Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Noritada Kaji Last modified date:2021.06.23

Professor / Department of Applied Chemistry / Faculty of Engineering


Papers
1. H. Takahashi, T. Yasui, H. Kashida, K. Makino, K. Shinjo, Q. Liu, T. Shimada, S. Rahong, N. Kaji, H. Asanuma, Y. Baba, Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA, Nanotechnology, 32, 255301, 2021.04.
2. S. Ryuzaki, T. Yasui, M. Tsutsui, K. Yokota, Y. Komoto, P. Paisrisarn, N. Kaji, D. Ito, K. Tamada, T. Ochiya, M. Taniguchi, Y. Baba, Rapid discrimination of extracellular vesicles by shape distribution analysis, Analytical Chemistry, 93, 7037-7044, 2021.04.
3. A. M. Zeid, J. J. M. Nasr, F. Belal, M. Walash, N. Kaji, Y. Baba, Microfluidic fast chiral separation of baclofen and phenylalanine enantiomers based on cyclodextrin-electrokinetic chromatography, Microchemical Journal, 160, 105770, 2021.06.
4. A. M. Zeid, J. J. M. Nasr, F. Belal, M. I. Walash, Y. Baba, N. Kaji, Determination of three antiepileptic drugs in pharmaceutical formulations using microfluidic chips coupled with light-emitting diode induced fluorescence detection, Spectrochimica Acta. Part A: Molecular and Biomolecular Spectroscopy, 246, 119021, 2021.02.
5. N. Yogo, T. Hase, T. Kasama, K. Nishiyama, N. Ozawa, T. Hatta, H. Shibata, M. Sato, K. Komeda, N. Kawabe, K. Matsuoka, T. F. Chen-Yoshikawa, N. Kaji, M. Tokeshi, Y. Baba, Y. Hasegawa, Development of an immuno-wall device for the rapid and sensitive detection of egfr mutations in tumor tissues resected from lung cancer patients, PloS One, 15, e0241422, 2020.11.
6. Hiroshi Suzuki, Kentaro Fujiyoshi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System, Analytical chemistry, 10.1021/acs.analchem.0c01417, 92, 13, 9132-9137, 2020.07, Conformational transitions from secondary (e.g., B- to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several single-molecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil-globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression..
7. Taisuke Shimada, Takao Yasui, Akihiro Yonese, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Mechanical rupture-based antibacterial and cell-compatible ZnO/SiO2 nanowire structures formed by bottom-up approaches, Micromachines, 10.3390/mi11060610, 11, 6, 2020.06, There are growing interests in mechanical rupture-based antibacterial surfaces with nanostructures that have little toxicity to cells around the surfaces; however, current surfaces are fabricated via top-down nanotechnologies, which presents difficulties to apply for bio-surfaces with hierarchal three-dimensional structures. Herein, we developed ZnO/SiO2 nanowire structures by using bottom-up approaches and demonstrated to show mechanical rupture-based antibacterial activity and compatibility with human cells. When Escherichia coli were cultured on the surface for 24 h, over 99% of the bacteria were inactivated, while more than 80% of HeLa cells that were cultured on the surface for 24 h were still alive. This is the first demonstration of mechanical rupture-based bacterial rupture via the hydrothermally synthesized nanowire structures with antibacterial activity and cell compatibility..
8. Taiki Suzuki, Noritada Kaji, Hirotoshi Yasaki, Takao Yasui, Yoshinobu Baba, Mechanical Low-Pass Filtering of Cells for Detection of Circulating Tumor Cells in Whole Blood, Analytical chemistry, 10.1021/acs.analchem.9b03939, 92, 3, 2483-2491, 2020.02, The detection of circulating tumor cells (CTCs) from liquid biopsies using microfluidic devices is attracting a considerable amount of attention as a new, less-invasive cancer diagnostic and prognostic method. One of the drawbacks of the existing antibody-based detection systems is the false negatives for epithelial cell adhesion molecule detection of CTCs. Here we report a mechanical low-pass filtering technique based on a microfluidic constriction and electrical current sensing system for the novel CTC detection in whole blood without any specific antigen-antibody interaction or biochemical modification of the cell surface. The mechanical response of model cells of CTCs, such as HeLa, A549, and MDA-MB-231 cells, clearly demonstrated different behaviors from that of Jurkat cells, a human T-lymphocyte cell line, when they passed through the 6-μm wide constriction channel. A 6-μm wide constriction channel was determined as the optimum size to identify CTCs in whole blood with an accuracy greater than 95% in tens of milliseconds. The mechanical filtering of cells at a single cell level was achieved from whole blood without any pretreatment (e.g., dilution of lysing) and prelabeling (e.g., fluorophores or antibodies)..
9. Mamiko Sano, Noritada Kaji, Amy C. Rowat, Hirotoshi Yasaki, Long Shao, Hidefumi Odaka, Takao Yasui, Tetsuya Higashiyama, Yoshinobu Baba, Microfluidic Mechanotyping of a Single Cell with Two Consecutive Constrictions of Different Sizes and an Electrical Detection System, Analytical chemistry, 10.1021/acs.analchem.9b02818, 91, 20, 12890-12899, 2019.10, The mechanical properties of a cell, which include parameters such as elasticity, inner pressure, and tensile strength, are extremely important because changes in these properties are indicative of diseases ranging from diabetes to malignant transformation. Considering the heterogeneity within a population of cancer cells, a robust measurement system at the single cell level is required for research and in clinical purposes. In this study, a potential microfluidic device for high-throughput and practical mechanotyping were developed to investigate the deformability and sizes of cells through a single run. This mechanotyping device consisted of two different sizes of consecutive constrictions in a microchannel and measured the size of cells and related deformability during transit. Cell deformability was evaluated based on the transit and on the effects of cytoskeleton-affecting drugs, which were detected within 50 ms. The mechanotyping device was able to also measure a cell cycle without the use of fluorescent or protein tags..
10. Maged F. Serag, Aimaiti Aikeremu, Ryoko Tsukamoto, Hubert Piwoński, Maram Abadi, Noritada Kaji, Jason R. Dwyer, Yoshinobu Baba, Satoshi Habuchi, Geometry-Based Self-Assembly of Histone-DNA Nanostructures at Single-Nucleotide Resolution, ACS nano, 10.1021/acsnano.9b03259, 13, 7, 8155-8168, 2019.07, Histones are basic protein monomers capable of interacting with DNA, providing the mechanism of DNA compaction inside the cell nucleus. The well-ordered assembly process of histone and DNA is a potential candidate as the approach for building DNA-protein nanostructures. Here, utilizing the sequence-independent histone-DNA interaction, we present an approach to self-assemble histones and single-stranded DNA (ssDNA) to form well-defined histone-DNA (sHD) nanoparticles and their multidimensional cross-linked complexes (cHD). By using various molecular biology and microscopy techniques, we elucidate the structure of these complexes, and we show that they are formed at carefully controlled conditions of temperature, ionic strength, concentration, and incubation time. We also demonstrate using a set of ssDNA molecular rulers and a geometric accommodation model that the assembly of sHD and cHD particles proceeds with precise geometry so that the number of ssDNA in these particles can be programmed by the length of ssDNA. We further show that the formation of cHD amplifies the effect of the length of ssDNA on the self-assembly, allowing for distinguishing ssDNA of different lengths at single nucleotide resolution. We envision that our geometry-directed approach of self-assembling histone-DNA nanostructures and the fundamental insights can serve as a structural platform to advance building precisely ordered DNA-protein nanostructures..
11. Yuya Hattori, Taisuke Shimada, Takao Yasui, Noritada Kaji, Yoshinobu Baba, Micro- and Nanopillar Chips for Continuous Separation of Extracellular Vesicles, Analytical chemistry, 10.1021/acs.analchem.8b05538, 91, 10, 6514-6521, 2019.05, Micro- and nanopillar chips are widely used to separate and enrich biomolecules, such as DNA, RNA, protein, and cells, as an analytical technique and to provide a confined nanospace for polymer science analyses. Herein, we demonstrated a continuous accurate and precise separation technique for extracellular vesicles (EVs), nanometer-sized vesicles (typically 50-200 nm) currently recognized as novel biomarkers present in biofluids, based on the principle of electroosmotic flow-driven deterministic lateral displacement in micro- and nanopillar array chips. Notably, the easy-to-operate flow control afforded by electroosmotic flow allowed nanoparticles 50-500 nm in size, including EVs, to be precisely separated and enriched in a continuous manner. By observation of the flow behavior of nanoparticles, we found that electroosmotic flow velocity in the nanopillar arrays did not solely depend on counterion mobility on the surface of nanopillar chips, but rather showed a parabolic flow profile. This hydrodynamic pressure-free and easy-to-use separation and enrichment technique, which requires only electrode insertion into the reservoirs and electric field application, may thus serve as a promising technique for future precise and accurate EV analysis, reflecting both size and composition for research and potential clinical diagnostic applications..
12. Keine Nishiyama, Kanako Sugiura, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Development of a microdevice for facile analysis of theophylline in whole blood by a cloned enzyme donor immunoassay, Lab on a Chip, 10.1039/c8lc01105b, 19, 2, 233-240, 2019.01, We have developed a microdevice for therapeutic drug monitoring. In this device, dispensing of sample and reagent was accomplished by simple manual operation of a syringe. Moreover, for a simple and rapid measurement, we used cloned enzyme donor immunoassay as a detection principle. These features and the reagent that is enclosed in microdevice beforehand make it possible to complete the facile analysis. In this paper, our model analyte was 1,3-dimethylxanthine (theophylline), a kind of bronchodilator. The fluorescence measurement of theophylline in whole blood was achieved with the limit of detection of 0.73 μg mL−1. This microdevice provides rapid analysis (4 min), requires only a small volume of sample (2 μL) and features simple operation; hence, it is readily applicable to point of care testing..
13. Takao Yasui, Takeshi Yanagida, Taisuke Shimada, Kohei Otsuka, Masaki Takeuchi, Kazuki Nagashima, Sakon Rahong, Toyohiro Naito, Daiki Takeshita, Akihiro Yonese, Ryo Magofuku, Zetao Zhu, Noritada Kaji, Masaki Kanai, Tomoji Kawai, Yoshinobu Baba, Engineering Nanowire-Mediated Cell Lysis for Microbial Cell Identification, ACS Nano, 10.1021/acsnano.8b08959, 2019.01, Researchers have demonstrated great promise for inorganic nanowire use in analyzing cells or intracellular components. Although a stealth effect of nanowires toward cell surfaces allows preservation of the living intact cells when analyzing cells, as a completely opposite approach, the applicability to analyze intracellular components through disrupting cells is also central to understanding cellular information. However, the reported lysis strategy is insufficient for microbial cell lysis due to the cell robustness and wrong approach taken so far (i.e., nanowire penetration into a cell membrane). Here we propose a nanowire-mediated lysis method for microbial cells by introducing the rupture approach initiated by cell membrane stretching; in other words, the nanowires do not penetrate the membrane, but rather they break the membrane between the nanowires. Entangling cells with the bacteria-compatible and flexible nanowires and membrane stretching of the entangled cells, induced by the shear force, play important roles for the nanowire-mediated lysis to Gram-positive and Gram-negative bacteria and yeast cells. Additionally, the nanowire-mediated lysis is readily compatible with the loop-mediated isothermal amplification (LAMP) method because the lysis is triggered by simply introducing the microbial cells. We show that an integration of the nanowire-mediated lysis with LAMP provides a means for a simple, rapid, one-step identification assay (just introducing a premixed solution into a device), resulting in visual chromatic identification of microbial cells. This approach allows researchers to develop a microfluidic analytical platform not only for microbial cell identification including drug- and heat-resistance cells but also for on-site detection without any contamination..
14. Hiroshi Yukawa, Kaoru Suzuki, Keita Aoki, Tomoko Arimoto, Takao Yasui, Noritada Kaji, Tetsuya Ishikawa, Takahiro Ochiya, Yoshinobu Baba, Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells, Scientific Reports, 10.1038/s41598-018-24563-0, 8, 1, 2018.12, Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment..
15. Hirotoshi Yasaki, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, A real-time simultaneous measurement on a microfluidic device for individual bacteria discrimination, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2018.01.079, 260, 746-752, 2018.05, Rapid detection of pathogenic bacteria is one of the important social issues for preventing and identifying cause of problems affecting human health. Ionic current sensing through pores has shown the ability to electrically measure bacteria. However, there is an inherent limitation to discriminate chemical characteristics of bacteria for existing ionic current sensing methods Here we propose a real-time simultaneous measurement method, which combines ionic current sensing and fluorescence observation on a microfluidic device. Our method can detect not only the size of individual bacteria passing through a micropore but also observe their stainability based on chemical properties of bacterial cell surface within 300 ms. We succeeded in discriminating each bacterium in a solution mixture including contaminant particles by combining highly accurate ionic current sensing which can detect a size difference of 70 nm, and fluorescence observation which can discriminate internal structures such as those that define bacteria as gram-positive or gram-negative. Our method can be applied to not only on-site bacteria detection but also to screening technology..
16. Mamiko Sano, Noritada Kaji, Qiong Wu, Toyohiro Naito, Takao Yasui, Masateru Taniguchi, Tomoji Kawai, Yoshinobu Baba, Quantitative evaluation of dielectric breakdown of silicon micro- and nanofluidic devices for electrophoretic transport of a single DNA molecule, Micromachines, 10.3390/mi9040180, 9, 4, 2018.04, In the present study, we quantitatively evaluated dielectric breakdown in silicon-based micro- and nanofluidic devices under practical electrophoretic conditions by changing the thickness of the insulating layer. At higher buffer concentration, a silicon nanofluidic device with a 100 nm or 250 nm silicon dioxide layer tolerated dielectric breakdown up to ca. 10 V/cm, thereby allowing successful electrophoretic migration of a single DNA molecule through a nanochannel. The observed DNA migration behavior suggested that parameters, such as thickness of the insulating layer, tolerance of dielectric breakdown, and bonding status of silicon and glass substrate, should be optimized to achieve successful electrophoretic transport of a DNA molecule through a nanopore for nanopore-based DNA sequencing applications..
17. Hirotoshi Yasaki, Taisuke Shimada, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Robust Ionic Current Sensor for Bacterial Cell Size Detection, ACS Sensors, 10.1021/acssensors.8b00045, 3, 3, 574-579, 2018.03, Ionic current sensing methods are useful tools for detecting sub- to several-micron scale particles such as bacteria. However, conventional commercially available ionic current sensing devices are not suitable for on-site measurement use because of inherent limitations on their robustness. Here, we proposed a portable robust ionic current sensor (Robust-ICS) using a bridge circuit that offers a high signal-to-noise (S/N) ratio by suppressing background current. Because the Robust-ICS can tolerate increased noise in current sensing, a simple, lightweight electromagnetic shield can be used and measurements under large electromagnetic noise conditions can be made. The weight of the device was lowered below 4 kg and outdoor particle detection measurements were completed successfully. Accuracy of size detection of Staphylococcus aureus (S. aureus) was equivalent to that obtained by SEM imaging..
18. Hirotoshi Yasaki, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Effect of channel geometry on ionic current signal of a bridge circuit based microfluidic channel, Chemistry Letters, 10.1246/cl.171139, 47, 3, 350-353, 2018.01, Bridge circuit based ionic current sensing in a microfluidic channel has attracted attention as a highly sensitive analytical method for bio-related molecules and particles. However, channel geometry which greatly influences the detected ionic current has not been investigated. Here, we investigate experimentally and theoretically the effect of differences in the microfluidic channel geometry on shapes and amplitude of signals in ionic current sensing. Our results clarify the geometrical effect of the channel in the bridge circuit based ionic current sensing method..
19. Taisuke Shimada, Takao Yasui, Asami Yokoyama, Tatsuro Goda, Mitsuo Hara, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Yuji Miyahara, Tomoji Kawai, Yoshinobu Baba, Biomolecular recognition on nanowire surfaces modified by the self-assembled monolayer, Lab on a Chip, 10.1039/c8lc00438b, 18, 21, 3225-3229, 2018.01, Molecular recognition is one of the key factors in designing biosensors due to which nanowires functionalized with molecular recognition have attracted a lot of attention as promising candidates for nanostructures embedded in biosensors. However, the difficulty in real-world applications with analytical specificity is that molecular recognition on nanowires mainly depends on antibody modification with multistep modification procedures. Furthermore, the antibody modification suffers from nonspecific adsorption of undesired proteins in body fluid on the nanowires, which causes false responses and lowers sensitivity. Herein, we propose biomolecular recognition using surface-modified nanowires via thiolated 2-methacryloxyethyl phosphorylcholine (MPC-SH). MPC-SH enables self-assembled monolayer (SAM) modification, which contributes to the reduction of nonspecific adsorption of biomolecules onto the nanowires, and the specific capture of a target protein is attained in the presence of calcium ions. Our concept demonstrates the recognition of the biomarker protein on nanowire surfaces modified by MPC-SH SAM with a single step modification procedure..
20. Abdallah M. Zeid, Noritada Kaji, Jenny Jeehan M. Nasr, Fathalla Belal, Mohamed I. Walash, Yoshinobu Baba, Determination of baclofen and vigabatrin by microchip electrophoresis with fluorescence detection
Application of field-enhanced sample stacking and dynamic pH junction, New Journal of Chemistry, 10.1039/c8nj00829a, 42, 12, 9965-9974, 2018.01, A simple, rapid and sensitive microchip electrophoretic (MCE) method with fluorescence detection is described for the simultaneous determination of two GABA analogue drugs, baclofen (BCN) and vigabatrin (VGN). Pre-microchip derivatization of both analytes with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was performed in a basic borate buffer. The NBD-fluorescent derivatives of the studied drugs (λex/em 470/540) were baseline separated in a dynamically-coated poly(methyl methacrylate) microfluidic channel within 120 s using a 40 mM borate buffer containing 0.4% methylcellulose as the background solution. The ability of methylcellulose to form a network sieve of small pore size allowed the labelled analytes to be separated efficiently according to their molecular size variations with a resolution factor equal to 7.8 and a number of theoretical plates of more than 50 0000 per meter. The MCE method was applied to assay BCN and VGN in tablets using 6-aminohexanoic acid as an internal standard. The method sensitivity was enhanced by application of combined stacking and a dynamic pH junction. The method was applied to assay BCN in human plasma and human urine samples with a detection limit lower than 0.3 ng mL-1 and mean extraction recoveries of more than 95% (% RSD < 7) after protein precipitation with methanol..
21. Taisuke Shimada, Hirotoshi Yasaki, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, PM2.5 Particle detection in a microfluidic device by using ionic current sensing, Analytical Sciences, 10.2116/analsci.18C018, 34, 12, 1347-1349, 2018.01, We have demonstrated a PM2.5 analysis method that adds information on the number concentration and size by using microfluidic-based ionic current sensing with a bridge circuit. The bridge circuit allows for suppression of the background current and the detection of small PM2.5 particles, even if a relatively large micropore is used. This is the first demonstration of the detection of PM2.5 particles via ionic current sensing; our method enables analyses of both the number concentration and size..
22. T. Suzuki, N. Kaji, H. Yasaki, T. Yasui, Y. Baba, Cell deformability measurement device for labeled-free cancer cells discriminating using ionic current detection, 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, 347-348, 2018.01, In this study, we have developed ionic current detection device that can discriminate cells by measurement of cell deformability for detecting circulating tumor cells (CTC). We successfully discriminated CTC model from leukocyte model without any fluorescent labels by using the device. Moreover, we could detect relatively small CTCs compared to blood cells, which are difficult to detect by conventional filter devices. This device is expected as a new cancer diagnostic device that is simple and highly sensitive..
23. Hiromi Takahashi, Takao Yasui, Keiko Shinjo, Noritada Kaji, Akimitsu Okamoto, Yoshinobu Baba, Microheater-nanowires device for detection of cell-free circulating methylated DNA, 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, 1886-1888, 2018.01, Detecting of cell-free circulating methylated DNA could be a promising method for early diagnosis of cancer. Here we have developed the microheater - zinc oxide nanowires (ZnO-NWs) structures embedded in the microchannel device for capturing DNA via electrostatic interaction. We found that 60% of DNA could be captured on ZnO-NWs. Additionally, the microheater, which constantly enables to generate heat, can denature double stranded DNA (dsDNA) to single stranded DNA (ssDNA). Finally, ICON Probe, which is an artificial ssDNA probe for DNA methylation, was introduced inside a device to bind to ssDNA for DNA methylation detection..
24. Taisuke Shimada, Hirotoshi Yasaki, Takao Yasui, Akihide Hibara, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, PM2.5 analysis in liquid phase via water film-based collection and microfluidics-based electrical detection, 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, 270-271, 2018.01, Toward evaluation of health risk of particulate matter with size less than or equal to 2.5 µm (PM2.5), we propose a methodology for realizing the PM2.5 analysis in liquid phase via water film-based collection and microfluidics-based electrical detection. A hydrophilic nanowire surface was utilized in the collection for forming a water film, which enabled continuous collection of air-floating PM2.5 into liquid phase and followed-by detection. The collected PM2.5 was electrically detected and analyzed via ionic current sensing with a micropore. We believe proposed PM2.5 analysis methodology in liquid phase would allow to realize health risk evaluation and reduce health damage..
25. Makusu Tsutsui, Takeshi Yoshida, Kazumichi Yokota, Hirotoshi Yasaki, Takao Yasui, Akihide Arima, Wataru Tonomura, Kazuki Nagashima, Takeshi Yanagida, Noritada Kaji, Masateru Taniguchi, Takashi Washio, Yoshinobu Baba, Tomoji Kawai, Discriminating single-bacterial shape using low-aspect-ratio pores, Scientific Reports, 10.1038/s41598-017-17443-6, 7, 1, 2017.12, Conventional concepts of resistive pulse analysis is to discriminate particles in liquid by the difference in their size through comparing the amount of ionic current blockage. In sharp contrast, we herein report a proof-of-concept demonstration of the shape sensing capability of solid-state pore sensors by leveraging the synergy between nanopore technology and machine learning. We found ionic current spikes of similar patterns for two bacteria reflecting the closely resembled morphology and size in an ultra-low thickness-to-diameter aspect-ratio pore. We examined the feasibility of a machine learning strategy to pattern-analyse the sub-nanoampere corrugations in each ionic current waveform and identify characteristic electrical signatures signifying nanoscopic differences in the microbial shape, thereby demonstrating discrimination of single-bacterial cells with accuracy up to 90%. This data-analytics-driven microporescopy capability opens new applications of resistive pulse analyses for screening viruses and bacteria by their unique morphologies at a single-particle level..
26. Takao Yasui, Takeshi Yanagida, Satoru Ito, Yuki Konakade, Daiki Takeshita, Tsuyoshi Naganawa, Kazuki Nagashima, Taisuke Shimada, Noritada Kaji, Yuta Nakamura, Ivan Adiyasa Thiodorus, Yong He, Sakon Rahong, Masaki Kanai, Hiroshi Yukawa, Takahiro Ochiya, Tomoji Kawai, Yoshinobu Baba, Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires, Science advances, 10.1126/sciadv.1701133, 3, 12, 2017.12, Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer..
27. Masatoshi Maeki, Yuka Fujishima, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi, Understanding the formation mechanism of lipid nanoparticles in microfluidic devices with chaotic micromixers, PLoS One, 10.1371/journal.pone.0187962, 12, 11, 2017.11, Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60–80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15–25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment..
28. Hirotoshi Yasaki, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Substantial Expansion of Detectable Size Range in Ionic Current Sensing through Pores by Using a Microfluidic Bridge Circuit, Journal of the American Chemical Society, 10.1021/jacs.7b06440, 139, 40, 14137-14142, 2017.10, Measuring ionic currents passing through nano- or micropores has shown great promise for the electrical discrimination of various biomolecules, cells, bacteria, and viruses. However, conventional measurements have shown there is an inherent limitation to the detectable particle volume (1% of the pore volume), which critically hinders applications to real mixtures of biomolecule samples with a wide size range of suspended particles. Here we propose a rational methodology that can detect samples with the detectable particle volume of 0.01% of the pore volume by measuring a transient current generated from the potential differences in a microfluidic bridge circuit. Our method substantially suppresses the background ionic current from the μA level to the pA level, which essentially lowers the detectable particle volume limit even for relatively large pore structures. Indeed, utilizing a microscale long pore structure (volume of 5.6 × 104 aL; height and width of 2.0 × 2.0 μm; length of 14 μm), we successfully detected various samples including polystyrene nanoparticles (volume: 4 aL), bacteria, cancer cells, and DNA molecules. Our method will expand the applicability of ionic current sensing systems for various mixed biomolecule samples with a wide size range, which have been difficult to measure by previously existing pore technologies..
29. Ivan Adiyasa Thiodorus, Noritada Kaji, Takao Yasui, Yoshinobu Baba, A zinc oxide nanowires device for extracellular vesicles isolation and purification, 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017, 886-887, 2017.10, In this study, we have developed extracellular vesicles (EVs) isolation and purification using a ZnO nanowires microfluidic device with the principle of ion exchange chromatography. We had successfully purify EVs in large concentration without any chemical or physical damage on EVs membrane..
30. N. Kaji, Y. Hattori, H. Yasaki, T. Yasui, Y. Baba, Extracellular vesicels separation by electroosmotic flow-driven deterministic lateral displacement in nanopillar chips, 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017, 904-905, 2017.10, Here we first demonstrate extracellular vesicles (EVs) separation by electroosmotic flow (EOF)-driven deterministic lateral displacement (DLD) in a nanopillar array. The proposed EOF-based separation technique solved the issue associated with high input pressure more than a few hundred kPa to drive the solution in the nanopillar array and offered an easy-to-use and high-throughput DLD separation technique for nanometer-scale objects..
31. N. Kaji, S. Ito, H. Yasaki, T. Yasui, H. Yukawa, Y. Baba, Millisecond mechanophenotyping of stem cells by electrical detection in microfluidic constrictions, 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017, 832-833, 2017.10, A microfluidic device based on electrical detection for high-throughput and practical mechanophenotyping was developed to investigate the deformability and sizes of cells in a single run. Deformability changes associated with passage of adipose tissue-derived stem cells (ASCs) were successfully detected by the device without any chemical or biological modification..
32. Tsuyoshi Naganawa, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Nanowires enabling cancer diagnosis from 1 mL urine, 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017, 1271-1272, 2017.10, We have far developed the device with ZnO nanowires for in-situ miRNA collection from 1 mL urine. Here, we collected 48 miRNA samples from 24 healthy subjects and 24 cancer subjects. And then, we conducted two kinds of analisis: heatmap and AUC (Area Under the Curve) analysis. After the analysis, we could show the results that miRNA types are different between before and after cancer formation, and that some biomarker candidates were identified. This methodology will be expected to be a pioneer for the new cancer diagnosis from non-invasive miRNA analysis..
33. Taisuke Shimada, Takao Yasui, Akihide Hibara, Hirotoshi Yasaki, Takeshi Yanagida, Mitsuo Hara, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, PM2.5 collection into aqueous solution via hydrophilic oxide nanowire surface, 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017, 1433-1434, 2017.10, For collection of particulate matters with the size less than or equal to 2.5 μm (PM2.5) into aqueous solutions, we propose a methodology for realizing the PM2.5 collection into aqueous solution via hydrophilic surface of oxide nanowires. The developed surface allowed to form a water film over 300 days, and to collect PM2.5 into a water film from air and recover the solution for followed-by PM2.5 analysis, continuously. This device would be a new platform to collect air-floating PM2.5 into aqueous solution for the subsequent detection of PM2.5 by using online micropore detector [1,2]..
