Researcher Database

Manabu Tokeshi
Faculty of Engineering Applied Chemistry Chemistry of Functional Molecules
Professor

Researcher Profile and Settings

Affiliation

  • Faculty of Engineering Applied Chemistry Chemistry of Functional Molecules

Job Title

  • Professor

Degree

  • Ph.D.(1997/03 Kyushu University)

URL

J-Global ID

Research Areas

  • Nanotechnology/Materials / Analytical chemistry
  • Nanotechnology/Materials / Nano/micro-systems
  • Nanotechnology/Materials / Nanomaterials

Academic & Professional Experience

  • 2021/01 - Today 名古屋大学 未来社会創造機構 客員教授
  • 2019/03 - Today Obihiro University of Agriculture and Veterinary Medicine National Research Center for Protozoan Diseases
  • 2018/01 - Today Northwest Univeristy Visiting Professor
  • 2015/06 - Today Hokkaido University Division of Applied Chemisty, Faculty of Engineering Professor
  • 2014/04 - Today Nagoya University Institute of Innovative for Future Society Visiting Professor
  • 2013/12 - Today Nagoya University Innovative Research Center for Preventive Medical Engineering Visiting Professor
  • 2014/04 - 2019/03 Nagoya University ImPACT Research Center for Advanced Nanobiodevices Visiting Professor
  • 2010/04 - 2016/03 愛知県「知の拠点」重点プロジェクト 研究員
  • 2011/11 - 2015/05 Hokkaido University Faculty of Engineering, Division of Biotechnology and Macromolecular Chemistry Professor
  • 2011/11 - 2014/03 Nagoya University FIRST Research Center for Innovative Nanobiodevice
  • 2011/09 - 2012/02 Karolinska Institutet
  • 2011/01 - 2011/10 Nagoya University FIRST Research Center for Innovative Nanobiodevice, Division of Single Molecule Device Research
  • 2005/12 - 2011/10 Nagoya University Graduate School of Engineering, Graduate School of Engineering
  • 2004 - 2009 Kanagawa Academy of Science and Technology
  • 2007 - 2008 愛知県産業技術研究所 客員研究員
  • 2004 - 2005 Institute of Microchemical Technology
  • 2005 Institute of Microchemical Technology
  • 2004 Kanagawa Academy of Science and Technology
  • 1999 - 2003 Kanagawa Academy of Science and Technology
  • 1998 - 1999 Kanagawa Academy of Science and Technology
  • 1997 - 1998 日本学術振興会 特別研究員(PD)
  • 1996 - 1997 日本学術振興会 特別研究員(DC)

Association Memberships

  • アメリカ化学会   THE SOCIETY FOR CHEMISTRY AND MICRO-NANO SYSTEMS   日本分光学会   日本分析化学会   日本化学会   Japan Society of Molecular Science   American Chemical Society   The Institute of Electrical Engineering of Japan   The Spectroscopical Society of Japan   The Japan Society for Analytical Chemistry   The Chemical Society of Japan   Royal Society of Chemistry   

Research Activities

Published Papers

  • Hiroki Tanaka, Nae Takata, Yu Sakurai, Tokuyuki Yoshida, Takao Inoue, Shinya Tamagawa, Yuta Nakai, Kota Tange, Hiroki Yoshioka, Masatoshi Maeki, Manabu Tokeshi, Hidetaka Akita
    Pharmaceutics 13 (4) 2021/04/13 
    The world-first success of lipid nanoparticle (LNP)-based siRNA therapeutics (ONPATTRO®) promises to accelerate developments in siRNA therapeutics/gene therapy using LNP-type drug delivery systems (DDS). In this study, we explore the optimal composition of an LNP containing a self-degradable material (ssPalmO-Phe) for the delivery of oligonucleotides. siRNA or antisense oligonucleotides (ASO) were encapsulated in LNP with different lipid compositions. The hepatic knockdown efficiency of the target genes and liver toxicity were evaluated. The optimal compositions for the siRNA were different from those for ASO, and different from those for mRNA that were reported in a previous study. Extracellular stability, endosomal escape and cellular uptake appear to be the key processes for the successful delivery of mRNA, siRNA and ASO, respectively. Moreover, the compositions of the LNPs likely contribute to their toxicity. The lipid composition of the LNP needs to be optimized depending on the type of nucleic acids under consideration if the applications of LNPs are to be further expanded.
  • Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Hideaki Hisamoto, Manabu Tokeshi
    ACS omega 6 (12) 8340 - 8345 2021/03/30 
    Analytical methods with fluorescence detection are in widespread use for detecting low abundance analytes. Here, we report a simple method for fluorescence signal amplification utilizing a structure of an azide-unit pendant water-soluble photopolymer (AWP) in a microchannel. The AWP is a poly(vinyl alcohol)-based photocross-linkable polymer, which is often used in biosensors. We determined that the wall-like structure of the AWP (AWP-wall) constructed in a microchannel functioned as an amplifier of a fluorescence signal. When a solution of fluorescent molecules was introduced into the microchannel having the AWP-wall, the fluorescent molecules accumulated inside the AWP-wall by diffusion. Consequently, the fluorescence intensity inside the AWP-wall increased locally. Among the fluorescent molecules considered in this paper, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) (DDAO) showed the highest efficiency of fluorescence signal amplification. We prepared a calibration curve for DDAO using the fluorescence intensity inside the AWP-wall, and the sensitivity was 5-fold that for the microchannel without the AWP-wall. This method realizes the improved sensitivity of fluorescence detection easily because the fluorescence signal was amplified only by injecting the solution into the microchannel having the AWP-wall. Furthermore, since this method is not limited to only the use of microchannel, we expect it to be applicable in various fields.
  • Takeshi Komatsu, Ryoga Maeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS sensors 6 (3) 1094 - 1102 2021/03/26 
    The development of low-cost, user-friendly paper-based analytical devices (PADs) that can easily measure target chemicals is attracting attention. However, most PADs require manipulation of the sample using sophisticated micropipettes for quantitative analyses, which restricts their user-friendliness. In addition, immobilization of detection molecules to cellulose fibers is essential for achieving good measuring ability as it ensures the homogeneity of color development. Here, we have described a dip-type PAD that does not require pipette manipulation for sample introduction and immobilization of detection molecules to cellulose fibers and its application to ascorbic acid (AA) and pH assays. The PAD consisted of a dipping area and two channels, each with two detection zones. The developed PADs show color distribution in the two detection zones depending on the sample flow from the dipping area. In comparison with a PAD that has one detection zone at the end of the channel, our developed device achieved higher sensitivity (limit of detection (LOD), 0.22 mg/mL) and reproducibility (maximum coefficient of variation (CV), 2.4%) in AA detection. However, in pH detection, the reproducibility of the PAD with one detection zone at the end of the channel (maximum CV, 21%) was worse than that with two zones (maximum CV, 11%). Furthermore, a dipping time over 3 s did not affect color formation or calibration curves in AA detection: LODs at 3 and 30 s dipping time were 18 and 5.8 μg/mL, respectively. The simultaneous determination of AA and pH in various beverages was performed with no significant difference compared to results of the conventional method.
  • Yuichi Suzuki, Haruno Onuma, Risa Sato, Yusuke Sato, Akari Hashiba, Masatoshi Maeki, Manabu Tokeshi, Mohammad Enamul Hoque Kayesh, Michinori Kohara, Kyoko Tsukiyama-Kohara, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 330 61 - 71 2021/02/10 [Refereed]
     
    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.
  • Keine Nishiyama, Yohei Takeda, Kazuki Takahashi, Mao Fukuyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Haruko Ogawa, Manabu Tokeshi
    Analytical and bioanalytical chemistry 2021/02/05 
    Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses.
  • Takeshi Komatsu, Yuki Sato, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytica chimica acta 1144 85 - 95 2021/02/01 [Refereed]
     
    Competitive immunoassays comprise the standard means of detecting small molecules. However, conventional methods using microwells are difficult to apply during point-of-care tests (POCT) because they require complicated handling and are time consuming. Although paper-based analytical devices (PAD) have received considerable focus because of their rapid and straightforward operation, only a few devices have been proposed for competitive immunoassays. Herein, we describe a novel universal PAD format with a 3-dimensional configuration for competitive immunoassays that rapidly and sensitively detects small molecules. The proposed device comprised a layered structure with uniform color formation and high capture efficiency between antigen and antibody that results in rapid and reproducible results. The device rapidly (90 s) assayed biotin as a model target, with a limit of detection (LOD) of 5.08 ng mL-1, and detected progesterone with an LOD of 84 pg mL-1 within 5 min. Moreover, sample volumes and reagent consumption rates were minimized. Thus, our device could be applied to competitive immunoassays of various small molecules in POCT.
  • Yubo Wang, Jose Enrico Q Quinsaat, Tomoko Ono, Masatoshi Maeki, Manabu Tokeshi, Takuya Isono, Kenji Tajima, Toshifumi Satoh, Shin-Ichiro Sato, Yutaka Miura, Takuya Yamamoto
    Nature communications 11 (1) 6089 - 6089 2020/11/30 [Refereed]
     
    Nano-sized metal particles are attracting much interest in industrial and biomedical applications due to the recent progress and development of nanotechnology, and the surface-modifications by appropriate polymers are key techniques to stably express their characteristics. Herein, we applied cyclic poly(ethylene glycol) (c-PEG), having no chemical inhomogeneity, to provide a polymer topology-dependent stabilization for the surface-modification of gold nanoparticles (AuNPs) through physisorption. By simply mixing c-PEG, but not linear counterparts, enables AuNPs to maintain dispersibility through freezing, lyophilization, or heating. Surprisingly, c-PEG endowed AuNPs with even better dispersion stability than thiolated PEG (HS-PEG-OMe). The stronger affinity of c-PEG was confirmed by DLS, ζ-potential, and FT-IR. Furthermore, the c-PEG system exhibited prolonged blood circulation and enhanced tumor accumulation in mice. Our data suggests that c-PEG induces physisorption on AuNPs, supplying sufficient stability toward bio-medical applications, and would be an alternative approach to the gold-sulfur chemisorption.
  • Akari Hashiba, Manaya Toyooka, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 327 467 - 476 2020/11/10 [Refereed]
     
    Although great advances have been made in the delivery of short RNAs by lipid nanoparticles (LNPs), the optimal formulation composition and physicochemical properties of LNPs for long RNA (including mRNA) remain unclear. In the present study, we optimized the lipid composition of liver-targeted mRNA-loaded LNPs that were prepared with pH-sensitive cationic lipids that had been previously designed for siRNA delivery through a two stepped design of experiment (DoE). Multiple responses including physicochemical properties, gene expression, and liver-specificity were analyzed in order, not only to understand the role of each formulation parameter, but also to examine parameters that would be difficult to predict. We found that particle size and the PEG-to-phospholipid (PEG/PL) ratio were additional key factors for liver-specific gene expression in addition to the other formulation factors. The optimized formulation showed a better gene expression compared to other lipid formulations from industry leaders. These findings suggest that a "DoE with multiple responses" approach can be used to predict significant parameters and permit optimized formulations to be prepared more efficiently.
  • Mao Fukuyama, Ayano Nakamura, Keine Nishiyama, Ayuko Imai, Manabu Tokeshi, Koji Shigemura, Akihide Hibara
    Analytical chemistry 92 (21) 14393 - 14397 2020/11/03 [Refereed]
     
    Fluorescent polarization immunoassay (FPIA) is a single-step immunoassay method that is applicable to point-of-care testing; however, its applicability to large biomolecules has been restricted because ordinary FPIA is a competitive assay. Here, we report a noncompetitive FPIA using the variable domain from the heavy chain of a camelid antibody (VHH antibody). FPIA with VHH was successfully used to quantitate rabbit immunoglobulin G (IgG) and demonstrated a wider response range than that observed with antibody-binding (Fab) fragment. Then, using a portable FPIA instrument, a VHH-based immunoassay of human IgG in a human serum certified reference material was demonstrated.
  • Keine Nishiyama, Yohei Takeda, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Yutaka Yonezawa, Kunitoshi Imai, Haruko Ogawa, Manabu Tokeshi
    Sensors and actuators. B, Chemical 316 128160 - 128160 2020/08/01 [Refereed][Not invited]
     
    A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 μL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS applied materials & interfaces 12 (30) 34011 - 34020 2020/07/29 [Refereed]
     
    Microfluidic methodologies for preparation of lipid nanoparticles (LNPs) based on an organic solvent injection method enable precise size control of the LNPs. After preparation of LNPs, the organic solvent injection method needs some post-treatments, such as overnight dialysis or direct dilution with a buffer solution. LNP production using the microfluidic-based organic solvent injection method is dominated by kinetics rather than thermodynamics. Kinetics of ethanol removal from the inner and outer membranes of LNPs could induce a structural change in the membrane that could lead to fusion of LNPs. However, the effects of microfluidic post-treatment on the final size of LNPs have not been sufficiently understood. Herein, we investigated the effect of the post-treatment processes on the final product size of LNPs in detail. A simple baffle device and a model lipid system composed of a neutral phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) and cholesterol were used to produce the LNPs. We demonstrated that flow conditions of the post-treatment diluting the remaining ethanol in the LNP suspension affected the final product size of LNPs. Based on the findings, we developed an integrated baffle device composed of an LNP production region and a post-treatment region for a microfluidic-based LNP production system; this integrated baffle device prevented the undesirable aggregation or fusion of POPC LNPs even for the high-lipid-concentration condition. Finally, we applied our concept to small interfering RNA (siRNA) delivery and confirmed that no significant effects due to the continuous process occurred on the siRNA encapsulation efficiency, biological distribution, and knockdown activity. The microfluidic post-treatment method is expected to contribute to the production of LNPs for practical applications and the development of novel LNP-based nanomedicines.
  • Masatoshi Maeki, Shohei Yamazaki, Reo Takeda, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS omega 5 (28) 17199 - 17206 2020/07/21 [Refereed]
     
    Preparation of high-quality protein crystals is a major challenge in protein crystallography. Natural convection is considered to be an uncontrollable factor of the crystallization process at the ground level as it disturbs the concentration gradient around the growing crystal, resulting in lower-quality crystals. A microfluidic environment expects an imitated microgravity environment because of the small Gr number. However, the mechanism of protein crystal growth in the microfluidic device was not elucidated due to limitations in measuring the crystal growth process within the device. Here, we demonstrate the real-time measurement of protein crystal growth rates within the microfluidic devices by laser confocal microscopy with differential interference contrast microscopy (LCM-DIM) at the nanometer scale. We confirmed the normal growth rates in the 20 and 30 μm-deep microfluidic device to be 42.2 and 536 nm/min, respectively. In addition, the growth rate of crystals in the 20 μm-deep microfluidic device was almost the same as that reported in microgravity conditions. This phenomenon may enable the development of more accessible alternatives to the microgravity environment of the International Space Station.
  • Hiroshi Suzuki, Kentaro Fujiyoshi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical chemistry 92 (13) 9132 - 9137 2020/07/07 [Refereed][Not invited]
     
    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.
  • Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ACS sensors 5 (5) 1287 - 1294 2020/05/22 [Refereed]
     
    Lithium carbonate is an effective medicine for the treatment of the bipolar disorder, but the concentration of lithium in the patient's blood must be frequently monitored because of its toxicity. To date, no colorimetric methods of lithium ion detection in whole blood without pretreatment have been reported. Here, we report a colorimetric paper-based device that allows point-of-care testing in one step. This device is composed of two paper-based elements linked to each other: a blood cell separation unit and a colorimetric detection unit. After a portion of whole blood has been placed on the end of the separation unit, plasma in the sample is automatically transported to the detection unit, which displays a diagnostic color. The key feature of this device is its simple, user-friendly operation. The limit of detection is 0.054 mM and the coefficient of variance is below 6.1%, which are comparable to those of conventional instruments using the same colorimetric reaction. Furthermore, we achieved high recovery (>90%) and reproducibility (<9.8%) with spiked human blood samples. Thus, the presented device provides an alternative method for the regular monitoring of lithium concentrations in the treatment of bipolar disorder by augmenting the coefficient of variation (maximum value, 6.1%).
  • Takashi Nakamura, Minori Kawai, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Molecular pharmaceutics 17 (3) 944 - 953 2020/03/02 [Refereed][Not invited]
     
    Because the lymph node (LN) is a critical organ for inducing immune responses against pathogens and cancers, the transport of immune functional molecules such as antigens and adjuvants to LNs by delivery systems is a useful strategy for the effective outcome of an immune response. The size and charge of a delivery system largely affect the transitivity to and distribution within LN. Although pH-sensitive lipid nanoparticles (LNPs) prepared by microfluidic mixing are the latest delivery system to be applied clinically, the effects of their size and charge on the transitivity to and distribution within LN are currently unknown. We investigated the size and charge effect of LNPs prepared by microfluidic mixing on transitivity to and distribution within LNs. A 30 nm-sized LNP (30-LNP) was efficiently translocated to LNs and was taken up by CD8+ dendritic cells, while the efficiency was drastically decreased in the cases of 100 and 200 nm-sized LNPs. Furthermore, a comparative study between neutral, positively, and negatively charged 30-LNP revealed that the negative 30-LNP moved to the LN more efficiently than the other LNPs. Interestingly, the negative 30-LNP reached the deep cortex, namely, the T cell zone. Our findings provide informative insights for designing LN-targeting LNPs prepared by microfluidic mixing and for the translocation of nanoparticles in LNs.
  • Yusuke Sato, Nana Okabe, Yusuke Note, Kazuki Hashiba, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Acta biomaterialia 102 341 - 350 2020/01/15 [Refereed][Not invited]
     
