cm2/(Vs) was observed in a low ionic strength sample solution. It was revealed that such temporarily enhanced EOF in the sample zone worked as the driving force to remove the sample matrix in LVSEP. To evaluate the analytical performance of LVSEP-MCZE, oligosaccharides were analyzed in the PVA-coated straight channel. As a result, both the glucose ladder and oligosaccharides obtained from bovine ribonuclease B were well enriched and separated with up to 2200-2900-fold sensitivity enhancement compared to those in a conventional MCZE analysis. The run-to-run repeatabilities of the migration time and peak height were good with relative standard deviations of 1.1% and 7.2%, respectively, which were better than those of normal MCZE. By applying the LVSEP technique to MCZE, a complicated voltage program for fluidic control could be simplified from four channels for two steps to two channels for one step. © 2010 American Chemical Society..
25. Atsushi Yokoyama, Bin-Hai Shi, Takayuki Kawai, Hiroaki Konishi, Ryota Andoh, Hiroyuki Tachikawa, Sayoko Ihara, Yasuhisa Fukui, Muc4 is required for activation of ErbB2 in signet ring carcinoma cell lines, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2007.01.133, 355, 1, 200-203, 2007.03, Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinorna cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GaINac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of Erb132 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis. (c) 2007 Elsevier Inc. All rights reserved..