34. Abdallah M. Zeid, Noritada Kaji, Jenny Jeehan M. Nasr, Fathalla F. Belal, Yoshinobu Baba, Mohamed I. Walash, Stacking-cyclodextrin-microchip electrokinetic chromatographic determination of gabapentinoid drugs in pharmaceutical and biological matrices, Journal of Chromatography A, 10.1016/j.chroma.2017.04.049, 1503, 65-75, 2017.06, A facile, rapid, and highly sensitive microchip-based electrokinetic chromatographic method was developed for the simultaneous analysis of two gabapentinoid drugs, gabapentin (GPN) and pregabalin (PGN). Both drugs were first reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) via nucleophilic substitution reactions to yield highly fluorescent products with λex/em 470/540 nm. Analyses of both fluorescently labeled compounds were achieved within 200 s in a poly(methyl methacrylate) (PMMA) microchip with a 30 mm separation channel. Optimum separation was achieved using a borate buffer (pH 9.0) solution containing methylcellulose and β-cyclodextrin (β-CD) as buffer additives. Methylcellulose acted as a dynamic coating to prevent adsorption of the studied compounds on the inner surfaces of the microchannels, while β-CD acted as a pseudo-stationary phase to improve the separation efficiency between the labeled drugs with high resolution (Rs > 7). The fluorescence intensities of the labeled drugs were measured using a light emitting diode-induced fluorescence detector at 540 nm after excitation at 470 nm. The sensitivity of the method was enhanced 14- and 17-fold for PGN and GPN, respectively by field-amplified stacking relative to traditional pinched injection so that it could quantify 10 ng mL−1 for both analytes, with a detection limit lower than 3 ng mL−1. The developed method was efficiently applied to analyze PGN and GPN in their pharmaceutical dosage forms and in biological fluids. The extraction recoveries of the studied drugs from plasma and urine samples were more than 89% with%RSD values lower than 6.2..
35. Qiong Wu, Noritada Kaji, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Manabu Tokeshi, Tomoji Kawai, Yoshinobu Baba, A millisecond micro-RNA separation technique by a hybrid structure of nanopillars and nanoslits, Scientific Reports, 10.1038/srep43877, 7, 2017.03, A millisecond micro-RNA separation of a mixture of total RNA and genomic DNA, extracted from cultured HeLa cells, was successfully achieved using a hybrid structure of nanopillars and nanoslits contained inside a microchannel. The nanopillars, 250-nm in diameter and 100-nm in height, were fabricated with a 750-nm space inside the nanoslits, which were 100-nm in height and 25-μm in width; the nanopillars were then applied as a new sieve matrix. This ultra-fast technique for the separation of miRNA can be an effective pretreatment for semiconductor nanopore DNA sequencing, which has an optimum reading speed of 1 base/ms to obtain effective signal-to-noise ratio and discriminate each base by ion or tunneling current during the passage of nucleic acids..
36. Xiaoyin Sun, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Sakon Rahong, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Effect of DNA methylation on the velocity of DNA translocation through a nanochannel, Analytical Sciences, 10.2116/analsci.33.727, 33, 6, 727-730, 2017.01, Here, we report the effect of DNA methylation on the velocity of DNA translocation through a nanochannel, as determined by measuring differences in translocation velocities between methylated and non-methylated DNA molecules. We found that the velocity of translocation of methylated DNA was faster than that of non-methylated DNA, which we attributed to variation in the coefficients of diffusion and friction with the nanochannel wall, due to the increased molecular weight and stiffness, respectively, of methylated DNA..
37. Toshihiro Kasama, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Fabrication and evaluation of microfluidic immunoassay devices with antibody-immobilized microbeads retained in porous hydrogel micropillars, Methods in Molecular Biology, 10.1007/978-1-4939-6734-6_4, 49-56, 2017.01, Due to the inherent characteristics including confinement of molecular diffusion and high surface-to-volume ratio, microfluidic device-based immunoassay has great advantages in cost, speed, sensitivity, and so on, compared with conventional techniques such as microtiter plate-based ELISA, latex agglutination method, and lateral flow immunochromatography. In this paper, we explain the detection of C-reactive protein as a model antigen by using our microfluidic immunoassay device, so-called immuno-pillar device. We describe in detail how we fabricated and used the immuno-pillar devices..
38. Xiaoyin Sun, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Sakon Rahong, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Nanostructures integrated with a nanochannel for slowing down DNA translocation velocity for nanopore sequencing, Analytical Sciences, 10.2116/analsci.33.735, 33, 6, 735-738, 2017.01, Here, we developed a device integrated with a nanochannel and nanostructures to slow DNA translocation velocity. We found that translocation velocity of a single DNA molecule inside a nanochannel was decreased by pre-elongating it using some nanostructures, such as a shallow channel or nanopillars. This decrease of the translocation velocity was associated with the DNA mobility change, which is an intrinsic parameter of DNA molecules and unaffected by an electric field..
39. Takao Yasui, Kensuke Ogawa, Noritada Kaji, Mats Nilsson, Taiga Ajiri, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating, Scientific Reports, 10.1038/srep31642, 6, 2016.08, Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications..
40. Takao Yasui, Jumpei Morikawa, Noritada Kaji, Manabu Tokeshi, Kazuo Tsubota, Yoshinobu Baba, Microfluidic autologous serum eye-drops preparation as a potential dry eye treatment, Micromachines, 10.3390/mi7070113, 7, 7, 2016.07, Dry eye is a problem in tearing quality and/or quantity and it afflicts millions of persons worldwide. An autologous serum eye-drop is a good candidate for dry eye treatment; however, the eye-drop preparation procedures take a long time and are relatively troublesome. Here we use spiral microchannels to demonstrate a strategy for the preparation of autologous serum eye-drops, which provide benefits for all dry eye patients; 100% and 90% removal efficiencies are achieved for 10 μm microbeads and whole human blood cells, respectively. Since our strategy allows researchers to integrate other functional microchannels into one device, such a microfluidic device will be able to offer a new one-step preparation system for autologous serum eye-drops..
41. Yusuke Sato, Yusuke Note, Masatoshi Maeki, Noritada Kaji, Yoshinobu Baba, Manabu Tokeshi, Hideyoshi Harashima, Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery, Journal of Controlled Release, 10.1016/j.jconrel.2016.03.019, 229, 48-57, 2016.05, Because nanoparticles with diameters less than 50 nm penetrate stromal-rich tumor tissues more efficiently, the synthesis of small-sized nanoparticles encapsulating short interfering RNA (siRNA) is important in terms of realizing novel siRNA medicine for the treatment of various cancers. Lipid nanoparticles (LNPs) are the leading systems for the delivery of siRNA in vivo. Limit size LNPs were successfully synthesized using a microfluidic mixing technique. However, the physicochemical properties and potential for in vivo siRNA delivery of the limit-size LNPs have not been examined in detail. In the present study, we prepared LNPs with different diameters from 32 to 67 nm using a microfluidic mixing devise and examined the physicochemical properties of the particles and the potential for their use in delivering siRNA in vitro and in vivo to liver. Reducing the size of the LNPs causes poor-packing and an increased surface area, which result in their instability in serum. Moreover, it was revealed that the ability of endosomal escape (cytosolic siRNA release) of the smaller LNPs is subject to inhibition by serum compared to that of larger counterparts. Taken together, an increase in packing and avoiding the adsorption of serum components are key strategies for the development of next-generation highly potent and small-sized LNPs..
42. Akane Yamamichi, Toshihiro Kasama, Fumiharu Ohka, Hiromichi Suzuki, Akira Kato, Kazuya Motomura, Masaki Hirano, Melissa Ranjit, Lushun Chalise, Michihiro Kurimoto, Goro Kondo, Kosuke Aoki, Noritada Kaji, Manabu Tokeshi, Toshio Matsubara, Takeshi Senga, Mika K. Kaneko, Hidenori Suzuki, Masahito Hara, Toshihiko Wakabayashi, Yoshinobu Baba, Yukinari Kato, Atsushi Natsume, An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas, Science and Technology of Advanced Materials, 10.1080/14686996.2016.1227222, 17, 1, 618-625, 2016.01, World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69–80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83–90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice..
43. Abdallah M. Zeid, Jenny Jeehan M. Nasr, Fathalla F. Belal, Shinya Kitagawa, Noritada Kaji, Yoshinobu Baba, Mohamed I. Walash, Determination of six anti-Parkinson drugs using cyclodextrin-capillary electrophoresis method
Application to pharmaceutical dosage forms, RSC Advances, 10.1039/c5ra26473a, 6, 21, 17519-17530, 2016.01, A novel capillary electrophoretic method was developed for the assay of two quaternary anti-Parkinson mixtures, entacapone, levodopa, carbidopa, and benserazide (mixture I), and selegiline, levodopa, carbidopa, and benserazide (mixture II), by using α-methyldopa as an internal standard. Furthermore, the method was extended for the determination of another anti-Parkinson drug, lisuride, as well as a psychoactive antihypertensive drug, α-methyldopa, without any modification of the general method. Separation and analyses of all compounds were simply achieved in an untreated fused-silica capillary tube (42.0 cm effective length and 50 μm internal diameter) within 7 minutes under an applied voltage of 20 kV. Optimum separation and analyses were obtained using 25 mM borate buffer (pH 9.5) containing 5 mM β-cyclodextrin as the background electrolyte. The apparatus was equipped with a diode array detector (DAD) to identify lisuride at 240 nm and all other drugs at 200 nm. The addition of 5 mM β-cyclodextrin to the borate buffer has a significant effect on the separation of entacapone and benserazide in mixture I, and on the separation of selegiline and benserazide in mixture II, which cannot be achieved without it. The proposed method was successfully applied to analyse the studied drugs in their multi-component and single-component pharmaceutical dosage forms. The analytical results proved the linearity (r2 ≥ 0.9997), accuracy, precision (% RSD < 2), and selectivity of the proposed capillary electrophoretic method..
44. Xiaoyin Sun, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Sakon Rahong, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Identifying DNA methylation in a nanochannel, Science and Technology of Advanced Materials, 10.1080/14686996.2016.1223516, 17, 1, 644-649, 2016.01, DNA methylation is a stable epigenetic modification, which is well known to be involved in gene expression regulation. In general, however, analyzing DNA methylation requires rather time consuming processes (24–96 h) via DNA replication and protein modification. Here we demonstrate a methodology to analyze DNA methylation at a single DNA molecule level without any protein modifications by measuring the contracted length and relaxation time of DNA within a nanochannel. Our methodology is based on the fact that methylation makes DNA molecules stiffer, resulting in a longer contracted length and a longer relaxation time (a slower contraction rate). The present methodology offers a promising way to identify DNA methylation without any protein modification at a single DNA molecule level within 2 h..
45. Toyohiro Naito, Makoto Nakamura, Noritada Kaji, Takuya Kubo, Yoshinobu Baba, Koji Otsuka, Three-dimensional fabrication for microfluidics by conventional techniques and equipment used in mass production, Micromachines, 10.3390/mi7050082, 7, 5, 2016, This paper presents a simple three-dimensional (3D) fabrication method based on soft lithography techniques and laminated object manufacturing. The method can create 3D structures that have undercuts with general machines for mass production and laboratory scale prototyping. The minimum layer thickness of the method is at least 4 μm and bonding strength between layers is over 330 kPa. The performance reaches conventional fabrication techniques used for two-dimensionally (2D)-designed microfluidic devices. We fabricated some 3D structures, i.e., fractal structures, spiral structures, and a channel-in-channel structure, in microfluidic channels and demonstrated 3D microfluidics. The fabrication method can be achieved with a simple black light for bio-molecule detection; thus, it is useful for not only lab-scale rapid prototyping, but also for commercial manufacturing..
46. Toshihiro Kasama, Mai Ikami, Wanchun Jin, Keiko Yamada, Noritada Kaji, Yusuke Atsumi, Makoto Mizutani, Atsushi Murai, Akira Okamoto, Takao Namikawa, Michio Ohta, Manabu Tokeshi, Yoshinobu Baba, Rapid, highly sensitive, and simultaneous detection of staphylococcal enterotoxins in milk by using immuno-pillar devices, Analytical Methods, 10.1039/c5ay00698h, 7, 12, 5092-5095, 2015.06, Staphylococcal enterotoxins (SEs) have repeatedly caused food poisoning incidents worldwide. Some of the challenges associated with food poisoning outbreaks are that traditional detection methods are expensive and require long processing times and trained technicians. Microchannel devices represent a potential detection method by which these difficulties can be overcome. In this paper, we propose that immuno-pillar devices may represent a rapid, highly sensitive, and low-cost analytical system for the simultaneous detection of staphylococcal enterotoxin types A, B, and D (SEA, SEB, and SED) in milk. To prepare milk samples simulating food contaminated with SEs, commercial milk was spiked with equal amounts of SEA, SEB, and SED. A quantitative analysis of the milk samples was performed within 15 min by using the microchannel device. The analysis required only 0.5 μL of untreated milk sample. The resultant limit of detection was 15.6 pg mL-1 for each SE, and the total assay time and sensitivity were markedly shorter and higher, respectively, than those for commercially available assay kits. The detection range of each enterotoxin using these devices was estimated as 15.6 pg mL-1 to 100 ng mL-1, which completely covers the SE concentrations that can lead to foodborne diseases based on the U.S. Food and Drug Administration's criterion for the infectious SE dose in SE poisoning (1 μg SE). Using our devices, frequent assessment of food potentially contaminated with SE is possible..
47. Sakon Rahong, Takao Yasui, Takeshi Yanagida, Kazuki Nagashima, Masaki Kanai, Gang Meng, Yong He, Fuwei Zhuge, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Three-dimensional Nanowire Structures for Ultra-Fast Separation of DNA, Protein and RNA Molecules, Scientific reports, 10.1038/srep10584, 5, 2015.06, Separation and analysis of biomolecules represent crucial processes for biological and biomedical engineering development; however, separation resolution and speed for biomolecules analysis still require improvements. To achieve separation and analysis of biomolecules in a short time, the use of highly-ordered nanostructures fabricated by top-down or bottom-up approaches have been proposed. Here, we reported on the use of three-dimensional (3D) nanowire structures embedded in microchannels fabricated by a bottom-up approach for ultrafast separation of small biomolecules, such as DNA, protein, and RNA molecules. The 3D nanowire structures could analyze a mixture of DNA molecules (50-1000 bp) within 50 s, a mixture of protein molecules (20-340 kDa) within 5 s, and a mixture of RNA molecules (100-1000 bases) within 25 s. And, we could observe the electrophoretic mobility difference of biomolecules as a function of molecular size in the 3D nanowire structures. Since the present methodology allows users to control the pore size of sieving materials by varying the number of cycles for nanowire growth, the 3D nanowire structures have a good potential for use as alternatives for other sieving materials..
48. Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Arrangement of a nanostructure array to control equilibrium and nonequilibrium transports of macromolecules, Nano Letters, 10.1021/acs.nanolett.5b00783, 15, 5, 3445-3451, 2015.05, Exploiting the nonequilibrium transport of macromolecules makes it possible to increase the separation speed without any loss of separation resolution. Here we report the arrangement of a nanostructure array in microchannels to control equilibrium and nonequilibrium transports of macromolecules. The direct observation and separation of macromolecules in the nanopillar array reported here are the first to reveal the nonequilibrium transport, which has a potential to overcome the intrinsic trade-off between the separation speed and resolution..
49. Shiori Ito, Noritada Kaji, Takao Yasui, Yoshinobu Baba, A microchamber array device for studying genomic diversity in cancer cells at a single cell level, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 534-536, 2015.01, We have developed microchamber array device for direct quantitative analysis of messenger ribonucleic acids (mRNA) inside a single cell with two types of fluorescent probes. Genomic diversity in HeLa cancer cells at a single cell level was successfully observed through the experiments of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA detection and monitoring over 3 hours..
50. Noritada Kaji, Ryo Koyama, Takao Yasui, Tetsuya Higashiyama, Yoshinobu Baba, A single cell nucleus array to monitor messenger-RNA molecule transportation through nuclear membrane, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 832-834, 2015.01, In this study, a microchamber array device was designed to capture a single cell, lyse a cell membrane, and isolate a single cell nucleus, and then mRNA in a single cell nucleus were directly quantitated by using highly sensitive In-Stem Molecular Beacons (ISMB). As a result, the expression of three frequently used house-keeping genes were successfully quantitated to an accuracy of 100 molecules within a single cell nucleus..
51. Masatoshi Maeki, Tatsuyoshi Saito, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi, A strategy for synthesis of lipid nanoparticles using microfluidic devices with a mixer structure, RSC Advances, 10.1039/c5ra04690d, 5, 57, 46181-46185, 2015.01, Formation behavior of lipid nanoparticles (LNPs) in microfluidic devices with a staggered herringbone micromixer (SHM) structure was investigated. The fundamental role for SHMs in LNP formation was demonstrated by determining such factors as the limiting SHM cycle numbers and the effect of flow rate. The SHM cycle numbers and the position of the first SHM were as significant as factors as the flow rate condition for producing the small-size LNPs..
52. Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Carbon nanotubes and modern nanoagriculture, Nanotechnology and Plant Sciences Nanoparticles and Their Impact on Plants, 10.1007/978-3-319-14502-0_10, 183-201, 2015.01, Since their discovery, carbon nanotubes have been prominent members of the nanomaterial family. Owing to their extraordinary physical, chemical, and mechanical properties, carbon nanotubes have been proven to be a useful tool in the field of plant science. They were frequently perceived to bring about valuable biotechnological and agricultural applications that still remain beyond experimental realization. An increasing number of studies have demonstrated the ability of carbon nanotubes to traverse different plant cell barriers. These studies, also, assessed the toxicity and environmental impacts of these nanomaterials. The knowledge provided by these studies is of practical and fundamental importance for diverse applications including intracellular labeling and imaging, genetic transformation, and for enhancing our knowledge of plant cell biology. Although different types of nanoparticles have been found to activate physiological processes in plants, carbon nanotubes received particular interest. Following addition to germination medium, carbon nanotubes enhanced root growth and elongation of some plants such as onion, cucumber and rye-grass. They, also, modulated the expression of some genes that are essential for cell division and plant development. In addition, multi-walled carbon nanotubes were evidenced to penetrate thick seed coats, stimulate germination, and to enhance growth of young tomato seedlings. Multi-walled carbon nanotubes can penetrate deeply into the root system and further distribute into the leaves and the fruits. In recent studies, carbon nanotubes were reported to be chemically entrapped into the structure of plant tracheary elements. This should activate studies in the fields of plant defense and wood engineering. Although, all of these effects on plant physiology and plant developmental biology have not been fully understood, the valuable findings promises more research activity in the near future toward complete scientific understanding of the behavior of carbon nanotubes in plants. This chapter focuses on the impact of carbon nanotubes on plants and the potential use of these unique nanomaterials in crop management and plant biotechnology..
53. Mamiko Sano, Noritada Kaji, Takao Yasui, Yoshinobu Baba, Cell deformability measurements for single cancer cells by ionic current in microfluidic devices, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 455-457, 2015.01, To realize high-throughput, easy to use, and high-accuracy cell deformability measurements, we have developed ionic current detection system combined with constriction structure inside microchannel. In this system, when cells are clogged in constriction, the resistivity of detection area increases and the current decreases by ionic current blockade. We successfully detected unique signal shapes derived from cell deformability during the cell passage at the constriction. The signal shapes might provide an information on cell deformation and nucleus position inside a cell..
54. Yukihiro Okamoto, Ayato Hibino, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Development of a micro RNA extraction chip from human tumor cells, bunseki kagaku, 10.2116/bunsekikagaku.64.9, 64, 1, 9-13, 2015.01, The importance of micro RNA analysis has been significantly increasing because the role of micro RNA in the human body has been gradually revealed. Despite its importance, the analysis has suffered from several troublesome pretreatments, which hamper any easy analysis. Therefore, an easy and high-throughput pretreatment method has been demanded. In this paper, we focused on the great advantages of microchip pretreatments over conventional manual pretreatments and developed a microchip for micro RNA extraction. To simplify microchip fabrication, we adopted poly(dimethyl siloxane) (PDMS) microchip and a silica membrane, which has rolls in RNA extraction fields. With silica membranes and the adhesion nature of PDMS, we could easily fabricate RNA extraction fields in the microchip. With this microchip, we successfully extracted micro RNA from cancer tumor cells. Though this is a preliminary experiment, and still has many improvement points, our device is expected to be applied for easy and fast micro RNA extraction from biological samples..
55. Atsunori Hattori, Takao Yasui, Noritada Kaji, Yoshinobu Baba, High-throughput methylation mapping by detecting fluorescently stained methylation sites at a single molecule level, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 861-863, 2015.01, We detected methylation sites on elongated lambda DNA molecules as high fluorescent distinct peaks via double collar imaging. We used methyl-CpG-binding domain 2 protein (MBD2p) combined with the quantum dots (QDs) for methylation imaging, and YOYO-1 for DNA imaging..
56. Toyohito Naito, Noritada Kaji, Manabu Tokeshi, Takuya Kubo, Yoshinobu Baba, Koji Otsuka, Hydrodynamic nonadhesive cell retention in a microfluidic circuit for stressless suspension culture, Analytical Methods, 10.1039/c5ay00485c, 7, 17, 7264-7269, 2015.01, Cell collection based on deterministic lateral displacement (DLD) and cell circulation with a loop channel are two component technologies for stressless cell retention which have been developed with a view to working toward suspension culture in a microfluidic channel. DLD devices with low array angles collect floating S. cerevisiae through an array effectively. The DLD device with an array angle of 2.6°showed an efficiency of 91.7%. Two types of loop channels with a piezoelectrie pump make a stable two-phase laminar flow of a pre-filled fluid and supplied fluid. A loop channel with a connection between an inlet and outlet on the opposite side replaces a filled fluid in the channel with a supplied fluid, which is essential for supplying nutrient rich medium to cells in microfluidic channels as well as retaining cells in a microenvironment without external stresses..
57. M. Maeki, T. Saito, Y. Node, Y. Sato, T. Yasui, N. Kaji, A. Ishida, H. Tani, Y. Baba, H. Harashima, M. Tokeshi, Microfluidic approach for production of lipid nanoparticles-based nano medicine, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 838-840, 2015.01, This paper described a simple preparation method for small-size and monodispersed lipid nanoparticles (LNPs) by using microfluidic devices. The fundamental role and importance of chaotic micromixer in the microfluidic device was demonstrated. The suitable cycle number of chaotic micromixer was confirmed for precise controlling LNPs size with narrow distribution under the any flow rate conditions. In addition, LNPs containing siRNA was synthesized for evaluation of penetration efficiency via in vivo experiment. The PEGylated LNPs containing siRNA with a diameter of 30 nm could penetrate to the mouse parenchymal liver cells rather than the LNPs with a diameter of 50 nm..
58. Hirotoshi Yasaki, Daisuke Onoshima, Takao Yasui, Hiroshi Yukawa, Noritada Kaji, Yoshinobu Baba, Microfluidic transfer of liquid interface for parallel stretching and stamping of terminal-unmodified single DNA molecules in zigzag-shaped microgrooves, Lab on a Chip, 10.1039/c4lc00990h, 15, 1, 135-140, 2015.01, The molecular stretching of DNA is an indispensable tool for the optical exploration of base sequences and epigenomic changes of DNA at a single molecule level. In stretching terminal-unmodified DNA molecules parallel to each other on solid substrate, the receding meniscus assembly and capillary force through the dewetting process are quite useful. These can be achieved by pulling the substrate out of the DNA solution or sliding a droplet of DNA solution between a pair of substrates. However, currently used methods do not allow control over liquid interface motion and single-molecule DNA positioning. Here, we show a microfluidic device for stretching DNA molecules by syringing through microgrooves. The device can trap single DNA molecules at vertices of the microgrooves, which were designed as parallel zigzag lines. Different zigzag pattern depths, sizes, and shapes were studied to evaluate the adsorption possibility of DNA on the surface. The microfluidic transfer of the liquid interface stretched over 1500 DNA molecules simultaneously. The stretched DNA molecules could be stamped to a silanized surface. The device will therefore serve as a template preparation for high-resolution DNA imaging studies. This journal is.
59. Sakon Rahong, Takao Yasui, Takeshi Yanagida, Kazuki Nagashima, Masaki Kanai, Gang Meng, Yong He, Fuwei Zhuge, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Self-assembled nanowire arrays as three-dimensional nanopores for filtration of DNA molecules, analytical sciences, 10.2116/analsci.31.153, 31, 3, 153-157, 2015.01, Molecular filtration and purification play important roles for biomolecule analysis. However, it is still necessary to improve efficiency and reduce the filtration time. Here, we show self-assembled nanowire arrays as three-dimensional (3D) nanopores embedded in a microfluidic channel for ultrafast DNA filtration. The 3D nanopore structure was formed by a vapor-liquid-solid (VLS) nanowire growth technique, which allowed us to control pore size of the filtration material by varying the number of growth cycles. λ DNA molecules (48.5 kbp) were filtrated from a mixture of T4 DNA (166 kbp) at the entrance of the 3D nanopore structure within 1 s under an applied electric field. Moreover, we observed single DNA molecule migration of T4 and λ DNA molecules to clarify the filtration mechanism. The 3D nanopore structure has simplicity of fabrication, flexibility of pore size control and reusability for biomolecule filtration. Consequently it is an excellent material for biomolecular filtration..
60. X. Sun, T. Yasui, S. Rahong, T. Yanagida, N. Kaji, M. Kanai, K. Nagashima, T. Kawai, Y. Baba, Conformation and dynamic behavior of single DNA molecules in nanofluidic channels for detection of DNA methylation, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1115-1117, 2015, In this paper, the single DNA molecules were linearized and stretched by using the nanochannel with a dimension of about 300 × 300 nm2 under applying electric fields. After that, the single DNA molecules methylation level was detected by estimating the contraction processes of both non-methylated and methylated single DNA molecules in nanochannel after switching off the electric fields. And furthermore, we also found that their translocation velocities were changed between non-methylated and methylated single DNA molecules in nanochannel..
61. Hirotoshi Yasaki, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Naritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Highly sensitive ionic current sensing system with optical observation for discriminating a wide diversity of sizes of bacteria with contaminants, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 314-316, 2015, We realized highly sensitive ionic current sensing system with optical observation for discriminating a wide diversity of sizes of bacteria (0.15∼10 μm) in biological samples with many contaminants. The proposed system enables us to discriminate 1% size difference of each sample within 400 ms. This highly sensitive ionic current sensing system with optical observation might provide a new sensing scheme to detect a single bacteria based on their sizes, shapes, colors, and stainability..
62. T. Kasama, T. Hase, N. Nishiwaki, N. Yogo, M. Sato, M. Kondo, N. Kaji, M. Tokeshi, Y. Hasegawa, Y. Baba, Immuno-wall lab-on-chip companion diagnostic devices for rapid and low-cost detection of mutant epidermal growth factor receptors (EGFR) from cytological samples in lung cancer patients, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 925-927, 2015, In the present study, we propose immuno-wall lab-on-a-chip companion diagnostic devices for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib. The lysates of cytological samples including pleural effusion in lung cancer patients were successfully analyzed within 20 minutes. This is the first experiment demonstrating the detection of mutated EGFRs in the pleural effusion by microdevices. Our devices have a great potential to become the next generation companion diagnostic devices which overcome the problems of currently available methods..
63. Takao Yasui, Satoru Ito, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Micropillars fabricated on poly(methyl methacrylate) substrates for separation of microscale objects, analytical sciences, 10.2116/analsci.31.1197, 31, 11, 1197-1200, 2015, Development of polymeric microfluidic devices has played an important role in the recent, rapid progress of biomedical research. Here we report a fabrication method for micropillars on poly(methyl methacrylate) (PMMA) substrates for separation of microscale objects. The fabricated micropillars enable continuous separation of microparticles only by introducing fluids. The present method offers a new strategy to fabricate polymeric prototype devices for R&D work..
64. Keiko Tabuchi, Takao Yasui, He Yong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Nanowire devices for extracellular vesicles analysis towards elucidation of intercellular communication, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1510-1512, 2015, We fabricate ZnO nanowires inside microchannels on Si substrates. Since the ZnO nanowires have positively charged surface [1] and most vesicles have negatively charged surface, it is able to capture the vesicles on the nanowire surface by electrostatic interaction. MicroRNA is extracted from the captured vesicles by introducing lysis buffer. It is expected that our fabricate devices would lead to fundamental understanding of cell-cell communication..
65. Takao Yasui, Kohei Otsuka, Masaki Takeuchi, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Sakon Rahong, Kazuki Nagashima, Toyohiro Naito, Tomoji Kawai, Yoshinobu Baba, Nanowire lysis and DNA extracrtion from a single bacterium for bacteria analysis, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 320-322, 2015, Exploiting the method to extract DNA molecules from bacteria makes it possible to analyze bacteria in a short time. Here, we realized DNA extraction from a single bacterium via physical interactions between nanowires and bacteria; nanowire lysis. The combination of nanowire lysis and heating at 94°C could help us to extract genome DNA from bacteria, efficiently..