    Despite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, their poor stability and intracellular trafficking significantly hinders their use as potent small-sized LNPs. It has been reported that both the diffusion of lipid components from LNPs and the adsorption of proteins on the surface of LNPs are responsible for their decreased potency. To overcome this issue, we focused on the chemical structure of hydrophobic scaffolds of pH-sensitive cationic lipids with various lengths and shapes. LNPs composed of a pH-sensitive cationic lipid with long, linear scaffolds induced gene silencing in a dose-dependent manner, while LNPs with a classical scaffold length (C18) failed. Replacing the helper lipid from cholesterol to egg sphingomyelin (ESM) resulted in the formation of smaller LNPs with a diameter of ~22 nm and enhanced gene silencing activity. Most of the ESMs were located in the outer layer and functioned to stabilize the LNPs. Long, linear scaffolds contributed to immiscibility with phosphocholine-containing lipids including ESM. This contribution was dependent on the scaffold length of pH-sensitive cationic lipids. Although phosphocholine-containing lipids usually inhibit membrane fusion-mediated endosomal escape, long, linear scaffolds contributed to avoiding the inhibitory effect and to enhance the potency of the LNPs. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and the selection of appropriate helper lipids and will facilitate the development of highly potent small-sized LNPs. STATEMENT OF SIGNIFICANCE: Despite the fact that small-sized lipid nanoparticles (LNPs) are important for improved tissue penetration and efficient drug delivery, the size reduction-associated decrease in the stability and intracellular trafficking significantly hinders the development of potent small-sized LNPs. Our limited understanding of the mechanism underlying the reduced potency has also hindered the development of more potent small-sized LNPs. The findings of the present study indicate that long and linear hydrophobic scaffolds of pH-sensitive cationic lipids could overcome the loss of efficiency for nucleic acid delivery. In addition, the long hydrophobic scaffolds led to immiscibility with neutral phospholipids, resulting in efficient endosomal escape. These findings provide useful information needed for the rational design of pH-sensitive cationic lipid structures and will facilitate the development of highly potent small-sized LNPs.
  • Naoyuki Yogo, Tetsunari Hase, Toshihiro Kasama, Keine Nishiyama, Naoya Ozawa, Takahiro Hatta, Hirofumi Shibata, Mitsuo Sato, Kazuki Komeda, Nozomi Kawabe, Kohei Matsuoka, Toyofumi Fengshi Chen-Yoshikawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba, Yoshinori Hasegawa
    PloS one 15 (11) e0241422  2020 [Refereed]
     
    Detecting molecular targets in specimens from patients with lung cancer is essential for targeted therapy. Recently, we developed a highly sensitive, rapid-detection device (an immuno-wall device) that utilizes photoreactive polyvinyl alcohol immobilized with antibodies against a target protein via a streptavidin-biotin interaction. To evaluate its performance, we assayed epidermal growth factor receptor (EGFR) mutations, such as E746_A750 deletion in exon 19 or L858R substitution in exon 21, both of which are common in non-small cell lung cancer and important predictors of the treatment efficacy of EGFR tyrosine kinase inhibitors. The results showed that in 20-min assays, the devices detected as few as 1% (E746_A750 deletion) and 0.1% (L858R substitution) of mutant cells. Subsequent evaluation of detection of the mutations in surgically resected lung cancer specimens from patients with or without EGFR mutations and previously diagnosed using commercially available, clinically approved genotyping assays revealed diagnostic sensitivities of the immuno-wall device for E746_A750 deletion and L858R substitution of 85.7% and 87.5%, respectively, with specificities of 100% for both mutations. These results suggest that the immuno-wall device represents a good candidate next-generation diagnostic tool, especially for screening of EGFR mutations.
  • Donny Nugraha Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 35 (11) 1221 - 1226 2019/11/10 [Refereed][Not invited]
     
    In this study, we developed an electrochemical sensor for ochratoxin A (OTA) by using an aptamer having a dithiol-based anchor, which exhibited higher stability on a gold electrode than a monothiol-based aptamer because of its two anchors. The sensor was also based on a signal-on scheme that produces a signal current resulting from structure-switching of the aptamer upon interaction with OTA. For simple fabrication of this sensor, the non-covalent interaction of methylene blue with the aptamer was also employed as an electrochemical indicator. In this study, the performance of the sensor, including the dissociation constant of the aptamer-OTA complex, was characterized. The proposed sensor exhibited high reproducibility and enough sensitivity to detect the minimum amount of OTA required for the analysis of real food samples with a limit of detection of 113 pM.
  • Vitsarut Primpray, Orawon Chailapakul, Manabu Tokeshi, Theerasak Rojanarata, Wanida Laiwattanapaisal
    Analytica chimica acta 1078 16 - 23 2019/10/31 [Refereed][Not invited]
     
    The adulteration of herbal medicines by dexamethasone or prednisolone is regarded as a serious problem in many communities. Herein, a novel platform for the separation and quantification of both target steroids in herbal medicines based on electrochemical paper-based analytical devices (ePADs) has been created. The ePAD was composed of Whatman SG81 chromatography paper, 3D-printed devices and a commercial screen-printed electrode. Whatman SG81 silica-coated paper was used for the separation of dexamethasone and prednisolone based on the difference in their partition coefficients during the flow of the mobile phase. The optimal mobile phase was composed of 60% ethyl acetate in cyclohexane and required 7 min for separation. The separated steroids on the paper were then quantified by electrochemical detection using differential pulse voltammetry, in which the 3D-printed devices facilitated the measurement. Analytical detection ranges of 10-500 μg mL-1 were obtained for both dexamethasone and prednisolone (r2 = 0.988 and 0.994, respectively). The limits of detection for dexamethasone and prednisolone were 3.59 and 11.98 μg mL-1, respectively, whereas the limits of quantification were 6.00 and 20.02 μg mL-1, respectively. The amounts of both target steroids derived from real herbal medicine samples determined by the proposed method were comparable to those obtained with assays using standard high-performance liquid chromatography. In addition, a simple evaporation step can be used to increase the concentration of the samples before analysis. These ePADs are simple, low-cost, rapid, and very promising for on-site quantitative detection.
  • Kenia Chávez Ramos, Keine Nishiyama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Toshihiro Kasama, Yoshinobu Baba, Manabu Tokeshi
    ACS omega 4 (15) 16683 - 16688 2019/10/08 [Refereed][Not invited]
     
    Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23-100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.
  • Osamu Wakao, Ken Satou, Ayano Nakamura, Polina A Galkina, Keine Nishiyama, Ken Sumiyoshi, Fumio Kurosawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Mikhail A Proskurnin, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Lab on a chip 19 (15) 2581 - 2588 2019/08/07 [Refereed][Not invited]
     
    High-throughput fluorescence polarization immunoassays (FPIAs) for mycotoxin were conducted using a portable FP analyzer with a microdevice. Simultaneous FPIA measurements for 8 different deoxynivalenol (DON) concentrations in 12 chambers (total of 96 samples) and high-throughput FPIA measurements for single DON concentrations in more than 500 chambers were conducted. The results indicated that simultaneous FPIAs for 96 independent samples and for 500 samples were possible by FP imaging. The FP analyzer has a size of 65 cm (W 35 cm × D 15 cm × H 15 cm) and costs less than $5000. The sample volume was 1 nL. Furthermore, it is expected that sample reaction and FP detection can be automatically conducted with the analyzer by changing the microdevice and the software. Its features such as low cost and portability will contribute to on-site measurement and point-of-care testing. Additionally, the high-throughput feature will contribute to the study of molecular interactions based on FP measurements.
  • Keine Nishiyama, Toshihiro Kasama, Seiya Nakamata, Koya Ishikawa, Daisuke Onoshima, Hiroshi Yukawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    The Analyst 144 (15) 4589 - 4595 2019/08/07 [Refereed][Not invited]
     
    We present an ultrasensitive immunoassay system for disease biomarkers utilizing the immuno-wall device and an enzymatic amplification reaction. The immuno-wall device consisted of 40 microchannels, each of which contained an antibody-modified wall-like structure along the longitudinal axis of the microchannel. The wall was fabricated with a water-soluble photopolymer containing streptavidin by photolithography, and biotinylated capture antibodies were immobilized on the sides through streptavidin-biotin interaction. For an assay, introducing the target biomarker and secondary and labeled antibodies produced a sandwich complex anchored on the sides of the wall. A conventional immuno-wall device uses a fluorescence-labeled antibody as a labeling antibody. To achieve an ultrasensitive detection of a trace biomarker, we used an enzyme label and amplified the signal with the enzymatic reaction with a fluorogenic substrate in the microchannel. The highest signal/background ratio was obtained by using alkaline phosphatase-labeled antibody and 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate. To evaluate the device performance, we detected human C-reactive protein (CRP) as a model biomarker. The detection limit (LOD) of CRP in phosphate-buffered saline was 2.5 pg mL-1 with a sample volume of 0.25 μL. This LOD was approximately 3 orders of magnitude lower than that obtained with fluorescent-dye (DyLight 650)-labeled antibody. In addition, the present device provided a wide detection range of 0.0025-10 ng mL-1 for CRP. We successfully developed an ultrasensitive immunoassay system with simple operation and only a small sample volume.
  • Mitsue Hibino, Yuma Yamada, Naoki Fujishita, Yusuke Sato, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Journal of pharmaceutical sciences 108 (8) 2668 - 2676 2019/08 [Refereed][Not invited]
     
    A number of drugs that are currently on the market, as well as new candidates for drugs, are poorly water soluble. Because of this, a need exists to develop drug formulations that will permit the expanded use of such drugs. The use of liposomes and lipid nanoparticles for drug delivery has attracted attention as a technique for solubilizing molecules that are poorly water soluble, but this technique faces serious scale-up risks. In this study, we report on attempts to encapsulate Coenzyme Q10 (CoQ10) as a model of a poorly water-soluble drug in an MITO-Porter, a liposome for mitochondrial delivery using a microfluidic device (a CoQ10-MITO-Porter [μ]). The physical properties of the CoQ10-MITO-Porter [μ] including homogeneity, size, and preparation volume were compared with those for a CoQ10-MITO-Porter prepared by the ethanol dilution method (a CoQ10-MITO-Porter [ED]). In the case where a microfluidic device was used, a small-sized CoQ10-MITO-Porter was formed homogeneously, and it was possible to prepare it on a large scale. Intracellular observations using HeLa cells showed that the CoQ10-MITO-Porter [μ] was efficiently internalized by cells to reach mitochondria. These results indicate that the CoQ10-MITO-Porter [μ] represents a potential candidate for use in mitochondrial nanomedicine.
  • A Concentric Ring Array Electrode for a Wall-Jet Cell in a Microfluidic Device
    Keine Nishiyama, Koki Hoshikawa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Electroanalysis 31 1736 - 1743 2019/06 [Refereed][Not invited]
  • Sensitive Fluorescent Polarization Immunoassay by Optimizing Synchronization Mismatch Condition
    Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    Sensors and Actuators B 285 418 - 422 2019/04 [Refereed][Not invited]
  • Yusuke Sato, Kazuki Hashiba, Kosuke Sasaki, Masatoshi Maeki, Manabu Tokeshi, Hideyoshi Harashima
    Journal of controlled release : official journal of the Controlled Release Society 295 140 - 152 2019/02/10 [Refereed][Not invited]
     
    Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering short interfering RNA (siRNA) in vivo. A pH-sensitive cationic lipid that facilitates the targeting of hepatocytes and endosomal escape, strongly influences the availability of siRNA, thus making it a key material for efficient siRNA delivery. A systematic knowledge regarding lipid structure-activity relationships would greatly facilitate the development of sophisticated pH-sensitive cationic lipids for use in siRNA-based therapeutics. The systemic derivatization of a hydrophilic head group and hydrophobic tails of YSK12-C4, a pH-sensitive cationic lipid that was developed in our laboratory, revealed that hydrophilic head significantly affected the apparent pKa of the final product, a key factor in both intrahepatic distribution and endosomal escape. The clogP value of a hydrophilic head group was found to be associated with the apparent pKa of the product. In contrast, the hydrophobic tail structure strongly affected intrahepatic distribution without depending on apparent pKa. A structure-activity relationship study enabled the selection of an adequate combination of a hydrophilic head group and hydrophobic tails and permitted a potent LNP composed of a pH-sensitive cationic lipid CL4H6 (CL4H6-LNPs) to be developed that showed efficient gene silencing activity (50% effective dose: 0.0025 mg/kg), biodegradability and was tolerated. In vivo experiments revealed that the CL4H6-LNPs showed a superior efficiency for endosomal escape, cytosolic release and the RNA-induced silencing for the complex-loading of siRNAs compared to the previously developed LNPs.
  • Development of a Microdevice for Facile Analysis of Theophylline in Whole Blood by a Cloned Enzyme Donor Immunoassay
    Keine Nishiyama, Kanako Sugimura, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Lab on a Chip 19 233 - 240 2019/02 [Refereed][Not invited]
  • Label-Free Electrochemical Sensor for Ochratoxin A Uisng a Microfabricated Electrode with Immobilized Aptamer
    Donny N. Mazaafrianto, Akihiko Ishida, Masatoshi Maeki, Hirofumi Tani, Manabu Tokeshi
    ACS Omega 3 16823 - 16830 2018/12 [Refereed][Not invited]
  • Development of the immuno-wall device for rapid detection of ALK and ROS1 fusions in lung cancer
    Yogo N, Hase T, Kasama T, Hatta T, Ozawa N, Sato M, Kaji N, Tokeshi M, Baba Y, Hasegawa Y
    ANNALS OF ONCOLOGY 29 .  0923-7534 2018/11 [Not refereed][Not invited]
  • Niko Kimura, Masatoshi Maeki, Yusuke Sato, Yusuke Note, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi
    ACS Omega 3 (5) 5044 - 5051 2470-1343 2018/05/09 [Refereed][Not invited]
     
    The precise size control of the lipid nanoparticle (LNP)-based nanodrug delivery system (DDS) carriers, such as 10 nm size tuning of LNPs, is one major challenge for the development of next-generation nanomedicines. Size-controlled LNPs would realize size-selective tumor targeting and deliver DNA and RNA to target tumor tissues effectively by passing through the stromal cells. Herein, we developed a baffle mixer device named the invasive lipid nanoparticle production device, or iLiNP device for short, which has a simple two-dimensional microchannel and mixer structure, and we achieved the first reported LNP size tuning at 10 nm intervals in the size range from 20 to 100 nm. In comparison with the conventional LNP preparation methods and reported micromixer devices, our iLiNP device showed better LNP size controllability, robustness of device design, and LNP productivity. Furthermore, we prepared 80 nm sized LNPs with encapsulated small interfering RNA (siRNA) using the iLiNP device these LNPs effectively performed as nano-DDS carriers in an in vivo experiment. We expect iLiNP devices will become novel apparatuses for LNP production in nano-DDS applications.
  • Recent Microdevice-Based Aptamer Sensors
    D. N. Mazaafrianto, M. Maeki, A. Ishida, H. Tani, M. Tokeshi
    Micromachines 9 1 - 25 2018/04 [Refereed][Not invited]
  • Manabu Tokeshi
    Advanced Drug Delivery Reviews 128 1 - 2 1872-8294 2018/03/15 [Refereed][Not invited]
  • Masatoshi Maeki, Niko Kimura, Yusuke Sato, Hideyoshi Harashima, Manabu Tokeshi
    Advanced Drug Delivery Reviews 128 84 - 100 1872-8294 2018/03/15 [Refereed][Not invited]
     
    Lipid-based nanobiomaterials as liposomes and lipid nanoparticles (LNPs) are the most widely used nanocarriers for drug delivery systems (DDSs). Extracellular vesicles (EVs) and exosomes are also expected to be applied as DDS nanocarriers. The performance of nanomedicines relies on their components such as lipids, targeting ligands, encapsulated DNA, encapsulated RNA, and drugs. Recently, the importance of the nanocarrier sizes smaller than 100 nm is attracting attention as a means to improve nanomedicine performance. Microfluidics and lab-on-a chip technologies make it possible to produce size-controlled LNPs by a simple continuous flow process and to separate EVs from blood samples by using a surface marker, ligand, or electric charge or by making a mass or particle size discrimination. Here, we overview recent advances in microfluidic devices and techniques for liposomes, LNPs, and EVs and their applications for DDSs.
  • Osamu Wakao, Ken Satou, Ayano Nakamura, Ken Sumiyoshi, Masanori Shirokawa, Chikaaki Mizokuchi, Kunihiro Shiota, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi
    Review of Scientific Instruments 89 (2) 024103  1089-7623 2018/02/01 [Refereed][Not invited]
     
    Fluorescence polarization (FP) offers easy operation and rapid processing, making it implementable in molecular interaction analysis. Previously we have developed a unique FP measurement system using a liquid crystal (LC) layer and an image sensor. The system is based on a principle of synchronized detection between the switching rate of the LC layer and the sampling rate of the CCD. The FP system realized simultaneous multiple sample detection however, the measurement precision was lower than that of the conventional FP apparatus. The main drawbacks were low light transmittance of the LC layer and insufficient synchronization between the LC layer and CCD. In this paper, we developed a new FP analyzer based on LC-CCD synchronization detection. By using a newly designed LC with high transmittance and improving synchronization, the performance of the system has been dramatically improved. Additionally, we reduced the cost by using an inexpensive CCD and an LED as the excitation source. Simultaneous FP immunoassay of multiple samples of prostaglandin E2 was performed. The error rate of the FP system is reduced from 16.9% to 3.9%, as comparable to the commercial conventional FP system.
  • Takeshi Komatsu, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytical Sciences 34 (1) 39 - 44 1348-2246 2018 [Refereed][Invited]
     