66. Daiki Takeshita, Takao Yasui, He Yong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Pdms-anchored nanowires for high throughput micro-RNA extraction from extracellular vesicles in body fluid, 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015 MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1516-1518, 2015, We anchored zinc oxide nanowires (ZnO) in PDMS microchannel for high throughput capture of extracellular vesicles and microRNA extraction in them from body fluids. Using this device, we achieved vesicles capture from urine sample within 10 min, which is faster than using the conventional methods, such as precipitation kit or ultracentrifugation. And we demonstrated microRNA extraction from the captured vesicles. These results highlighted that we realized high throughput microRNA extraction from body fluid..
67. Hiroshi Yukawa, Shingo Nakagawa, Yasuma Yoshizumi, Masaki Watanabe, Hiroaki Saito, Yoshitaka Miyamoto, Hirofumi Noguchi, Koichi Oishi, Kenji Ono, Makoto Sawada, Ichiro Kato, Daisuke Onoshima, Momoko Obayashi, Yumi Hayashi, Noritada Kaji, Tetsuya Ishikawa, Shuji Hayashi, Yoshinobu Baba, Novel positively charged nanoparticle labeling for in vivo imaging of adipose tissue-derived stem cells, PloS one, 10.1371/journal.pone.0110142, 9, 11, 2014.11, Stem cell transplantation has been expected to have various applications for regenerative medicine. However, in order to detect and trace the transplanted stem cells in the body, non-invasive and widely clinically available cell imaging technologies are required. In this paper, we focused on magnetic resonance (MR) imaging technology, and investigated whether the trimethylamino dextran-coated magnetic iron oxide nanoparticle -03 (TMADM-03), which was newly developed by our group, could be used for labeling adipose tissue-derived stem cells (ASCs) as a contrast agent. No cytotoxicity was observed in ASCs transduced with less than 100 μg-Fe/mL of TMADM-03 after a one hour transduction time. The transduction efficiency of TMADM-03 into ASCs was about four-fold more efficient than that of the alkali-treated dextrancoated magnetic iron oxide nanoparticle (ATDM), which is a major component of commercially available contrast agents such as ferucarbotran (Resovist), and the level of labeling was maintained for at least two weeks. In addition, the differentiation ability of ASCs labeled with TMADM-03 and their ability to produce cytokines such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2), were confirmed to be maintained. The ASCs labeled with TMADM-03 were transplanted into the left kidney capsule of a mouse. The labeled ASCs could be imaged with good contrast using a 1T MR imaging system. These data suggest that TMADM-03 can therefore be utilized as a contrast agent for the MR imaging of stem cells..
68. Sakon Rahong, Takao Yasui, Takeshi Yanagida, Kazuki Nagashima, Masaki Kanai, Annop Klamchuen, Gang Meng, Yong He, Fuwei Zhuge, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Ultrafast and wide range analysis of DNA molecules using rigid network structure of solid nanowires, Scientific reports, 10.1038/srep05252, 4, 2014.06, Analyzing sizes of DNA via electrophoresis using a gel has played an important role in the recent, rapid progress of biology and biotechnology. Although analyzing DNA over a wide range of sizes in a short time is desired, no existing electrophoresis methods have been able to fully satisfy these two requirements. Here we propose a novel method using a rigid 3D network structure composed of solid nanowires within a microchannel. This rigid network structure enables analysis of DNA under applied DC electric fields for a large DNA size range (100 bp-166 kbp) within 13 s, which are much wider and faster conditions than those of any existing methods. The network density is readily varied for the targeted DNA size range by tailoring the number of cycles of the nanowire growth only at the desired spatial position within the microchannel. The rigid dense 3D network structure with spatial density control plays an important role in determining the capability for analyzing DNA. Since the present method allows the spatial location and density of the nanostructure within the microchannels to be defined, this unique controllability offers a new strategy to develop an analytical method not only for DNA but also for other biological molecules..
69. T. Naito, M. Nakamura, T. Kubo, T. Yasui, Noritada Kaji, Y. Baba, K. Otuska, Additive manufacturing based on injection molding for three dimensional microfluidics, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 1686-1688, 2014.01, This paper describes three dimensional (3D) microfluidic devices fabricated by an injection molding-based additive manufacturing with standard equipment for soft-lithography. The method achieves 3D fabrication without specific instruments or water-soluble support material. It provides the same level of minimum membrane thickness as a high end model commercial 3D printer, and the bond strength between the membranes have a sufficient pressure resistance to the flow of water with a syringe pump. Finally, two types of 3D microstructures were fabricated by the method: the 3D fractal structure called Menger sponge, and a channel within a channel structure for 3D sheath flow..
70. Toshihiro Kasama, Yutaka Hasegawa, Haruyasu Kondo, Tsutomu Ozawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Development of immuno-wall devices and a mobile fluorescence reader for on-site sample-to-answer immunoassay, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 935-937, 2014.01, We have developed a novel immunoassay device which has a long and thin wall structure inside a microchannel, hence the name 'immuno-wall device'. Unreacted antigens and antibodies were completely removed by just immersing the device in a washing buffer for 1 minute. The long structure also allowed us to analyze fluorescence intensity by using inexpensive desktop fluorescence reader ($6,000) instead of expensive fluorescence microscopes. By using the immuno-wall devices and the fluorescence reader, the high-sensitivity C-reactive protein assays were performed with sample-in-answer-out in 15 minutes..
71. Jun Wang, Michihiko Aki, Daisuke Onoshima, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba, Microfluidic biosensor for the detection of DNA by fluorescence enhancement and the following streptavidin detection by fluorescence quenching, Biosensors and Bioelectronics, 10.1016/j.bios.2013.07.058, 51, 280-285, 2014.01, We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA-biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA-biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1. pM, equating to an absolute detection limit of 60. amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions..
72. Daiki Takeshita, Daisuke Onoshima, Yukawa Hiroshi, Takao Yasui, Noritada Kaji, Yoshinobu Baba, Microfluidic stretching of DNA with fluorescent gold nanoparticle for optical/electron microscopic imaging of a single DNA methylation, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 2348-2350, 2014.01, We combined a microfluidic stretch of a single DNA molecule and a visualization technique with fluorescent gold nanoparticle labeling, which can be observed with fluorescence microscope and scanning electron microscope (SEM) for imaging of methylated site on a single DNA molecule. It will realize bimodal microscope imaging of methylated site and enhance the imaging resolution down to nanometer level toward epigenetic analysis on the basis of high-resolution mapping of methylation sites..
73. Noritada Kaji, Yoshinobu Baba, Nanobiodevice-based single biomolecule analysis, single-cell analysis, and in vivo imaging for cancer diagnosis, cancer theranostics, and iPS cell-based regenerative medicine, analytical sciences, 10.2116/analsci.30.859, 30, 9, 859-864, 2014.01, Numerous nanobiodevices have been developed for cancer diagnosis through the analysis of cancer cells and cancer-related biomolecules, cancer gene therapy, and iPS cell-based regenerative medicine. A microchamber array, which is fabricated on a plastic chip, enables to develop a reliable circulating tumor cell (CTC) detection technique for cancer metastasis diagnosis. A nanopillar-array or a nanowire-array on a quartz chip allows ultrafast analysis of DNA and microRNA within 1 s for the molecular diagnosis of cancer. Immunopillar devices, which contain a tremendous amount of antibody-immobilized microparticles inside an immunopillar structure, have realized fast and low invasive "from blood to analysis" type biomarker detection of cancer with a pM detection sensitivity within 5 min. Quantum dots and gene delivery nanodevices are applied to single cancer cell diagnosis, in vivo imaging for iPS cell based regenerative medicine, and theranostic devices for cancer diagnosis and therapy..
74. Noritada Kaji, Yoshinobu Baba, Nanobiodevices for single DNA and cell analysis, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 264-266, 2014.01, Nanopore-based DNA sequencing is an emerging technology that may soon overcome even the current next-generation sequencing from the viewpoint of DNA sequencing cost and throughput. In this paper, we will show that recent advances of the "pretreatment" process using various nanostructures such as nanopillars, nanowires, and nanoslits structures inside microchannel. In addition, another type of micro and nanostructures such as micro and nanometer-scale chamber array on a chip becomes a powerful new tool for bioanalysis since it could stochastically capture and measure biomolecules at a single molecule level. The applications of these chamber array structures for single cell analysis will be also described..
75. Qiong Wu, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, A millisecond micro-RNA extraction technique for nanopore-based nucleic acid sequencing, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 233-235, 2014, A ms(millisecond) miRNA(micro RNA) extraction from a mixture of total RNA and genomic DNA was successfully achieved by the combination of nanopillar and nanoslit structures inside a microchannel. This ultra-fast miRNA extraction technique especially useful for exosome-derived miRNA sequencing by nanopore-based RNA sequencer since exosomes contain miRNA and mRNA..
76. Yuki Konakade, Takao Yasui, Takeshi Yanagida, Noritada Kaji, Yong He, Masaki Kanai, Kazuki Nagashima, Hiroshi Yukawa, Tomoji Kawai, Yoshinobu Baba, Exosomal membrane protein detection by nanowire device, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 2017-2019, 2014, We analyzed exosomal membrane proteins using ZnO nanowire devices, quantitatively and qualitatively. The nanowire devices could capture plenty of exosomes quickly, resulting in rapid analysis of exosomal membrane proteins in response to exosome concentrations, rather than conventional methods. A potential of the nanowire devices would open a window for early cancer detection..
77. Takao Yasui, Satoru Ito, Takeshi Yanagida, Yong He, Sakon Rahong, Masaki Kanai, Kazuki Nagashima, Hiroshi Yukawa, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Exosomal micro-RNA analysis in urine or serum using nanowire structures, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 680-682, 2014, We report a highly-efficient separation of exosomes in 1 mL urine or serum using nanowire structures, and an extraction of exosomal micro-RNA(miRNA) from the separated exosomes. A series of procedures can be demonstrated with no any troublesome techniques, just only by introducing solution into nanowire structures embedded in microchannels. Such an easy analysis method for exosomal miRNA, especially from 1mL urine, could contribute to less invasive diagnosis and unknown biomarkers discovery..
78. Xiaoyin Sun, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Geometrical control of a single DNA molecule translocation velocity in nanopore channels, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 1338-1340, 2014, The geometrical control of translocation velocity for single DNA molecules in nanoscale space was studied in this paper. We fabricated the nanopore channels which have 250 μm in length, 300 nm in width and 300 nm in depth for confinement of single DNA molecules, and measurement of single DNA molecules translocation velocities. Single DNA molecules translocation velocities in nanopore channels were controlled by fabricating the different nanostructures in front of nanopore channels, such as shallow channels and nanopillars. Based on these strategies, we successfully decreased the single DNA molecules translocation velocities to 7.7 ± 0.8×10-3 cm/s in nanopore channels..
79. Yasuhiro Hayashi, Erina Suemitsu, Kazuaki Kajimoto, Yusuke Sato, Afsana Akhter, Yu Sakurai, Hiroto Hatakeyama, Mamoru Hyodo, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima, Hepatic monoacylglycerol O-acyltransferase 1 as a promising therapeutic target for steatosis, obesity, and type 2 diabetes, Molecular Therapy - Nucleic Acids, 10.1038/mtna.2014.4, 3, e154, 2014, Over the past decade, considerable advances have been made in the discovery of gene targets in metabolic diseases. However, in vivo studies based on molecular biological technologies such as the generation of knockout mice and the construction of short hairpin RNA vectors require considerable effort and time, which is a major limitation for in vivo functional analysis. Here, we introduce a liver-specific nonviral small interfering RNA (siRNA) delivery system into rapid and efficient characterization of hepatic gene targets in metabolic disease mice. The comparative transcriptome analysis in liver between KKAy diabetic and normal control mice demonstrated that the expression of monoacylglycerol O-acyltransferase 1 (Mogat1), an enzyme involved in triglyceride synthesis and storage, was highly elevated during the disease progression. The upregulation of Mogat1 expression in liver was also found in other genetic (db/db) and diet-induced obese mice. The silencing of hepatic Mogat1 via a liver-specific siRNA delivery system resulted in a dramatic improvement in blood glucose levels and hepatic steatosis as well as overweight with no apparent overall toxicities, indicating that hepatic Mogat1 is a promising therapeutic target for metabolic diseases. The integrated approach with transcriptomics and nonviral siRNA delivery system provides a blueprint for rapid drug discovery and development..
80. Hirotoshi Yasaki, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Micropore channell-based simultaneous electrical and optical sensing from single biomolecules, single exosomes to single cells, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 2161-2163, 2014, We realized simultaneous measurements of ionic current and fluorescent imaging in microscale devices towards wide variety analysis of biomolecules, exosomes (<200 nm), circulating tumor cells (<20 μm), and white blood cells (<30 μm). The detection system based on ionic current would distinguish object sizes ranging from 500 nm to 3 μm diameter and fluorescence imaging enabled to detect target objects based on the optical properties. This electrical/optical approach might provide a new sensing scheme to analyze single biomolecules, single exosomes, and single cells with various sizes, shapes, and characteristics..
81. Mamiko Sano, Takao Yasui, Noritada Kaji, Masateru Taniguchi, Tomoji Kawai, Yoshinobu Baba, Quantitative evaluation of insulating performance on silicon nanobiodevices for fully integrated nanopore single DNA sequencing, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 1479-1481, 2014, To realize a silicon-based semiconductor nanopore DNA sequencer, we have quantitatively evaluated insulating performance of silicon-based nanoslit array structures to stretch a random-coiled DNA molecule by electrokinetic force as a step prior to nanopore DNA sequencing. We found critical electric fields which trigger electric breakdown as a function of buffer concentration and successfully observed a single DNA molecular stretching in the nanoslit array structures under the explored conditions..
82. Sakon Rahong, Takao Yasui, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Ultrafast separation of small biomolecules by three-dimensional nanowire structure, 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014, 2432-2434, 2014, We report on fast biomolecules separation using three-dimensional nanowires structure devices. The three-dimensional nanowire structure can be synthesized and embedded in microchannel by Vapor-Liquid-Solid (VLS) technique. We decorate Au catalyst along the nanowires by DC sputtering and growth nanowire as a cycle to prepare the branch of the nanowire structure. This technique allows researchers to control the pore size of the three-dimensional nanowires by increasing the number of growth time. We separated small DNA molecules mixture (50-100 bp) within 35 seconds and proteins mixture (20-66 kDa) within 2.5 seconds under DC electric fields..
83. Shinya Iida, Hiroyuki Kamiya, Akihiro Nakaya, Yasuhiro Hayashi, Akihiro Sawada, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima, Reduced plasma glucose by asparagine synthetase knockdown in the mouse liver, Biological and Pharmaceutical Bulletin, 10.1248/bpb.b13-00414, 36, 12, 2009-2011, 2013.12, Expression of the asparagine synthetase gene is dependent on extracellular glucose concentration. This gene was knocked-down in livers of male Balb/c mice by a hydrodynamic tail vein injection of small interfering RNA (siRNA) against the gene. This knockdown resulted in a significant decrease in plasma glucose concentration. These results suggested that asparagine synthetase is a novel protein that regulates plasma glucose levels..
84. Hidetaka Akita, Soichiro Ishii, Naoya Miura, Sharif Mohammad Shaheen, Yasuhiro Hayashi, Takashi Nakamura, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima, A DNA microarray-based analysis of immune-stimulatory and transcriptional responses of dendritic cells to KALA-modified nanoparticles, Biomaterials, 10.1016/j.biomaterials.2013.08.003, 34, 35, 8979-8990, 2013.11, Technologies for the transfection of antigen-encoding genes into the dendritic cells, and subsequent immune-activation are both prerequisites for a successful DNA vaccine. We herein report on the density-dependent enhancement of transgene expression by the simple modification by stearyl-conjugated KALA, an α-helical peptide (STR-KALA), onto a lipid envelope-type nanoparticle (the R8-MEND, an octaarginine-modified multifunctional envelope-type nano device). The enhanced transgene expression in the KALA-modified R8-MEND (R8/KALA-MEND) cannot be explained by cellular uptake and nuclear delivery efficacy. Thus, the post-nuclear delivery process (i.e. transcription), but not intracellular trafficking processes attributed the enhanced transfection efficacy. Microarray analyses revealed that transfection with the R8/KALA-MEND resulted in a greater perturbation in host genes expression in comparison with the R8-MEND and that this effect was time-dependent. Further pathway analyses in the category of transcription-related genes and a gene ontology analysis indicated that the R8/KALA-MEND stimulated the expression of transcription factors that are closely related to immune-activation (i.e. NF- kB and STAT). Inhibition of the transfection efficacy by blockage of the STAT pathways revealed that the enhanced transcription activity is the result of immune-stimulation. Collectively, the R8/KALA-MEND mounts a "switch-on" function that triggers signal transduction forward to the immune-stimulation analogous to an adjuvant, and consequently elicits active transcription..
85. Takao Yasui, Noritada Kaji, Yoshinobu Baba, Nanobiodevices for biomolecule analysis and imaging, Annual Review of Analytical Chemistry, 10.1146/annurev-anchem-062012-092619, 6, 83-96, 2013.06, Nanobiodevices have been developed to analyze biomolecules and cells for biomedical applications. In this review, we discuss several nanobiodevices used for disease-diagnostic devices, molecular imaging devices, regenerative medicine, and drug-delivery systems and describe the numerous advantages of nanobiodevices, especially in biological, medical, and clinical applications. This review also outlines the fabrication technologies for nanostructures and nanomaterials, including top-down nanofabrication and bottom-up molecular self-assembly approaches. We describe nanopillar arrays and nanowall arrays for the ultrafast separation of DNA or protein molecules and nanoball materials for the fast separation of a wide range of DNA molecules, and we present examples of applications of functionalized carbon nanotubes to obtain information about subcellular localization on the basis of mobility differences between free fluorophores and fluorophore-labeled carbon nanotubes. Finally, we discuss applications of newly synthesized quantum dots to the screening of small interfering RNA, highly sensitive detection of disease-related proteins, and development of cancer therapeutics and diagnostics..
86. Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Nanopillar array chip integrated with on-line stacking for fast DNA separation with high sensitivity and high resolution, Microfluidics and Nanofluidics, 10.1007/s10404-012-1103-7, 14, 6, 961-967, 2013.06, Recent developments of nanofabrication techniques have created a trend switching from randomly ordered polymeric matrices, such as gel, to highly ordered sieving nanostructures in the separation of biomolecules. These nanostructures have enormous potential for fast separation of biomolecules, while nanostructure-based separation techniques suffer from critical scaling problems; they are efficient in handling less than nanoliter amounts of sample fluids, but most biomolecule samples are available in a liquid volume that is over several microliter, leading to a reduction in sensitivity and resolution. In this study, we developed a nanopillar array chip integrated with an easy and rapid on-line stacking method and achieved fast DNA separation with high sensitivity and high resolution. The developed on-line stacking method is based on the balance of two forces driven by electric fields: electroosmotic flow (EOF) and electrophoresis. The EOF mobility from the microchannel to the nanopillar-channel is drastically decreased, while, on the other hand, electrophoresis has constant mobilities in the whole length of the channels. The on-line stacking was realized at the well-balanced position of the two forces, and the on-line stacking using the nanopillar array chip can also be achieved within 10 s by just applying electric voltages without any other special reagents and materials. After applying on-line stacking using the nanopillar array chip, the relative fluorescence intensity increased 1,000-fold, and the resolution was twice as good as that without on-line stacking..
87. Takao Yasui, Sakon Rahong, Koki Motoyama, Takeshi Yanagida, Qiong Wu, Noritada Kaji, Masaki Kanai, Kentaro Doi, Kazuki Nagashima, Manabu Tokeshi, Masateru Taniguchi, Satoyuki Kawano, Tomoji Kawai, Yoshinobu Baba, DNA manipulation and separation in sublithographic-scale nanowire array, ACS nano, 10.1021/nn4002424, 7, 4, 3029-3035, 2013.04, Electrokinetic manipulations of biomolecules using artificial nanostructures within microchannels have proven capability for controlling the dynamics of biomolecules. Because there is an inherent spatial size limitation to lithographic technology, especially for nanostructures with a small diameter and high aspect ratio, manipulating a single small biomolecule such as in DNA elongation before nanopore sequencing is still troublesome. Here we show the feasibility for self-assembly of a nanowire array embedded in a microchannel on a fused silica substrate as a means to manipulate the dynamics of a single long T4-DNA molecule and also separate DNA molecules. High-resolution optical microscopy measurements are used to clarify the presence of fully elongated T4-DNA molecules in the nanowire array. The spatial controllability of sublithographic-scale nanowires within microchannels offers a flexible platform not only for manipulating and separating long DNA molecules but also for integrating with other nanostructures to detect biomolecules in methods such as nanopore sequencing..
88. Maged F. Serag, Noritada Kaji, Satoshi Habuchi, Alberto Bianco, Yoshinobu Baba, Nanobiotechnology meets plant cell biology
Carbon nanotubes as organelle targeting nanocarriers, RSC Advances, 10.1039/c2ra22766e, 3, 15, 4856-4862, 2013.04, For years, nanotechnology has shown great promise in the fields of biomedical and biotechnological sciences and medical research. In this review, we demonstrate its versatility and applicability in plant cell biology studies. Specifically, we discuss the ability of functionalized carbon nanotubes to penetrate the plant cell wall, target specific organelles, probe protein-carrier activity and induce organelle recycling in plant cells. We also, shed light on prospective applications of carbon nanomaterials in cell biology and plant cell transformation..
89. Wanchun Jin, Keiko Yamada, Mai Ikami, Noritada Kaji, Manabu Tokeshi, Yusuke Atsumi, Makoto Mizutani, Atsushi Murai, Akira Okamoto, Takao Namikawa, Yoshinobu Baba, Michio Ohta, Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products, Journal of Microbiological Methods, 10.1016/j.mimet.2013.01.001, 92, 3, 323-331, 2013.03, Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2. ng/ml within 15. min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1. ng/ml in milk; the SEs were detected within 12. min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products..
90. Takao Yasui, Koki Motoyama, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Enzyme-catalysed reaction for long-term fluorescent observation of single DNA molecules, RSC Advances, 10.1039/c3ra22999h, 3, 10, 3237-3240, 2013.03, The technical development of long-term fluorescent observation of single DNA molecules is central to fields ranging from molecular biological detection to understanding the physical properties of them under a microscope. Here, we address this challenge using protocatechuic acid and protocatechuate-3,4- dioxygenase (PADase) and demonstrate fluorescent lifetimes of dyed single DNA molecules of 150-180 s, three times longer than those without any treatments..
91. Toyohiro Naito, Rerngchai Arayanarakool, Séverine Le Gac, Takao Yasui, Noritada Kaji, Manabu Tokeshi, Albert Van Den Berg, Yoshinobu Baba, Temperature-driven self-actuated microchamber sealing system for highly integrated microfluidic devices, Lab on a Chip, 10.1039/c2lc41030c, 13, 3, 452-458, 2013.02, We present here a novel microchamber sealing valve that is self-actuated by a pressure change during the temperature change in the thermal activation of reactions. Actuation of our valve requires only the use of the same heating device as employed for the reactions. A thermoplastic UV-curable polymer is used as a device material; the polymer allows realization of the temperature-driven valve actuation as well as the fabrication of multi-layered devices. The self-actuated valve achieves effective sealing of the microchamber for the polymerase chain reaction (PCR) even at 90 °C, which is essential for developing highly parallel PCR array devices without the need for complicated peripherals to control the valve operation..
92. Mitsuhiro Horade, Masahiro M. Kanaoka, Motoki Kuzuya, Tetsuya Higashiyama, Noritada Kaji, A microfluidic device for quantitative analysis of chemoattraction in plants, RSC Advances, 10.1039/c3ra42804d, 3, 44, 22301-22307, 2013.01, To precisely quantitate the effect of chemoattractants on directional pollen tube growth, a new microdevice was developed. Torenia fournieri pollen tubes, which generally grow freely on agarose medium, were funneled through a narrow flow channel that splits into a T-shaped channel leading to two reservoirs. The main channel was thus divided in two so that pollen tubes could choose their growth in either the left or right direction. Liquid solution or plant tissues were loaded into the reservoir, and diffusible molecules from the materials gradually spread in the narrow channel, leading to a concentration gradient. When egg-cell containing ovules were placed in one reservoir, pollen tubes grew selectively in that direction, suggesting that materials secreted from the ovules attracted the pollen tubes. Furthermore, UV-irradiation of female gametophytes in ovules decreased their ability to attract pollen tube growth. These results suggest that this novel device provides a unique platform for screening materials that may attract pollen tubes and for quantitatively analyzing the chemical features of attractants..
93. Noritada Kaji, Daisuke Shigenaka, Masami Ukawa, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba, High throughput purification devices for in vivo applications of gene-delivery multifunctional envelope-type nanodevices, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 1170-1172, 2013.01, We developed microfluidic continuous purification devices based on free-flow electrophoresis for gene-delivery multifunctional envelope-type nanodevices (MEND) which consists of DNA core and phospholipid bilayer envelope. Various impurities, such as DNA, DNA-peptide complex, and liposomes, produced during the fabrication process were removed in an efficient manner and over 75% collection of the input plasmid DNA which corresponds to purified MEND was achieved..
94. Takao Yasui, Kensuke Ogawa, Noritada Kaji, Mats Nilsson, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Label-free detection and quantification of real-time DNA amplification using one-dimensional photonic crystal, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 1123-1125, 2013.01, We could achieve label-free detection and quantification of real-time DNA amplification using a one-dimensional (1D) photonic crystal embedded in microchannels. Our method could be applicable to ultra-highly sensitive detection of human papillomavirus (HPV) and tubercle bacillus (TB) from 1 zmol to 1 amol. The limit of detection (LOD) of TB sequence in our system was around 500 ymol..
95. Hirotoshi Yasaki, Daisuke Onoshima, Takao Yasui, Toyohiro Naito, Noritada Kaji, Yoshinobu Baba, Microfluidic parallel stretching and stamping of single DNA molecules for super resolution microscope imaging, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 8-10, 2013.01, We developed a method for making high-density arrayed DNA molecules on PDMS and glass substrates. Arrayed DNA could be applied to various observation methods, and were elongated and immobilized on cover plates without any modification to DNA molecules. The depth, size and shape of the devices were optimized. It enables researchers to make such a high-density array of DNA molecules in a simple way..
96. D. Onoshima, Noritada Kaji, M. Tokeshi, Y. Baba, Microfluidic single-molecule nuclease digestion reveals rate-enhancing off-and-on molecular encountering function for site-specific DNA break, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 820-822, 2013.01, An intermittent molecular encounter leading to a site-specific DNA-break was unveiled under a limited protein's sliding-free condition. It reflects a transient molecular action by which the reaction rate and efficiency of restriction enzyme in bacterial cells is enhanced. This was experimentally verified for the first time in the world by using our microfluidic device..
97. Sakon Rahong, Takao Yasui, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Annop Klamchuen, Meng Gang, He Yong, Fuwei Zhuge, Noritada Kaji, Yoshinobu Baba, Tomoji Kawai, Christmas-tree nanowire chips for dna separation, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 164-166, 2013, We demonstrated the DNA separation by using the three dimensional Christmas-tree nanowire chips embedded in the microfluidic system to enhance the separation range and decrease the time consuming also. We synthesize the Christmas-tree nanowire by decorating Au catalyst along the nanowires structure by sputtering and growing the branch of nanowires by Vapor-Liquid-Solid (VLS) technique as a cycle. The DNA electrophoretic mobility in the Christmas- tree nanowire decreases when the pore size between nanowire decreases by growth time. The DNA migration in the Christmas-tree nanowire chip is faster than gel electrophoresis due to the rigidity of 3D nanowire structure. Consequently, we succeeded to separate the wide range of DNA molecules by mobility difference in 3D nanowire structure in short time..
98. Xiaoyin Sun, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Control of DNA translocation velocities for nanopore-based DNA sequencing, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 1117-1119, 2013, Reducing translocation velocity of DNA molecules in a nanopore is an urgent issue for rapid single identification. Here we fabricated a nanopore-channel, which has 300 × 300nm (width and depth) dimensions for electrophoretically driven transport of single DNA molecule, and decreased the single DNA molecule's translocation velocity by changing length of the nanopore-channels or electric fields. We obtained a 180 kbp/s translocation velocity for 10 kbp DNA through the 250 μm long nanopore-channel by applying 3 V..
99. Mayu Mizutani, Takao Yasui, Nortada Kaji, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, DNA methylation mapping in nanoslit devices at a single molecule level, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 29-31, 2013, We demonstrated methylation mapping of DNA molecules specifically in nanoslit devices by using the quantum dots (QDs) combined with methyl-CpG-binding domain protein (MBDp) at a single molecule level. We fabricated nanoslit devices for fully elongation of DNA molecules. We measured the methylation sites using QDs-MBDp complexes in the nanoslit devices at a single molecule level. We found that these methylation sites showed a good agreement with the enzymatic sites of the methyltransferase..
100. Toshihiro Kasama, Yutaka Hasegawa, Hiroyuki Matsumoto, Haruyasu Kondo, Tsutomu Ozawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Establishment of portable immunoassay system for early diagnosis, 2013 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2013 2013 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2013, 10.1109/MHS.2013.6710451, 2013, We have developed immunopillar devices for rapid and easy-to-use immunoassay with pM-fM detection sensitivity, but long total assay time (sample-in-answer-out) and the compatibility with a portable detection system have still remained as major problems toward the point-of-care testing (POCT). We report here next generation immunopillar devices and portable detection system suitable for them. Thin structure of those devices allowed us to wash nonspecifically bound antigens and fluorescent-labeled secondary antibodies in a minute, resulting in the dramatic reduction in the total assay time. In addition, by using the portable detection system, we gained the concentrations of C-reactive protein in human sera while preserving pM detection sensitivity. The total assay time was 20 minutes per sample. Our immunoassay system possesses the potential for use in POCT..
101. Qiong Wu, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, High-speed micro-RNA isolation from DNA fragments by nanopiller array chip, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 1206-1208, 2013, A novel nanopillar chip, which combines pillar structures (nanopillars) and dammed structures (nanoslits) at the nanometer scale inside a microchannel, was fabricated and applied to micro-RNA isolation from a mixture of nucleic acids. Electrophoretic behaviors of micro-RNA and DNA fragments in the nanopillar chip were carefully investigated and the isolation condition was optimized for the mixture of 10-kbp, lambda (48.5-kbp) and T4 DNA (165.5-kbp)..
102. Satoru Ito, Takao Yasui, He Yong, Takeshi Yanagida, Sakon Rahong, Masaki Kanai, Kazuki Nagashima, Hiroshi Yukawa, Noritada Kaji, Tomoji Kawai, Yoshinobu Baba, Nanowire devices for exosomal MicroRNA extraction, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 1887-1889, 2013, We fabricated nanowire devices, which had zinc oxide (ZnO) nanowires embedded in microchannels on PMMA substrate, to isolate exosomes from cell culture supernatant and extract MicroRNA (miRNA) from the isolated exosomes. The exosomes could be trapped by nanowires without any capturing molecules, such as antibodies against the exosomes. The exosomal miRNA could be extracted efficiently with little loss of the miRNA amount, which is comparable to that using a conventional method to extract miRNA from the exosomes..
103. Toyohiro Naito, Ai Yatsuhashi, Noritada Kaji, Taeko Ando, Kazuo Sato, Hisao Moriya, Hiroaki Kitano, Takao Yasui, Manabu Tokeshi, Yoshinobu Baba, Parallel real-time PCR on a chip for genetic tug-of-war (gTOW) method, analytical sciences, 10.2116/analsci.29.367, 29, 3, 367-371, 2013, A microchip-based real-time polymerase chain reaction (PCR) device has been developed for the genetic tug-of-war (gTOW) method that provides quantitative data for research on biorobustness and systems biology. The device was constructed of a silicon glass chip, a temperature controlling Peltier element, and a microscope. A parallel real-time amplification process of target genes on the plasmids and the housekeeping genes in a model eukaryote Saccharomyces cerevisiae were detected simultaneously, and the copy number of the target genes were estimated. The device provides unique quantitative data that can be used to augment understanding of the system-level properties of living cells..
104. Kouhei Ootsuka, Takao Yasui, Noritada Kaji, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Single DNA molecule extraction from single bacterium using nanowire structures, 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013, 479-481, 2013, There are many methods to extract cellular DNA molecule. But these methods need troublesome procedures such as adding lysis reagent and heating samples. However, nanowire embedded microchannel can extract DNA molecule only by injecting cell sample and applying electric field. This nanowire has needle-like shaped and can damage cellular membrane because of physical interaction between the nanowire s and the cellular membrane. We damaged cell and extracted DNA molecules from the damaged-membrane by applying electric field..
105. Daisuke Onoshima, Jun Wang, Michihiko Aki, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba, A deep microfluidic absorbance detection cell replicated from a thickly stacked SU-8 dry film resist mold, Analytical Methods, 10.1039/c2ay26099a, 4, 12, 4368-4372, 2012.12, Dry film resist SU-8 was used to make a thick mold for soft lithography of a poly(dimethylsiloxane) (PDMS) microfluidic chip with deep channels. The stacking of the SU-8 film enabled an ultra-thick (up to 500 μm) resist process on Si wafer. This process was fast and highly reproducible compared with the conventional liquid SU-8 process. The deep channel in the PDMS chip was utilized as a micro-flow cell for sensitive absorbance measurement. Sunset Yellow FCF dye was used to demonstrate absorption spectroscopy in the deep channel. Since the channel depth was proportional to the optical path length, which was proportional to the absorbance value, the PDMS chip achieved a detection limit (15.9 μM) comparable to U- or Z-shaped microfabricated absorbance detection cells in glass. Calibration curves for different solution concentrations were obtained with good R2 values (∼1)..
106. Y. Miyamoto, V. Y. Koshidaka, H. Noguchi, K. Oishi, H. Saito, H. Yukawa, N. Kaji, T. Ikeya, H. Iwata, Y. Baba, K. Murase, S. Hayashi, Polysaccharide functionalized magnetic nanoparticles for cell labeling and tracking
A new three-dimensional cell-array system for toxicity testing, Nanomaterials for Biomedicine, 10.1021/bk-2012-1119.ch009, 191-208, 2012.12, Magnetic resonance imaging (MRI) using contrast agents has been widely used to diagnose vascular diseases, to visualize the internal structure of organs, and to monitor transplanted cells and tissues. We recently developed a novel contrast agent for MRI using polysaccharide-modified magnetic iron oxide nanoparticles. The polysaccharide-magnetic particle complex has an advantage of low toxicity to cells and tissues and slow blood clearance over conventional contrast agents. However, because the surface charge of the particles is negative, transplanted cells such as pancreatic islet cells are less labeled. To overcome this problem, a complex of a polysaccharide and a positively charged magnetic metallic compound has been developed. These newly designed magnetic nanoparticles were efficiently transduced into various cells (for example pancreatic islet cells, MIN6 cells, HepG2 cells, hepatocytes, and somatic stem cells). The present chapter mainly describes the establishment of a cytotoxicity test system using our polysaccharide-based magnetic iron oxide nanoparticles. As a promising cell evaluation system, a new three-dimensional cell culture system "cell-array system" has been established. In the future, the system will be useful as a powerful tool for drug development..
107. Maged F. Serag, Kevin Braeckmans, Satoshi Habuchi, Noritada Kaji, Alberto Bianco, Yoshinobu Baba, Spatiotemporal visualization of subcellular dynamics of carbon nanotubes, Nano Letters, 10.1021/nl3029625, 12, 12, 6145-6151, 2012.12, To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards..
108. Takao Yasui, Yosuke Inoue, Toyohiro Naito, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Inkjet injection of DNA droplets for microchannel array electrophoresis, Analytical chemistry, 10.1021/ac3020565, 84, 21, 9282-9286, 2012.11, We demonstrated DNA droplets could be injected with an inkjet injector for microchannel array electrophoresis and attained high throughput analysis of biomolecules. This injection method greatly reduced both analysis time and sample amount, compared with a conventional microchip electrophoresis method, and allowed high parallelization of a microchannel array on a small substrate. Since we do not need to use complicated electric programs or microchannel design, our injection method should facilitate omics analyses and contribute to high performance clinical assays..
109. Yeon Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Size-selective synthesis of ultrasmall hydrophilic CdSe nanoparticles in aqueous solution at room temperature, Nanoparticles in Biology and Medicine Methods and Protocols, 10.1007/978-1-61779-953-2_9, 125-141, 2012.08, Hydrophilic semiconductor nanoparticles are very attractive for various biological applications, such as in optical sensing, tracing, and imaging of biological molecules-of-interest, because of their broad excitation wavelength, tunable emission wavelength, strong photoluminescence, and relatively high stability against photobleaching and chemicals. Compared to organic phase synthesis and subsequent surface modification, aqueous phase synthesis approaches provide multiple advantages for obtaining hydrophilic semiconductor nanoparticles. Here, we describe methods for the size-selective growth and stabilization of ultrasmall hydrophilic CdSe nanoparticles in aqueous solution at room temperature by using amino acid cysteine or one of its derivatives as a surface capping agent..
110. Yusuke Omoto, Takashi Kato, Norikazu Suzuki, Takao Yasui, Keiko Osato, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Yasuhiko Sakai, Eiji Shamoto, Development of mass-producible rapid mixer based on baker's transformation, Nihon Kikai Gakkai Ronbunshu, B Hen/Transactions of the Japan Society of Mechanical Engineers, Part B, 10.1299/kikaib.78.762, 78, 788, 762-768, 2012.06, We developed a new passive-type lamination mixer for high viscosity fluid with a low Reynolds number, based on the baker's transformation (BT). BT is the best transformation for mixing fluids of laminar flow. However, there was difficulty in mass-producing the BT structure especially for micro devices like MicroTAS, Lab-on-a-Chip and Micro-Reactors, because conventional BT mixers require three-dimensional (3D) piping structures. We have successfully developed the easy-to-massproduce BT mixer by changing that concept of 3D piping structures to 3D channel structures. The 3D channel structures are not easy to produce by photolithography unlike the conventional mixers, while they can be easily mass-produced by molding once their 3D molds are produced. In this report, we newly developed a miniature scale BT mixer to meet the needs for mixing high viscosity fluids in food processing, resin blending, etc. An experiment for mixing different colored hardening silicone elastomers was performed by using the prototype mixer made of aluminum alloy, and the good BT mixing result was obtained, with observing several cross sectional patterns. The numerical fluid analysis also gave similar results of the patterns to those observed in the experiment..
111. Katsuma Kitazoe, Yeon Su Park, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba, Fabrication of functionalized double-Lamellar multifunctional envelope-type nanodevices using a microfluidic chip with a Chaotic mixer array, PloS one, 10.1371/journal.pone.0039057, 7, 6, 2012.06, Multifunctional envelope-type nanodevices (MENDs) are very promising non-viral gene delivery vectors because they are biocompatible and enable programmed packaging of various functional elements into an individual nanostructured liposome. Conventionally MENDs have been fabricated by complicated, labor-intensive, time-consuming bulk batch methods. To avoid these problems in MEND fabrication, we adopted a microfluidic chip with a chaotic mixer array on the floor of its reaction channel. The array was composed of 69 cycles of the staggered chaotic mixer with bas-relief structures. Although the reaction channel had very large Péclet numbers (>105) favorable for laminar flows, its chaotic mixer array led to very small mixing lengths (<1.5 cm) and that allowed homogeneous mixing of MEND precursors in a short time. Using the microfluidic chip, we fabricated a double-lamellar MEND (D-MEND) composed of a condensed plasmid DNA core and a lipid bilayer membrane envelope as well as the D-MEND modified with trans-membrane peptide octaarginine. Our lab-on-a-chip approach was much simpler, faster, and more convenient for fabricating the MENDs, as compared with the conventional bulk batch approaches. Further, the physical properties of the on-chip-fabricated MENDs were comparable to or better than those of the bulk batch-fabricated MENDs. Our fabrication strategy using microfluidic chips with short mixing length reaction channels may provide practical ways for constructing more elegant liposome-based non-viral vectors that can effectively penetrate all membranes in cells and lead to high gene transfection efficiency..
112. Hiroshi Yukawa, Masaki Watanabe, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshitaka Miyamoto, Hirofumi Noguchi, Yoshinobu Baba, Shuji Hayashi, Monitoring transplanted adipose tissue-derived stem cells combined with heparin in the liver by fluorescence imaging using quantum dots, Biomaterials, 10.1016/j.biomaterials.2011.12.009, 33, 7, 2177-2186, 2012.03, Adipose tissue-derived stem cell (ASC) transplantation, when used in combination with heparin, has proven to be an effective treatment for acute liver failure in mice. However, the behavior and organ-specific accumulation of transplanted ASCs alone or in combination with heparin is poorly understood. In this paper, we investigated whether quantum dots (QDs) labeling using octa-arginine peptide (R8) for ASCs could be applied for invivo fluorescence imaging in mice with acute liver failure, and analyzed the behavior and organ-specific accumulation of ASCs that were transplanted alone or in combination with heparin using an IVIS ® Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10min without heparin. However, when heparin was used in combination with the ASCs, the accumulation of the transplanted ASCs was found not only in the lungs but also in the liver. The region of interest (ROI) analysis of exvivo fluorescence imaging showed that the accumulation rate of transplanted ASCs in the liver increased to about 30%. In the time course analysis, the accumulation rate of ASCs in the liver was about 10% in 1 day and was maintained at that level for at least 2 day. We observed that heparin was effective for increasing the accumulation of transplanted ASCs in the liver using fluorescence imaging technology. We suggest that fluorescence imaging by means of QDs labeling using R8 can be useful for tracing the transplanted cells..
113. Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Alberto Bianco, Yoshinobu Baba, The plant cell uses carbon nanotubes to build tracheary elements, Integrative Biology, 10.1039/c2ib00135g, 4, 2, 127-131, 2012.02, Since their discovery, carbon nanotubes (CNTs) have been eminent members of the nanomaterial family. Because of their unique physical, chemical and mechanical properties, they are regarded as new potential materials to bring enormous benefits in cell biology studies. Undoubtedly, the first step to prove the advantages of CNTs is to understand the basic behavior of CNTs inside the cells. In a number of studies, CNTs have been demonstrated as new carrier systems for the delivery of DNA, proteins and therapeutic molecules into living cells. However, post-uptake behavior of CNTs inside the cells has not received much consideration. Utilizing the plant cell model, we have shown in this study that the plant cells, differentiating into tracheary elements, incorporate cup-stacked carbon nanotubes (CSCNTs) into cell structure via oxidative cross-linking of monolignols to the nanotubes surface during lignin biosynthesis. This finding highlights the fate of CNTs inside plant cells and provides an example on how the plant cell can handle internalized carbon nanomaterials..
114. Yoshiyuki Miyazaki, Takao Yasui, Kazumi Inagaki, Yukihiro Okamoto, Noritada Kaji, Tomonari Umemura, Manabu Tokeshi, Yoshinobu Baba, Detection of metallic elements in a single cancer cell using microfluidic devices coupled with ICP-MS, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 767-769, 2012.01, This paper presents a new single cancer cell analysis technique to combine microfluidic devices with inductively coupled plasma-mass spectrometry (ICP-MS). Inherent signals of cancer cells were quantitatively evaluated by launching the cells into ICP-MS via the microfluidic devices..
115. Kouhei Ootsuka, Yukihiro Okamoto, Tetsunari Hase, Manabu Tokeshi, Noritada Kaji, Yoshinori Hasegawa, Yoshinobu Baba, Develpoement of novel circulating tumor cells separation and non-labeling detection by circulating tumor cells' specific properties, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1123-1125, 2012.01, We developed non-labeling detection and easy separation method of circulating tumor cells (CTCs) based on CTCs' specific properties. Our developed method requires no special apparatus and tedious pretreatment before separation and detection of CTCs. In addition, our methods can also separate and detect epithelial cell adhesion molecule (EpCAM) negative CTCs, which cannot be attained by present methods. Therefore, our method would become accurate CTCs' analysis method and useful for point-of-care testing of CTCs..
116. Satoru Ito, Takao Yasui, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Fabrication of PMMA micropillars by reactive ion etching towards separation of white and red blood cells, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1234-1236, 2012.01, Fine, high aspect ratio, and smooth surface micropillars, which never realized by conventional hot-embossing fabrication method, were fabricated on poly(methyl methacrylate) (PMMA) substrates by reactive ion etching (RIE). The separation ability of PMMA micropillars for microbeads as pseudo erythrocytes and leukocytes are comparable to Si micropillars, while the features of PMMA are superior to Si in terms of disposability, cost, and surface treatment..
117. Yukihiro Okamoto, Hitoshi Watanabe, Kazutoshi Kubo, Hiroki Kondo, Noritada Kaji, Manabu Tokeshi, Masaru Hori, Yoshinobu Baba, Graphene forest devices as cell scaffolds for stem cells, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1633-1635, 2012.01, We developed carbon nanowalls devices (CNWs), on which graphenes vertically stand in the nanometer spacing like "graphene forest", with different wettability. CNWs permitted cell adhesion and proliferation, and especially super hydrophobic CNWs enabled easy and less invasive cell collection. Furthermore, collagen coated CNWs successfully enhanced the differential ability of the human mesenchymal stem cells (hMSC) to osteoblast cells compared to collagen coated polystyrene culture dishes. Thus, CNWs have superior many properties as cells scaffolds and are expected to be useful for regenerative medicine..
118. Daisuke Shigenaka, Masami Ukawa, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba, High throughput purification device for gene delivery multifunctional envelope-type nanodevice, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1546-1548, 2012.01, Multifunctional envelope-type nanodevices (MEND) developed by Harashima et al. is one of the novel non-viral DNA vectors expected as a safe gene delivery system. Our group has already attained fast and easy preparation of MEND by using the microfluidic device. However, purification of MEND should be performed in a high-throughput manner because prepared MEND contains impurities such as plasmid-cationic polymer complex. In this paper, we report a novel purification method for MEND by free flow electrophoresis based on microfluidic device, which purification principle is based on the zeta potential difference between MEND and impurities..
119. Yukihiro Okamoto, Tatsuki Sano, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Identification of single molecular DNA methylation points by microfluidic DNA molecule stretching and quantum dot detection, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1939-1941, 2012.01, We report a rapid and simple epigenetic analysis based on microfluidic single DNA molecular methylation detection. We developed a microfluidic device to stretch a single DNA molecule and subsequent detection of a methylated DNA base by quantum dot (QD)-immobilized methyl binding domain (MBD) protein. A QD-MBD complex specifically bound to a methylated base of stretched DNA and was detected by a total internal reflection fluorescence microscope (TIRFM). Our method enables us to detect a number of methylation site and identify the methylated positions simultaneously in the simple and rapid manner compared with the conventional methods..
120. Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Introducing carbon nanotubes into living walled plant cells through cellulase-induced nanoholes, RSC Advances, 10.1039/c1ra00760b, 2, 2, 398-400, 2012.01, Carbon nanotubes can intracellularly transport through different cellular barriers. However, their use in plant cells is limited by the cellulosic cell wall surrounding these cells. Here we show that cup-stacked carbon nanotubes with cellulase immobilized on their sidewalls and tips penetrate the cell wall and transport intracellularly through cellulase-induce nanoholes..
121. Kensuke Ogawa, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Mats Nilsson, Yoshinobu Baba, Label-free detection of real-time DNA amplification using nanowall array structures, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 25-27, 2012.01, We performed label-free detection of real-time DNA amplification inside nanowall array structures, which have never realized in other techniques. The label-free and real-time detection of linear amplification of DNA molecules by circle-to-circle amplification (C2CA) was achieved because our label-free detection system could recognize the length of DNA molecules..
122. Yukihiro Okamoto, Yukinori Nakakita, Takahiro Sano, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Microfluidic rare cancer cell collection with anti-EpCAM antibody modified euglena by phototaxis inside microchannels, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1108-1110, 2012.01, We develop a microfluidic cell separation method which combined microfluidic devices and multifunctional Euglena. Multifunctional Euglena for cell separation was successfully prepared by the surface modification of Euglena cell membrane and its performances were confirmed. Mutlifunctional Euglena in the inlet attached cells, migrated inside the microchannel by phototaxis towards the outlet chamber, and brought specific cells to the outlet. After moving to the outlet, cells in the outlet were detached from Euglena. Since it is based on the unique characters of Euglena, our method needs only simple device and can deal with small to large amount variety kinds of samples including cells..
123. Takao Yasui, Koki Motoyama, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Nanopillar parallel-array structure with DNA trapping and torque-assisted escape mode for DNA separation, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1186-1188, 2012.01, We revealed that the nanopillar parallel-array structure has two modes for DNA separation; DNA trapping and torque-assisted escape mode. Single DNA molecule observation revealed that the separation of DNA molecules could be achieved by two modes in the wide range of DNA molecules; 166 kbp to 100 bp..
124. Mitsuhiro Horade, Yoko Mizuta, Noritada Kaji, Tetsuya Higashiyama, Hideyuki Arata, Plant-on-a-chip microfluidic-system for quantitative analysis of pollen tube guidance by signaling molecule
Towards cell-to-cell communication study, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1027-1029, 2012.01, This paper reports the first observation and quantitative analysis of single cell pollen tube guidance by the signaling molecule realized on the microfluidic device. Design and dimensions of the microfluidic device were optimized for handling individual pollen tube of Torenia Fournieri. Concentration of LURE peptide, the signaling molecule for pollen tube guidance, in the reaction chamber was proved to have appropriate gradient for the guidance. Finally, pollen tube guidance by LURE peptide was successfully observed and analyzed under quantitatively controlled conditions..
125. Jumpei Morikawa, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kazuo Tsubota, Yoshinobu Baba, Plasma separation from human blood using spiral microchannels for dry eye treatment, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 464-466, 2012.01, We demonstrated that spiral microchannels could efficiently separate plasma from whole human blood toward autologous serum eye drop, which provides benefits for all dry eye patients. The optimized spiral microchannels realized 100% separation of 10 μm particles and over 90% separation of blood samples..
126. Toyohiro Naito, Rerngchai Arayanarakool, Noritada Kaji, Séverine Le Gac, Manabu Tokeshi, Albert Van Den Berg, Yoshinobu Baba, A highly efficient 3D micromixer fabricated by standard soft-lithography equipment, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 662-664, 2012, This paper reports a stereolithography-like 3D fabrication method based on soft-lithography techniques. It only requires standard equipment for photolithography, but it makes true 3D structures fabrication possible. We developed a rotating partition by this method in a microfluidic channel, which cannot be achieved by conventional soft-lithography, and demonstrated a prototyping three-dimensional flow mixer..
127. Yeon Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Aqueous phase synthesized CdSe magic-sized clusters
Solution composition dependence of adsorption layer structure, Journal of nanoscience and nanotechnology, 10.1166/jnn.2012.5354, 12, 1, 539-546, 2012, We report dispersion solution composition dependence of the adsorption layer structure and the physical and optical properties of aqueous phase-synthesized semiconductor nanoparticles (NPs). We synthesized cysteine (Cys)-capped CdSe NPs with well-defined core structures, dispersed them in a series of aqueous solutions with different compositions, and then investigated their adsorption layer structure and physical and optical properties. Each CdSe NP consisted of a (CdSe) 33 or (CdSe) 3434 magic-sized cluster (d ∼ 1.45 nm) core, a ligand-Cys shell, and an adsorption layer. The dispersion solution composition strongly affected the adsorption layer structure of the CdSe NPs. The solution with a composition close to that of the as-prepared solution stabilized the physical and optical properties of the NPs. The solution with a composition different from that of the as-prepared solution, however, resulted in large changes in their adsorption layer structure and thus their physical and optical properties. The solution composed of neutral or weakly charged Cys and Cd-Cys complexes led to the adsorption layer with low charge density and that destabilized the NPs. The solution containing only neutral or weakly charged forms of Cys, without Cd-Cys complexes, was favorable to the formation of a thick adsorption layer with low charge density and that destabilized the NPs. The amount of adsorbed Cys in the adsorption layer depended on the dispersion solution composition. However, the amount of adsorbed Cd-Cys complexes in the adsorption layer was almost constant regardless of the dispersion solution composition..
128. Takao Yasui, Yusuke Omoto, Keiko Osato, Noritada Kaji, Norikazu Suzuki, Toyohiro Naito, Yukihiro Okamoto, Manabu Tokeshi, Eiji Shamoto, Yoshinobu Baba, Confocal microscopic evaluation of mixing performance for three-dimensional microfluidic mixer, analytical sciences, 10.2116/analsci.28.57, 28, 1, 57-60, 2012, We developed a confocal microscopic method for a quantitative evaluation of the mixing performance of a three-dimensional microfluidic mixer. We fabricated a microfluidic baker's transformation (MBT) mixer as a three-dimensional passive-type mixer for the efficient mixing of solutions. Although the MBT mixer is one type of ideal mixers, it is hard to evaluate its mixing performance, since the MBT mixer is based on several cycles of complicated three-dimensional microchannel structures. We applied the method developed here to evaluate the mixing of water and a fluorescein isothiocyanate (FITC; diffusion coefficient, 4.9 × 10 -10 m 2 s -1 ) solution by the MBT mixer. This method enables us to capture vertical section images for the fluid distributions of FITC and water at different three-dimensional microchannel structures of the MBT device. These images are in good agreement with those of mixing images based on numerical simulations. The mixing ratio could be calculated by the fluorescence intensity at each pixel of the vertical section image; complete mixing is recognized by a mixing ratio of more than 90%. The mixing ratios are measured at different cycles of the MBT mixer by changing the flow rate; the mixing performance is evaluated by comparisons with the mixing ratio of the straight microchannel without the MBT mixer..
129. Toyohiro Naito, Noritada Kaji, Séverine Le Gac, Manabu Tokeshi, Albert Van Den Berg, Yoshinobu Baba, On-chip electric power generation system from sound of portable music plyers and smartphones towerd portable μTAS, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 1852-1854, 2012, This paper demonstrates electric generation from sound to minimize and integrate microfluidic systems for point of care testing or in-situ analysis. In this work, 5.4 volts and 50 mW DC was generated from sound through an earphone cable, which is a versatile system and able to actuate small size and low power consumption devices like an electro osmotic pump..
130. Miaomiao Sun, Toshihiro Kasama, Noritada Kaji, Shin Ichi Akiyama, Yukio Yuzawa, Seiichi Matsuo, Manabu Tokeshi, Yoshinobu Baba, Rapid urine-based clinical diagnosis of diabetic nephropathy with femto-molar sensitivity by immuno-pillar devices, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 812-814, 2012, We have developed immuno-pillar devices for rapid and easy-to-use immunoassay, but to improve the speed of assay (5-10 min) and the detection sensitivity (nM-pM level) has still remained as major problems toward the clinical applications of immuno-pillar devices. We report here the second-generation immuno-pillar devices with faster assay within 2 min and pM-fM detection sensitivity. New devices enable us to apply them to the clinical trials for the detection of multiple biomarkers (monocyte chemotactic protein 1 (MCP-1), angiotensinogen (AGT), liver-type fatty acid binding protein (L-FABP)) of diabetic nephropathy in human urine without any pretreatments..
131. Takao Yasui, Sakon Rahong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kentaro Doi, Manabu Tokeshi, Satovuki Kawano, Tomoji Kawai, Yoshinobu Baba, Single DNA manipulation in sublithographic nanowire array chips, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 228-230, 2012, We show the feasibility of self-assembled nanowire post-array embedded in niicrochamiels to manipulate the dynamics of single long T4-DNA molecule. DNA molecules in the spot-array of nanowires are fully elongated, and interestingly they exhibit not only the dynamics inside nanopillar array but also that inside gel matrix..