    We report on the effects of fabrication methods, photolithography, wax printing, screen printing, and craft cutting, on selected properties of microfluidic paper-based analytical devices (μPADs): cost, fabrication precision, wicking rate, and analytical accuracy. Photolithography requires numerous fabrication steps, and an oxygen plasma treatment is necessary when using an aqueous solution. Although the boundary between the hydrophobic and hydrophilic areas in the μPAD is sharpest, the obtained K-scale intensity in measuring of protein concentrations is lower than those of the devices by other methods. Wax printing offers the simplest and fastest fabrication, although solution leakage measures should be taken to improve the wicking rate and to prevent cross-contamination. Screen printing also offers easy fabrication. The screenprinted μPAD has a good wicking performance and shows a high detection intensity. Craft cutting allows automated fabrication of many μPADs at once. The craft cut μPAD has the fastest wicking rate among the four μPADs due to bare cellulose fibers. We consider that the detection intensity of this μPAD can be raised by optimizing the evaporation rate.
  • Mao Fukuyama, Manabu Tokeshi, Mikhail A. Proskurnin, Akihide Hibara
    Lab on a Chip 18 (2) 356 - 361 1473-0189 2018 [Refereed][Not invited]
     
    We herein report the preparation of a surface that behaves in a hydrophobic manner but does not undergo protein adsorption in an aqueous/organic two-phase system. We found that polyethylene-glycol (PEG)-modified poly(dimethylsiloxane) (PDMS) exhibits hydrophobic properties when the surface is immersed in an organic solution, while the PEG moiety prevents protein adsorption on the PDMS surface in an aqueous solution at high protein concentrations due to the dynamic behaviour of the PEG moiety. As such, we demonstrated the in-well droplet formation of an aqueous solution containing a high protein concentration. In addition, to demonstrate the feasibility of this method in single cell analyses, a droplet array of a liquid medium containing 10% fetal bovine serum and HeLa cells was formed. The preparation of a droplet array using our PDMS-PEG surface to promote in-well droplet formation avoided the use of flow control equipment and complicated microstructures. We therefore expect that the dynamic wettability of our reported surface will be applicable in single cell and biochemical analyses, such as protein characterisation using crystallography or immunoassays.
  • Development of the immuno-wall device for rapid, low-cost detection of EGFR mutations in tumor samples from patients with lung cancer
    Yogo N, Hase T, Kasama T, Ozawa N, Sato M, Kaji N, Tokeshi M, Baba Y, Hasegawa Y
    ANNALS OF ONCOLOGY 28 .  0923-7534 2017/11 [Not refereed][Not invited]
  • Masatoshi Maeki, Yuka Fujishima, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    PLoS ONE 12 (11) e0187962  2017/11 [Refereed][Not invited]
     
    © 2017 Maeki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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.
  • Taiga Ajiri, Haruya Kasa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Junji Nishi, Manabu Tokeshi
    Analytical Sciences 33 (10) 1197 - 1199 0910-6340 2017/10 [Refereed][Not invited]
     
    Recently, we developed a label-free detection method based on optical diffraction, and implemented it in on our fabricated micro- and nanofluidic device. This detection method is simple and useful for detecting biomolecules, but the device fabrication consists of complicated processes. In this paper, we propose a simple method for fabricating the micro- and nanofluidic device; the fabrication combines laser interference lithography with conventional photolithography. The performance of a device fabricated by the proposed method is comparable to the performance of the device in our previous study.
  • Taiga Ajiri, Takao Yasui, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi
    Sensors and Actuators B-Chemical 250 39 - 43 0925-4005 2017/10 [Refereed][Not invited]
     
    Single-molecule detection of the biomolecules in a label-free manner has a tremendous impact for various fields. Recently, we developed a label-free detection method without any pretreatment procedures, which is based on optical diffraction derived from a nanofluidic channel array (in other words, a nanowall array). However, the single-molecule detection is hampered by the inherent sensitivity of the method. We propose a solution to improve the sensitivity of the method by adjusting the height of the nanowall array. Numerical simulations showed that a larger nanowall array height could provide better sensitivity, but a lower nanowall array height could provide better sensitivity difference, contrary to what we would intuitively expect. We used a 250 nm height nanowall array to achieve a label-free detection of 0.18 DNA molecules to verify the simulation prediction. These results demonstrate our method has a potential to be implemented in a highly sensitive refractometer with small sample consumption. (C) 2017 Elsevier B.V. All rights reserved.
  • Qiong Wu, Noritada Kaji, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Kazuki Nagashima, Manabu Tokeshi, Tomoji Kawai, Yoshinobu Baba
    Scientific Reports 7 43877  2045-2322 2017/03 [Refereed][Not invited]
     
    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-mu m 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.
  • Toshihiro Kasama, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Methods in Molecular Biology 1547 49 - 56 1064-3745 2017 [Not refereed][Not invited]
     
    © Springer Science+Business Media LLC 2017. 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.
  • Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytical Sciences 32 (12) 1359 - 1362 0910-6340 2016/12 [Refereed][Not invited]
     
    We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an important marker for chronic kidney disease in cats, using immuno-pillar chips. The required amount of reagent solution is 200 times smaller than that for the conventional ELISA in the 96-well microplate (0.5 mu L versus 100 mu L). In addition, the total assay time in the proposed method is more than 12 times shorter than in the conventional method (20 min versus 240 min). The limit of detection in the new method of 3 ng mL(-1) is comparable to that of the conventional method (1 ng mL(-1)) and it is in the clinically relevant range.
  • Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analytical and Bioanalytical Chemistry 408 (27) 7559 - 7563 1618-2642 2016/11 [Refereed][Not invited]
     
    A novel washing technique for microfluidic paper-based analytical devices (mu PADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow mu PADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model mu PAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 mu g mL(-1).
  • Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Sensors and Actuators B-Chemical 236 433 - 441 0925-4005 2016/11 [Not refereed][Not invited]
     
    This article describes the development of a simple, portable assay system using microfluidic paper-based analytical devices (mu PADs) coupled with colorimetric detection for rapid measurements. The properties of different paper substrates were first investigated to determine which type of paper would be the most suitable for the fabrication of the mu PADs. Simultaneous detection of horseradish peroxidase (HRP) utilizing a 5 mu L sample analytical volume was demonstrated using a single mu PAD. Hydrophilic test regions were separated by hydrophobic barriers, which were fabricated through photolithography. These test regions were immobilized with 10 mM of 3,3',5,5'-tetramethylbenzidine for HRP assay. The detection range obtained with the proposed system covered HRP concentrations from 0.37 to 124 fmol (or 31000 ng mL(-1)). The detection limit (blank + 3 sigma) for HRP was calculated to be 0.69 fmol (or 5.58 ng mL(-1)) through a 4-parameter logistic nonlinear regression using results obtained within a 15 min assay time. The findings obtained using the developed system suggest that mu PAD assay systems for simple but highly sensitive measurements can be designed to give on-site determinations of target compounds using peroxidase-conjugated molecules. ((c)) 2016 Elsevier B.V. All rights reserved.
  • Takao Yasui, Kensuke Ogawa, Noritada Kaji, Mats Nilsson, Taiga Ajiri, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    Scientific Reports 6 31642  2045-2322 2016/08 [Refereed][Not invited]
     
    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.
  • Lori Shayne Alamo Busa, Takeshi Komatsu, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES 32 (8) 815 - 818 0910-6340 2016/08 [Refereed][Not invited]
     
    We report on the colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide using horseradish peroxidase on photolithography-fabricated (P-PAD) and wax-printed (W-PAD) paper-based analytical devices. Fabricating PADs via photolithography exposes the hydrophilic areas to polymers (photoresists) and solvents, not only reducing the hydrophilicity, but also affecting the TMB-H2O2 assay system with an unavoidable incomplete elimination of photoresist during fabrication. Detection signals are then observed in the presence of photoresist residues on the P-PAD, even at a blank HRP concentration.
  • Takao Yasui, Jumpei Morikawa, Noritada Kaji, Manabu Tokeshi, Kazuo Tsubota, Yoshinobu Baba
    Micromachines 7 (7) 113  2072-666X 2016/07 [Refereed][Not invited]
     
    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 mu m 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.
  • Yusuke Sato, Yusuke Note, Masatoshi Maeki, Noritada Kaji, Yoshinobu Baba, Manabu Tokeshi, Hideyoshi Harashima
    Journal of Controlled Release 229 48 - 57 0168-3659 2016/05 [Refereed][Not invited]
     
    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. (C) 2016 Elsevier B.V. All rights reserved.
  • Hidekatsu Tazawa, Shohei Sunaoshi, Manabu Tokeshi, Takehiko Kitamori, Ritsuko Ohtani-Kaneko
    Analytical Sciences 32 (3) 349 - 353 0910-6340 2016/03 [Refereed][Not invited]
     
    In this study, we developed an integrated, low-cost microfluidic cell culture system that is easy to use. This system consists of a disposable polystyrene microchip, a polytetrafluoroethylene valve, an air bubble trap, and an indium tin oxide temperature controller. Valve pressure resistance was validated with a manometer to be 3 MPa. The trap protected against bubble contamination. The temperature controller enabled the culture of Macaca mulatta RF/6A 135 vascular endothelial cells, which are difficult to culture in glass microchips, without a CO2 incubator. We determined the optimal coating conditions for these cells and were able to achieve stable, confluent culture within 1 week. This practical system is suitable for low-cost screening and has potential applications as circulatory cell culture systems and research platforms in cell biology.
  • Takao Yasui, Jumpei Morikawa, Noritada Kaji, Manabu Tokeshi, Kazuo Tsubota, Yoshinobu Baba
    Micromachines 7 (7) 113 - 113 2072-666x 2016 [Refereed][Not invited]
     
    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.
  • Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Micromachines 7 (5) 1 - 21 2072-666X 2016 [Refereed][Not invited]
     
    Food and water contamination cause safety and health concerns to both animals and humans. Conventional methods for monitoring food and water contamination are often laborious and require highly skilled technicians to perform the measurements, making the quest for developing simpler and cost-effective techniques for rapid monitoring incessant. Since the pioneering works of Whitesides' group from 2007, interest has been strong in the development and application of microfluidic paper-based analytical devices (μPADs) for food and water analysis, which allow easy, rapid and cost-effective point-of-need screening of the targets. This paper reviews recently reported μPADs that incorporate different detection methods such as colorimetric, electrochemical, fluorescence, chemiluminescence, and electrochemiluminescence techniques for food and water analysis.
  • Masatoshi Maeki, Hiroshi Yamaguchi, Manabu Tokeshi, Masaya Miyazaki
    Analytical Sciences 32 (1) 3 - 9 1348-2246 2016 [Refereed][Not invited]
     
    This review summarizes two microfluidic-based protein crystallization methods, protein crystallization behavior in the microfluidic devices, and their applications for X-ray crystal structure analysis. Microfluidic devices provide many advantages for protein crystallography they require small sample volumes, provide high-throughput screening, and allow control of the protein crystallization. A droplet-based protein crystallization method is a useful technique for high-throughput screening and the formation of a single crystal without any complicated device fabrication process. Well-based microfluidic platforms also enable effective protein crystallization. This review also summarizes the protein crystal growth behavior in microfluidic devices as, is known from viewpoints of theoretical and experimental approaches. Finally, we introduce applications of microfluidic devices for on-chip crystal structure analysis.
  • Lori Shayne Alamo Busa, Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analyst 141 (24) 6598 - 6603 0003-2654 2016 [Refereed][Not invited]
     
    The development of a competitive immunoassay system for colorimetric detection on microfluidic paper-based analytical devices (mu PADs) is reported. The mu PADs were fabricated via photolithography to define hydrophilic flow channels and consisted of three main elements: the control and test zones, where target detection was performed, the sample introduction zone, and the competitive capture zone located between the sample introduction zone and the test zone. The chromogenic substrate 3,3', 5,5'-tetra-methylbenzidine (TMB) was deposited at the control and test zones. mu PAD surface modification was performed at the capture zone first via chitosan activation, then the BSA-conjugated target compound was immobilized. The sample solution consisting of the target compound, the peroxidase-conjugated antibody, and the hydrogen peroxide oxidizing agent was introduced into the device and competition occurred at the capture zone, allowing only the target-bound peroxidase-conjugated antibody to travel past the capture zone and into the test zone via capillary action. The developed competitive immunoassay system was successfully demonstrated on the mu PAD detection of biotin as a model compound with a detection limit of 0.10 mu g mL(-1). The applicability of the proposed immunoassay system for point-of-need testing was further demonstrated using aflatoxin B-1, a highly toxic foodborne substance, with a detection limit of 1.31 ng mL(-1). The mu PAD with the competitive immunoassay format showed promising results for practical applications in point-of-need testing involving small molecular weight targets in food and water safety and quality monitoring, environmental analysis, and clinical diagnostics.
  • Takeshi Komatsu, Saeed Mohammadi, Lori Shayne Alamo Busa, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analyst 141 (24) 6507 - 6509 0003-2654 2016 [Refereed][Not invited]
     
    The combination of a microfluidic paper-based analytical device (mu PAD) and digital image analysis is widely used for quantitative analysis with mu PADs because of its easy and simple operation. Herein, we have demonstrated a quantitative analysis based on multiple color changes on a mu PAD. The CIE L*a*b* color system was employed to analyse the digital images obtained with the mu PAD. We made pH measurements using a universal pH-indicator showing multiple color changes for various pH values of aqueous test solutions. The detectable pH range of this method was wider than the typical grayscale-based image analysis, and we succeeded in the measurements for a wide pH range of 2-9.
  • Masatoshi Maeki, Shohei Yamazaki, Ashtamurthy S. Pawate, Akihiko Ishida, Hirofumi Tani, Kenichi Yamashita, Masakazu Sugishima, Keiichi Watanabe, Manabu Tokeshi, Paul J. A. Kenis, Masaya Miyazaki
    CrystEngComm 18 (40) 7722 - 7727 1466-8033 2016 [Refereed][Not invited]
     
    Protein crystallization and subsequent X-ray diffraction analysis of the three-dimensional structure are necessary for elucidation of the biological functions of proteins and effective rational drug design. Therefore, controlling protein crystallization is important to obtain high resolution X-ray diffraction data. Here, a simple microfluidic method using chips with 10 and 50 mu m high crystallization chambers to selectively form suitable single protein crystals for X-ray analysis is demonstrated. As proof of concept, three different types of proteins: lysozyme, glucokinase from Pseudoalteromonas sp. AS-131 (PsGK), and NADPH-cytochrome P450 oxidoreductase-heme oxygenase complex were crystallized. We demonstrate that the crystal growth orientation depends on the height of the crystallization chamber regardless of the protein type. Our results suggest that the confined micro space induces the protein molecules to adhere to a specific crystal face and affects the growth kinetics of each crystal face. The present microfluidic-based protein crystallization method can reform a suitable single protein crystal for X-ray analysis from aggregates of needle-shaped protein crystals.
  • 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
    Science and Technology of Advanced Materials 17 (1) 618 - 625 1468-6996 2016 [Refereed][Not invited]
     
    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 mu l 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.
  • Akihiko Ishida, Mitsutaka Fujii, Takehiro Fujimoto, Shunsuke Sasaki, Ichiro Yanagisawa, Hirofumi Tani, Manabu Tokeshi
    ANALYTICAL SCIENCES 31 (11) 1163 - 1169 0910-6340 2015/11 [Refereed][Not invited]
     
    A compact and lightweight liquid chromatography system is presented with overall dimensions of 26 cm width x 18 cm length x 21 cm height and weight of 2 kg. This system comprises a battery-operated compact electroosmotic pump, a manual injector, a microfluidic chip device containing a packed column and an electrochemical detector, and a USB bus-powered potentiostat. The pumping system was designed for microfluidic-based reversed-phase liquid chromatography in which an electroosmotically generated water stream pushes the mobile phase via a diaphragm for the output. The flow rate ranged from 0 to 10 mu L/min and had a high degree of precision. The pumping system operated continuously for over 24 h with dry batteries. The column formed in the microfluidic device was packed with 3-mu m ODS particles with a length of 30 mm and a diameter of 0.8 mm. The results presented herein demonstrate the performance of the pumping system and the column using alkylphenols, catecholamine, catechin, and amino acids.
  • Takao Yasui, Satoru Ito, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 31 (11) 1197 - 1200 0910-6340 2015/11 [Refereed][Not invited]
     
    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.
  • Osamu Wakao, Yusaku Fujii, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi
    Analytical Chemistry 87 (19) 9647 - 9652 0003-2700 2015/10 [Refereed][Not invited]
     
    The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.
  • Hidekatsu Tazawa, Kenjiro Sato, Atsuhiro Tsutiya, Manabu Tokeshi, Ritsuko Ohtani-Kaneko
    Thrombosis Research 136 (2) 328 - 334 0049-3848 2015/08 [Refereed][Not invited]
     
    Endothelial damage induced by a highly elevated body temperature is crucial in some diseases including viral hemorrhagic fevers. Here, we report the heat-induced sequential changes of endothelial cells under shear stress, which were determined with a microfluidic culture system. Although live cell imaging showed only minor changes in the appearance of heat-treated cells, Hsp70 mRNA expression analysis demonstrated that the endothelial cells in channels of the system responded well to heat treatment. F-actin staining also revealed clear changes in the bundles of actin filaments after heat treatment. Well-organized bundles of actin filaments in control cells disappeared in heat-treated cells cultured in the channel. Furthermore, the system enabled detection of sequential changes in plasminogen activator inhibitor-1 (PAI-1) secretion from endothelial cells. PAI-1 concentration in the effluent solution was significantly elevated for the first 15 min after initiation of heat treatment, and then decreased subsequently. This study provides fundamental information on heat-induced endothelial changes under shear stress and introduces a potent tool for analyzing endothelial secretions. (C) 2015 Elsevier Ltd. All rights reserved.
  • Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    Nano Letters 15 (5) 3445 - 3451 1530-6984 2015/05 [Refereed][Not invited]
     
    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 micro-channels 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.
  • Masatoshi Maeki, Ashtamurthy S. Pawate, Kenichi Yamashita, Masahide Kawamoto, Manabu Tokeshi, Paul J. A. Kenis, Masaya Miyazaki
    Analytical Chemistry 87 (8) 4194 - 4200 0003-2700 2015/04 [Refereed][Not invited]
     
    We demonstrate a seamless and contactless method from protein crystallization to X-ray analysis using a microfluidic chip with the aim of obtaining a complete crystallographic data set of a protein crystal under cryogenic conditions. Our microfluidics-based approach did not require direct manipulation of the protein crystal. Therefore, the microfluidic chip approach is suitable for novices of X-ray analysis of protein crystals. We also investigated the effect of stepwise cryoprotection on the quality of protein crystals. Protein crystals with cryoprotection via on-chip manipulation did not show deterioration of crystallographic quality of the protein crystal. The complete diffraction data set of a protein crystal, which is required for determining the 3D structure of the target protein, is obtainable by a simple manipulation.
  • Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Nanotechnology and Plant Sciences: Nanoparticles and Their Impact on Plants 183 - 201 2015/01/01 [Refereed][Not invited]
     
    © Springer International Publishing Switzerland 2015. 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.
  • Akihiko Ishida, Mitsutaka Fujii, Takehiro Fujimoto, Shunsuke Sasaki, Ichiro Yanagisawa, Hirofumi Tani, Manabu Tokeshi
    Analytical Sciences 31 (11) 1163 - 1169 1348-2246 2015 [Refereed][Not invited]
     
    A compact and lightweight liquid chromatography system is presented with overall dimensions of 26 cm width × 18 cm length × 21 cm height and weight of 2 kg. This system comprises a battery-operated compact electroosmotic pump, a manual injector, a microfluidic chip device containing a packed column and an electrochemical detector, and a USB buspowered potentiostat. The pumping system was designed for microfluidic-based reversed-phase liquid chromatography in which an electroosmotically generated water stream pushes the mobile phase via a diaphragm for the output. The flow rate ranged from 0 to 10 μL/min and had a high degree of precision. The pumping system operated continuously for over 24 h with dry batteries. The column formed in the microfluidic device was packed with 3-μm ODS particles with a length of 30 mm and a diameter of 0.8 mm. The results presented herein demonstrate the performance of the pumping system and the column using alkylphenols, catecholamine, catechin, and amino acids.
  • Saeed Mohammadi, Masatoshi Maeki, Reza M. Mohamadi, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Analyst 140 (19) 6493 - 6499 0003-2654 2015 [Refereed][Not invited]
     
    This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 mu m, as obtained by this method. Fabricated microfluidic paper-based analytical devices (mu PADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 mu M, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for mu PADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.
  • Toyohito Naito, Noritada Kaji, Manabu Tokeshi, Takuya Kubo, Yoshinobu Baba, Koji Otsuka
    Analytical Methods 7 (17) 7264 - 7269 1759-9660 2015 [Refereed][Not invited]
     
    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 degrees 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.
  • 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
    Analytical Methods 7 (12) 5092 - 5095 1759-9660 2015 [Refereed][Not invited]
     
    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 mu L 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 mu g SE). Using our devices, frequent assessment of food potentially contaminated with SE is possible.
  • Masatoshi Maeki, Tatsuyoshi Saito, Yusuke Sato, Takao Yasui, Noritada Kaji, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Hideyoshi Harashima, Manabu Tokeshi
    RSC Advances 5 (57) 46181 - 46185 2046-2069 2015 [Refereed][Not invited]
     
    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.
  • NISHIWAKI Nanako, KASAMA Toshihiro, ISHIDA Akihiko, TANI Hirofumi, BABA Yoshinobu, TOKESHI Manabu
    BUNSEKI KAGAKU 日本分析化学会 64 (5) 329 - 335 0525-1931 2015 [Refereed][Not invited]
     
    In order to realize ultra-early diagnosis of disease in practical applications, we have fabricated next-generation immuno-pillar devices with higher sensitivity. In the newly developed devices, capture antibodies were immobilized on affinity beads based on chemical bonding, while in the previous-generation ones, polystyrene beads were used for physical adsorption-based immobilization. To evaluate the sensitivity of the next-generation immuno-pillar device, we quantitatively analyzed C-reactive protein (CRP). The limit of detection was estimated to be 0.1 ng mL−1 (total assay time, 23 min), which was twoorders of magnitude lower than that obtained by using the previous-generation immuno-pillar device, and was low enough to perform the CRP test. In addition, we investigated the storage stability of the immuno-pillar device, and confirmed that the device can retain its performance for over 9 months.
  • OKAMOTO Yukihiro, HIBINO Ayato, KAJI Noritada, TOKESHI Manabu, BABA Yoshinobu
    BUNSEKI KAGAKU 日本分析化学会 64 (1) 9 - 13 0525-1931 2015 [Refereed][Not invited]
     
    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.
  • Toshiya Yoshiiwa, Satoshi Umezu, Manabu Tokeshi, Yoshinobu Baba, Mitsuru Shindo
    Journal of Flow Chemistry 4 (4) 180 - 184 2062-249X 2014/12 [Refereed][Not invited]
     
    We report a method for the generation and subsequent reaction of ynolates in a flow microreactor via a stop-flow process. This procedure allowed for the synthesis of ynolates at ambient temperature within 11 min via a Li-Br exchange reaction with sec-butyllithium, whereas the corresponding batch process generally requires low temperature control and extended reaction times of up to 1 h. The stop-flow method is especially useful for optimizing the reaction time without having to use various microtube lengths. The resulting ynolates were applied to the olefination of carbonyl compounds and to pyrrole synthesis. These results indicate the practical utility of the ynolate reaction and should contribute to progress in flash chemistry.
  • Yusuke Nakatani, Chiaki Shido, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Luminescence 29 83 - 84 1522-7235 2014/08 [Refereed][Not invited]
  • Ryoko Kurishiba, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi
    Luminescence 29 78 - 78 1522-7235 2014/08 [Refereed][Not invited]
  • Hirofumi Tani, Ai Masuyama, Akihiko Ishida, Manabu Tokeshi
    Luminescence 29 99 - 100 1522-7235 2014/08 [Refereed][Not invited]
  • Satoshi Umezu, Toshiya Yoshiiwa, Manabu Tokeshi, Mitsuru Shindo
    Tetrahedron Letters 55 (10) 1822 - 1825 0040-4039 2014/03 [Refereed][Not invited]
     
    A new method has been developed for the generation and subsequent reaction of ynolates in a micro flow reactor system. This new procedure allowed for ynolates to be prepared at 0 degrees C or ambient temperature within 1 min via a reductive lithiation reaction, whereas the corresponding batch processes generally require low temperature control and extended reaction times of up to 1 h. The resulting ynolates were applied to the olefination of carbonyl compounds, with the reactions reaching completion in a much shorter reaction time in the continuous flow reactor than the batch reactor. These results highlight the practical utility of the ynolate reaction, and represent the first reported example of the use of lithium naphthalenide in a flow microreactor, which would contribute to progress of the flash chemistry. (C) 2014 Elsevier Ltd. All rights reserved.
  • Jun Wang, Michihiko Aki, Daisuke Onoshima, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba
    Biosensors and Bioelectronics 51 280 - 285 0956-5663 2014/01 [Refereed][Not invited]
     
    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. (C) 2013 Elsevier B.V. All rights reserved.
  • Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    Microfluidics and Nanofluidics 14 (6) 961 - 967 1613-4982 2013/06 [Refereed][Not invited]
     
    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.
  • Takehiko Tsukahara, Hiroyasu Hotokezaka, Masayuki Harada, Yoshikuni Kikutani, Manabu Tokeshi, Yasuhisa Ikeda
    Microfluidics and Nanofluidics 14 (6) 989 - 994 1613-4982 2013/06 [Refereed][Not invited]
     
    We have developed a novel microchip equipped with a microchannel and Pt microelectrode array for electrochemically controlling valences of actinide (An) species. The square wave voltammograms of the redox reaction of potassium hexacyanoferrate(II) in the microchannel were measured. We found that the fabricated Pt microelectrode array has superior performances for the detection of the electrochemically active species in the microchannel. Therefore, the potentiostatic electrolysis experiments of uranium ions were carried out in the microchannel, and the concentration changes of uranium ions accompanied by the potentiostatic electrolysis were examined using thermal lens microscope. The results showed that the redox reactions between U(VI) and U(IV) can be performed completely in a microchannel in a few minutes, that is, the microscale reaction is accelerated by a factor of more than 10 compared with the bulk solution reactions taking hours mostly. The developed microchip was found to have enough performances for realizing rapid and highly efficient redox reactions for An species, which are impossible in the bulk reactions.
  • 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
    ACS Nano 7 (4) 3029 - 3035 1936-0851 2013/04 [Refereed][Not invited]
     
    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. © 2013 American Chemical Society.
  • Toyohiro Naito, Ai Yatsuhashi, Noritada Kaji, Taeko Ando, Kazuo Sato, Hisao Moriya, Hiroaki Kitano, Takao Yasui, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 29 (3) 367 - 371 0910-6340 2013/03 [Refereed][Not invited]
     
    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.
  • Wanchun Jin, Keiko Yamada, Mai Ikami, Noritada Kaji, Manabu Tokeshi, Yusuke Atsumi, Makoto Mizutani, Atsushi Murai, Akira Okamoto, Takao Namikawa, Yoshinobu Baba, Michio Ohta
    Journal of Microbiological Methods 92 (3) 323 - 31 2013/03 [Refereed][Not invited]
     
    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.
  • Quantum Dots Conjugated with Transferrin for Brain Tumor Cell Imaging
    Hiroshi Yukawa, Ryoko Tsukamoto, Ayano Kano, Yukihiro Okamoto, Manabu Tokeshi, Tetsuya Ishikawa, Masaaki Mizuno, Yoshinobu Baba
    Journal of Cell Science & Therapy 4 150-1 - 150-7 2013 [Refereed][Not invited]
  • Takao Yasui, Koki Motoyama, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    RSC Advances 3 (10) 3237 - 3240 2046-2069 2013 [Refereed][Not invited]
     
    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.
  • Toyohiro Naito, Rerngchai Arayanarakool, Severine Le Gac, Takao Yasui, Noritada Kaji, Manabu Tokeshi, Albert van den Berg, Yoshinobu Baba
    Lab on a Chip 13 (3) 452 - 458 1473-0197 2013 [Refereed][Not invited]
     
    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 degrees C, which is essential for developing highly parallel PCR array devices without the need for complicated peripherals to control the valve operation.
  • Toshihiro Kasama, Yutaka Hasegawa, Hiroyuki Matsumoto, Haruyasu Kondo, Tsutomu Ozawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    2013 International Symposium on Micro-NanoMechatronics and Human Science, MHS 2013 1 - 3 2013 [Refereed][Not invited]
     
    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. © 2013 IEEE.
  • 加地 範匡, 渡慶次 学, 馬場 嘉信
    現代化学 東京化学同人 1 (502) 50 - 54 0386-961X 2013/01 [Not refereed][Not invited]
  • Takao Yasui, Yosuke Inoue, Toyohiro Naito, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Chemistry 84 (21) 9282 - 9286 0003-2700 2012/11 [Refereed][Not invited]
     
    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.
  • Katsuma Kitazoe, Yeon-Su Park, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba
    PLOS ONE 7 (6) e39057-1 - 8 1932-6203 2012/06 [Refereed][Not invited]
     
    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 Peclet numbers (>10(5)) 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.
  • Hiroshi Yukawa, Masaki Watanabe, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshitaka Miyamoto, Hirofumi Noguchi, Yoshinobu Baba, Shuji Hayashi
    Biomaterials 33 (7) 2177 - 2186 0142-9612 2012/03 [Refereed][Not invited]
     
    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 in vivo 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 (R) Spectrum analysis. Almost all of the transplanted ASCs were observed to accumulate in the lungs within 10 min 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 ex vivo 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. (C) 2011 Elsevier Ltd. All rights reserved.
  • Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    RSC Advances 2 (2) 398 - 400 2046-2069 2012 [Refereed][Not invited]
     
    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.
  • Takao Yasui, Yusuke Omoto, Keiko Osato, Noritada Kaji, Norikazu Suzuki, Toyohiro Naito, Yukihiro Okamoto, Manabu Tokeshi, Eiji Shamoto, Yoshinobu Baba
    Analytical Sciences 28 (1) 57 - 59 0910-6340 2012/01 [Refereed][Not invited]
     
    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 x 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.
  • Maged F. Serag, Noritada Kaji, Manabu Tokeshi, Alberto Bianco, Yoshinobu Baba
    Integrative Biology 4 (2) 127 - 131 1757-9694 2012 [Refereed][Not invited]
     
    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.
  • Yeon-Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Nanoscience and Nanotechnology 12 (1) 539 - 546 1533-4880 2012/01 [Refereed][Not invited]
     
    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)(34) magic-sized cluster (d similar to 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.
  • Yusuke OMOTO, Takashi KATO, Norikazu SUZUKI, Takao YASUI, Keiko OSATO, Noritada KAJI, Manabu TOKESHI, Yoshinobu BABA, Yasuhiko SAKAI, Eiji SHAMOTO
    TRANSACTIONS OF THE JAPAN SOCIETY OF MECHANICAL ENGINEERS Series B 一般社団法人 日本機械学会 78 (788) 762 - 768 0387-5016 2012 [Refereed][Not invited]
     
    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.
  • Daisuke Onoshima, Jun Wang, Michihiko Aki, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba
    Analytical Methods 4 (12) 4368 - 4372 1759-9660 2012 [Refereed][Not invited]
     
    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 mu m) 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 mu M) comparable to U- or Z-shaped microfabricated absorbance detection cells in glass. Calibration curves for different solution concentrations were obtained with good R-2 values (similar to 1).
  • Yeon-Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Methods in molecular biology (Clifton, N.J.) 906 125 - 41 2012 [Refereed][Not invited]
     
    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.
  • Takao Yasui, Mohamad Reza Mohamadi, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
    Biomicrofluidics 5 (4) 044114-1 - 9 1932-1058 2011/12 [Refereed][Not invited]
     
    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. (C) 2011 American Institute of Physics. [doi:10.1063/1.3668233]
  • 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
    Analytical Chemistry 83 (21) 8252 - 8258 0003-2700 2011/11 [Refereed][Not invited]
     
    Adipose tissue-derived stein 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 (Qps-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 HNO3 and H2O2 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.
  • Sonoko Inoue, Noritada Kaji, Masatoshi Kataoka, Yasuo Shinohara, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
    ELECTROPHORESIS 32 (22) 3241 - 3247 0173-0835 2011/11 [Refereed][Not invited]
     
    We have developed a separation technique for DNAprotein complex based on electrophoretic mobility shift assay (EMSA) by microchip electrophoresis, which we call microchip electrophoretic mobility shift assay (mu EMSA). To evaluate the mu EMSA, we employed recombinant human nuclear factor-?B (rhNF-B) and its consensus double-stranded oligonucleotide (dsOligo) fluorescently labeled with Cy5. We carried out the electrophoretic separation of the consensus dsOligo-rhNF-B complex and the unbound dsOligo in methylcellulose solution and confirmed rapid (similar to 200s) and reliable identification and semi-quantitation of the specific interaction between dsOligo and rhNF-B. The binding specificity of rhNF-?B 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-B complex formation reached equilibrium (ca. 90% of the dsOligo was bound to rhNF-B) after 5min. Furthermore, without any purification process, even crude NF-?B in nuclear extracts from HeLa cells was specifically detected within 120s by the mu EMSA.
  • Maged F. Serag, Noritada Kaji, Enrica Venturelli, Yukihiro Okamoto, Kazuyoshi Terasaka, Manabu Tokeshi, Hajime Mizukami, Kevin Braeckmans, Alberto Bianco, Yoshinobu Baba
    ACS Nano 5 (11) 9264 - 9270 1936-0851 2011/11 [Refereed][Not invited]
     
    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.
  • Yeon-Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Nanoparticle Research 13 (11) 5781 - 5798 1388-0764 2011/11 [Refereed][Not invited]
     