132. Qiong Wu, Koki Motoyama, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba, Sub-millisecond separation of DNA and micro-RNA by nanopillar array chips, 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012, 599-601, 2012, In this study, we fabricated a novel nanostructure that combined the pillar structures (nanopillar) and dammed structures (nanoslit) in nanometer-scale inside microchannels to realize ultra-fast separation. Nanopilliar chips were fabricated by using electron-beam lithography, photolithography and plasma etching. We used the nanopillar chip to realize ultra-fast separation of T4 DNA (165.5 kbp) and microRNA (22 b). By decreasing height down to 100 nm, we succeeded in separating DNA and microRNA in sub-milliseconds..
133. Maged F. Serag, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, A DNA size probe based on energy migration in crosslinked chromatin, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 1, 58-60, 2011.12, DNA sizing has a central role in virtually every aspect of physical genomic analysis. Although capillary gel electrophoresis adequately separates DNA samples, accurate sizing of short sequences is problematic. Here we introduce a simple methodology that affords sizing of short DNA sequences at 2nt resolution via wrapping and linking histone subunits with fluorophore-tagged ssDNA to extract a wrapping and linking ratio that is unique to the reported DNA size..
134. Koki Motoyama, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, DNA trapping at the interface of nanopillar and nanopillar-free regions for long DNA separation, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 647-649, 2011.12, We report a technique of DNA separation based on DNA dynamics at the interface of nanopillar and nanopillar-free regions in nanopillar array chips. We investigated the residence time of DNA molecules at the interface of nanopillar and nanopillar-free regions, and revealed that DNA molecules were trapped at the interface by entirely introducing DNA molecules from nanopillar-free to nanopillar regions. The separation of DNA molecules could be achieved within five seconds based on the difference of residence time of each DNA..
135. Tatsuki Sano, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Epigenetic analysis of a single DNA molecule by microfluidic device with quantum dot, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 1, 299-301, 2011.12, We report a rapid and simple epigenetic analysis based on microfluidic single DNA molecular methylation detection. We developed a microfluidic device to stretch a single DNA molecule and subsequent detection of a methylated DNA base by quantum dot (QD)-immobilized methyl binding domain (MBD) protein. A QD-MBD complex specifically bound to a methylated base of stretched DNA and was detected by a total internal reflection fluorescence microscope (TIRFM). Our method enables us to detect a number of methylation sites and identify the positions methylated simultaneously in the simple and rapid manner compared with the conventional methods..
136. Toyohiro Naito, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Long-time stress less cell culture chips for non-adhesive cells, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 816-818, 2011.12, We present a novel non-adhesive cell culture microdevice that has a loop channel with a micropost array for cell handling. Non-adhesive cells are focused on the upper side in the channel by the tilted micropost array, and they circulate around the same channel while medium was exchange. Our device enables us to culture non-adhesive cells for longer-term without stressful cell trapping..
137. Yukihiro Okamoto, Yukinori Nakakita, Takahiro Sano, Jumpei Morikawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Microfluidic cell separation with antibody modified euglena by using phototaxis mediated migration, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 1909-1911, 2011.12, We develop a microfluidic cell separation method which combined microfluidic devices and multifunctional Euglena. Multifunctional Euglena for cell separation was successfully prepared by the surface modification of Euglena cell membrane and its performances were confirmed. Mutlifunctional Euglena in the inlet attached cells, migrated inside the microchannel by phototaxis towards the outlet chamber, and brought specific cells to the outlet. After moving to the outlet, cells in the outlet were detached from Euglena. Since it is based on the unique characters of Euglena, our method needs only simple device and can deal with small to large amount variety kinds of samples including cells..
138. D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba, Microfluidic visualization of encounter complex in enzymatic digestion of single DNA molecule by dual molecular tagging, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 2061-2063, 2011.12, We combined molecular tagging with microfluidics to study kinematics in a single DNA molecular digestion. Moving tags of both DNA ends revealed initial breakup of a single DNA molecule into fragments induced by limited enzymatic digestion and subsequent rapid formation of transient structure in encounter complex. The method enables us to analyze rapid dissociation of weakly bound complexes derived from intramolecular transfer..
139. Toyohiro Naito, Rerngchai Arayanarakool, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Séverine Le Gac, Albert Van Den Berg, Y. Baba, Temperature-driven self-actuated microvalve for PCR, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 248-250, 2011.12, We report here a novel chamber-sealing valve that is self-actuated by pressure change during the cyclic temperature changes in PCR processes. Actuation of our valve requirs only a heating device employed for PCR. An UV-curable polymer is used as a device material and it allowed us to realize temperature-driven valve actuation as well as to fabricate a 3D device. The self-actuated microvalve achieves the effective sealing of microchamber for PCR even at 90°C, which is essential to develop highly parallel PCR array device without any complicated actuator circuits..
140. Yeon Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Aqueous phase-synthesized small CdSe quantum dots
Adsorption layer structure and strong band-edge and surface trap emission, Journal of Nanoparticle Research, 10.1007/s11051-011-0273-7, 13, 11, 5781-5798, 2011.11, We synthesized, in aqueous solution at room temperature, small water-soluble CdSe quantum dots (QDs) with strong photoluminescence (PL) and then correlated the PL with their adsorption layer structure. For synthesizing the QDs, their initial synthesis condition was controlled to form small Cdcontaining species capable of passivating dangling bonds on the CdSe core surface. Each CdSe QD (d ∼ 2.5 nm) consisted of a CdSe core (d ∼ 2.1 nm), a cysteine (cys)-ligand shell, and an adsorption layer composed of Cd-cys complexes (mainly CdL(-H) -, cys ≡ H 2L), cys (as L 2-), Cd(OH) 2, and CdO x (x ≥ 1). Our CdSe QDs showed strong blue bandedge PL as well as strong green surface trap PL. Their PL quantum yield (QY) of ∼ 18% was unexpectedly high, considering their extremely small core size and their absence of any wide-bandgap inorganic shell. We attributed the QY to their adsorption layer species. The small weakly charged Cd-cys complex and the small neutral cadmium oxides in the adsorption layer could relatively readily diffuse into the unprotected surface sites on the core. These wide-bandgap species coalesced selectively on the unprotected surface sites with minimal spatial disturbance to the preexisting surface Cd-ligand coordination, and passivated them effectively. These decreased nonradiative recombination of the excitons significantly and thus led to the unexpectedly high QYs..
141. Takao Yasui, Mohamad Reza Mohamadi, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Characterization of low viscosity polymer solutions for microchip electrophoresis of non-denatured proteins on plastic chips, Biomicrofluidics, 10.1063/1.3668233, 5, 4, 2011.11, In this paper, we study characteristics of polymers (methylcellulose, hypromellose ((hydroxypropyl)methyl cellulose), poly(vinylpyrrolidone), and poly(vinyl alcohol)) with different chemical structures for microchip electrophoresis of non-denatured protein samples in a plastic microchip made of poly(methyl methacrylate) (PMMA). Coating efficiency of these polymers for controlling protein adsorption onto the channel surface of the plastic microchip, wettability of the PMMA surface, and electroosmotic flow in the PMMA microchannels in the presence of these polymers were compared. Also relative electrophoretic mobility of protein samples in solutions of these polymers was studied. We showed that when using low polymer concentrations (lower than the polymer entanglement point) where the sieving effect is substantially negligible, the interaction of the samples with the polymer affected the electrophoretic mobility of the samples. This effect can be used for achieving better resolution in microchip electrophoresis of protein samples..
142. Yuka Takasaki, Masaki Watanabe, Hiroshi Yukawa, Akhmad Sabarudin, Kazumi Inagaki, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshitaka Miyamoto, Hirofumi Noguchi, Tomonari Umemura, Shuji Hayashi, Yoshinobu Baba, Hiroki Haraguchi, Estimation of the distribution of intravenously injected adipose tissue-derived stem cells labeled with quantum dots in mice organs through the determination of their metallic components by ICPMS, Analytical chemistry, 10.1021/ac202053y, 83, 21, 8252-8258, 2011.11, Adipose tissue-derived stem cells (ASCs) have shown promise in cell therapy because of their ability to self-renew damaged or diseased organs and easy harvest. To ensure the distribution and quantification of the ASCs injected from tail vein, several whole-body imaging techniques including fluorescence optical imaging with quantum dots (QDs) have been employed, but they may suffer from insufficient sensitivity and accuracy. Here, we report quantitative distribution of ASCs in various organs (heart, lung, liver, spleen, and kidney) of mice, which were intravenously injected with QDs-labeled ASCs (QDs-ASCs), through the detection of QDs-derived metallic components by inductively coupled plasma mass spectrometry (ICPMS). For accurate and precise determination, each organ was harvested and completely digested with a mixture of HNO 3 and H 2O 2 in a microwave oven prior to ICPMS measurement, which was equipped with a microflow injection system and a laboratory-made capillary-attached micronebulizer. After optimization, 16 elements including major components (Cd, Se, and Te) of QDs and essential elements (Na, K, Mg, Ca, P, S, Mn, Fe, Co, Cu, Zn, Se, Sr, and Mo) were successfully determined in the organs. As compared to untreated mice, QDs-ASCs-treated mice showed significantly higher levels of Cd and Te in all organs, and as expected, the molar ratio of Cd to Te in each organ was in good agreement with the molar composition ratio in the QDs. This result indicates that the increment of Cd (or Te) can be used as a tracer for calculating the distribution of ASCs in mice organs. As a result of the calculation, 36.8%, 19.1%, 0.59%, 0.49%, and 0.25% of the total ASCs injected were estimated to be distributed in the liver, lung, heart, spleen, and kidney, respectively..
143. Maged F. Serag, Noritada Kaji, Enrica Venturelli, Yukihiro Okamoto, Kazuyoshi Terasaka, Manabu Tokeshi, Hajime Mizukami, Kevin Braeckmans, Alberto Bianco, Yoshinobu Baba, Functional platform for controlled subcellular distribution of carbon nanotubes, ACS nano, 10.1021/nn2035654, 5, 11, 9264-9270, 2011.11, As nanoparticles can cross different cellular barriers and access different tissues, control of their uptake and cellular fate presents a functional approach that will be broadly applicable to nanoscale technologies in cell biology. Here we show that the trafficking of single-walled carbon nanotubes (SWCNTs) through various subcellular membranes of the plant cell is facilitated or inhibited by attaching a suitable functional tag and controlling medium components. This enables a unique control over the uptake and the subcellular distribution of SWCNTs and provides a key strategy to promote their cellular elimination to minimize toxicity. Our results also demonstrate that SWCNTs are involved in a carrier-mediated transport (CMT) inside cells; this is a phenomenon that scientists could use to obtain novel molecular insights into CMT, with the potential translation to advances in subcellular nanobiology..
144. Sonoko Inoue, Noritada Kaji, Masatoshi Kataoka, Yasuo Shinohara, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Rapid qualitative evaluation of DNA transcription factor NF-κB by microchip electrophoretic mobility shift assay in mammalian cells, ELECTROPHORESIS, 10.1002/elps.201100261, 32, 22, 3241-3247, 2011.11, We have developed a separation technique for DNA-protein complex based on electrophoretic mobility shift assay (EMSA) by microchip electrophoresis, which we call microchip electrophoretic mobility shift assay (μEMSA). To evaluate the μEMSA, we employed recombinant human nuclear factor-κB (rhNF-κB) and its consensus double-stranded oligonucleotide (dsOligo) fluorescently labeled with Cy5. We carried out the electrophoretic separation of the consensus dsOligo-rhNF-κB complex and the unbound dsOligo in methylcellulose solution and confirmed rapid (~200s) and reliable identification and semi-quantitation of the specific interaction between dsOligo and rhNF-κB. The binding specificity of rhNF-κB was confirmed by introducing non-fluorescently labeled consensus oligonucleotide as a competitor. The progression of the binding reaction under various incubation times was monitored, and it was found that the dsOligo and rhNF-κB complex formation reached equilibrium (ca. 90% of the dsOligo was bound to rhNF-κB) after 5min. Furthermore, without any purification process, even crude NF-κB in nuclear extracts from HeLa cells was specifically detected within 120s by the μEMSA..
145. Tomoya Tachi, Tetsunari Hase, Yukihiro Okamoto, Noritada Kaji, Takeshi Arima, Hiroyuki Matsumoto, Masashi Kondo, Manabu Tokeshi, Yoshinori Hasegawa, Yoshinobu Baba, A clinical trial for therapeutic drug monitoring using microchip-based fluorescence polarization immunoassay, Analytical and Bioanalytical Chemistry, 10.1007/s00216-011-5304-9, 401, 7, 2301-2305, 2011.10, Microchip analysis is a promising method for therapeutic drug monitoring. This led us to evaluate a microchip-based fluorescence polarization immunoassay (FPIA) system for point-of-care testing on patients being treated with theophylline. The sera were collected from 20 patients being treated with theophylline. Fluorescence polarization was measured on the microchip and theophylline concentrations in serum were obtained. Regression analysis of the correlations was done between the results given by the microchip-based FPIA and the conventional cloned enzyme donor immunoassay (CEDIA), and between the results given by the microchip-based FPIA and the conventional particle-enhanced turbidimetric inhibition immunoassay (PETINIA). We successfully carried out a quantitative analysis of theophylline in serum at values near its therapeutic range in 65 s. The results obtained by the microchip-based FPIA correlated well with CEDIA and PETINIA results; the correlation coefficients (R 2) were 0.986 and 0.989, respectively. The FPIA system is a simple and rapid method for point-of-care testing of drugs in serum, and its accuracy is the same as the conventional CEDIA and PETINIA. It is essential to use real samples from patients and to confirm good correlations with conventional methods for a study on the realization of microchip..
146. Katsuma Kitazoe, Jun Wang, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba, A touch-and-go lipid wrapping technique in microfluidic channels for rapid fabrication of multifunctional envelope-type gene delivery nanodevices, Lab on a Chip, 10.1039/c1lc20392d, 11, 19, 3256-3262, 2011.10, Multifunctional envelope-type gene delivery nanodevices (MENDs) are promising non-viral vectors for gene therapy. Though MENDs remain strong in prolonged exposure to blood circulation, have low immunogenic response, and are suitable for gene targeting, their fabrication requires labor-intensive processes. In this work, a novel approach has been developed for rapid fabrication of MENDs by a touch-and-go lipid wrapping technique in a polydimethylsiloxane (PDMS)/glass microfluidic device. The MEND was fabricated on a glass substrate by introduction of a condensed plasmid DNA core into microfluidic channels that have multiple lipid bilayer films. The principle of the MEND fabrication in the microfluidic channels is based on electrostatic interaction between the condensed plasmid DNA cores and the coated lipid bilayer films. The constructed MEND was collected off-chip and characterized by dynamic light scattering. The MEND was constructed within 5 min with a narrow size distribution centered around 200 nm diameter particles. The size of the MEND showed strong dependence on flow velocity of the condensed plasmid DNA core in the microfluidic channels, and thus, could be controlled to provide the optimal size for medical applications. This approach was also proved possible for fabrication of a MEND in multiple channels at the same time. This on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. Our results strongly indicated that MENDs fabricated with our microfluidic device have a good potential for medical use. Moreover, MENDs fabricated by this microfluidic device have a great potential for clinical use because the devices are autoclavable and all the fabrication steps can be completed inside closed microfluidic channels without any external contamination..
147. Takao Yasui, Noritada Kaji, Mohamad Reza Mohamadi, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Electroosmotic flow in microchannels with nanostructures, ACS nano, 10.1021/nn2030379, 5, 10, 7775-7780, 2011.10, Here we report that nanopillar array structures have an intrinsic ability to suppress electroosmotic flow (EOF). Currently using glass chips for electrophoresis requires laborious surface coating to control EOF, which works as a counterflow to the electrophoresis mobility of negatively charged samples such as DNA and sodium dodecyl sulfate (SDS) denatured proteins. Due to the intrinsic ability of the nanopillar array to suppress the EOF, we carried out electrophoresis of SDS-protein complexes in nanopillar chips without adding any reagent to suppress protein adsorption and the EOF. We also show that the EOF profile inside a nanopillar region was deformed to an inverse parabolic flow. We used a combination of EOF measurements and fluorescence observations to compare EOF in microchannel, nanochannel, and nanopillar array chips. Our results of EOF measurements in micro- and nanochannel chips were in complete agreement with the conventional equation of the EOF mobility (μEOF-channel = αCi-0.5, where Ci is the bulk concentration of the i-ions and α differs in micro- and nanochannels), whereas EOF in the nanopillar chips did not follow this equation. Therefore we developed a new modified form of the conventional EOF equation, μEOF-nanopillar ≈ β[Ci - (Ci 2/Ni)], where Ni is the number of sites available to i-ions and β differs for each nanopillar chip because of different spacings or patterns, etc. The modified equation of the EOF mobility that we proposed here was in good agreement with our experimental results. In this equation, we showed that the charge density of the nanopillar region, that is, the total number of nanopillars inside the microchannel, affected the suppression of EOF, and the arrangement of nanopillars into a tilted or square array had no effect on it..
148. Takao Yasui, Yusuke Omoto, Keiko Osato, Noritada Kaji, Norikazu Suzuki, Toyohiro Naito, Masaki Watanabe, Yukihiro Okamoto, Manabu Tokeshi, Eiji Shamoto, Yoshinobu Baba, Microfluidic baker's transformation device for three-dimensional rapid mixing, Lab on a Chip, 10.1039/c1lc20342h, 11, 19, 3356-3360, 2011.10, We developed a new passive-type micromixer based on the baker's transformation and realized a fast mixing of a protein solution, which has lower diffusion constant. The baker's transformation is an ideal mixing method, but there is no report on the microfluidic baker's transformation (MBT), since it is required to fabricate the complicated three-dimensional (3D) structure to realize the MBT device. In this note, we successfully fabricate the MBT device by using precision diamond cutting of an oxygen-free copper substrate for the mould fabrication and PDMS replication. The MBT device with 10.4 mm mixing length enables us to achieve complete mixing of a FITC solution (D = 2.6 × 10-10 m2 s-1) within 51 ms and an IgG solution (D = 4.6 × 10-11 m2 s-1) within 306 ms. Its mixing speed is 70-fold higher for a FITC solution and 900-fold higher for an IgG solution than the mixing speed by the microchannel without MBT structures. The Péclet number to attain complete mixing in the MBT device is estimated to be 6.9 × 104..
149. Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, DNA separation in nanowall array chips, Analytical chemistry, 10.1021/ac201184t, 83, 17, 6635-6640, 2011.09, A nanowall array structure was fabricated on a quartz chip as a separation matrix of DNA fragments, and a 30 s separation was realized for a mixture of DNA fragments (48.5 and 1 kbp fragments) by applying the electric voltage. A longer DNA fragment migrates faster than a shorter one in a nanowall array chip, and it is completely different from the separation of DNA based on gel electrophoresis, nanopillar chips, and nanoparticle array chips. Although the result is similar to DNA separation by entropic trapping, it could not be fully explained by entropic trapping phenomena. Direct observation of single-DNA molecular dynamics inside a nanowall array structure indicates that both confined elongation and relaxation recoiling of a DNA molecule occur, and an elongated DNA molecule migrates faster than a recoiled DNA molecule. Numerical fitting of DNA molecular dynamics reveals that the balance between times for the transverse of a DNA molecule in the nanowall array chip and the relaxation-recoiling of a DNA molecule governs the separation of DNA..
150. Hiroto Hatakeyama, Erika Ito, Momoko Yamamoto, Hidetaka Akita, Yasuhiro Hayashi, Kazuaki Kajimoto, Noritada Kaji, Yoshinobu Baba, Hideyoshi Harashima, A DNA microarray-based analysis of the host response to a nonviral gene carrier
A strategy for improving the immune response, Molecular Therapy, 10.1038/mt.2011.24, 19, 8, 1487-1498, 2011.08, The purpose of this study was to investigate the host response to systemically administered lipid nanoparticles (NPs) encapsulating plasmid DNA (pDNA) in the spleen using a DNA microarray. As a model for NPs, we used a multifunctional envelope-type nano device (MEND). Microarray analysis revealed that 1,581 of the differentially expressed genes could be identified by polyethylene glycol (PEG)-unmodified NP using a threefold change relative to the control. As the result of PEGylation, the NP treatment resulted in the reduction in the expression of most of the genes. However, the expression of type I interferon (IFN) was specifically increased by PEGylation. Based on the microarray and a pathway analysis, we hypothesize that PEGylation inhibited the endosomal escape of NP, and extended the interaction of toll-like receptor-9 (TLR9) with CpG-DNA accompanied by the production of type I IFN. This hypothesis was tested by introducing a pH-sensitive fusogenic peptide, GALA, which enhances the endosomal escape of PEGylated NP. As expected, type I IFN was reduced and interleukin-6 (IL-6) remained at the baseline. These findings indicate that a carrier design based on microarray analysis and the manipulation of intracellular trafficking constitutes a rational strategy for reducing the host immune response to NPs..
151. Hiroto Hatakeyama, Hidetaka Akita, Erika Ito, Yasuhiro Hayashi, Motoi Oishi, Yukio Nagasaki, Radostin Danev, Kuniaki Nagayama, Noritada Kaji, Hiroshi Kikuchi, Yoshinobu Baba, Hideyoshi Harashima, Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid, Biomaterials, 10.1016/j.biomaterials.2011.02.045, 32, 18, 4306-4316, 2011.06, Previously, we developed a multifunctional envelope-type nano device (MEND) for efficient delivery of nucleic acids. For tumor delivery of a MEND, PEGylation is a useful method, which confers a longer systemic circulation and tumor accumulation via the enhanced permeability and retention (EPR) effect. However, PEGylation inhibits cellular uptake and subsequent endosomal escape. To overcome this, we developed a PEG-peptide-DOPE (PPD) that is cleaved in a matrix metalloproteinase (MMP)-rich environment. In this study, we report on the systemic delivery of siRNA to tumors by employing a MEND that is modified with PPD (PPD-MEND). An in vitro study revealed that PPD modification accelerated both cellular uptake and endosomal escape, compared to a conventional PEG modified MEND. To balance both systemic stability and efficient activity, PPD-MEND was further co-modified with PEG-DSPE. As a result, the systemic administration of the optimized PPD-MEND resulted in an approximately 70% silencing activity in tumors, compared to non-treatment. Finally, a safety evaluation showed that the PPD-MEND showed no hepatotoxicity and innate immune stimulation. Furthermore, in a DNA microarray analysis in liver and spleen tissue, less gene alternation was found for the PPD-MEND compared to that for the PEG-unmodified MEND due to less accumulation in liver and spleen..
152. Jun Wang, Daisuke Onoshima, Michihiko Aki, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Label-free detection of DNA-binding proteins based on microfluidic solid-state molecular beacon sensor, Analytical chemistry, 10.1021/ac200236r, 83, 9, 3528-3532, 2011.05, A solid-state molecular beacon using a gold support as a fluorescence quencher is combined with a polydimethylsiloxane (PDMS) microfluidic channel to construct an optical sensor for detecting single-stranded DNA binding protein (SSBP) and histone protein. The single-stranded DNA-Cy3 probe or double-stranded DNA-Cy3 probe immobilized on the gold surface is prepared for the detection of SSBP or histone, respectively. Due to the different quenching ability of gold to the immobilized single-stranded DNA-Cy3 probe and the immobilized double-stranded DNA-Cy3 probe, low fluorescence intensity of the attached single-stranded DNA-Cy3 is obtained in SSBP detection, whereas high fluorescence intensity of the attached double-stranded DNA-Cy3 is obtained in histone detection. The amounts of SSBP in sample solutions are determined from the degree of fluorescence recovery of the immobilized single-stranded DNA-Cy3 probe, whereas that of histone in sample solutions is determined from the degree of fluorescence quenching of the immobilized double-stranded DNA-Cy3 probe. Using this approach, label-free detection of target proteins at nanomolar concentrations is achieved in a convenient, general, continuous flow format. Our approach has high potential for the highly sensitive label-free detection of various proteins based on binding-induced conformation changes of immobilized DNA probes..
153. Jun Wang, Yong Zhang, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip, Analyst, 10.1039/c0an00434k, 136, 6, 1142-1147, 2011.03, Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method..
154. Maged F. Serag, Noritada Kaji, Claire Gaillard, Yukihiro Okamoto, Kazuyoshi Terasaka, Mohammad Jabasini, Manabu Tokeshi, Hajime Mizukami, Alberto Bianco, Yoshinobu Baba, Trafficking and subcellular localization of multiwalled carbon nanotubes in plant cells, ACS nano, 10.1021/nn102344t, 5, 1, 493-499, 2011.01, Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies..
155. Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Label-free detection of DNA using diffracted laser in nanowall array structures, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 54-56, 2011, We developed a new label-free system using nanowall array structures and realized label-free detection of small molecules and DNA. For the achievement of label-free detection, we used the diffracted laser beam, which was produced by the nanowall array structures when the laser was passed through the nanowall array structures perpendicularly. The diffracted signals showed a good linear relationship between the signal intensity and refractive indices, concentrations of molecules, or DNA length..
156. Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Nanowall array chips for DNA separation, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 638-640, 2011, A 25 s separation of DNA fragments (48.5 and 1 kbp fragments) was achieved by a nanowall array structure, although the separation of 1 kbp and 100 bp DNA fragments was not. Direct observation of a single DNA molecule indicates that it takes over 20 s for an elongated DNA molecule to relax inside the nanowall array structure. Numerical fitting of DNA molecular dynamics reveals that the balance between times for the transverse of an elongated DNA molecule and the relaxation process of a DNA molecule inside the nanowall array structure governs the separation of DNA..
157. K. Iijima, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba, Single molecule enzymatic kinetics in subcellularsized nanospaces using pneumatic valve-assisted atto-liter chamber array devices, 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011, 1092-1094, 2011, We developed the pneumatic valve-assisted atto-liter chamber array devices for quantitative investigation of enzymatic kinetics at the single molecular level. We fabricated chambers in the size ranges from a cell (fL) down to the subcellular organelle (aL) with and without inner-surface coating. The devices enable us to investigate quantitatively the effects of molecular confinement and surface coating on the single molecular enzymatic kinetics even at aL level..
158. K. Kitazoe, Y. Okamoto, N. Kaji, M. Tokeshi, K. Kogure, H. Harashima, Y. Baba, A microdevice with chaotic mixer to constract multifunctional envelope-type nanodevice for delivery system, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 827-829, 2010.12, We report a new microdevice with a chaotic mixer for preparing multifunctional envelope-type nanodevice (MEND) for gene delivery system. Our developed device affords MEND with narrow size distribution, needs no troublesome procedures, and greatly reduces preparation time from 3 hours to 30 minutes compared to conventional MEND preparation method. Therefore, MEND prepared with our microdevice is expected to be applied in clinical use..
159. K. Sugiura, Noritada Kaji, Y. Okamoto, M. Tokeshi, Y. Baba, Development of three-step consolidating microchip for therapeutic drug monitoring, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 806-808, 2010.12, We developed easy-to-use microchip for therapeutic drug monitoring (TDM). Our assay platform consolidates three steps; preparation of reagents, quantitative liquid dispensing of sample and assay. The assay platform uses cloned enzyme donor immunoassay (CEDIA). The structure of the microchip permit liquid reagents and samples to be dispensed quantitatively [1]. Assay reagents were dispensed and lyophilized in the microchip. This microchip would permit one-step and rapid (5 min) assay, requiring only small volumes of reagent and sample (1.5 μl)..
160. Mai Ikami, Ayako Kawakami, Masaya Kakuta, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Immuno-pillar chip
A new platform for rapid and easy-to-use immunoassay, Lab on a Chip, 10.1039/c0lc00241k, 10, 24, 3335-3340, 2010.12, We present a new rapid and easy-to-use immunoassay chip which we have named the immuno-pillar chip. It has hydrogel pillars, fabricated inside a microchannel, with many antibody molecules immobilized onto 1 m diameter polystyrene beads. To evaluate the chip performance, we applied it to the sandwich assay of C-reactive protein (CRP), α-fetoprotein (AFP) and prostate-specific antigen (PSA), a cardiac and inflammation marker, tumors and prostate cancer markers, respectively. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time of about 4 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 L each). Moreover, multiplex assay for three biomarkers was also possible. Additionally, the immuno-pillar chip has a big advantage of having hardly any influence on the assay results even if the introduction quantities of the sample or reagents are different..