    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 Cd-containing species capable of passivating dangling bonds on the CdSe core surface. Each CdSe QD (d similar to 2.5 nm) consisted of a CdSe core (d similar to 2.1 nm), a cysteine (cys)-ligand shell, and an adsorption layer composed of Cd-cys complexes (mainly CdL(-H)(-), cys a parts per thousand H2L), cys (as L2-), Cd(OH)(2), and CdO (x) (x a parts per thousand yen 1). Our CdSe QDs showed strong blue band-edge PL as well as strong green surface trap PL. Their PL quantum yield (QY) of similar to 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.
  • Yasutoshi Ban, Yoshikuni Kikutani, Manabu Tokeshi, Yasuji Morita
    Journal of Nuclear Science and Technology 48 (10) 1313 - 1318 0022-3131 2011/10 [Refereed][Not invited]
     
    Extraction of Am(III) was performed at the interface of organic-aqueous two-layer flow in a microchannel having an asymmetric cross section. A solution of 3 mol/dm(3) nitric acid containing Am-243(III) and octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphineoxide diluted with n-dodecane were introduced into the microchannel as the aqueous phase and organic phase, respectively. The two phases formed a stable two-layer flow with an interface parallel to the sidewall of the microchannel, and they were separated from each other at the divergence point of the microchannel. The extraction reaction of Am(III) proceeded at the interface of the two phases, and reached the equilibrium state while the two phases passed through the microchannel.
  • Tomoya Tachi, Tetsunari Hase, Yukihiro Okamoto, Noritada Kaji, Takeshi Arima, Hiroyuki Matsumoto, Masashi Kondo, Manabu Tokeshi, Yoshinori Hasegawa, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 401 (7) 2301 - 2305 1618-2642 2011/10 [Refereed][Not invited]
     
    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.
  • Takao Yasui, Noritada Kaji, Mohamad Reza Mohamadi, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    ACS Nano 5 (10) 7775 - 7780 1936-0851 2011/10 [Refereed][Not invited]
     
    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 (mu(EOF-channel) = alpha C-i(-0.5), where C-i is the bulk concentration of the i-ions and alpha 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, mu(EOF-nanopillar) approximate to beta[C-i -(C-i(2)/N-i)], where N-i is the number of sites available to i-ions and beta differs for each nanopiliar 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 nanopiliar 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.
  • Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    Analytical Chemistry 83 (17) 6635 - 6640 0003-2700 2011/09 [Refereed][Not invited]
     
    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.
  • YASUI Takao, KAJI Noritada, OKAMOTO Yukihiro, TOKESHI Manabu, HORIIKE Yasuhiro, BABA Yoshinobu
    電気学会研究会資料. BMS, バイオ・マイクロシステム研究会 = The papers of Technical Meeting on Bio Micro Systems, IEE Japan 2011 (8) 43 - 45 2011/06/30 [Not refereed][Not invited]
  • Jun Wang, Daisuke Onoshima, Michihiko Aki, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Chemistry 83 (9) 3528 - 3532 0003-2700 2011/05 [Refereed][Not invited]
     
    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.
  • Maged F. Serag, Noritada Kaji, Claire Gaillard, Yukihiro Okamoto, Kazuyoshi Terasaka, Mohammad Jabasini, Manabu Tokeshi, Hajime Mizukami, Alberto Bianco, Yoshinobu Baba
    ACS Nano 5 (1) 493 - 499 1936-0851 2011/01 [Refereed][Not invited]
     
    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.
  • Jun Wang, Yong Zhang, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analyst 136 (6) 1142 - 1147 0003-2654 2011 [Refereed][Not invited]
     
    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.
  • Kazuma Mawatari, Toshinori Ohashi, Tomohiko Ebata, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 11 (17) 2990 - 2993 1473-0197 2011 [Refereed][Not invited]
     
    A thermal lens detection device was developed to realize an easy-to-use, portable and sensitive detector for nonfluorescent molecules. Two laser diodes (658 nm for excitation and 785 nm for probe) were made coaxial in an optical unit and were coupled to a single-mode optical fiber. On a microfluidic chip, a small holder for the optical fiber was fixed, and micro-lenses (numerical aperture of 0.2) were also integrated inside the holder. The micro-lenses were designed to realize an adequate chromatic aberration (50 mm), which was essential for sensitive thermal lens detection. Compared with conventional thermal lens detection systems which required very laborious and accurate optical alignment with the microchannel, the new device needed just attachment-detachment of the optical fiber, which was important for practical application. The lower limit of detection was 10 nM for nickel (II) phthalocyaninetetrasulfonic acid tetrasodium salt solutions (model sample), and the absorbance was 9 x 10(-6) AU. The absolute number of molecules detected was less than 200 zmol. The coefficient of variance for 5-time attachment-detachment of the optical probe was as small as 3.6%. The technical development allowed integration of the thermal lens detection devices inside a microsystem (e. g. enzyme-linked immuno-sorbent assay system), and practical microsystems were realized with sensitivities several-orders higher than absorptiometry.
  • Katsuma Kitazoe, Jun Wang, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba
    Lab on a Chip 11 (19) 3256 - 3262 1473-0197 2011 [Refereed][Not invited]
     
    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.
  • Takao Yasui, Yusuke Omoto, Keiko Osato, Noritada Kaji, Norikazu Suzuki, Toyohiro Naito, Masaki Watanabe, Yukihiro Okamoto, Manabu Tokeshi, Eiji Shamoto, Yoshinobu Baba
    Lab on a Chip 11 (19) 3356 - 3360 1473-0197 2011 [Refereed][Not invited]
     
    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. TheMBTdevice with 10.4 mm mixing length enables us to achieve complete mixing of a FITC solution (D = 2.6 x 10(-10) m(2) s(-1)) within 51 ms and an IgG solution (D = 4.6 x 10(-11) m(2) 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 Peclet number to attain complete mixing in the MBT device is estimated to be 6.9 x 10(4).
  • Maged F. Serag, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
    15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011 1 58 - 60 2011 [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Yeon-Su Park, Andriy Dmytruk, Igor Dmitruk, Atsuo Kasuya, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Physical Chemistry C 114 (44) 18834 - 18840 1932-7447 2010/11 [Refereed][Not invited]
     
    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.
  • Keisuke Kondo, Noritada Kaji, Sayaka Toita, Yukihiro Okamoto, Manabu Tokeshi, Kazunari Akiyoshi, Yoshinobu Baba
    Biomicrofluidics 4 (3) 032210-1 - 10 1932-1058 2010/09 [Refereed][Not invited]
     
    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. (C) 2010 American Institute of Physics. [doi:10.1063/1.3479997]
  • YASUI Takao, KAJI Noritada, OKAMOTO Yukihiro, TOKESHI Manabu, HORIIKE Yasuhiro, BABA Yoshinobu
    Micro Total Analysis System 2009 2010 (7) 33 - 35 2010/06/18 [Not refereed][Not invited]
  • 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
    Biomaterials 31 (14) 4094 - 4103 0142-9612 2010/05 [Refereed][Not invited]
     
    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 x 10(4) 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. (C) 2010 Elsevier Ltd. All rights reserved.
  • Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
    Chemical Society Reviews 39 (3) 948 - 56 2010/03 [Not refereed][Not invited]
     
    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.
  • Yasuko Yoshida, Kousuke Niwa, Kazunari Yamada, Manabu Tokeshi, Yoshinobu Baba, Yoshio Saito, Akimitsu Okamoto, Isao Saito
    Chemistry Letters 39 (2) 116 - 117 0366-7022 2010/02 [Refereed][Not invited]
     
    We have developed a new probe containing two base-discriminating fluorescent (BDF) nucleosides for single nucleotide polymorphism (SNP) typing. It is possible to determine the SNP type using just one probe. We confirmed suitability of the probe from hybridization assay results obtained for the ALDH2 gene.
  • Yeon-Su Park, Andriy Dmytruk, Igor Dmitruk, Atsuo Kasuya, Motohiro Takeda, Noriaki Ohuchi, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    ACS Nano 4 (1) 121 - 128 1936-0851 2010/01 [Refereed][Not invited]
     
    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 short and long-term stabilities, whereas those having relatively compact spatial geometry near the interface led to NIPS 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 dearly showed general trends for the possibility of selective growth of stable semiconductor NPs with particular sizes in aqueous solution.
  • Sugiura Kanako, Kaji Noritada, Okamoto Yukihiro, Tokeshi Manabu, Baba Yoshinobu
    IEEJ Transactions on Sensors and Micromachines 一般社団法人 電気学会 130 (10) 471 - 475 1341-8939 2010 [Refereed][Not invited]
     
    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.
  • Mai Ikami, Ayako Kawakami, Masaya Kakuta, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Lab on a Chip 10 (24) 3335 - 3340 1473-0197 2010 [Refereed][Not invited]
     
    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 mu m diameter polystyrene beads. To evaluate the chip performance, we applied it to the sandwich assay of C-reactive protein (CRP), alpha-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 mu 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.
  • Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
    Bio-Inspired Nanomaterials and Nanotechnology 41 - 58 2010/01/01 [Refereed][Not invited]
     
    © 2010 Nova Science Publishers, Inc. 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.
  • Jun Wang, Yong Zhang, Molhamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Electrophoresis 30 (18) 3250 - 3256 0173-0835 2009/09 [Refereed][Not invited]
     
    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-gamma-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.
  • Masaya Murata, Yukihiro Okamoto, Yeon-Su Park, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 394 (1) 277 - 283 1618-2642 2009/05 [Refereed][Not invited]
     
    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.
  • Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Lab on a Chip 9 (7) 966 - 71 2009/04/07 [Refereed][Not invited]
     
    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 microm-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.
  • Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Chemistry 81 (8) 3194 - 3198 0003-2700 2009/04 [Refereed][Not invited]
     
    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.
  • Arata Aota, Kazuma Mawatari, Susumu Takahashi, Teruki Matsumoto, Kazuteru Kanda, Ryo Anraku, Akihide Hibara, Manabu Tokeshi, Takehiko Kitamori
    Microchimica Acta 164 (3-4) 249 - 255 0026-3672 2009/03 [Refereed][Not invited]
     
    Phase separation of gas-liquid and liquid-liquid microflows in microchannels were examined and characterized by interfacial pressure balance. We considered the conditions of the phase separation, where the phase separation requires a single phase flow in each output of the microchannel. As the interfacial pressure, we considered the pressure difference between the two phases due to pressure loss in each phase and the Laplace pressure generated by the interfacial tension at the interface between the separated phases. When the pressure difference between the two phases is balanced by the Laplace pressure, the contact line between the two phases is static. Since the contact angle characterizing the Laplace pressure is restricted to values between the advancing and receding contact angles, the Laplace pressure has a limit. When the pressure difference between the two phases exceeds the limiting Laplace pressure, one of the phases leaks into the output channel of the other phase, and the phase separation fails. In order to experimentally verify this physical picture, microchips were used having a width of 215 mu m and a depth of 34 mu m for the liquid-liquid microflows, a width of 100 mu m and a depth of 45 mu m for the gas-liquid microflows. The experimental results of the liquid-liquid microflows agreed well with the model whilst that of the gas-liquid microflows did not agree with the model because of the compressive properties of the gas phase and evaporation of the liquid phase. The model is useful for general liquid-liquid microflows in continuous flow chemical processing.
  • Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 25 (2) 149 - 51 2009/02 [Refereed][Not invited]
     
    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 beta-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 microg mL(-1). Our CEDIA using a microfluidic device was very simple and rapid, unlike microchip-based heterogeneous immunoassays and CEDIA on a well-type microchip.
  • Toshinori Ohashi, Kazuma Mawatari, Kae Sato, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 9 (7) 991 - 995 1473-0197 2009 [Refereed][Not invited]
     
    We have developed a novel, practical micro-ELISA system for sensitive and rapid allergy diagnosis. The enzymatic reactions occurred under stopped-flow conditions, resulting in both high precision and high sensitivity. A BSA-biotin-avidin linker was introduced for the immobilization of water-soluble allergens on polystyrene microbeads, enabling immobilization of allergens in sufficient density to provide high sensitivity. Evaluation of the system's performance showed a good detection limit (2 ng/mL) for total IgE measurement. In addition, a good correlation with a conventional method (CAP method) was demonstrated using human serum samples from 85 allergy patients. Importantly, sample volumes (5 mu L) were 10 times smaller and analysis time (12 min) was > 20 times faster than the conventional method. All procedures were automatically regulated with our simple microfluidic system, and all the fluidic, optic and electronic components were integrated for portability. We believe that our system has the potential to become a very powerful tool, particularly for point-of-care diagnosis.
  • Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba
    Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences 174 - 176 2009 [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Hiroshi Yukawa, Shogo Mizufune, Chiharu Mamori, Yukimasa Kagami, Koichi Oishi, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Hirofumi Noguchi, Yoshinobu Baba, Michinari Hamaguchi, Nobuyuki Hamajima, Shuji Hayashi
    Cell Transplantation 18 (5-6) 591 - 599 0963-6897 2009 [Not refereed][Not invited]
     
    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.
  • Manabu Tokeshi, Takehiko Kitamori
    Advances in Flow Analysis 149 - 166 2008/09/16 [Refereed][Not invited]
  • Manabu Tokeshi, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 391 (8) 2693 - 2694 1618-2642 2008/08 [Refereed][Not invited]
  • Hiroshi Kuramoto, Yeon-Su Park, Noritada Kaji, Manabu Tokeshi, Kentaro Kogure, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 391 (8) 2729 - 2733 1618-2642 2008/08 [Not refereed][Not invited]
     
    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.
  • Daisuke Kuroda, Yong Zhang, Jun Wang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 391 (7) 2543 - 2549 1618-2642 2008/08 [Not refereed][Not invited]
     
    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 25C, 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.
  • Yoshikuni Kikutani, Kazuma Mawatari, Kenji Katayama, Manabu Tokeshi, Takashi Fukuzawa, Mitsuo Kitaok, Takehiko Kitamori
    Sensors and Actuators B-Chemical 133 (1) 91 - 96 0925-4005 2008/07 [Refereed][Not invited]
     
    A novel micro-flow velocimeter, the flowing thermal lens micro-flow velocimeter (FTL-MFV) is developed in which a photothermally generated local refractive index change (a thermal lens) is utilized as a micro-tracer of flow. Flow velocity is measured from the time required for the thermal lens to travel between two points. Generation and detection of thermal lenses are carried out optically without contacting the flow. By choosing the wavelength of the excitation beam pulse so that it coincides with the absorption band of the solvent used, thermal lenses can be generated without adding anything to the liquid. Synchronous detection using a lock-in amplifier makes detection of thermal lens with a very small temperature rise possible. Thus, with the FTL-MFV, non-contact in situ measurement of flow can be carried out with only slight disturbance to the microfluid. In order to make the sensor small, optical fibers and SELFOC(TM) microlenses are used in focusing the excitation and probe beams. A dynamic range of 25-300 mu L/min is realized in measurement of flow rates in a microchannel. (C) 2008 Elsevier B.V. All rights reserved.
  • Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Electrophoresis 29 (12) 2533 - 2538 0173-0835 2008/06 [Refereed][Not invited]
     
    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.
  • Maged Fouad, Mohammad Jabasini, Noritada Kaji, Kazuyoshi Terasaka, Manabu Tokeshi, Hajime Mizukami, Yoshinobu Baba
    Electrophoresis 29 (11) 2280 - 2287 0173-0835 2008/06 [Refereed][Not invited]
     
    We describe a new and selective analytical method for the separation and quantitation of plant glucosinolates. The new method, which utilizes microchip CE (mu-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 glucosinotates-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 mu g/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 mu-CE in plant metabolomics analyses without use of labeling fluorophores.
  • Ryo Anraku, Kazuma Mawatari, Manabu Tokeshi, Masatoshi Nara, Takahiro Asai, Akihiko Hattori, Takehiko Kitamori
    Electrophoresis 29 (9) 1895 - 1901 0173-0835 2008/05 [Refereed][Not invited]
     
    Thermal lens microscope (TLM) is a sensitive detection method for nonfluorescent molecules and widely applied to detection in a capillary or on a microchip. In this paper, we developed a flexible design tool for TLM systems to meet various applications utilizing a microspace. The TL effect was modeled, including signal processing, and calculated by combining fluidic dynamics and wave optics software. The coincidence of the calculations and experiments was investigated by measuring the effects of optical path length or focus positions of the excitation beams on TL signals which are quite difficult to calculate by a conventional method. Good agreement was shown and the applicability of the TLM design tool was verified.
  • Eiki Maeda, Masatoshi Kataoka, Shouki Yatsushiro, Kazuaki Kajimoto, Mami Hino, Noritada Kaji, Manabu Tokeshi, Mika Bando, Jun-Ichi Kido, Mitsuru Ishilkawa, Yasuo Shinohara, Yoshinobu Baba
    Electrophoresis 29 (9) 1902 - 1909 0173-0835 2008/05 [Not refereed][Not invited]
     
    A high-performance determination system for alpha-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 relationship between amylase activity and fluorescence intensity in the range of 5-500 U/L of and the LOD was 4.38 U/L. Amylase activities in 13 amylase activity (r(2) = 0.9995 p < 0.01), 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.
  • Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Chromatography A 1192 (2) 289 - 293 0021-9673 2008/05 [Not refereed][Not invited]
     
    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 x 10(-5) cm(2) 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. (c) 2008 Elsevier B.V. All rights reserved.
  • Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba
    Analytical Chemistry 80 (7) 2483 - 2490 0003-2700 2008/04 [Not refereed][Not invited]
     
    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 degrees 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.
  • Laili Mahmoudian, Jonas Melin, Mohamad Reza Mohamadi, Keiko Yamada, Michio Ohta, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba
    Analytical Sciences 24 (3) 327 - 332 0910-6340 2008/03 [Not refereed][Not invited]
     