161. Kazutoshi Kubo, Yukihiro Okamoto, Masaya Yamamoto, Noritada Kaji, Manabu Tokeshi, Yasuhiko Tabata, Yoshinobu Baba, Influence of nanostructure on proliferation and differentiation processes of stem cell, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 181-183, 2010.12, In this work, we used nanometer sized grooves which fabricated by nano imprinting method combined with a sol-gel process and observed the effect of nanostructure on mesenchymal stem cells (MSCs). MSCs on the nanostructures spread along the grooves in the first stage of proliferation, and then aggregated after differentiation to osteoblasts. In addition, the proliferation rate of MSCs on the nanostructures was suppressed compared to that on the flat surfaces. These results indicate that nanostructures change the environment around MSCs and affect the behavior of MSCs..
162. Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Label-free detection of biomolecules with nanowall arrays, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 485-487, 2010.12, In this paper, we developed a new method for label-free detection of biomolecules by using the nanowall array chips. Our method is based on the detection of diffracted light by nanostructures inside a microchannel, and also has a great potential not only to distinguish the difference between water and buffer solution but detect biomolecules without any fluorescent molecules. Moreover, as for the detection limit, DNA molecules as much as zepto-mole level could be detected by using our label-free method..
163. J. Wang, M. Aki, D. Onoshima, K. Arinaga, N. Kaji, M. Tokeshi, S. Fujita, N. Yokoyama, Y. Baba, Microfluidic sensor for the detection of DNA or protein by hybridization-based fluorescence enhancement or immunoassay-based fluorescence quenching, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 1514-1516, 2010.12, We have developed a novel optical DNA/protein sensor which consists of single-stranded (ss) DNA-Cy3 probes on gold surface and one line-shape polydimethylsiloxane (PDMS) microfluidic channel. This novel ssDNA-Cy3 probes, which does not require hairpin-like stem-loop conformation, show similar advantages as traditional molecular beacon technique in this microfluidic sensor. This chip-based optical sensor showed strong fluorescence enhancement by the binding of cDNA or cDNA-biotin. After introduction of cDNA-biotin, streptavidin can be determined by fluorescence quenching. This sensor showed strong affinity and high sensitivity toward streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 50 amol in this sensor..
164. H. Suzuki, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba, Real-time observation of dna comformational transitions at a single-molecule level by microfluidic devices, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 1880-1882, 2010.12, We have developed a precise fluid control and a solution exchange system by a combination of novel microchannel designs and electroosmotic pumps for real-time observation of DNA conformational transitions at a single-molecule level. By using the microfluidic devices, we revealed the stepwise conformational transitions induced by ethanol solution..
165. D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba, Tracking of single DNA and protein molecules undergoing enzymatic degradation in fluid, 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010, 1439-1441, 2010.12, Here we demonstrate that a molecular motion detector in microfluidic channel can track nucleolytic and proteolytic degradations at a single molecule level. Central to these results is our observation that the microfluidic control worked well to visualize transient molecular processes clearly. A geometric analysis of the time course of molecular trajectories indicated hydrolysis rates and induction periods of each reaction..
166. Yeon Su Park, Andriy Dmytruk, Igor Dmitruk, Atsuo Kasuya, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Aqueous phase synthesized CdSe nanoparticles with well-defined numbers of constituent atoms, Journal of Physical Chemistry C, 10.1021/jp107608b, 114, 44, 18834-18840, 2010.11, We report aqueous phase synthesized semiconductor nanoparticles with well-defined numbers of constituent atoms. Aqueous phase synthesis provides many advantages over organic phase synthesis for producing such high-quality semiconductor nanoparticles. We synthesized CdSe nanoparticles with excellent colloidal and optical stabilities directly in aqueous solution at room temperature and then identified them as selectively grown (CdSe)33 and (CdSe)34 magic-sized clusters. These clusters displayed extremely sharp excitonic absorption and emission peaks because of their practically monodispersed size distribution. Their X-ray diffraction pattern and Raman spectral features were considerably different from the corresponding pattern and features for typical crystalline CdSe nanoparticles. Growth of our magic-sized clusters was very slow and proceeded via the formation of different sizes of progressively larger CdSe nanoparticle intermediates with time. Our results demonstrated that aqueous phase synthetic routes could be successfully adopted for producing high-quality semiconductor nanoparticles..
167. Hiroshi Yukawa, Yukimasa Kagami, Masaki Watanabe, Koichi Oishi, Yoshitaka Miyamoto, Yukihiro Okamoto, Manabu Tokeshi, Noritada Kaji, Hirofumi Noguchi, Kenji Ono, Makoto Sawada, Yoshinobu Baba, Nobuyuki Hamajima, Shuji Hayashi, Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells, Biomaterials, 10.1016/j.biomaterials.2010.01.134, 31, 14, 4094-4103, 2010.05, Quantum dots (QDs) have been used to study the effects of fluorescent probes for biomolecules and cell imaging. Adipose tissue-derived stem cells, which carry a relatively lower donor site morbidity, while yielding a large number of stem cells at harvest, were transduced with QDs using the octa-arginine peptide (R8) cell-penetrating peptide (CPP). The concentration ratio of QDs:R8 of 1 × 104 was optimal for delivery into ASCs. No cytotoxicity was observed in ASCs transduced with less than 16 nM of QDs655. In addition, >80% of the cells could be labeled within 1 h and the fluorescent intensity was maintained at least for 2 weeks. The ASCs transduced with QDs using R8 could be differentiated into both adipogenic and osteogenic cells, thus suggesting that the cells maintained their stem cell potency. The ASCs labeled with QDs using R8 were further transplanted subcutaneously into the backs of mice or into mice through the tail vein. The labeled ASCs could be imaged with good contrast using the Maestro in vivo imaging system. These data suggested that QD labeling using R8 could be utilized for the imaging of ASCs..
168. Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Nanopillar, nanoball, and nanofibers for highly efficient analysis of biomolecules, Chemical Society Reviews, 10.1039/b900410f, 39, 3, 948-956, 2010.02, DNA separation technologies combined with micro- and nanofabrication technologies found a breakthrough in genotyping and DNA sequencing. This tutorial review outlines the fabrication technologies for nano-scaled structures inside microchannels and how the precisely designed structures contribute to obtaining higher performances in DNA separations from the viewpoint of the fabrication process, “top-down” nanofabrication and “bottom-up” molecular self-assembly approaches. It was found that these nanofabricated structures generated the unique separation modes that could not be achieved by random-sized pores of conventional gel or polymer systems. Furthermore, it was found that nanoscale-specific phenomena such as electroosmotic flow should be taken into consideration for further development of nanofabricated structures in DNA analysis. These separation technologies will contribute as a core technology for a future integrated biomolecule anlaysis chip..
169. Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Nano-fabricated structures for biomolecule analysis, Bio-Inspired Nanomaterials and Nanotechnology, 41-58, 2010.01, In this chapter, the recent development of biomolecule analysis, especially biomolecule separation using nano-fabricated structures was reviewed. Fundamental fabrication techniques for micro- and nano-structures on silicon or glass substrates, various approaches for biomolecule separation based on different separation mechanisms, and typical applications such as DNA separation will be included, and practical applications such as DNA separation are described from the aspect of ?nanomaterials and nanotechnology for bio-analytical chemistry..
170. Yeon Su Park, Andriy Dmytruk, Igor Dmitruk, Atsuo Kasuya, Motohiro Takeda, Noriaki Ohuchi, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Size-selective growth and stabilization of small CdSe nanoparticles in aqueous solution, ACS nano, 10.1021/nn901570m, 4, 1, 121-128, 2010.01, Using cysteine and its derivatives as capping molecules, we investigated the influence of the physical structure and chemical nature of capping molecules on the selective growth and stabilization of small CdSe nanoparticles (NPs) in aqueous solution at room temperature. Our investigations revealed specific roles for each functional group of cysteine, and we could correlate this structure and nature of the capping molecules with the size, size restriction, size distribution, and stability of the NPs. For selective growth and stabilization of the NPs in aqueous solution, their capping molecules should have at least one functional group with strong nucleophilicity as well as another free, charged functional group. Capping molecules acting as a monodentate ligand were more effective than those acting as a bidentate ligand for restricting the NPs to a smaller size, whereas the former was less effective than the latter for getting a narrower NP size distribution. Capping molecules with relatively bulky spatial geometry near the ligand-NP interface resulted in the formation of NPs with poor shortand long-term stabilities, whereas those having relatively compact spatial geometry near the interface led to NPs with at least moderate short-term stability. We saw that capping molecules having relatively compact outermost spatial geometry led to NPs with excellent long-term stability, whereas those having relatively bulky outermost spatial geometry produced NPs with at most only moderate long-term stability. Our results clearly showed general trends for the possibility of selective growth of stable semiconductor NPs with particular sizes in aqueous solution..
171. Yasuhiro Hayashi, Kazuaki Kajimoto, Shinya Iida, Yuichiro Sato, Shogo Mizufune, Noritada Kaji, Hiroyuki Kamiya, Yoshinobu Baba, Hideyoshi Harashima, DNA microarray analysis of whole blood cells and insulin-sensitive tissues reveals the usefulness of blood RNA profiling as a source of markers for predicting type 2 diabetes, Biological and Pharmaceutical Bulletin, 10.1248/bpb.33.1033, 33, 6, 1033-1042, 2010, To determine if gene expression profiling of whole blood cells is a useful source of markers for the early diagnosis of the onset of type 2 diabetes, we examined gene expression profiling of whole blood cells and type 2 diabetes-related organs, such as liver, adipose tissue, and skeletal muscle, of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. At the age of 6 weeks, RNA was isolated from tissues of fasted OLETF and control Long-Evans Tokushima Otsuka (LETO) rats. Gene expression was analyzed using the Agilent rat oligo microarray. Gene ontology analysis showed that gene expression of biologically meaningful groups of genes in liver, adipose tissue, and skeletal muscle, which are involved in the pathogenesis of type 2 diabetes, differed between OLETF and LETO rats. Three hundred genes of whole blood cells were differentially expressed. Four out of these 300 genes were related to the insulin-signaling pathway and 57 out of 300 genes were up- or down-regulated in at least one tissues in OLETF rats. These results support our hypothesis that gene expression profiling of whole blood cells might be a useful source of markers to predict the onset of type 2 diabetes..
172. Keisuke Kondo, Noritada Kaji, Sayaka Toita, Yukihiro Okamoto, Manabu Tokeshi, Kazunari Akiyoshi, Yoshinobu Baba, DNA separation by cholesterol-bearing pullulan nanogels, Biomicrofluidics, 10.1063/1.3479997, 4, 3, 2010, We present an application of a novel DNA separation matrix, cholesterol-bearing pullulan (CHP) nanogels, for microchip electrophoresis. The solution of the CHP showed a unique phase transition around 30 mg/ml and formed gel phase over this critical concentration. This gel phase consists of the weak hydrophobic interactions between the cholesterols could be easily deformed by external forces, and thus, loading process of the CHP nanogels into microchannels became easier. The high concentration of the CHP nanogels provided excellent resolutions especially for small DNA fragments from 100 to 1500 bp. The separation mechanism was discussed based on Ogston and Reptation models which had developed in gels or polymer solutions. The result of a single molecule imaging gave us an insight of the separation mechanism and the nanogel structures as well..
173. Kanako Sugiura, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Development of an easy and high-throughput cell assay system with a culture chip and an assay chip, IEEJ Transactions on Sensors and Micromachines, 10.1541/ieejsmas.130.471, 130, 10, 471-475, 2010, High throughput cell assay is significantly important in drug screening, assessment of toxicity etc. Cell assay with a microchip is one of the candidates for high throughput cell assay. However, reported cell assay system with the microchip requires expensive apparatus for refluxing medium and investigation of optimum experimental condition for steady data. For an inexpensive, easy and high throughput cell assay, we introduce a new cell assay system combined with a culture chip and an assay chip made of poly(dimethyl siloxane). Cell culture chips enabled cell to proliferate along the microchannel without refluxing medium and permitted to prepare cell patterning easily. Also, assay chips formed concentration gradient inside the chip and allowed the cell assay with different concentrations of drug at the same time. Thus, our developed cell assay system can overcome the problems of the present cell assay and would promote the drug discovery, assessment of toxicity etc..
174. Yousuke Inoue, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Luminescence of cup-stacked carbon nanotubes and its application to microchip electrophoresis, bunseki kagaku, 10.2116/bunsekikagaku.58.517, 58, 6, 517-521, 2009.12, Microchip electrophoresis (MCE) is one of the most suitable methods for biomolecular separation and analysis because of its superior characteristics. In biomolecular separation and analysis, laser induced fluorescent (LIF) detection is frequently employed in MCE and attains high sensitive detection. However, LIF detection requires the labeling of analytes, which contains troublesome procedures and results in decreasing of the separation efficiency. In addition, the labeling reaction at low concentration is significantly difficult, and thus true high sensitive detection is not easily attained and has been desired. Here, we report on the possibility of cup-stacked carbon nanotubes (CSCNTs) for high sensitive label-free detection and high separation efficiency. At first we investigated fluorescence properties of CSCNTs. Our investigation revealed that supernatant solution of CSCNTs after centrifugation had fluorescence at around 500 nm, while CSCNTs suspension did not. The application of CSCNTs for MCE enabled us to successfully separate and detect DNA without labeling it. Compared to the direct detection method, our method resulted in poor separation efficiency. However, by controlling of the CSCNTs' aspect ratio and the immobilization of functional molecules on the CSCNTs surfaces, we could improve separation efficiency and attain the separation of various samples without labeling samples..
175. Jun Wang, Yong Zhang, Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Exceeding 20 000-fold concentration of protein by the on-line isotachophoresis concentration in poly(methyl methacrylate) microchip, ELECTROPHORESIS, 10.1002/elps.200900111, 30, 18, 3250-3256, 2009.09, In this research, a simple on-line microchip gel electrophoresis with ITP was applied for the concentration and separation of BSA and its immunoassay complex with mAb in a single cross form PMMA microchip. We investigated the ITP concentration effect in PMMA MCE using combination of leading electrolytes, terminating electrolytes and other factors. We realized an ITP-based concentration and separation of BSA and its immunoassay complexes in standard cross-channel microchip gel electrophoresis, which exceeded 2000-fold concentration of BSA immunocomplex using Tris-H3PO4 as a leading electrolyte and Tris-γ-amino butyric acid as a terminating electrolyte. In addition, we also realized concentration of BSA sample in water, which was more than 20 000-fold and was the result of the concentration effect from combining ITP and the sample stacking techniques..
176. Fuquan Dang, Takeshi Hasegawa, Vasudevanpillai Biju, Mitsuru Ishikawa, Noritada Kaji, Takao Yasui, Yoshinobu Baba, Spontaneous adsorption on a hydrophobic surface governed by hydrogen bonding, Langmuir, 10.1021/la900850u, 25, 16, 9296-9301, 2009.08, Spontaneous adsorption from solution onto solid surface is a common phenomenon in nature, but the force that governs adsorption is still a matter of considerable debate.1,2 We found that surfactants and cellulose adsorb from solution onto a poly(methyl methacrylate) (PMMA) surface in an ordered and cooperative way governed by hydrogen bonding. The glucose rings of n-dodecyl-β-D-maltoside (DDM) and hydroxyethylcellulose (HEC) stand perpendicular to the surface, H-bond to the surface COOMe groups with their C=O and Me-O bonds parallel to the surface, and form a tight monolayer. The non-H-bonded COOMe groups orient their C=O bonds perpendicular to the surface. In contrast, the glucose rings of hydrophobically modified hydroxyethylcellulose (HMHEC) lie flat with the side chains perpendicular to the surface and H-bond to the perpendicular-oriented C=O groups. The non-H-bonded COOMe groups orient their C=O bonds parallel but Me-O bonds near-perpendicular to the surface for stabilizing HMHEC. The current work provides a detailed picture of how surface-active molecules interact with a solid surface and selfassemble into greatly different architectures..
177. Masaya Murata, Yukihiro Okamoto, Yeon Su Park, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Cell separation by the combination of microfluidics and optical trapping force on a microchip, Analytical and Bioanalytical Chemistry, 10.1007/s00216-009-2648-5, 394, 1, 277-283, 2009.05, We investigated properties of cells affecting their optical trapping force and successfully established a novel cell separation method based on the combined use of optical trapping force and microfluidics on a microchip. Our investigations reveal that the morphology, size, light absorption, and refractive index of cells are important factors affecting their optical trapping force. A sheath flow of sample solutions created in a microchip made sample cells flow in a narrow linear stream and an optical trap created by a highly focused laser beam captured only target cells and altered their trajectory, resulting in high-efficiency cell separation. An optimum balance between optical trapping force and sample flow rate was essential to achieve high cell separation efficiency. Our investigations clearly indicate that the on-chip optical trapping method allows high-efficiency cell separation without cumbersome and time-consuming cell pretreatments. In addition, our on-chip optical trapping method requires small amounts of sample and may permit high-throughput cell separation and integration of other functions on microchips..
178. Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Simultaneous separation, metering, and dilution of plasma from human whole blood in a microfluidic system, Analytical chemistry, 10.1021/ac802434z, 81, 8, 3194-3198, 2009.04, In about a 3 min period, we have simultaneously separated plasma from human whole blood and metered and diluted the plasma using a microchip with an interchannel microstructure. The plasma separation was based on both cross-flow filtration and sedimentation of red blood cells in the microchannels. Metering and diluting operations of the plasma were based on volume control of liquid in the microchannels by syringe pumps. On this microchip, we produced plasma diluted by a factor of 6 from whole blood containing theophylline and we observed very little hemolysis. It is possible to separate plasma from one or just several drops of whole blood by using this microchip..
179. Yong Zhang, Jun Wang, Yukihiro Okamoto, Manabu Tokeshi, Noritada Kaji, Yoshinobu Baba, Velocity gap theory developed for magnifying resolutions without changing separation mechanisms or separation lengths, Analytical Chemistry, 10.1021/ac802671m, 81, 7, 2745-2750, 2009.04, Separation techniques, such as chromatography and electrophoresis, form the basis in many fields and are continually developed for better separation efficiency. The efforts normally involve a new mechanism together with sufficient separation length. We develop a velocity gap theory to make things simple. The theory is based on the discovery that the velocity gap (VG) effect could enlarge the distance between two moving objects. Mathematical deduction certified that the resolution may be magnified infinitely without changing the separation mechanism or the separation length. DNA separation confirmed its practical feasibility by achieving 2-5 times higher resolution on a microchip. Our results indicate that VG effect could enlarge the distance between two moving objects and may potentially be utilized to ameliorate separation efficiency..
180. Katsuma Kitazoe, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba, A new microdevice to rapidly constract Multifunctional Envelope-type Nanodevice for gene therapy, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1267-1269, 2009.01, Multifunctional Envelope-type Nanodevice (MEND) is a promising gene delivery system. However, the application of MEND for the medical use has been prevented due to labor-intensive and time-consuming procedures in the preparation of MEND. In this paper, we report a microfluidic device which realizes to prepare MEND with narrow size distribution in 5 min (the conventional method needs 2 days). Our results strongly indicate that MEND prepared with our microfluidic device has a potential for medical use..
181. Hiroshi Suzuki, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, A single molecule analysis for the conformational transition of DNA using microfluidics, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 827-829, 2009.01, We have developed a real time monitoring system of DNA conformational transition at a single molecule level by using a microfluidic device. We revealed that the conformational transition process of a DNA induced by ethanol solution is totally depending on the concentration of ethanol..
182. Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Electroosmotic flow in nanopillar array structures, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 174-176, 2009.01, In this paper, we found that ions in nanospace would be concentrated according to electric field gradient by using nanopillar array structures. Due to the ion-enrichment inside the nanopillar region, electroosmotic flow (EOF) in the nanopillar chip was suppressed dramatically. And also, we revealed that EOF suppression by ion-enrichment generated EOF profile deformation from a plug flow to an inverse parabolic flow..
183. Yukihiro Okamoto, Yousuke Inoue, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, High-throughput microchip electrophoresis analysis with inkjet-injection method, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 794-796, 2009.01, We established an inkjet injection method for robust microchip electrophoresis (MCE) separation and attained high-throughput analysis of biomolecules by automated inkjet injector for microchannel array. This injection method greatly reduces both analysis time and sample amount compared with conventional microchip electrophoresis, and allows us to realize highly parallelization of microchannel array on a smaller chip, since we do not need to use complicated arrays of cross channel sample injection [1]. Our method would facilitate omics and contribute to the highperformance clinical assay..
184. Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Microchip-based homogeneous immunoassay using fluorescence polarization spectroscopy, Lab on a Chip, 10.1039/b813640h, 9, 7, 966-971, 2009.01, We have realized fluorescence polarization immunoassay (FPIA) on a microchip in about 1 minute. FPIA is a homogeneous competitive immunoassay which is based on measuring fluorescence polarization after competitive binding of an analyte and a tracer to an antibody. We constructed a microfluidic FPIA system composed of a newly designed microchip, a laser, a CCD camera and an optical microscope with two specially installed polarizers - one fixed and one rotatable. Theophylline, a typical small drug molecule, was used as a model analyte. Theophylline and fluorescence-labeled theophylline were introduced through different inlets and combined in a 100 m-wide microchannel where anti-theophylline antibody was added. To optimize the microchip design for FPIA, we investigated the diffusion time of theophylline and the mixing time of theophylline and antibody in this channel, which were 6 s and 36 s, respectively. We successfully carried out a quantitative analysis of theophylline in serum near the therapeutic range in 65 s. In FPIA, a larger tracer-antibody complex emits more polarized fluorescence than the tracer, and therefore, by increasing the antigen concentration in a sample, more polarization relaxation is observed since the tracer-antibody complex concentration is decreased and the tracer concentration is increased. Tracer binding to an antibody is directly measured by spectroscopic techniques without any separation process. This microchip-based FPIA is very simple and rapid, unlike microchip-based heterogeneous immunoassay, because it does not require several processes such as washing and reflowing and immobilizing of antibodies or antigens in the channel. In the future, microchip-based FPIA should find frequent use for point-of-care testing in the clinical field, where conventional FPIA has been used for laboratory tests..
185. Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba, Non-equilibrium DNA transport in nanopillar array structures, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1198-1200, 2009.01, In this paper, a novel DNA separation mechanism by nanopillar array structures was elucidated via single DNA molecule observation. We revealed that this new separation mechanism was based on non-equilibrium DNA transport under high electric field conditions. In fact, 10 seconds DNA separation was achieved without any loss of resolution..
186. Jun Wang, Yong Zhang, Yeon Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Poly (methyl methacrylate) microchip gel electrophoresis for the concentration and separation of aptamer and aptamer-thrombin complex with discontinuous buffers, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 791-793, 2009.01, A simple continuous isotachophoresis (ITP)-based microchip gel electrophoresis (MGE) was firstly applied for the concentration and separation of aptamer and its thrombin complex in a single cross-form poly (methyl methacrylate) (PMMA) microchip. The aptamer and its thrombin complex can be simultaneously concentrated and separated by this method which does not require time-consuming steps and complicated chip design. We found that different terminating electrolyte (TE) ions lead to different concentration enhancement factors and that lower mobility of TE ions can get higher concentration results. After optimization of various impact factors, we successfully achieved about 2000-fold concentration of aptamer-thrombin complex..
187. Mitsuru Shibata, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Precisely size controlled liposome formation with a microfluidic device, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 624-626, 2009.01, We greatly improved our previous liposome preparation method [1] in terms of the flow direction and liposome solvent, and could prepare precisely size controlled liposomes (97-139 nm in diameter) with narrow size distribution (2.9-6.5% CV) easily and rapidly, which cannot be attained with conventional bulk methods and our previous one. Liposomes with specific sizes and narrow size distribution would greatly enhance drug delivery and gene therapy efficacy..
188. Kiichi Sato, Keisuke Aono, Noritada Kaji, Yoshinobu Baba, Etsuro Yoshimura, Rapid two-step Southern hybridization analysis using a microchip, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1862-1864, 2009.01, Microchip-based Southern hybridization analysis system was developed, which consists of DNA separation by microchip electrophoresis and successive hybridization in a micro square space. The system realized both DNA separation by size and identification by hybridization with simple operations and short analysis time..
189. Yong Zhang, Jun Wang, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Velocity gap theory developed for magnifying resolutions without changing separation mechanisms or separation lengths, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 788-790, 2009.01, We developed velocity gap theory for enhancing separation resolutions. The theory is based on the discovery that velocity gap (VG) effect could enlarge the distance between two moving objects. DNA separation confirmed its practical feasibility by achieving 2-5 times higher resolution on a microchip. Our results indicate that VG effect could enlarge the distance between two moving objects and may potentially be utilized to ameliorate separation efficiency..
190. Daisuke Onoshima, Jun Wang, Michihiko Aki, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba, A simple and low-cost technique for fabricating microchannels and microstructures with high aspect ratio, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1587-1589, 2009, We developed a simple method for creating high-aspect-ratio (> 4) structures with sharp edge by using the lamination forming of a commercially available dry film photoresist in a conventional photolithography process. This type of fluidic structure is attractive to achieve a good separation and an ultra-sensitive detection of the liquid in microchannels readily..
191. Kazuki Iijima, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Atto- and femto-litre microfabrcated structures for investigating depression of enzyme activity at a single molecule reaction, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 854-856, 2009, In this work, we successfully measured a single enzyme reaction in a microfabricated chamber -(510 aL, 7.2 fL, 61 fL and 624 fL) and estimated the effect of enzyme adsorption and denaturation on the reaction rate with inner-surface coated and non-coated chambers. Our results indicate that a single enzyme reaction rate decreased with decreasing the chamber size and that reaction rate in coating chambers slightly increased compared to that in non-coated one..
192. Kanako Sugiura, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Easy cell assay device using two microchannels, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1243-1245, 2009, We developed a new cell assay device. The assay device consists of two microchennels; a culture channel and a assay channel. In the culture channel, cells proliferated without continuous medium exchange and formed spots in line. And in the assay channel, a concentration gradient of reagent was formed by diffusion and the observation of cell spots permits cell assays with different concentration reagent at the same time. This device would permit easy and high throughput cell assay..
193. Ayako Kawakami, Mai Ikami, Yukihiro Okamoto, Noritada Kaji, Jin Wanchun, Keiko Yamada, Michio Ohata, Hidekatsu Tazawa, Tomohiko Ebata, Manabu Tokeshi, Yoshinobu Baba, Immuno-pillar chip
Multiplex detection of proteins in real samples, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 436-438, 2009, We present a multiplex detection of Staphylococcus aureus enterotoxins (SEA, SEB and SED) in milk and disease markers (α-fetoprotein (AFP), C-reactive protein (CRP) and prostate-specific antigen (PSA)) in whole blood using immuno-pillar chips within 12 min or less..
194. Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Microchip-based homogeneous immunoassay using a cloned enzyme donor, analytical sciences, 10.2116/analsci.25.149, 25, 2, 149-151, 2009, We have realized a cloned enzyme donor immunoassay (CEDIA) on a microchip in 96 s. CEDIA is a homogeneous immunoassay, based on the bacterial enzyme β-galactosidase, which was genetically engineered into two inactive fragments: an enzyme donor and an enzyme acceptor. A model analyte was theophylline, and the detectable concentration range was from 0 to 40 μg mL -1 . Our CEDIA using a microfluidic device was very simple and rapid, unlike microchipbased heterogeneous immunoassays and CEDIA on a well-type microchip..
195. Toyohiro Naito, Ai Yatsuhashi, Noritada Kaji, Taeko Ando, Kazuo Sato, Hisao Moriya, Hiroaki Kitano, Yukihiro Okamoto, Manabu Tokeshi, Yosinobu Baba, On-chip real-time PCR for genetic tug-of-war (g-TOW) experiment, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 627-629, 2009, A micro-chip based real-time PCR device has been developed for biorobustness research and systems biology. The device is constructed of a silicon-glass chip, a Peltier element and a microscopy. With the device, amplification of target gene on a plasmid and housekeeping gene were detected simultaneously, and the copy number of the plasmid could be estimated. We had optimized the device, and achieved detection of fluorescent intensity increase in reaction channels..