    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 degrees 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 I 10 s. Purification was found to be an indispensable step for the analysis of the C2CA product of genomic DNA samples.
  • Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 24 (3) 321 - 325 0910-6340 2008/03 [Not refereed][Not invited]
     
    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.
  • Hiroko Ueno, Jun Wang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Separation Science 31 (5) 898 - 903 1615-9306 2008/03 [Not refereed][Not invited]
     
    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-benzoxacliazole, which has a short labeling time for amino acids, was used as the fluorescently labeled dye. This NICE 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.
  • Daisuke Onoshima, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 24 (2) 181 - 183 0910-6340 2008/02 [Not refereed][Not invited]
     
    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).
  • Yukihiro Okamoto, Yeon Su Park, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference 892 - 894 2008 [Refereed][Not invited]
     
    We have developed an easy and versatile method for the surface modification of the microchannels in polymer microchips. Our method employed N-5-azido-2- nitrobenzoyloxysuccinimide, a photo-reactive azide, for the surface modification. Unlike the previously developed methods, it allows us to immobilize biomolecules without sacrificing their activity as well as to coat polymers which inhibit protein adsorption. Moreover, it does not require specific equipment, highly toxic chemicals, or labor-intensive procedures. © 2008 CBMS.
  • H. Okada, N. Kaji, M. Tokeshi, Y. Baba
    12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference 251 - 253 2008 [Refereed][Not invited]
     
    We developed a new surface modification method using poly(methyl methacrylate) (PMMA) with a film of a cellulose derivative to ensure the long-term stability of the modification. For electrophoretic protein separation using PMMA chips as well as other polymeric chips, protein adsorption is so inevitable that analytical errors (band broadening, low detection sensitivity) are unavoidable. However, our new method overcame these drawbacks of polymeric materials and provided much better analytical performances (higher theoretical plates and consecutive runs in a single channel) than other reported methods of surface modification. © 2008 CBMS.
  • Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Chemistry 80 (1) 312 - 316 0003-2700 2008/01 [Not refereed][Not invited]
     
    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 mu/mu(0)) is correlated with the ratio of cross-linking agent to PVP ([c(Mg)(2+)/c(PVP)]) as In mu/mu(0) = A'[c(Mg)(2+)/c(PVP)](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%.
  • Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Electrophoresis 28 (24) 4582 - 9 0173-0835 2007/12 [Refereed][Not invited]
     
    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 degrees ) and EOF was significantly suppressed (up to 4.0 x 10(-6) cm2.V(-1)s(-1)). Three proteins (20.5, 68.0, and 114.6 kDa) were successfully 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 x 10(5)).
  • Yong Zhang, Guichen Ping, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Electrophoresis 28 (18) 3308 - 14 0173-0835 2007/09 [Refereed][Not invited]
     
    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 degrees on a pristine PMMA surface to 37.5 degrees 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, DeltaDi-diS(B)/DeltaDi-diS(D) and DeltaDi-0S/DeltaDi-HA, was achieved in Tris-borate buffer within 130 s by MCE.
  • Yong Zhang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Expert Review of Proteomics 4 (4) 565 - 72 2007/08 [Refereed][Not invited]
     
    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.
  • Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Chemistry 79 (10) 3667 - 72 0003-2700 2007/05/15 [Refereed][Not invited]
     
    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.
  • Akihiro Sawada, Shogo Mizufune, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 387 (8) 2645 - 54 1618-2642 2007/04 [Refereed][Not invited]
     
    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.
  • Yong Zhang, Guichen Ping, Bingmei Zhu, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Electrophoresis 28 (3) 414 - 21 0173-0835 2007/02 [Refereed][Not invited]
     
    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, beta-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 monosulfate and trisulfate unsaturated disaccharides (DeltaDi-UA2S, DeltaDi-4S, DeltaDi-6S, and DeltaDi-triS) derivatized with 2-aminoacridone hydrochloride. A mixture of monosulfate disaccharide isomers (DeltaDi-UA2S, DeltaDi-4S, and DeltaDi-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 E(sep) of 558 V/cm. The theoretical plate was in the range of 5 x 10(5) to 1 x 10(6) m-1.
  • Ryo Anraku, Takahiro Asai, Kenji Uchiyama, Akihiko Hattori, Manabu Tokeshi, Takehiko Kitamori
    WORLD CONGRESS ON MEDICAL PHYSICS AND BIOMEDICAL ENGINEERING 2006, VOL 14, PTS 1-6 14 318 - + 1680-0737 2007 [Refereed][Not invited]
     
    In this study, we compared the observation experiment of a liquid-liquid interface with a simulation result about the three-phase micro channel used for an extraction reaction. As a result, each flow rate ratio greatly influences the formation of the three-phase flow. And the result of computing this multiphase flow model is corresponding to the experiment well, so the development of the unit chemistry operation model will become possible with the simulation in the future.
  • Shinichiro Hiki, Manabu Tokeshi, Masaya Kakuta, Kazuma Mawatari, Yoshikuni Kikutani, Kiichi Sato, Akihide Hibara, Kiyohito Shimura, Naoyuki Uchida, Takehiko Kitamori
    Bunseki Kagaku 56 (1) 1 - 7 0525-1931 2007 [Refereed][Not invited]
     
    A UV excitation thermal-lens microscope (UV-TLM) with an excitation beam wavelength of 266 nm and liquid chromatograph (LC) were connected to build a new system (LC/UV-TLM) for the separation and highly sensitive label-free determination of biomolecules, such as peptides. A variation in the composition of the mobile phase with time, as in gradient elution, is very commonly utilized in LC for improved separations. Thermal-lens signals are generally highly dependent on the properties of the media surrounding the photo-absorber. This effect must be carefully considered in such situations where the composition of a solution drastically changes in time. Therefore, we tested the developed LC/UV-TLM system operated in a gradient elution mode, by observing the effects of the variation in the solution composition upon TLM signal intensities. In water/acetonitrile gradient elution, no serious effects were observed as long as the acetonitrile content was less than 40% v/v. We then tested the developed system in the separation of a standard sample solution of mixed synthetic peptides we found that the developed system was 10-times more sensitive than a conventional system with the spectrophotometer used as a detector. © 2007 The Japan Society for Analytical Chemistry.
  • Laili Mahmoudian, Mohamad Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, Third Edition 1527 - 1542 2007/01/01 [Refereed][Not invited]
     
    © 2008 by Taylor & Francis Group, LLC. 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.4 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.5 In conventional gel electrophoresis, DNA molecules move in the electric field based on a size-dependent mobility through pores in a gel matrix.6 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.
  • Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Chemical Record (New York, N.Y.) 7 (5) 295 - 304 1527-8999 2007 [Refereed][Not invited]
     
    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. 10.1002/tcr.20128.
  • Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Analytical Sciences 23 (1) 21 - 4 0910-6340 2007/01 [Refereed][Not invited]
     
    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.
  • M. Kakuta, H. Takahashi, S. Kazuno, K. Murayama, T. Ueno, M. Tokeshi
    Measurement Science and Technology 17 (12) 3189 - 3194 1361-6501 2006/12/01 [Refereed][Not invited]
     
    A microchip-based semi-automated heterogeneous immunoassay was developed wherein a flow injection-like system and repeatable-use gel were employed. Microfluidics used a pump with highly accurate control of flow rate, two injector valves and a microchip. The results of a system suitability test based on dye injection for microfluidics including a thermal lens microscope as a detector were good. A regeneration buffer for antigen-antibody interaction of brain natriuretic peptide (BNP) was found which did not lose binding activity of the antibody on the gels and integration of such an immunoassay scheme into a semi-automated microfluidics system would facilitate repeated-use heterogeneous immunoassay. Good reproducibility of BNP assay was obtained with the selected regeneration buffer. The concentration of BNP in the range of 0.1-100 pg mL -1 (n ≤ 3) was successfully determined using the system and protocol developed here. © 2006 IOP Publishing Ltd.
  • Eiichiro Tamaki, Akihide Hibara, Haeng-Boo Kim, Manabu Tokeshi, Takehiko Kitamori
    JOURNAL OF CHROMATOGRAPHY A 1137 (2) 256 - 262 0021-9673 2006/12 [Refereed][Not invited]
     
    We developed a novel flow control system for a nanofluidic chemical process. Generally, flow control in nanochannels is difficult because of its high-pressure loss with very small volume flow rate. In our flow control method, liquid pressure in a microchannel connected to the nanochannels is regulated by utilizing a backpressure regulator. The flow control method was verified by using simple structured microchip, which included parallel nanochannels. We found that the observed flow rate was three times lower than the value expected from Hagen-Poiseuille's equation. That implied a size-dependent viscosity change in the nanochannels. Then, we demonstrated mixing of two different fluorescent solutions in a Y-shaped nanochannel and also a proton exchange reaction in the Y-shaped nanochannel. The flow control method will contribute to further integration of nanochemical systems. (c) 2006 Elsevier B.V. All rights reserved.
  • Noritada Kaji, Ryo Ogawa, Akio Oki, Yasuhiro Horiike, Manabu Tokeshi, Yoshinobu Baba
    Analytical and Bioanalytical Chemistry 386 (3) 759 - 64 1618-2642 2006/10 [Refereed][Not invited]
     
    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.
  • Hajime Miyaguchi, Manabu Tokeshi, Yoshikuni Kikutani, Akihide Hibara, Hiroyuki Inoue, Takehiko Kitamori
    JOURNAL OF CHROMATOGRAPHY A 1129 (1) 105 - 110 0021-9673 2006/09 [Refereed][Not invited]
     
    A microchip-based liquid-liquid extraction for the gas chromatography analysis of urine for amphetamine-type stimulants has been developed. Partially modified microchannels with the capillarity restricted modification (CARM) method were employed for stabilizing the interface consisting of 1-chlorobutane and alkalized urine. Reliability of the microchip-based extraction was evaluated with respect to linearity, trueness and precision. As a practical demonstration, methoxyphenamine hydrochloride (50 mg) was administered to three healthy volunteers, and the concentration of methoxyphenamine in their urine was determined by both methods for comparison. This study showed the potential of pressure-driven microfluidics to contribute to the rapid automation analysis in forensic toxicology. (c) 2006 Elsevier B.V. All rights reserved.
  • Mohammad Jabasini, Ashraf Abdulazim Ewis, Maged Fouad, Fuquan Dang, Guichen Ping, Toshikatsu Shinka, Yutaka Nakahori, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Biological & Pharmaceutical Bulletin 29 (7) 1487 - 9 0918-6158 2006/07 [Refereed][Not invited]
     
    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.
  • Fuquan Dang, Wenhao Li, Lihua Zhang, Mohammad Jabasini, Tatsuhiro Ishida, Hiroshi Kiwada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Chromatography. A 1118 (2) 218 - 25 0021-9673 2006/06/23 [Refereed][Not invited]
     
    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 (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.
  • Kazuma Mawatari, Manabu Tokeshi, Takehiko Kitamori
    Analytical Sciences 22 (5) 781 - 784 0910-6340 2006/05 [Refereed][Not invited]
     
    A detection and fixation method of single and multiple gold nanoparticles on the wall of a microfluidic channel is demonstrated. A thermal lens microscope (TLM) with continuous-wave excitation (wavelength, 532 nm) and probe (wavelength, 670 nm) laser beams was used to realize the sensitive detection of heat generated by light absorption of individual gold nanoparticles (50 nm in diameter) fixation of the individual nanoparticles was realized simultaneously. The fixation mechanism was investigated and attributed to an absorption-based optical force. In addition to single nanoparticle detection, multiple-nanoparticle detection and fixation was demonstrated. An acceleration of fixation was observed when the number of fixed particles was increased. TLM is expected to be a powerful tool for both the quantitative detection and precise fixation of individual nanoparticles. 2006 © The Japan Society for Analytical Chemistry.
  • Masayo Yamauchi, Kazuma Mawatari, Akihide Hibara, Manabu Tokeshi, Takehiko Kitamori
    Analytical Chemistry 78 (8) 2646 - 2650 0003-2700 2006/04/15 [Refereed][Not invited]
     
    A novel chiral detector, a circular-dichroism thermal lens microscope (CD-TLM), was developed to realize sensitive and selective detection of small volume chiral samples on a microchip. To realize chiral recognition on TLM, an excitation beam was phase-modulated at a frequency of 1.2 kHz, and left-circularly polarized light (LCPL) and right-circularly polarized light (RCPL) were generated. Then, the differential light absorption between LCPL and RCPL, which is the CD effect, was detected as thermal lens signal intensity and phase. As a standard sample, optically active tris(ethylenediamine)cobalt(III) [Co-(en) 3] 3+I 3 - aqueous solutions were used for performance evaluations. First, we verified the basic principle for selective chiral analysis by comparing the signals in intensity-modulation and phase-modulation modes of the excitation beam. Also, we found that the g-factor, which is significant for determining enantiomeric excess, agreed well with the value obtained by the CD spectrometer. The limit of detection (LOD) for enantiopure [Co-(en) 3] 3+I 3 - was 6.3 × 10 -5 M (1.9 × 10 -7 abs) for (-)-Co(en) 3 3+, and the sensitivity in absorbance units was more than 250 times higher than that in a CD spectrophotometer. Finally, we demonstrated enantiomeric excess determination on a microchip. The LOD was 1.7% (8.5 × 10 -7 abs) for (-)-Co(en) 3 3+ and at least one order superior to the LOD of a CD spectrometer. The applicability of CD-TLM for sensitive chiral analysis on a microchip was verified, and CD-TLM is expected to be promising for microchip-based chiral synthesis and analysis systems. © 2006 American Chemical Society.
  • Kiichi Sato, Akiko Egami, Tamao Odake, Manabu Tokeshi, Makoto Aihara, Takehiko Kitamori
    Journal of Chromatography A 1111 (2) 228 - 232 0021-9673 2006/04/14 [Refereed][Not invited]
     
    A cellular biochemistry analysis system was integrated on a quartz glass microchip with a microchamber for cell culture followed by a microchannel for detecting with a thermal lens microscope (TLM). Nerve cells from rat hippocampus were successfully cultured to form neural networks in the microchip. An aqueous solution of glutamate, which is known as a neurotransmitter, was introduced to stimulate the cultured neuron to release a retrograde messenger, arachidonate which is considered to be critical for neuronal plasticity, especially for long-term potentiation (LTP). After the introduction, the solution that flowed through the culture chamber was analyzed using the UV-TLM (excitation wavelength, 244 nm). The measured signal intensity was dependent on glutamate solution concentration, and the neurons were considered to release the retrograde messenger according to the glutamate concentration. This system is suitable for time-course monitoring of ultra trace amounts of chemicals released from very small amount of cultured cells. © 2005 Elsevier B.V. All rights reserved.
  • E Tamaki, A Hibara, HB Kim, M Tokeshi, T Ooi, M Nakao, T Kitamori
    ANALYTICAL SCIENCES 22 (4) 529 - 532 0910-6340 2006/04 [Refereed][Not invited]
     
    We developed a fabrication method and a liquid filling method for a nano chemical reactor that used Y-shaped nanochannels specially designed for mixing and reacting. In order to reduce the pressure loss and to utilize the characteristics of the nanochannel, inlet microchannels were fabricated just beside the nanochannels. We investigated all initial liquid filling method into the nanochannels that ensured there were no air bubbles that could cause a flow stack due to the capillary pressure. In our method, the micro- and nanochannels were filled with carbon dioxide and any remaining air during the initial liquid introduction was dissolved utilizing the high solubility of carbon dioxide. We propose that chemical reactions in nanospaces can be realized by utilizing these fabrication and liquid introduction techniques.
  • Kouichi Tsuji, Yousuke Hanaoka, Akihide Hibara, Manabu Tokeshi, Takehiko Kitamori
    Spectrochimica Acta - Part B Atomic Spectroscopy 61 (4) 389 - 392 0584-8547 2006/04 [Not refereed][Not invited]
     
    A chemical microchip, which has a flat region on the surface, was recently designed for total reflection X-ray fluorescence (TXRF) analysis. A sample solution was introduced from an inlet by a microsyringe and flowed into a microchannel. Finally it overflowed from the well-type microchannel on the flat region. The sample solution on this region was dried, and then measured by TXRF. The TXRF spectra could be measured with a low background level. This preliminary result indicated that the edge of the well-type channel would not cause a serious problem for TXRF analysis. In addition, a good linear relationship was obtained for Zn Kα in Zn standard solution. This suggests that quantitative analysis by TXRF is feasible in combination with a chemical microchip. © 2006.
  • Mohammad Jabasini, Yuji Murakami, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Biological & Pharmaceutical Bulletin 29 (4) 595 - 604 0918-6158 2006/04 [Refereed][Not invited]
     
    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.
  • Fuquan Dang, Kazuaki Kakehi, Kazuki Nakajima, Yasuo Shinohara, Mitsuru Ishikawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Journal of Chromatography. A 1109 (2) 138 - 43 0021-9673 2006/03/24 [Refereed][Not invited]
     
    A novel method for fast profiling of complex oligosaccharides released from glycoproteins based on microchip electrophoresis (mu-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 ( G2'), maltriose (G3) and panose (G3') as oligosaccharide isomer models. In mu-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, alpha1-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 mu-CE, indicating that the present mu-CE-based method is a promising alternative for characterization of the N-linked oligosaccharides in glycoproteins.
  • M Yamauchi, M Tokeshi, J Yamaguchi, T Fukuzawa, A Hattori, A Hibara, T Kitamori
    JOURNAL OF CHROMATOGRAPHY A 1106 (1-2) 89 - 93 0021-9673 2006/02 [Refereed][Not invited]
     
    We have developed a miniaturized two-way detection system using thermal lens and fluorescence spectroscopies for microchip chemistry. The system was composed of laser diode (LD) modules, fiber-based optics combined with a gradient index lens. and miniaturized detection units for thermal lens and fluorescence signals. The detection limits in the thermal lens and fluorescence spectroscopies were 6.3 x 10(-9) M for Ni(II) phthalocyanine tetrasulfonic acid and 3.0 x 10(-9) M for cy5, respectively. The performance of the system with the miniaturized thermal lens was equivalent to that of a conventional thermal lens microscope. The fluorescence sensitivity was comparable to sensitivities offered by conventional miniaturized systems. (c) 2005 Elsevier B.V. All rights reserved.
  • Manabu Tokeshi
    Shinku/Journal of the Vacuum Society of Japan 49 (7) 400 - 403 0559-8516 2006 [Refereed][Not invited]
  • Hidematsu Ikeda, Manabu Tokeshi, Hiroyasu Hotokezaka, Yasuhisa Ikeda, Takehiko Kitamori
    Transactions of the Atomic Energy Society of Japan 5 (3) 209 - 220 1347-2879 2006 [Refereed][Not invited]
     
    Analytical equipment, which consists of a microchemical chip and a desktop-sized thermal lens microscope (DT-TLM), is being developed to analyze solutions in PUREX reprocessing of spent nuclear fuels. Radiation degradation by gamma-rays of the microchemical chip and capillary tubes used in this equipment were studied. The decreased thermal lens signal of a colored microchemical chip made of Pyrex (Corning#7740) glass by the irradiation can be corrected by using empirical correlations of the light transmittance. The usable dose of the EXLON PFA capillary tube was less than 30 kGy. The microchemical chip made of Pyrex (Corning#7740) glass and the EXLON PFA capillary tubes can be applied to the analyses of high radioactivity samples since the sample quantity required for analysis is very small. Radiation degradation of the microchemical chip made of synthetic quartz (SUPRASIL-P) and the VICTREX PEEK capillary tubes was not observed for the dose studied here. © Atomic Energy Society of Japan.
  • M Tokeshi, Y Ikeda, T Kitamori
    JOURNAL OF THE ATOMIC ENERGY SOCIETY OF JAPAN 48 (1) 38 - 43 0004-7120 2006/01 [Refereed][Not invited]
  • “UV-excitation thermal lens microscope for non-labeled and ultrasensitive detection of non-fluorescent molecules
    S. Hiki, K.Mawatari, A.Hibara, M.Tokeshi, T.Kitamori
    Analytical Chemistry 8 (78) 2859 - 2863 2006 [Refereed][Not invited]
  • Yong Zhang, Guichen Ping, Bingmei Zhu, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba
    Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences 395 - 397 2006 [Refereed][Not invited]
     
    The glycosaminoglycans (GAGs) labeled with 2-aminoacridone, hydrochloride (AMAC) were found to be prevented from adsorption to the surface of polymehylmethacrylate (PMMA) microchips by dynamic coating microchannel with methyl cellulose (MC). To obtain sufficient resolution of mono-sulfate GAG disaccharide isomers on microchip electrophoresis (MCE), buffer pH, β-cyclodextrin (β-CD), and 1,4-dioxane (DO) were systematically investigated. We found that the application of 1,4-dioxane (DO) dramatically improved the resolution of the analytes. ΔDi-triS, ΔDi-UA2S, ΔDi-4S, and ΔDi-6S were baseline separated within 75 sec. The theoretical plate was in the range of 5×105-1×10 6 m-1. © 2006 Society for Chemistry and Micro-Nano Systems.
  • Yoshikuni Kikutani, Masaharu Ueno, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori
    QSAR and Combinatorial Science 24 (6) 742 - 757 1611-020X 2005/08 [Refereed][Not invited]
     
    Chemical syntheses using microchemical chips have been intensively investigated in recent years, but they have still not fully reached the stage of practical use. Combinatorial synthesis has been expected to be one of their useful applications. Benefits of using microchips, such as reduction in quantity of reagents and wastes, are thought to be suited for combinatorial syntheses, where numerous variations of products have to be synthesized each in small amounts. There are two modes of combinatorial syntheses using microchemical chips. One is sequential synthesis, in which reactant solutions are introduced sequentially in various combinations to a reaction microchannel. The other is parallel synthesis, where the number of reaction channels is the same as the number of final products, and starting materials are distributed to all the reaction channels in different combinations. Sequential synthesis has been considered as the more favorable way, because, especially when libraries of starting materials are large, numerous microchannels and complicated pipings for distribution of reagents to the channels are required for the parallel mode. Therefore, research on parallel microcombinatorial syntheses has been done only rarely, whereas there have been many reports of sequential microcombinatorial syntheses. Against this trend, our research group has taken an alternative path and tried to integrate both parallel reaction microchannels onto a glass microchip and channels for distributing reactant solutions to each reaction channel in a combinatorial mode. Microchips fabricated by laminating multiple layers of glass substrates have been used to construct three-dimensional microchannel networks necessary for mixing reagent flows in different combinations. We utilized low Reynolds number multi-phase laminar flow and developed micro-unit operations (MUOs). By combining MUOs in the multi-phase continuous laminar flows, microchemical systems were constructed. We called this micro-integration methodology "continuous-flow chemical processing (CFCP)", and by using it, a sequence of various operations in chemical syntheses could be integrated on a microchemical chip. Not just simple mixing of two reagents, which was most commonly done in microreactors, but also multi-step processes including other chemical operations, such as extraction and separation, could be carried out via CFCP. In the first part of this short review article, we explain CFCP with an example. Then, a microchemical chip with a three-dimensional microchannel network for a parallel reaction system is introduced. Finally, we present a case in which CFCP was realized in a three-dimensional microchannel network. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA.
  • Hiroyasu Hotokezaka, Manabu Tokeshi, Masayuki Harada, Takehiko Kitamori, Yasuhisa Ikeda
    Progress in Nuclear Energy 47 (1-4) 439 - 447 0149-1970 2005/07 [Refereed][Not invited]
     
    In order to clarify the extraction behavior of U(VI) from aqueous phase to organic one in microchannel, we have carried out extraction experiments of U(VI) from HNO3 aqueous solution of 3 M (M = mol/dm3) to 30% or 100% TBP phase in microchannel. From the results of extraction experiments, it was found that the extraction of U(VI) in microchannel could be performed in a short time for approximately 1 s with a good extractability in both organic phases of 30% and 100% TBP, and suggested that the other nuclides could be extracted with high extraction efficiency in microchannel. Furthermore, it is expected that the innovative and sophisticated nuclide separation systems can be developed by using microchannel extraction with selective extractants for specific nuclide. © 2005 Published by Elsevier Ltd. All rights reserved.
  • Akihide Hibara, Shinobu Iwayama, Shinya Matsuoka, Masaharu Ueno, Yoshikuni Kikutani, Manabu Tokeshi, Takehiko Kitamori
    Analytical Chemistry 77 (3) 943 - 947 0003-2700 2005/02/01 [Refereed][Not invited]
     
    A capillarity restricted modification method for microchannel surfaces was developed for gas-liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic-hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 μm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.
  • Yoshikuni Kikutani, Keisuke Morishima, Manabu Tokeshi, Jun Yamaguchi, Takashi Fukuzawa, Akihiko Hattori, Yoshikazu Yoshida, Mitsuo Kitaoka, Takehiko Kitamori
    Digest of Technical Papers - International Conference on Solid State Sensors and Actuators and Microsystems, TRANSDUCERS '05 1 380 - 383 2005 [Refereed][Not invited]
     
    Flowing thermal lens microflow velocimeter, in which photothermally produced local refractive index change (a thermal lens) was used as a tracer, was developed for microchemical chips made of glass. A laser pulse was focused with a microlens on a liquid flow inside a microchannel, and a thermal lens was produced. The thermal lens drifted downstream and was detected by using another beam. Velocity was calculated from the time required for the thermal lens to travel between the two points. The microflow velocimeters enabled non-contact measurement with only slight disturbance to the microfluid possible and were suitable for microchemical systems. © 2005 IEEE.
  • Eiichiro Tamaki, Akihide Hibara, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 5 (2) 129 - 131 1473-0197 2005 [Refereed][Not invited]
     
    A tunable thermal lens spectrometry system was developed for microchip analysis. The system utilized a Xe lamp as an excitation source, instead of a laser. The system can measure the absorption spectrum of a turbid solution without disturbance of the light scattering background.
  • K Okubo, M Tokeshi, Y Yoshida, T Kitamori
    Micro Total Analysis Systems 2004, Vol 1 (296) 162 - 164 0260-6291 2005 [Refereed][Not invited]
     
    Integration of HPLC on microchips were tried in a few reports, but it was difficult to realize pressure driven HPLC systems on-chip. We have developed high-pressure proof microfluidic devises and succeeded in integrating packed channel HPLC systems on microchips. Two types of high-pressure proof micro connectors and two types of column termination frits were developed, and performance of packed-channel HPLC systems based on those microfluidic devises was evaluated using standard samples.
  • M Tokeshi, T Kitamori
    PROGRESS IN NUCLEAR ENERGY 47 (1-4) 434 - 438 0149-1970 2005 [Refereed][Not invited]
     
    The new methodology for integration of chemical processing on a microchip was proposed. This methodology, continuous flow chemical processing (CFCP), is based on a combination of microunit operations (MUOs) and a multiphase flow network. Chemical operations in microchannels, such as mixing, reaction, and extraction, were classified into several MUOs. The complete procedure for Co(II) wet analysis, including a chelating reaction, solvent extraction, and purification was decomposed into MUOs and reconstructed as CFCP on a microchip. Chemical reaction and molecular transport were realized in and between continuous liquid flows in a multiphase flow network, such as aqueous/aqueous, aqueous/organic, and aqueous/organic/aqueous flows. (C) 2005 Elsevier Ltd. All rights reserved.
  • K Uchiyama, M Tokeshi, Y Kikutani, A Hattori, T Kitamori
    ANALYTICAL SCIENCES 21 (1) 49 - 52 0910-6340 2005/01 [Refereed][Not invited]
     
    This paper presents a capillary-to-microchip connection, which can be used as an interface for coupling Capillary electrophoresis (CE) with a thermal lens microscope (TLM). It is difficult to directly apply TLM to samples in a capillary with a curved surface, and such an interface chip at the end of a CE separation column is needed for reliable TLM measurements. The dependence of the TLM signal intensity on the TLM detection point in the interface chip and the dependence of the theoretical plate number of CE separation on the channel dimensions of the interface chip were, investigated and optimized with a mixture of 4-dimethylaminoazobenzene-4'-sulfonyl(DABSYL)-derivatized amino acids (glycine, alanine, methionine, and proline) as a model sample. By using an optimized inter-face chip. theoretical plate numbers of DABSYL-glycine, -methionine, -alanine, and -pro line were obtained to be 104000, 95000, 104000, and 95000, respectively.
  • Y Sugii, K Okamoto, A Hibara, M Tokeshi, T Kitamori
    JOURNAL OF VISUALIZATION 8 (2) 117 - 124 1343-8875 2005 [Refereed][Not invited]
     
    Miscible liquid two-layer flow in a Y-shaped microfluidic device, which consists of microchannels with 120 mu m in width and 35 mu m in depth, is investigated by particle image velocimetry (PIV) to clarify the flow characteristics at fluid interfaces. The obtained velocities with a spatial resolution of 5.9 x 1.5 mu m(2) around the interface between water and ethanol indicate an imbalance in shear stress at interface. The reason of the imbalance is to be the Korteweg stress generated by interfacial tension gradient due to a concentration gradient by diffusion in a miscible two-layer flow. The stress may cause an interfacial instability and destroy a uniform mixing in two flowing fluids in the case of large concentration gradient.
  • XH Yang, A Hibara, K Sato, M Tokeshi, K Morishima, Y Kikutani, H Kimura, T Kitamori
    Micro Total Analysis Systems 2004, Vol 1 (296) 120 - 122 0260-6291 2005 [Refereed][Not invited]
     
    A microstructure similar to cross-flow micro-filter was developed for separation of plasma from whole blood without centrifugation, which consists of many shallow channels, like micro pores, located between two bigger channels (main channels), and acted as passages of plasma but dams for blood cells. Plasma without obvious hemolysis could be observed in main channels within 3 min. Such a microstructure has the potential to combine or integrate with other types of microstructure to form on-chip blood analyses devices. As a model, integrated with on-chip bead-bed immunoassay, a microchip was used for the determination of human alpha-fetoprotein on the range of 5-600 ng/mL.
  • Y Sugii, K Okamoto, A Hibara, M Tokeshi, T Kitamori
    Micro Total Analysis Systems 2004, Vol 1 (296) 228 - 230 0260-6291 2005 [Refereed][Not invited]
     
    Miscible liquid two-layer flow in a Y-shaped microfluidic device with 120 Pm in width and 33 mu m in depth, is investigated using particle image velocimetry (PIV) to clarify the flow characteristics at fluid interfaces. The obtained velocities with a spatial resolution of 5.9 x 1.5 mu m(2) around the interface between water and ethanol indicate an imbalance in shear stress at interface. The numerical simulation based on the Korteweg stress generated by interfacial tension gradient was carried out. The stress may cause an interfacial instability and destroy a uniform mixing in two flowing fluids in the case of large concatenation gradient.
  • A Hibara, S Iwayama, M Ueno, Y Kikutani, M Tokeshi, T Kitamori
    Micro Total Analysis Systems 2004, Vol 1 (296) 557 - 559 0260-6291 2005 [Refereed][Not invited]
     
    A surface modification control method for microchannels was developed for gas-liquid microchemical operations in microchips. In this method, shallow and deep microchannels were modified with hydrophobic and hydrophilic groups, respectively. This patterning was prepared based on principle of capillarity. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure agreed well with theoretical prediction from Young-Laplace equation. In order to demonstrate usefulness and effectiveness of the method, air bubble was introduced to the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with nitrogen-flow. The concentration of dissolved oxygen decreased with contact time and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method is promising to general microfluidic gas-liquid operations.
  • M Tokeshi, J Yamaguchi, A Hattori, T Kitamori
    ANALYTICAL CHEMISTRY 77 (2) 626 - 630 0003-2700 2005/01 [Refereed][Not invited]
     
    This paper describes two types of miniaturized thermal lens optical systems that use optical fibers, SELFOC microlenses and light sources. The first system consists of a compact diode pumped solid-state laser (532 nm) as an excitation light source, a laser diode (635 nm) as a probe light source, an acoustoptic modulator as an excitation light modulator, fiber-based and conventional optics, and a detection system that combines a pinhole, an interference filter, and a photodiode. The second system consists of two laser diodes as the excitation (658 nm) and probe (780 nm) light sources, fiber-based optics, and the same detection system as the first one. The performance of the two systems was evaluated by the limit of detection (LOD) using standard solutions of sunset yellow (SY) and nickel(II) phthalocyaninetetrasulfonic acid tetrasodium salt (NiP). The LODs of the first system for SY and second system for NiP were calculated to be 3.7 x 10(-8) (1.7 x 10(-6) AU) and 7.7 x 10(-9) M (3.4 x 10(-6) AU), respectively. These results were consistent with the expected values obtained from photothermal parameters.
  • E Tamaki, A Hibara, HB Kim, M Tokeshi, T Ooi, M Nakao, T Kitamori
    Micro Total Analysis Systems 2004, Vol 1 (296) 318 - 320 0260-6291 2005 [Refereed][Not invited]
     
    A pressure-driven flow control scheme for nano chemical reactor was developed. For control the pressure of flow, we utilized a back pressure regulator and U-shaped microchannel. Based on this scheme, we fabricated a nano chemical reactor including a Y-shaped nanochannel for reaction and an U-shaped microchannel for liquid introduction. After combining the nano chemical reactor and back pressure regulators, we confirmed that the system worked properly with the pressure up to 7 MPa. By utilizing this system, chemical reaction in nanospace is realized.
  • K Shinohara, Y Sugii, A Hibara, M Tokeshi, T Kitamori, K Okamoto
    EXPERIMENTS IN FLUIDS 38 (1) 117 - 122 0723-4864 2005/01 [Refereed][Not invited]
     
    The micro-LIF (laser-induced fluorescence) technique was applied to the measurement of pH distributions in a chemically reacting flow in a microfluidic device. Two liquid streams were combined at the junction of a Y-shaped microchannel (100-mum width and 33-mum depth), and allowed to diffuse into each other and react. The results for non-reacting fluids (hydrochloric acid and water) show good agreement with theoretical values calculated using conventional diffusion. When a reaction occurred (hydrochloric acid and sodium hydroxide), a large difference between the measurement results and the theoretical values was observed, indicating rapid proton diffusion compared with the theoretically calculated values.
  • E Tamaki, A Hibara, M Tokeshi, T Kitamori
    LAB ON A CHIP 5 (2) 129 - 131 1473-0189 2005 [Refereed][Not invited]
     
    A tunable thermal lens spectrometry system was developed for microchip analysis. The system utilized a Xe lamp as an excitation source, instead of a laser. The system can measure the absorption spectrum of a turbid solution without disturbance of the light scattering background.
  • K Mawatari, S Hiki, A Hibara, M Tokeshi, T Kitamori
    Micro Total Analysis Systems 2004, Vol 2 (297) 434 - 436 0260-6291 2005 [Refereed][Not invited]
     