196. Hiroshi Yukawa, Shogo Mizufune, Chiharu Mamori, Yukimasa Kagami, Koichi Oishi, Noritada Kaji, Yukihiro Okamoto, Manabu Takeshi, Hirofumi Noguchi, Yoshinobu Baba, Michinari Hamaguchi, Nobuyuki Hamajima, Shuji Hayashi, Quantum dots for labeling adipose tissue-derived stem cells, Cell Transplantation, 10.1177/096368970901805-615, 18, 5-6, 591-599, 2009, Adipose tissue-derived stem cells (ASCs) have a self-renewing ability and can be induced to differentiate into various types of mesenchymal tissue. Because of their potential for clinical application, it has become desirable to label the cells for tracing transplanted cells and for in vivo imaging. Quantum dots (QDs) are novel inorganic probes that consist of CdSe/ZnS-core/shell semiconductor nanocrystals and have recently been explored as fluorescent probes for stem cell labeling. In this study, negatively charged QDs655 were applied for ASCs labeling, with the cationic liposome, Lipofectamine. The cytotoxicity of QDs655-Lipofectamine was assessed for ASCs. Although some cytotoxicity was observed in ASCs transfected with more than 2.0 nM of QDs655, none was observed with less than 0.8 nM. To evaluate the time dependency, the fluorescent intensity with QDs655 was observed until 24 h after transfection. The fluorescent intensity gradually increased until 2 h at the concentrations of 0.2 and 0.4 nM, while the intensity increased until 4 h at 0.8 nM. The ASCs were differentiated into both adipogenic and osteogenic cells with red fluorescence after transfection with QDs655, thus suggesting that the cells retain their potential for differentiation even after transfected with QDs655. These data suggest that QDs could be utilized for the labeling of ASCs..
197. Tomoya Tachi, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba, Simultaneous separation, metering and dilution of plasma from human whole blood using a microchip with an interchannel microstructure, 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2009 Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 427-429, 2009, In this research, we designed and operated a microchip with an interchannel microstructure for separation of plasma from human whole blood and for metering and diluting the plasma. The plasma separation was based on both cross-flow filtration and sedimentation of red blood cells in the microchannels. Metering and diluting the plasma was based on volume control of liquid in the microchannels by syringe pumps. Simultaneous operations to separate, meter and dilute plasma obtained from whole blood were done in microchannels using this microchip, and plasma precisely diluted by a factor of 6 was generated without hemolysis in the microchannels in about 3 minutes. This microchip is easily connected with other microchannels and microfluidic devices for quantitative analyses..
198. Daisuke Kuroda, Yong Zhang, Jun Wang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, A viscosity-tunable polymer for DNA separation by microchip electrophoresis, Analytical and Bioanalytical Chemistry, 10.1007/s00216-008-2196-4, 391, 7, 2543-2549, 2008.08, A thermo-responsive separation matrix, consisting of Pluronic F127 tri-block copolymers of poly(ethylene oxide) and poly(propylene oxide), was used to separate DNA fragments by microchip electrophoresis. At low temperature, the polymer matrix was low in viscosity and allowed rapid loading into a microchannel under low pressure. With increasing temperatures above 25°C, the Pluronic F127 solution forms a liquid crystalline phase consisting of spherical micelles with diameters of 17-19 nm. The solution can be used to separate DNA fragments from 100 bp to 1500 bp on poly(methyl methacrylate) (PMMA) chips. This temperature-sensitive and viscosity-tunable polymer provided excellent resolution over a wide range of DNA sizes. Separation is based on a different mechanism compared with conventional matrices such as methylcellulose. To illustrate the separation mechanism of DNA in a Pluronic F127 solution, DNA molecular imaging was performed by fluorescence microscopy with F127 polymer as the separation matrix in microchip electrophoresis..
199. Hiroshi Kuramoto, Yeon Su Park, Noritada Kaji, Manabu Tokeshi, Kentaro Kogure, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba, On-chip fabrication of mutifunctional envelope-type nanodevices for gene delivery, Analytical and Bioanalytical Chemistry, 10.1007/s00216-008-2124-7, 391, 8, 2729-2733, 2008.08, Microfluidic devices may be highly beneficial to the rapid fabrication of small quantities of various nonviral vectors with different functionalities, which is indispensable for effective order-made gene therapy. We adapted a microfluidic chip-based approach for fabricating small quantities of nonviral vectors in a short time in preparation for order-made gene therapy applications. This approach permitted us to fabricate multifunctional envelope-type nanodevices (MENDs), composed of a compacted (or condensed) DNA core and a lipid bilayer membrane shell, which are considered as promising nonviral vectors for gene therapy applications. The on-chip fabrication of the MEND was very simple, rapid, convenient, and cost-effective compared with conventional methods. The size of the MEND showed strong dependence on the concentration and flow rate of the reaction precursors and could be controlled to be much smaller than that achievable by conventional methods. This, together with abovementioned merits, makes our microfluidic chip-based approach very attractive for the fabrication of MENDs for effective application to order-made gene therapy..
200. Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Highly sensitive double-fluorescent dye staining on microchip electrophoresis for analysis of milk proteins, ELECTROPHORESIS, 10.1002/elps.200700775, 29, 12, 2533-2538, 2008.06, We demonstrated a highly sensitive double-fluorescent dye staining in microchip electrophoresis (ME) for analysis of milk proteins. The detection sensitivity of ME was very limited so far and needed improvement. Our staining method consisted of two steps. First, in sample preparation before electrophoresis, protein was covalently bound to an amine-reactive fluorescent dye, Cy5. Then, the Cy5-attached protein was denatured with SDS and was further stained during electrophoresis, with Agilent fluorescent dye, which was non-covalently attached to hydrophobic regions of the SDS-protein complexes. This double-fluorescent staining enhanced fluorescent intensity and lowered the detection limit to 200 pg of protein. This provided higher sensitivity than silver- or SYPRO Ruby-staining methods, which have previously given the highest sensitivity in protein staining. In addition, we applied our staining method to analysis of milk proteins and achieved their successful detection, whereas it was difficult to analyze them by the unimproved method..
201. Maged Fouad, Mohammad Jabasini, Noritada Kaji, Kazuyoshi Terasaka, Manabu Tokeshi, Hajime Mizukami, Yoshinobu Baba, Microchip analysis of plant glucosinolates, ELECTROPHORESIS, 10.1002/elps.200700635, 29, 11, 2280-2287, 2008.06, We describe a new and selective analytical method for the separation and quantitation of plant glucosinolates. The new method, which utilizes microchip CE (μ-CE) with fluorescence detection, circumvents the multistep procedures characteristic of conventional methods. Glucosinolates form charge transfer complexes with the xanthene dyes phloxine-B and eosin-B. The glucosinolates-phloxine-B complex cannot be excited at 470 nm. Thus, the decrease in peak intensity of phloxine-B after complex formation is used to quantitatively measure total glucosinolates in Arabidopsis thaliana seeds. For qualitative analysis, complex formation with eosin-B is used. The sensitivity of eosin-B detection at excitation/emission 470 nm/540 nm was low. However, sensitivity increased following complex formation with sinigrin (≥3 μg/mL). A batch-learning, self-organizing map was applied to visualize and organize analytical data into 2-D matrix with similar and related data clustered together or near each other. This organized matrix was used to optimize electrophoretic conditions for the analysis. This study suggests potential applications of μ-CE in plant metabolomics analyses without use of labeling fluorophores..
202. Eiki Maeda, Masatoshi Kataoka, Shouki Yatsushiro, Kazuaki Kajimoto, Mami Hino, Noritada Kaji, Manabu Tokeshi, Mika Bando, Jun Ichi Kido, Mitsuru Ishikawa, Yasuo Shinohara, Yoshinobu Baba, Accurate quantitation of salivary and pancreatic amylase activities in human plasma by microchip electrophoretic separation of the substrates and hydrolysates couples with immunoinhibition, ELECTROPHORESIS, 10.1002/elps.200700688, 29, 9, 1902-1909, 2008.05, A high-performance determination system for α-amylase isoenzyme activities in human plasma involving microchip electrophoresis with a plastic chip was developed. The combination of microchip electrophoresis for substrate and hydrolysate separation and an immunoinhibition method for the differentiation of isoenzyme activities using antihuman salivary amylase (S-AMY) mAb allowed the highly selective determination of amylase isoenzyme (S-AMY and pancreatic amylase (P-AMY)) activities even in a complex matrix such as a crude plasma sample. We used 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltohexaose (G6) as a substrate. Amylase in a human plasma sample hydrolyzed APTS-G6 into APTS-maltotriose (G3) and G3, which was measured as the fluorescence intensity of APTS-G3 on microchip electrophoresis. A double logarithm plot revealed a linear relatioship between amylase activity and fluorescence intensity in the range of 5-500 U/L of amylase activity (r2 = 0.9995, p<0.01), and the LOD was 4.38 U/L. Amylase activities in 13 subjects determined by the present method were compared with the results obtained by conventional methods with nitrophenylated oligosaccharides as substrates, respectively. Good correlations were observed for each method on simple linear regression analysis (both p<0.01). The reproducibilities of within-days for total amylase and P-AMY were 2.98-6.27 and 3.83-6.39%, respectively, and these between-days were 2.88-5.66 and 3.64-5.63%, respectively. This system enables us to determine amylase isoenzyme activities in human plasma with high sensitivity and accuracy, and thus will be applicable to clinical diagnosis..
203. Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Rinse and evaporation coating of poly(methyl methacrylate) microchip for separation of sodium dodecyl sulfate-protein complex, Journal of Chromatography A, 10.1016/j.chroma.2008.03.012, 1192, 2, 289-293, 2008.05, We developed a novel channel wall coating on a poly(methyl methacrylate) (PMMA) microchip using methylcellulose (MC) as a coating reagent to suppress electroosmotic flow (EOF) following the strong analytes adsorption via hydrophobic interaction with channel walls of PMMA. Our coating was obtained by first rinsing channel walls with MC-containing aqueous solution followed by evaporation. The coating made the hydrophilic channel wall lowering EOF by two orders of magnitude (1.2 × 10-5 cm2 V-1 s-1) as well as reducing the hydrophobic adsorption. On the coated channel walls, we successfully separated sodium dodecyl sulfate-protein complexes with high reproducibility and efficiency using dextran as a lower viscosity protein separation medium..
204. Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba, Rolling circle amplification and circle-to-circle amplification of a specific gene integrated with electrophoretic analysis on a single chip, Analytical chemistry, 10.1021/ac702289j, 80, 7, 2483-2490, 2008.04, We have developed an integrated platform for rolling circle amplification (RCA) and circle-to-circle amplification (C2CA) of circular probe (padlock probe) and subsequent microchip electrophoretic detection of a specific gene on a poly(methyl methacrylate) microchip. RCA and C2CA were successfully carried out at a steady temperature of 37°C in the sample well of the microchip, and their respective product was detected on the same channel of the microchip, which was prefilled with a polymer separation matrix and fluorescent dye. Using a species-specific padlock probe for bacterial pathogen V. cholerae, a 25-ng bacterial genomic DNA could be detected in less than 65 min (including RCA and microchip electrophoresis) by this platform. Stable dsDNA C2CA product of genomic DNA for V. cholerae can be detected with the introduced integrated platform. Furthermore, the usefulness of this technique for the monitoring of RCA was demonstrated. This integrated platform provides a sensitive, fast, high-throughput, and reproducible method for signal amplification and detection of the padlock probes in the same microchip and is a promising tool for highly specific gene detection strategies..
205. Laili Mahmoudian, Jonas Melin, Mohamad Reza Mohamadi, Keiko Yamada, Michio Ohta, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba, Microchip electrophoresis for specific gene detection of the pathogenic bacteria V. cholerae by circle-to-circle amplification, analytical sciences, 10.2116/analsci.24.327, 24, 3, 327-332, 2008.03, We have developed a new method for a fast and precise analysis of circle-to-circle amplification (C2CA) product for specific gene detection by microchip electrophoresis. In this method, we have added a new enzymatic step to the C2CA reaction, which could be carried out isothermally at 37°C. Compared to the original single-stranded DNA, the double-stranded DNA that is produced by this enzymatic reaction is more reliable for analysis by microchip electrophoresis. C2CA product was detected within 55 s with high reproducibility by this method which was successfully applied to the detection of 10-ng genomic DNA of the pathogenic bacteria Vibrio. cholerae within 110 s. Purification was found to be an indispensable step for the analysis of the C2CA product of genomic DNA samples. 2008.
206. Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Poly(methylmethacrylate) microchip electrophoresis of proteins using linear-poly(acrylamide) solutions as separation matrix, analytical sciences, 10.2116/analsci.24.321, 24, 3, 321-325, 2008.03, Poly(methylmethacrylate) (PMMA) microchip electrophoresis of sodium dodecyl sulfate-protein complexes (SDS-PC) using linear-poly(acrylamide) (L-PA) as a separation matrix was investigated. Prior to electrophoresis, channel walls of PMMA were modified with methylcellulose (MC) to prevent adsorption between channel walls and SDS-PC. Size-based protein separation (SBPS) was successfully performed using the MC-coated microchips with Ferguson plot-fittings. The entangled L-PA solution provided high resolution of peaks of SDS-PC when the concentration of L-PA was increased. Some investigations into the separation mechanism, such as the plot of the logarithm of mobility of each SDS-PC versus the logarithm of the molecular weight of the complex exhibiting linear behavior, indicated that the separation mechanism was dependent on mass discrimination, in accordance with Ogston model. 2008.
207. Hiroko Ueno, Jun Wang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Quantitative determination of amino acids in functional foods by microchip electrophoresis, Journal of Separation Science, 10.1002/jssc.200700517, 31, 5, 898-903, 2008.03, Microchip electrophoresis (MCE), a first-generation micrototal analysis system, has emerged during the miniaturization phase of food analysis. Based on the micellar electrokinetic chromatography mode, a simple and fast MCE method with light emitting diode-induced fluorescence detection was developed for quantitative analysis of amino acids in three different kinds of functional foods, viz. sports beverages, jelly-form beverages, and tablet-form functional foods. In contrast to the glass microchip, we improved the separation of amino acids on a poly(methyl methacrylate) (PMMA) chip by addition of cationic starch derivatives. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole, which has a short labeling time for amino acids, was used as the fluorescently labeled dye. This MCE method takes less than 10 min of total analysis time including sample preparation and analysis of amino acids in functional foods on a PMMA chip. The results show that this approach has the potential to be a fast and simple method for amino acid analysis in functional foods..
208. Daisuke Onoshima, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Nuclease tolerant FRET probe based on DNA-quantum dot conjugation, analytical sciences, 10.2116/analsci.24.181, 24, 2, 181-183, 2008.02, We have developed a fluorescence resonance energy transfer (FRET) probe based on the conjugation of a quantum dot (QD) with dye (YOYO-3) intercalated DNA. The FRET-inducing electrostatic coupling of DNA and the QD made structural changes to the QD-DNA conjugates, which significantly prevented an enzymatic reaction between the DNA and a conventional restriction endonuclease (EcoRI). 2008.
209. Mohamad Reza Mohamadi, Noritada Kaji, Anabu Tokeshi, Yoshinobu Baba, Dynamic cross-linking effect of Mg2+ to enhance sieving properties of low-viscosity poly(vinylpyrrolidone) solutions for microchip electrophoresis of proteins, Analytical Chemistry, 10.1021/ac701974u, 80, 1, 312-316, 2008.01, We report a dynamic cross-linking effect of Mg2+ that enhances the sieving properties of low-viscosity poly-(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgCl2. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (In μ/μ0) is correlated with the ratio of cross-linking agent to PVP ([cMg2+/cPVP]) as In μ/μ0 = A′[cMg2+ /cPVP] b′. A′ was related to the protein radius (R), and b′ was found to be 0.72 for proteins with R = 2.4 nm and 0.82 for proteins with R = 1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg2+ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%..
210. Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Channel wall coating on a poly-(methyl methacrylate) CE microchip by thermal immobilization of a cellulose derivative for size-based protein separation, ELECTROPHORESIS, 10.1002/elps.200700105, 28, 24, 4582-4589, 2007.12, We demonstrate channel wall coating using a cellulose derivative on a poly-(methyl methacrylate) (PMMA) CE microchip to eliminate EOF disturbing protein separation. The channel walls were modified by preconditioning with a solution containing the cellulose derivative and then thermally evaporating the solution to produce hydrophilic channel walls which prevent adsorption of analytes via a hydrophobic interaction. When the PMMA substrate was coated with the cellulose derivative hydroxypropylmethylcellulose (HPMC) 90SH, the water contact angle on the coated substrate was decreased (up to 15°) and EOF was significantly suppressed (up to 4.0 × 10-6 cm2·V-1·s-1). Three proteins (20.5, 68.0, and 114.6 kDa) were successfidly separated on the 0.15% HPMC 90SH-coated channel walls with good reproducibility of migration time (RSD <1.75%) and high efficiency (theoretical plate number per meter: 2.62 × 105)..
211. Noritada Kaji, Akio Oki, Ryo Ogawa, Yuzuru Takamura, Takahiro Nishimoto, Hiroaki Nakanishi, Yasuhiro Horiike, Manabu Tokeshi, Yoshinobu Baba, Influences of electroosmotic flows in nanopillar chips on DNA separation
Experimental results and numerical simulations, Israel Journal of Chemistry, 10.1560/IJC.47.2.161, 47, 2, 161-169, 2007.12, The various potential factors affecting the performance of nanopillar chips on DNA separation were investigated from the viewpoints of both numerical calculations and actual experiments. To yield higher performance and replace the conventional DNA separation techniques such as microchip electrophoresis, the phenomenon specific to the nanopillar chips should be deeply understood. In this paper, although various factors affecting the performance of the nanopillar chips are considered, we focused on the effect of electroosmotic flow, which is particularly noticeable in quartz-made nanopillar chips. High-resolution separation of DNA was realized when an electroosmotic flow was suppressed by simply using a higher concentration of buffer, but DNA separation failed in the presence of an electroosmotic flow. It was confirmed from the numerical simulations and the direct observations that the deformation of DNA band during the injection process was induced by electroosmotic flow and consequently led to a poor resolution..
212. Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Microchip-based immunoassay, bunseki kagaku, 10.2116/bunsekikagaku.56.521, 56, 7, 521-534, 2007.11, Microchip-based immunoassay, which is immunoassay performed on a microchip, has recently been used in various fields owing to its advantages, such as reduction in sample and reagent consumption, short analysis time and simple operation. Different types of immunoassay have been applied to the miniaturization on a microchip. Material and surface modifications of a microchip, introducing and moving samples and detection must be taken into consideration in order to realize a microchip-based immunoassay. Also, the microchip system should be designed according to a type of immunoassay. In the present review article, we explain the types and classifications of immunoassays and describe important points for developing a microchip-based immunoassay. We then introduce several examples of microchip-based immunoassay..
213. Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Single-molecule measurements with a single quantum dot, Chemical Record, 10.1002/tcr.20128, 7, 5, 295-304, 2007.11, Recent progress of quantum dot (QD) applications in single-molecule measurements are reviewed in this paper. Bright fluorescence and anti-photobleaching properties of QDs have explored the way to conduct long-time trajectory tracking of transmembrane proteins both in vitro and in vivo. Coupled with diversities of chemical and biochemical modifications of QD surfaces, their application fields are expanding to multidiscipline fields including imaging on the basis of a single molecule. Currently, molecular interactions and conformational changes on the QD surface can be detected at a single-molecule level. These expansions of application fields also involve toxicity problems in cells since most commercially available QDs consist of cadmium selenide or cadmium telluride, which are inherently toxic. For widespread applications of QDs including in vivo and therapeutic use in place of current organic fluorophore, cytotoxicity is discussed as well in this paper..
214. Yong Zhang, Guichen Ping, Noritada Kaji, Manabu Tokeshi, Yoshi Baba, Dynamic modification of poly(methyl methacrylate) chips using poly(vinyl alcohol) for glycosaminoglycan disaccharide isomer separation, ELECTROPHORESIS, 10.1002/elps.200700088, 28, 18, 3308-3314, 2007.09, We describe a microchip electrophoresis (MCE) method for the assay of unsaturated disaccharides of chondroitin sulfates, dermatan sulfates, and hyaluronic acid (HA). Poly(vinyl alcohol) (PVA) could be irreversibly adsorbed onto poly(methyl methacrylate) (PMMA) substrates and this approach was applicable for dynamic coating. The characteristics of the PMMA surface with PVA coating were evaluated in terms of the wettability, EOF, and adsorption of 2-aminoacridone (AMAC)-labeled disaccharide. The water contact angle decreased from ∼73° on a pristine PMMA surface to ∼37.5° on a PVA-coated surface, indicating that the PVA coating increased hydrophilicity. EOF was reduced approximately twofold and was relatively stable. Scanning electron microscopy and fluorescence microscopy images showed that adsorption of AMAC-labeled disaccharides was dramatically suppressed. Using the PVA coating, baseline separation of two pairs of glycosaminoglycan (GAG) disaccharide isomers, ΔDi-diSB/ΔDi-diSD and ΔDi-0S/ΔDi-HA, was achieved in Tris-borate buffer within 130 s by MCE..
215. Eiki Maeda, Masatoshi Kataoka, Mami Hino, Kazuaki Kajimoto, Noritada Kaji, Manabu Tokeshi, Jun Ichi Kido, Yasuo Shinohara, Yoshinobu Baba, Determination of human blood glucose levels using microchip electrophoresis, ELECTROPHORESIS, 10.1002/elps.200600795, 28, 16, 2927-2933, 2007.08, A high-performance monitoring system for human blood glucose levels was developed using microchip electrophoresis with a plastic chip. The combination of reductive amination as glucose labeling with fluorescent 2-aminoacridone (AMAC) and glucose-borate complex formation realized the highly selective detection of glucose even in a complex matrix such as a blood sample. The migration time of a single peak, observed on an electropherogram of AMAC-labeled plasma, closely resembled that of glucose standard solution. The treatment of plasma with hexokinase or glucokinase for glucose phosphorylation resulted in a peak shift from approximately 145 to 70 s, corresponding to glucose and glucose-6-phosphate, respectively. A double-logarithm plot revealed a linear relationship between glucose concentration and fluorescence intensity in the range of 1-300 μM of glucose (r2 = 0.9963; p <0.01), and the detection limit was 0.92 μM. Furthermore, blood glucose concentrations estimated from the standard curves of three subjects were compared with results obtained by conventional colorimetric analysis using glucose dehydrogenase. Good correlation was observed between methods according to simple linear regression analysis (p <0.05). The reproducibility of the assay was about 6.3-9.1% (RSD) and the within-days and between-days reproducibility were 1.6-8.4 and 5.2-7.2%, respectively. This system enables us to determine blood glucose with high sensitivity and accuracy, and will be applicable to clinical diagnosis..
216. Yong Zhang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Nanobiotechnology
Quantum dots in bioimaging, Expert Review of Proteomics, 10.1586/14789450.4.4.565, 4, 4, 565-572, 2007.08, Many biological systems, including protein complexes, are natural nanostructures. To better understand these structures and to monitor them in real time, it is becoming increasingly important to develop nanometer-scale signaling markers. Single-molecule methods will play a major role in elucidating the role of all proteins and their mutual interactions in a given organism. Fluorescent semiconductor nanocrystals, known as quantum dots, have several advantages of optical and chemical features over the traditional fluorescent labels. These features make them desirable for long-term stability and simultaneous detection of multiple signals. Here, we review current approaches to developing a biological application for quantum dots..
217. Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Online preconcentration by transient isotachophoresis in linear polymer on a poly(methyl methacrylate) microchip for separation of human serum albumin immunoassay mixtures, Analytical Chemistry, 10.1021/ac0623890, 79, 10, 3667-3672, 2007.05, Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM..
218. Yasuhiro Hayashi, Shinya Iida, Yuichiro Sato, Akihiro Nakaya, Akihiro Sawada, Noritada Kaji, Hiroyuki Kamiya, Yoshinobu Baba, Hideyoshi Harashima, DNA microarray analysis of type 2 diabetes-related genes co-regulated between white blood cells and livers of diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, Biological and Pharmaceutical Bulletin, 10.1248/bpb.30.763, 30, 4, 763-771, 2007.04, In a previous study, we hypothesized that some type 2 diabetes mellitus susceptible genes may be up/down-regulated in white blood cells (WBC) of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, reflecting their up/down-regulation in major insulin-target tissues such as the liver before the onset of diabetes. We identified 57 potential candidate genes for predicting diabetes. In this study, we examined this hypothesis further by extending the experimental conditions from before the onset (6 weeks) to after the onset (24 weeks) of diabetes that type 2 diabetes mellitus susceptible genes are co-regulated in WBC, reflecting their expression in the liver. Using rat oligo DNA microarrays, we found that 48 genes are up/down-regulated in OLETF rats compared to control Long-Evans Tokushima Otsuka (LETO) rats in WBC and liver under fasting or insulin administration conditions. Twenty nine and 33 genes were up/down-regulated in both WBC and livers, respectively, under fasting and insulin administration conditions, respectively. Eight out of 29 genes in fasting condition and 12 out of 33 genes in insulin administration conditions have been reported to be type 2 diabetes mellitus susceptible genes and the remainder have not been reported to be related to type 2 diabetes mellitus. These results support our hypothesis that the expression levels of type 2 diabetes mellitus related genes in WBC are reflective of those in the liver after the onset of diabetes..
219. Akihiro Sawada, Shogo Mizufune, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Evaluation of amplified cRNA targets for oligonucleotide microarrays, Analytical and Bioanalytical Chemistry, 10.1007/s00216-007-1125-2, 387, 8, 2645-2654, 2007.04, Due to their hybridization specificity and capacity for systematic gene discovery, oligonucleotide-based microarray platforms offer numerous advantages over the cDNA microarrays currently widely used for comprehensive analysis of gene expression. Although fluorescently labeled amplified cRNA generated by T7 transcription is generally used in oligonucleotide microarrays, the feasibility of this combination (and that of cDNA microarrays) is yet to be studied systematically. In this paper, we performed a comparative study using a direct labeling method and T7 amplification to evaluate amplified cRNA targets for oligonucleotide microarrays. The efficiency of incorporation of Cy3- and Cy5-CTP into the target preparations, the reproducibility and the number of genes detected were investigated for each labeling approach and compared. The 12 genes that showed different expression profiles in the two labeling methods were evaluated by quantitative real-time PCR. In the 60-mer oligonucleotide microarray, amplified cRNA targets prepared by the T7 amplification method showed higher reproducibility and reliability than targets prepared by the direct labeling method in a comparative analysis of gene expression. This result also suggests the importance of fragmenting cRNA down to lengths of 50-200 bases before the hybridization process..
220. Ryo Ogawa, Noritada Kaji, Shingi Hashioka, Yoshinobu Baba, Yasuhiro Horiike, Fabrication and characterization of quartz nanopillars for DNA separation by size, Japanese Journal of Applied Physics, Part 1: Regular Papers and Short Notes and Review Papers, 10.1143/JJAP.46.2771, 46, 4 B, 2771-2774, 2007.04, We fabricated two different distributions of nanopillars-tilted and square distributions. There are difficulties in fabricating a chip containing these nanopillars, particularly in Ni plating and quartz bonding. We improved the Ni plating by applying stable electric current and the reliability of the quartz bonding. In nanopillars with a square distribution, DNA showed conformational changes, and did not show any reptile motion, but moved in a straight line in contrast with its behavior in nanopillars with a tilted distribution. Nanopillars with a tilted distribution acted effectively as sieves for DNA separation by size..
221. Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips, ELECTROPHORESIS, 10.1002/elps.200600373, 28, 5, 830-836, 2007.03, A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb (∼220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%)..
222. Laili Mahmoudian, Jonas Melin, Mohamad Reza Mohamadi, Keiko Yamada, Michio Ohta, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba, Microchip electrophoresis for detection of circle-to-circle amplification products towards sensitive and rapid DNA analysis, Chemistry Letters, 10.1246/cl.2007.396, 36, 3, 396-397, 2007.03, A new method for fast and precise analysis of circle-to-circle amplification (C2CA) products by microchip electrophoresis has been developed. Stable C2CA products were produced by applying a new enzymatic step to C2CA. Detection was carried out within 55s with RSD of migration time of 3.6% (n = 6) enabling reproducibility and high speed. A real sample of bacterial pathogen (V. Cholerae) at single nucleotide level was detected successfully based on this method..
223. Yong Zhang, Guichen Ping, Bingmei Zhu, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Enhanced electrophoretic resolution of monosulfate glycosaminoglycan disaccharide isomers on poly(methyl methacrylate) chips, ELECTROPHORESIS, 10.1002/elps.200600339, 28, 3, 414-421, 2007.02, To improve the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis, we found that addition of 1,4-dioxane (DO) dramatically improved analyte resolution, probably due to solvation effects. Methylcellulose (MC) was tested for the ability to suppress EOF and analyte adsorption to the chip. To improve analyte resolution, buffer pH, β-CD, and DO were systematically investigated. Fast separation was achieved by increasing the electric field strength, and field-amplified sample stacking occurred with increasing buffer concentrations. Therefore, based on our findings, we describe an efficient method for the separation of mono-sulfate and trisulfate unsaturated disaccharides (ΔDi-UA2S, ΔDi-4S, ΔDi-6S, and ΔDi-triS) derivatized with 2-aminoacridone hydrochloride. A mixture of monosulfate disaccharide isomers (ΔDi-UA2S, ΔDi-4S, and ΔDi-6S) was baseline-separated within 75 s on a poly(methyl methacrylate) chip using a mixed buffer (DO /running buffer 57:43 v:v), 0.5% MC, pH 6.81, with an Esep of 558 V/cm. The theoretical plate was in the range of 5 × 105 to 1 × 106 m-1..
224. Laili Mahmoudian, Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Nanoscale DNA analysis, Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, Third Edition, 1527-1542, 2007.01, Microchip electrophoresis has been developed as a promising technology for detection of DNA in samples.
1-3
However, the development of systems for the separation of long DNA strands [greater than several kilo basepairs (kbps)] using commercially available media, has not been successful.
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In recent years, efforts have been focused on the overcoming the limitations of current DNA electrophoresis methods. Those limitations largely result from an application base that only required the separation of DNA fragments in a narrow size range, one where using fundamental separation modes based on sieving using conventional methods.
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In conventional gel electrophoresis, DNA molecules move in the electric field based on a size-dependent mobility through pores in a gel matrix.
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However, length-dependent mobility vanishes for DNA molecules longer than 40 kbps, primarily due to tendency of DNA molecules to become stretched and oriented to the direction of electric field.
7,8
Buoyed by the knowledge of micro- and nanofabrication technologies, nanotechnology has been shown to be quite successful for long fragment DNA analysis. To achieve the required efficiency needed for this type of DNA electrophoresis, two directions in the field of nanotechnology have been established: one focused on providing artificial gel structures produced with nanofabrication technology, and the other on nanomaterials for DNA separation. In this chapter, we discuss the developments and capabilities of nanoscale methods for DNA electrophoresis, and provide some important technical information for each method. Considering the point that the mechanism of DNA separation in each nanotechnique is different (and sometimes unique) from other better-understood separation modes, these will be explained separately..
225. Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Quantum dots for single bio-molecule imaging, analytical sciences, 10.2116/analsci.23.21, 23, 1, 21-24, 2007.01, The emerging nanomaterial, quantum dots or QDs, offers numerous potential applications in the biological area. As cell labeling probes, QDs become now an alternative of existing organic fluorescent dyes and fluorescent proteins. In this short review, we cover typical and successful applications of QDs as fluorescent probes in cell labeling and genomic diagnosis. As a future important application, biomolecular detection at a single molecule level utilizing QDs is also discussed. 2007.
226. Lihua Zhang, Toshikatsu Shinka, Yutaka Nakahori, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Rapid and simultaneous multiple genotyping of human Y chromosome from a human blood sample by on-chip enzymatic digestion and microchannel array electrophoresis coupled with blood sample pretreatment, and microscale PCR, Sensors and Actuators, B: Chemical, 10.1016/j.snb.2006.09.020, 121, 1, 124-128, 2007.01, In this paper, with distinct six DNA markers, 47z, SRY, YAP, 12f2, TB4Y and UTY, as the judgement standard, a combined technique for the rapid haplotyping (multiple genotyping) of human Y chromosome was developed by a DNA analysis system. By Generation capture disk, 5 min were enough to extract and purify DNA from five samples with only 4.5 L blood for each one. Consequently, LightCycler was chosen for the amplification of the six fragments of each sample. Under the optimized conditions, the simutaneous amplification of DNA extracted from five samples could be finished within 25 min. Among the six genomic fragments of each sample, the polymorphisms of YAP and 12f2 could be distinguished directly from PCR products. The other four were single nucleotide polymorphisms. Therefore, microchip-based restriction fragment length polymorphism analysis was carried out. With a 12-channel microchip, in 8 min, two blood samples could be analyzed at the same time through on-chip digestion and the subsequent separation of DNA fragments in the channel. In summary, with the above-mentioned technique, microscale haplotyping of human Y chromosome could be finished at the average speed of 10 min/sample with only 4.5 μL blood..
227. M. R. Mohamadi, L. Mahmoudian, N. Kaji, M. Tokeshi, Y. Baba, 800 fold signal enhancements by transient isotachophoresis for immunoassay of HSA on standard cross channel microchips, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 1402-1404, 2007, A fully automated preconcentration and stacking method based on transient isotachophoresis (ITP) on a standard cross channel microchip made of poly (methylmethacrylate) PMMA has been reported. Previously reported ITP has been either on a special chip design or required a hydrodynamic pressure for sample injection and a pause in the electrophoresis process which caused low reproducibility in analysis. In this work sample injection, preconcentration and separation are done continuously and are controlled solely by a sequential voltage switching program. The ITP followed by nondenaturing gel electrophoresis was applied for immunoassay of HSA..
228. Hiroshi Kuramoto, Noritada Kaji, Kentarou Kogure, Manabu Tokeshi, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba, A more excellent construction method of a multi functional envelope-type nano device on microdevice for gene therapy, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 781-783, 2007, A multifunctional envelope-type nano device (MEND) is an extremely efficient technology for a non-viral gene delivery system. However, the construction processes of the MEND by the existing method are very complicated. Therefore we develop a new method to construct it quickly and easily in the previous work. In this work, we report a new on-chip construction method of the MEND which is much more suitable for the gene therapy..
229. Mai Ikami, Manabu Tokeshi, Noritada Kaji, Yoshinobu Baba, A new on-chip platform for rapid and easy-to-use immunoassay, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 655-657, 2007, We describe a new platform for chip-based immunoassay to create the effective reaction sites of immunological reaction between antigen and antibody. The reaction sites were fabricated by the UV photopolymerization of novel PEG-based polymer and the antibody-immobilized polystyrene beads. Our platform has many advantages; (i) rapid analysis (a few minutes), (ii) easy handling (no need pump and valve), (iii) low-cost (disposable), (iv) small volume (1 droplets (0.25µL) of sample and reagents), and (v) high sensitivity..
230. Masaya Murata, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Cell sorting of live and dead cells by laser radiation pressure and sheath flow in microchannel, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 116-118, 2007, We have developed a novel cell sorting technique for live and dead cells by using optical tweezers (1064 nm Nd; YAG Laser) and sheath flow in a PDMS microchip. The dead cells stained by Trypan Blue have low refraction index than the live cells. Therefore, by using appropriate laser power and flow rate only, the live cells could be trapped by optical tweezers..
231. T. Yasui, N. Kaji, R. Ogawa, S. Hashioka, M. Tokeshi, Y. Horiike, Y. Baba, DNA Separation by square patterned nanopillar chips, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 1207-1209, 2007, In this paper, the mechanism of DNA separation by different array-pattern nanopillar chips and the effect of nanopillar spacing were carefully studied. We revealed that array-pattern of nanopillars and nanopillar spacing dominates the separation mode, and furthermore, the square patterned nanopillar chips could achieve a high-resolution and high-speed separation..
232. Eiki Maeda, Masatoshi Kataoka, Yasuo Shinohara, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Determination of total and pancreatic amylase activities in human blood by use of microchip electrophoresis, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 65-67, 2007, In this study, a high-performance determination system for isoenzyme activities of human a-amylase was developed using microchip electrophoresis (ME) with a plastic chip. The combination of ME for substrate/hydrolysate separation and immunomhibition method for differentiation of isoenzymes using anti human salivary amylase (S-AMY) monoclonal antibody, Tu88E8, realized the highly selective determination of amylase isoenzyme activities even in a complex matrix such as a blood sample. This paper demonstrates that this system enables us to determine amylase isoenzyme activities with easy operation and high accuracy, and it will be applicable to clinical diagnosis for acute pancreatic disorders..
233. Keiko Osato, Manabu Tokeshi, Noritada Kaji, Ryu Anraku, Takahiro Asai, Akihiko Hattori, Kazuma Mawatri, Takehiko Kitamori, Eiji Yashima, Yoshinobu Baba, Dynamics measurement of structural change of helical polymer using thermal lens microscopy and microfluidic technique, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 1565-1567, 2007, In this paper, we report a new on-chip system for dynamics measurement of structural change of polymer. This experimental system consists of a thermal lens microscope (TLM) and a microchip. Using this system, we demonstrated that it can measure dynamically molecular structural change in a microchannel by time-resoled measurement..
234. Keiko Osato, Manabu Tokeshi, Noritada Kaji, Yusuke Omoto, Norikazu Suzuki, Yasuhiko Sakai, Eiji Shamoto, Yoshinobu Baba, Micromixer based on baker's transformation, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 309-311, 2007, Herein we report the development of a new passive micromixer with 3-D structure based on Baker's transformation. The 3-D structure was fabricated on an oxygen-free copper block by using an ultrasonic elliptical vibration cutting method [1] and was used as a mold master. Using this master mold, we fabricated a PDMS micromixer chip. The mixing efficiency of the micromixer was evaluated by measuring the fluorescence image that results from mixing of two liquids (FITC solution and water). From these measurements, we found that the mixing efficiency at relatively high flow rate conditions is much better than that of low flow rate conditions. The mixing behavior in the microchannel was observed by a confocal fluorescence microscopy..
235. H. Murahara, N. Kaji, M. Tokeshi, Y. Baba, Molecular crowding effect on enzymatic reaction in a fl-microchamber to mimic crowded intracellular environment, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 1003-1005, 2007, In this work, we investigated how the size of reaction space would affect enzyme activities to understand intracellular enzyme kinetics. Two approaches were used to create pseudo-intracellular environments: a high concentration of polymers in bulk state and microfabricated chambers. Both approaches were carefully compared and we found that an enzyme activity was inhibited in 7.2 fL chamber by molecular crowding effect as in the case of highly concentrated polymer solution..
236. D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba, Single DNA/protein interaction analysis using quantum dot conjugates and microfluidic systems, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 1619-1621, 2007, We have designed a microfluidic detection platform in which we can monitor and analyze single DNA/protein dynamic interaction using “single-molecule fluorescence tracing”. The use of quantum dot (QD) labelling on DNA, polydimethylsiloxane (PDMS) microchip, micro patterning of active enzymes, and total internal reflection fluorescence (TIRF) microscope was successfully applied for nano-space experiments in micrometer-sized channels. We used this detection platform to observe the enzymatic reaction of restriction endonuclease (EcoRI) and measured the duration of its cleavage reaction..
237. Daisuke Kuroda, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Viscosity-tunable polymer for microchip electrophoresis of DNA, 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007 Proceedings of the 11th International Conference on Miniaturized Systems for Chemistry and Life Sciences, uTAS 2007, 841-843, 2007, A versatile alternative to entangled and random-coiled polymers, Pluronic F127, has been developed for a DNA separation matrix in microchip electrophoresis. This temperature-sensitive and viscosity-tunable polymer provided excellent resolutions over a wide range of DNA sizes based on a different separation mechanism compared with conventional polymers such as cellulose-derivatives..
238. Shinya Iida, Yuichiro Sato, Akihiro Nakaya, Yasuo Shinohara, Yasuhiro Hayashi, Akihiro Sawada, Hideya Nagata, Noritada Kaji, Hiroyuki Kamiya, Yoshinobu Baba, Hideyoshi Harashima, Genome wide expression analysis of white blood cells and liver of pre-diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a cDNA microarray, Biological and Pharmaceutical Bulletin, 10.1248/bpb.29.2451, 29, 12, 2451-2459, 2006.12, In a prior study, we reported on a significant decrease in calpain10 gene expression in white blood cells (WBC) as well as the major insulin-target tissues including liver and adipose tissue, before the onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In this study, we extended our hypothesis that some type 2 diabetes mellitus (NIDDM) susceptible genes are up/down-regulated before the onset in WBC of OLETF rats, reflecting their up/down-regulation in major insulin-target tissues, such as the liver. We tested this hypothesis using rat cDNA microarrays. The findings show that 1080 genes are up/down-regulated by more than 2-fold compared to the controls, Long-Evans Tokushima Otsuka rats, before the onset in WBC and liver under fasted or insulin administered condition. Fifty-seven of the 1080 genes were up/down-regulated in both WBC and the liver. More than half have been reported to NIDDM susceptible genes and the remainder have not been reported to be related to NIDDM. These results indicate that there some NIDDM related genes are up/down-regulated in WBC before the onset of diabetes..
239. Noritada Kaji, Ryo Ogawa, Akio Oki, Yasuhiro Horiike, Manabu Tokeshi, Yoshinobu Baba, Study of water properties in nanospace, Analytical and Bioanalytical Chemistry, 10.1007/s00216-006-0469-3, 386, 3, 759-764, 2006.10, Here we report an anomalous behavior of water, especially its viscosity and hydrodynamic flow, in a nanometer-confined space. As a typical model of a nanometer-confined space, the nanopillar chip, which was developed for DNA size-based separation was used, and single-particle tracking (SPT) technique was applied to investigate water viscosity and hydrodynamic flow in the nanopillar chip. The diffusion coefficients of nanospheres were almost one-third of the theoretical value derived from the Stokes-Einstein equation. This result gave indirect proof that water viscosity in a nanometer-confined space is higher than in a bulk solution. In order to improve resolution and throughput of the nanopillar chip for DNA separation, these potential factors affecting performance should be seriously considered..
240. Mohammad Jabasini, Ashraf Abdulazim Ewis, Maged Fouad, Fuquan Dang, Guichen Ping, Toshikatsu Shinka, Yutaka Nakahori, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Rapid multiplexing and simultaneous detection of human spermatogenetic failure with a 12 lane microchip electrophoresis system, Biological and Pharmaceutical Bulletin, 10.1248/bpb.29.1487, 29, 7, 1487-1489, 2006.07, For the amplification and ultrafast separation of the genetic markers and DNA sequences that are related to human male infertility, a multiplex PCR for amplifying three DNA sequence-tagged sites (STS) located on the human Y chromosome with possible roles in the spermatogenesis process has been designed and applied followed by separation on a microchip. First, the optimum T m degree for the three DNA markers was optimized and determined experimentally, and the three DNA STS were amplified. These three DNA markers were then separated on a 12-lane microchip electrophoresis system, which can analyze the DNA markers on 12 channels simultaneously. The combination of these two technologies, multiplex PCR and microchip electrophoresis, allows the analysis of 36 DNA markers (12x3) within only 180 s..
241. Fuquan Dang, Wenhao Li, Lihua Zhang, Mohammad Jabasini, Tatsuhiro Ishida, Hiroshi Kiwada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes, Journal of Chromatography A, 10.1016/j.chroma.2006.03.120, 1118, 2, 218-225, 2006.06, In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (μ-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or μ-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems..
242. Mohammad Jabasini, Yuji Murakami, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Low viscous separation media for genomics and proteomics analysis on microchip electrophoresis system, Biological and Pharmaceutical Bulletin, 10.1248/bpb.29.595, 29, 4, 595-604, 2006.04, Microchip electrophoresis has widely grown during the past few years, and it has showed a significant result as a strong separation tool for genomic as well as proteomic researches. To enhance and expand the role of microchip electrophoresis, several studies have been proposed especially for the low viscous separation media, which is an important factor for the success of microchip with its narrow separation channels. In this paper we show an overview for the done researches in the field of low viscous media developed for the use in microchip electrophoresis. For genomic separation studies polyhydroxy additives have been used enhance the separation of DNA at low polymer concentration of HPMC (Hydroxypropylmethyl cellulose) which could keep the viscosity low. Mixtures of poly(ethylene oxide) as well as Hydroxyporpyl cellulose have been successfully introduced for chip separation. Furthermore high molecular mass polyacrylamides at low concentrations have been studied for DNA separation. A mixture of polymer nanoparticle with conventional polymers could show a better resolution for DNA at low concentration of the polymer. For the proteomic field isoelectric focusing on chip has been well overviewed since it is the most viscous separation media which is well used for the protein separation. The different types of isoelectric focusing such as the ampholyte-free type, the thermal type as well as the ampholyte-depended type have been introduced in this paper. Isoelectric focusing on chip with its combination with sodium dodecyl sulfate (SDS) page or free solution could give a better separation. Several application for this low viscous separation medias for either genomic or proteomic could clearly show the importance of this field..
243. Fuquan Dang, Kazuaki Kakehi, Kazuki Nakajima, Yasuo Shinohara, Mitsuru Ishikawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Rapid analysis of oligosaccharides derived from glycoproteins by microchip electrophoresis, Journal of Chromatography A, 10.1016/j.chroma.2005.11.133, 1109, 2, 138-143, 2006.03, A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (μ-CE) is presented here. The characterization of separation conditions, i.e., the composition, concentration and pH of running buffer as well as the applied voltage, has been performed using maltose (G2), cellobiose (G′2), maltriose (G3) and panose (G′3) as oligosaccharide isomer models. In μ-CE, much better separation of oligosaccharide isomers and oligosaccharide ladder was obtained in phosphate buffer than in borate buffer over a wide pH range. Under optimal conditions, high-performance separation of the N-linked complex oligosaccharides released from ribonuclease B, fetuin, α1-acid glycoprotein (AGP) and IgG was achieved using polymethylmethacrylate (PMMA) microchips with an effective separation channel of 30 mm. These results represent the first reported analysis of the N-linked oligosaccharides derived from glycoproteins by μ-CE, indicating that the present μ-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins..
244. Lihua Zhang, Fuquan Dang, Noritada Kaji, Yoshinobu Baba, Fast extraction, amplification and analysis of genes from human blood, Journal of Chromatography A, 10.1016/j.chroma.2005.10.071, 1106, 1-2, 175-180, 2006.02, In order to shorten the time spent on the sample preparation for gene analysis, a novel method was proposed through the combination of fast DNA extraction and purification by Generation capture disk, amplification by capillary polymerase chain reaction, and confirmation of amplification products by microchip electrophoresis. With this method, 3 μL blood was enough to obtain adequate target fragments in human genes. Under the optimal conditions in each step, the sample preparation for eight fragments in β-globin gene and four fragments in ras gene could be finished within 20 min. Since all the experiments were performed on commercial instruments, this method showed a wide range of applicability. In addition, other advantages such as fast speed and low consumption of samples were demonstrated. All these merits proved that such a combination method was of great potential for the clinical diagnostics..
245. Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Hiroshi Chuman, Yoshinobu Baba, Nanotechnology for genomics & proteomics, Nano Today, 10.1016/S1748-0132(06)70021-4, 1, 1, 38-45, 2006.02, This review, though not comprehensive, looks at recent developments in nanodevices for genomics and proteomics and some of the new applications in biomedicine..
246. Ryo Ogawa, Akio Oki, Shingi Hashioka, Noritada Kaji, Yoshinobu Baba, Yasuhiro Horiike, Allocation dependence of nano-pillars for DNA electrophoresis separation, 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2005 Micro Total Analysis Systems - Proceedings of MicroTAS 2005 Conference 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1012-1014, 2005.01, Different allocations of nano-pillars were investigated for DNA electrophoresis separation. The square allocation shows that DNA was extended and moved straight without disturbance of pillars, but DNA entangled around pillars in the case of the tilt allocation. The tilt allocation allowed us to separate DNA of 10 kbp and 38 kbp by their sizes, whereas square allocation demonstrated one broad peak alone. The separation in the tilt nano-pillars pattern is considered to result from the sieving effect for different sizes of DNA. These results suggest that any disturbance is necessary for electrophoretic separation of small size DNA..
247. Noritada Kaji, A. Oki, R. Ogawa, Y. Horiike, Y. Baba, Water viscosity and hydrodynamic flow in nanopillar chips, 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2005 Micro Total Analysis Systems - Proceedings of MicroTAS 2005 Conference 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1, 736-738, 2005.01, Here we report an anomalous behavior of water, especially viscosity and hydrodynamic flow, in nanopillar chips. These physicochemical properties of water were investigated by single-particle tracking (SPT) technique. The results of diffusion constants of nanospheres indicated that a higher viscosity and a less hydrodynamic flow in nanopillar chips compared with a bulk solution..
248. Noritada Kaji, Yojiro Tezuka, Yuzuru Takamura, Yasuhiro Horiike, Takahiro Nishimoto, Hiroaki Nakanishi, Yoshinobu Baba, Nano-biodevice for genomic drug discovery and genomic medicine, Proceedings - 2004 International Conference on MEMS, NANO and Smart Systems, ICMENS 2004 Proceedings - 2004 International Conference on MEMS, NANO and Smart Systems, ICMENS 2004, 498-499, 2004.12, We have developed nano-biodevices to accelerate genomic drug discovery and post genome sequencing researches. The nanopillar chips, which has an array of nanometer-sized pillars inside a microchannel, were successfully fabricated on quartz substrate and applied for DNA separation. The nanopillar chips showed great potential to realize fast genome analysis and integration of whole lab procedures on a single chip..
249. I. A. Khalil, S. Futaki, M. Niwa, Y. Baba, N. Kaji, H. Kamiya, H. Harashima, Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine
Enhanced cellular association by hydrophobic core formation, Gene Therapy, 10.1038/sj.gt.3302128, 11, 7, 636-644, 2004.04, The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors..
250. Mari Tabuchi, Masanori Ueda, Noritada Kaji, Yuichi Yamasaki, Yukio Nagasaki, Kenichi Yoshikawa, Kazunori Kataoka, Yoshinobu Baba, Nanospheres for DNA separation chips, Nature Biotechnology, 10.1038/nbt939, 22, 3, 337-340, 2004.03, We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations..
251. Noritada Kaji, Yoshinobu Baba, Microchip technology for SNPs analysis and proteomics, Seikagaku. The Journal of Japanese Biochemical Society, 76, 10, 1275-1282, 2004.01.
252. Noritada Kaji, Yojiro Tezuka, Yuzuru Takamura, Masanori Ueda, Takahiro Nishimoto, Hiroaki Nakanishi, Yasuhiro Horiike, Yoshinobu Baba, Separation of Long DNA Molecules by Quartz Nanopillar Chips under a Direct Current Electric Field, Analytical chemistry, 10.1021/ac030303m, 76, 1, 15-22, 2004.01, We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip. The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp). A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragments (1 - 38 kbp) and large DNA fragments (λ DNA, 48.5 kbp; T4 DNA, 165.6 kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed-field technique. DNA fragments ranging from 1 to 38 kbp were separated as clear bands, and furthermore, the mixture of λ DNA and T4 DNA was successfully separated by a 380-μm-long nanopillar channel within only 10 s even under a direct current (dc) electric field. Theoretical plate number N of the channel (380-1450 μm long) was 1000-3000 (0.7 × 106-2.1 × 106 plates/m). A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas λ DNA kept a rather spherical conformation. We demonstrated that, even under a dc electric field, the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time..
253. Noritada Kaji, Masanori Ueda, Yoshinobu Baba, Stretching of megabase-sized deoxyribonucleic acid molecules by tuning electric-field frequency, Applied Physics Letters, 10.1063/1.1621075, 83, 16, 3413-3415, 2003.10, The stretching of megabase-sized deoxyribonucleic acid molecules by tuning electric-field frequency was studied. Combination of optimal conditions for the stretching process and maintenance of the stretched conformation helped in accomplishing the efficient stretching method. It was found that any sized DNA could be maintained in stretched conformation under a 10 Hz ac field..
254. Gen Hashiguchi, Takushi Goda, Maho Hosogi, Ken Hirano, Noritada Kaji, Yoshinobu Baba, Kuniyuki Kakushima, Hiroyuki Fujita, DNA manipulation and retrieval from an aqueous solution with micromachined nanotweezers, Analytical chemistry, 10.1021/ac034501p, 75, 17, 4347-4350, 2003.09, We have demonstrated DNA handling with micromachined nanotweezers that consist of a pair of opposing nanoprobes and integrated thermal expansion microactuators for changing the probe gap. The probe tips coated with a thin Al layer were dipped into a droplet of a solution containing λ-DNA molecules labeled with fluorescence dye, and then an ac electric field was applied between probes for several seconds. DNA molecules were then captured between the probe tips and retrieved from the solution to the air. The DNA capture between the probe tips could be performed more successfully on the droplet surface than in the underwater region. We also conducted an observation of the retrieved DNA molecules by transmission electron microscope and found that the thickness of the retrieved DNA molecules under the condition of this experiment was ∼21 nm when the time of the applied ac power (1 MHz, 20 Vpp) was 20 s..
255. Fuquan Dang, Lihua Zhang, Mohammad Jabasini, Noritada Kaji, Yoshinobu Baba, Characterization of electrophoretic behavior of sugar isomers by microchip electrophoresis coupled with videomicroscopy, Analytical chemistry, 10.1021/ac034110a, 75, 10, 2433-2439, 2003.05, The electrophoretic behavior of oligosaccharide isomers was investigated by microchip electrophoresis (μ-CE) coupled with videomicroscopy using maltose, cellobiose, maltriose, and panose as oligosaccharide isomer models. The present study revealed for the first time that the formation of a carbohydrate - phosphate complex is a pH-independent rapid process, whereas the formation of a carbohydrate - borate complex is a highly pH-dependent slow process. As a result, phosphate buffer gave much better separation on oligosaccharide isomers than borate and borate-Tris buffers over a wide pH range μ-CE. The imaging analysis of the complete process of sample loading and injection with field-amplified stacking (FAS) demonstrated that FAS could be used as an efficient method for manipulating the shape of injected sample plugs, and thus improving the performance of μ-CE in the absence of electroosmotic flow. However, once the ionic strength mismatch between sample and running buffer reached a critical threshold, a further increase in ionic strength mismatch deteriorated the effect of FAS, resulting in a surprising decrease in separation efficiency and peak distortion. Under optimal conditions, high-resolution separation of some oligosaccharide isomers and a complex oligosaccharide mixture released from ribonuclease B was achieved using PMMA microchips with an effective separation channel of 30 mm..
256. Noritada Kaji, Masanori Ueda, Yoshinobu Baba, Molecular stretching of long DNA in agarose gel using alternating current electric fields, Biophysical Journal, 10.1016/S0006-3495(02)75398-8, 82, 1, 335-344, 2002, We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields. The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy. The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated. Stretching was only observed in the AC field when a frequency of -10 Hz was used. The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used. Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3%. Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose. Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb). These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell..
257. Noritada Kaji, Masanori Ueda, Yoshinobu Baba, Direct measurement of conformational changes on DNA molecule intercalating with a fluorescence dye in an electrophoretic buffer solution by means of atomic force microscopy, ELECTROPHORESIS, 10.1002/1522-2683(200109)22:16<3357::AID-ELPS3357>3.0.CO;2-C, 22, 16, 3357-3364, 2001, By means of atomic force microscopy (AFM), we performed the direct imaging of DNA molecules (200, 500, 1000 bp) in a Tris-borate buffer solution, and measured the contour length and the end-to-end distance of DNA. Processing the data according to the worm-like chain model, we calculated the persistence length of the double-stranded DNA. Based on the analysis of the contour length and the persistence length, we discussed the interactions between DNA and an intercalating fluorescence dye (YO-PRO-1). YO-PRO-1 stacks between the base pairs and extends the contour length of DNA, changing the electric charge and the persistence length of DNA. From AFM measurement, we investigated directly the relationship between the persistence length and the number of the YO-PRO-1 intercalating to DNA. We will discuss on the relationship between the effect of an intercalating dye on the electrophoretic behavior and the conformational changes of DNA with an intercalating dye..