    Thermal lens microscope (TLM) is an ultra-sensitive method for detecting non-fluorescent samples in microspace. TLM is a kind of optical absorption spectrophotometry with comparable sensitivity to the laser induced fluorescence method. In this presentation, the applicability of TLM was extended from visible to ultraviolet (UV) light absorbing samples by UV laser excitation. The flow velocity was investigated for optimizing the sensitivity. The detection limit of 9.2 x 10-7 in absorbance unit was obtained and about 2 orders of magnitude superior to spectrophotometry: In addition, TLM was further developed for individual nanoparticles counting with visible laser excitation. Individual gold nanoparticles of 15 nm in diameter could be detected, and the detection limit of 5 nm, that was about 1 order lower in diameter than laser scattering method, was obtained.
  • Development of a Microchip-Based Bioassay System Using Cultured Cells
    Makiko Goto, Kiichi Sato, Atsushi Murakami, Manabu Tokeshi, Takehiko Kitamori
    Analytical Chemistry 77 2125 - 2131 2005 [Not refereed][Not invited]
  • K Shinohara, Y Sugii, A Aota, A Hibara, M Tokeshi, T Kitamori, K Okamoto
    MEASUREMENT SCIENCE AND TECHNOLOGY 15 (10) 1965 - 1970 0957-0233 2004/10 [Refereed][Not invited]
     
    This paper reports a new technique of micro-resolution particle image velocimetry (PIV). To investigate transient phenomena in a microfluidic device, a high-speed micro-PIV technique was developed by combining a high-speed camera and a continuous wave (CW) laser. The technique was applied to a micro counter-current flow, consisting of water and butyl acetate. The velocity fields of water in the micro counter-current flow were visualized for a time resolution of 500 mus and a spatial resolution of 2.2 x 2.2 mum(2). Using the micro-PIV technique, the vortex-like motions of fluorescent particles around the water-butyl acetate interface were captured clearly.
  • K Shinohara, Y Sugii, K Okamoto, H Madarame, A Hibara, M Tokeshi, T Kitamori
    MEASUREMENT SCIENCE AND TECHNOLOGY 15 (5) 955 - 960 0957-0233 2004/05 [Refereed][Not invited]
     
    The interaction between chemical reactions and the flow field in microfluidic devices is investigated by a laser-induced fluorescence technique refined for use at microscopic spatial resolution. The pH distribution of chemically reacting flow at a Y-junction in a neutralization reaction in a microfluidic device is successfully visualized at a spatial resolution of 0.89 mum x 0.89 mum.
  • T Kitamori, M Tokeshi, A Hibara, K Sato
    ANALYTICAL CHEMISTRY 76 (3) 52A - 60A 0003-2700 2004/02 [Refereed][Not invited]
  • Kiichi Sato, Maho Yamanaka, Tomokazu Hagino, Manabu Tokeshi, Hiroko Kimura, Takehiko Kitamori
    Lab on a Chip 4 (6) 570 - 575 1473-0197 2004 [Refereed][Not invited]
     
    A microchip-based enzyme-linked immunosorbent assay (microELISA) system was developed and interferon-γ was successfully determined. The system was composed of a microchip with a Y-shaped microchannel and a dam structure, polystyrene microbeads, and a thermal lens microscope (TLM). All reactions required for the immunoassay were done in the microchannel by successive introduction of a sample and regents. The enzyme reaction product, in a liquid phase, was detected downstream in the channel using the TLM as substrate solution was injected. The antigen-antibody reaction time was shortened by the microchip integration. The limit of the determination was improved by adopting the enzyme label. Moreover, detection procedures were greatly simplified and required time for the detection was significantly cut. The system has good potential to be developed as a small and automated high throughput analyzer.
  • Yoshikuni Kikutani, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 4 (4) 328 - 332 1473-0197 2004 [Refereed][Not invited]
     
    A three-dimensional microchannel network with two-level crossings of channels was constructed in a glass microchip by sandwiching an insulating glass plate between two glass plates with microchannels followed by thermal bonding. Pressure-driven stable multi-phase laminar flows inside the three-dimensional channel network were realized by balancing flow rates of syringe pumps. Micro unit operations for mixing, reaction, solvent extraction, and detection were properly arranged in the multi-phase laminar flows, so that four parallel analyses, comprising twenty unit operations in total, could be integrated onto a single chip. Two chelating reagents and two sample solutions containing heavy metal ions (Fe(II) or Co(II)) were mixed and reacted in four different combinations using the three-dimensional channel network. After chelating reactions were completed, post processing (solvent extraction or addition of acid) was applied to each solution stream to remove the interferences of coexisting metal ions. Finally, target metal complexes were detected using a thermal lens microscope (TLM). Integrity of the micro system was confirmed by qualitative analysis of Fe(II) and Co(II). This is the first example of continuous flow chemical processing utilizing multi-phase laminar flow realized in a three-dimensional channel network.
  • H Hachiya, M Tokeshi, Y Yoshida, T Kitamori
    PROCEEDINGS OF THE IEEE SENSORS 2004, VOLS 1-3 166 - 169 1930-0395 2004 [Refereed][Not invited]
     
    We developed a novel microchannel structure comprised of deep/shallow/deep three-channels. Each end of the channel was split into three, and supplying and taking of fluids to/from the channels were possible. Aqueous solution tended to fill the shallow channel because of the capillary force. Thus by introducing gas/liquid/gas from one end of the channel, stable laminar three-phase flow was formed throughout the channel. We introduced liquid/gas/liquid to the channels then. The flow became turbulent, near the confluence, and settled into gas/liquid/gas laminar profile at downstream. The channel-design and flow rates were optimized to form this gas-liquid crossing flow continuously. The gas-liquid crossing flow was useful in micro-analysis and synthesis, because efficient gas-liquid contact at the turbulence promote extraction and reaction, and spontaneous separation of the two phases enabled post processes (e.g. detection). The crossing flows were also formed at the other microchannel-structures like a shallow/deep/shallow three channels and a deep/shallow two channels. A sensor for low-concentration ammonia gas by using this structure was also reported in this paper.
  • Y Kikutani, H Hisamoto, M Tokeshi, T Kitamori
    LAB ON A CHIP 4 (4) 328 - 332 1473-0189 2004 [Refereed][Not invited]
     
    A three-dimensional microchannel network with two-level crossings of channels was constructed in a glass microchip by sandwiching an insulating glass plate between two glass plates with microchannels followed by thermal bonding. Pressure-driven stable multi-phase laminar flows inside the three-dimensional channel network were realized by balancing flow rates of syringe pumps. Micro unit operations for mixing, reaction, solvent extraction, and detection were properly arranged in the multi-phase laminar flows, so that four parallel analyses, comprising twenty unit operations in total, could be integrated onto a single chip. Two chelating reagents and two sample solutions containing heavy metal ions (Fe(II) or Co(II)) were mixed and reacted in four different combinations using the three-dimensional channel network. After chelating reactions were completed, post processing ( solvent extraction or addition of acid) was applied to each solution stream to remove the interferences of coexisting metal ions. Finally, target metal complexes were detected using a thermal lens microscope (TLM). Integrity of the micro system was confirmed by qualitative analysis of Fe( II) and Co( II). This is the first example of continuous flow chemical processing utilizing multi-phase laminar flow realized in a three-dimensional channel network.
  • A Hibara, M Nonaka, M Tokeshi, T Kitamori
    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 125 (49) 14954 - 14955 0002-7863 2003/12 [Refereed][Not invited]
  • S Hiki, M Tokeshi, A Hibara, T Kitamori
    BUNSEKI KAGAKU 52 (8) 569 - 574 0525-1931 2003/08 [Refereed][Not invited]
     
    A desktop-sized thermal lens microscope (DT-TLM) having high sensitivity and wide applicability was developed. The operationality of the DT-TLM compared with that of a big-sized TLM has been remarkably improved by simplifying the optics and optimizing the optical configuration. In order to evaluate the performance, the DT-TLM was applied to the ultrasensitive detection of a non-fluorescent molecule (dye: Sunset Yellow) in water. The concentration dependence of the thermal lens signal (calibration curve) showed good linearity in the range of 1 x 10(-8)similar to1 x 10(-7) M. From the value of twice the standard deviation (2sigma) of this calibration curve, the lower limit of the quantitative determination was estimated to be 1.10 x 10(-8) M. The lower limit of detection was estimated as 8.27 x 10(-9) M from the conditions of the signal-to-noise ratio S/N=2. These obtained values were almost the same as that of the big-sized TLM.
  • Mikhail A. Proskurnin, Maksim N. Slyadnev, Manabu Tokeshi, Takehiko Kitamori
    Analytica Chimica Acta 480 (1) 79 - 95 0003-2670 2003/03/17 [Refereed][Not invited]
     
    The thermal lens optical scheme-design was optimised for microscopic measurements in microchannels. The efficient pathlength of the sample, irradiated volume, and the diameter of the thermal lens were estimated. Experimental time curves of development of the thermal lens and periodical oscillations of the signal due to convectional heat transfer are in good agreement with the theoretically expected behaviour. Noise sources (laser noises, instrumental flicker noise, convection, and flow noise) were studied. The possible effect of probe laser power on transient and steady-state thermal lens measurements were estimated. The effect of solvent absorption on the performance characteristics is shown. Under the optimum optical scheme-design, the limits of detection of ferroin and Sunset Yellow FCF at 488.0nm are 1×10-8 and 4×10-9moldm-3, respectively (corresponding quantities in the detection volume are 3×10-21 and 1×10-21mol). The total linear calibration range is n×10-8 to n×10-4moldm-3, the repeatability R.S.D. for this range is 3-7%. The optimised instrument was also used for the determination of characteristic rate constants of formation and dissociation of ferroin at the level of n × 10-8moldm-3. Some analytical applications are discussed. © 2003 Published by Elsevier Science B.V.
  • E Tamaki, A Hibara, M Tokeshi, T Kitamori
    JOURNAL OF CHROMATOGRAPHY A 987 (1-2) 197 - 204 0021-9673 2003/02 [Refereed][Not invited]
     
    Microchannel-assisted thermal lens spectrometry (MATLS) was developed for microchip analysis. This method utilized a photothermal effect in a very small space and rapid thermal conduction between a solid-liquid interface to produce a temperature gradient in the microchannel. In order to examine the mechanism experimentally, we constructed a detection system of laser defocus setup in which an excitation beam was not tightly focused, but it irradiated the microchannel homogeneously. The signal intensity dependence on modulation frequency of excitation and on solvent was investigated with the laser defocusing setup. The results of this investigation indicated that the mechanism of MATLS worked as expected. Since the mechanism of MATLS does not require directivity and coherence of the laser beam, other incoherent light sources can be used as excitation light for sensitive detections. Finally, we considered some future applications utilizing the mechanism. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Hideaki Hisamoto, Takayuki Horiuchi, Akihide Hibara, Manabu Tokeshi, Takehiko Kitamori
    IEEJ Transactions on Sensors and Micromachines 123 (4) 124 - 127 1347-5525 2003 [Refereed][Not invited]
     
    A new fluid flow inside the microchannel was successfully developed. The flow created here involves segmented flow injection of plural organic phases into a microchannel followed by contact with a single aqueous phase to form stable organic-aqueous two-layer flow inside the microchannel. Fundamental study on the developed flow inside the microchannel was performed by monitoring the dye-doped segmented organic phases by thermal lens microscopy (TLM). Excellent repeatability and very small injection volume in developing segmented flow were realized. The new fluid flow created here is expected to allow us multi-ion sensing, which is not easily demonstrated by conventional ion sensor technology using a solvent polymeric membrane, by combining with neutral ionophore-based ion pair extraction using plural numbers of organic phases containing different ionophore molecules. Keywords : Segmented flow injection, Microchip, μ-TAS, Multi-ion, Multiphase flow. © 2003, The Institute of Electrical Engineers of Japan. All rights reserved.
  • Yoshikuni Kikutani, Akihide Hibara, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori
    Lab-on-a-Chip: Miniaturized Systems for (Bio) Chemical Analysis and Synthesis 285 - 307 2003 [Refereed][Not invited]
     
    This chapter provides information on micro and nano-fabrication technologies that have been introduced into chemical and biotechnologies. It also explains molecular and energy transport between multiphase laminar flow in a micro channel. As for chemical synthesis, micro reactors with sub-millimeter-sized micro channels are now eagerly investigated. Though there are some examples of electrokinetically-driven flow control in micro reactors, pressure-driven flow control is more commonly used for micro reactors. The aim is to develop methodology to integrate as many chemical processes as possible on microchips and to make full use of characteristics of micro space. Therefore, pressure-driven flow and developed techniques to operate chemical processing in pressure-driven laminar flow in micro channels are selected and for this microchips made of glass are used. Glass vessels are commonly used in many chemical experiments because of their chemical inertness and optical transparency. In order to realize these micro chemical systems based on multiphase laminar flow, some stabilization techniques for the multiphase flow has been inevitable. A micro channel with guide structures and surface modification to stabilize multiphase flows has been used. The chapter also presents a micro integration methodology, continuous flow chemical processing (CFCP), based on spontaneous motion of molecules in multi-phase laminar flow network in micro channel circuits. © 2003 Elsevier B.V. All rights reserved.
  • H Hisamoto, Y Shimizu, K Uchiyama, M Tokeshi, Y Kikutani, A Hibara, T Kitamori
    ANALYTICAL CHEMISTRY 75 (2) 350 - 354 0003-2700 2003/01 [Refereed][Not invited]
     
    Here we report a design and synthesis of a chemically functional polymer membrane by an interfacial polycondensation reaction and multilayer flow inside a microchannel. Single and parallel dual-membrane structures are successfully prepared by using organic/aqueous two-layer flow and organic/aqueous/organic three-layer flow inside the microchannel followed by an interfacial polycondensation reaction. By using the inner-channel membrane, permeation of ammonia species through the inner-channel membrane is successfully achieved. Furthermore, horseradish peroxidase is immobilized on one side of the membrane surface to integrate the chemical transform function onto the inner-channel membrane. Here substrate permeation through the membrane and subsequent chemical transformation at the membrane surface are realized. The polymer membrane prepared inside the microchannel has an important role in ensuring stable contact of different phases such as gas/liquid or liquid/liquid and the permeation of chemical species through the membrane. Furthermore, membrane surface modification chemistry allows chemical transformation of permeated chemical species. These methods are expected to lead to development of complicated and sophisticated chemical systems involving membrane permeation and chemical reactions.
  • K Uchiyama, A Hibara, K Sato, H Hisamoto, M Tokeshi, T Kitamori
    ELECTROPHORESIS 24 (1-2) 179 - 184 0173-0835 2003/01 [Refereed][Not invited]
     
    A thermal lens microscope (TLM) detection of capillary electrophoresis (CE) utilizing microchip technology was developed. Fused-silica capillaries with an inner diameter of 50 pm were directly connected to a microchannel in a microchip. The detection limit by TLM was estimated as 2.8 x 10(-7) absorbance by measuring pure water. The detection limit of derivatized amino acids determined by CE-TLM was estimated as 2.4 x 10(-8) M, which was 100 times lower than that of conventional absorbance detection.
  • Yoshikuni Kikutani, Manabu Tokeshi, Kiichi Sato, Takehiko Kitamori
    Pure and Applied Chemistry 74 (12) 2299 - 2309 0033-4545 2002/12/01 [Refereed][Not invited]
     
    By analogy to unit operations (e.g., mixers, reactors, etc.) used in conventional chemical engineering, the concept of microunit operations permits the integration of complicated chemical systems onto a small microchip. A protocol for fabrication of such microchips is described, and its use is illustrated in several examples. In addition, the thermal lens microscope, which determines nonfluorescent species at the single-molecule level, is indispensable as an ultrasensitive detector for general use. Applications of microchip technology are given for chemical analysis, immunoassay, and full bioassay. Microchip analysis can provide very large enhancements in sensitivity and substantial reductions in measurement time as compared with conventional analytical methods.
  • A Hibara, T Saito, HB Kim, M Tokeshi, T Ooi, M Nakao, T Kitamori
    ANALYTICAL CHEMISTRY 74 (24) 6170 - 6176 0003-2700 2002/12 [Refereed][Not invited]
     
    We have fabricated nanometer-sized channels, demonstrated a technique for the introduction of liquid into the channels, and carried out time-resolved fluorescence measurements of aqueous solutions. In this study, 330-nm- and 850-nm-sized channels were fabricated on fused-silica substrates by fast atom beam etching and hydrofluoric acid bonding methods. A liquid introduction method utilizing capillary action was demonstrated. The liquid introduction was observed under an optical microscope, and the liquid velocity during the introduction was analyzed by surface energy and macroscale hydrodynamics. The liquid velocity due to capillary action in the nanometer-sized channel seemed more than four times slower than the estimation. Then, aqueous solutions of rhodamine 6G (R6G), sulforhodamine 101 (SR101), and rhodamine B (RB) in the channels were measured by time-resolved fluorescence spectroscopy; spectra of the same solution in a 250-mum-sized channel were also measured as a reference for the macrospace. Although the fluorescence spectra in the 330-nm-, 850-nm- and 250-mum-sized channels agreed with one another, the fluorescent decays in the nanometer-sized channels were faster for R6G and SR101 and slower for RB than the respective decays in the 250-mum-sized channels. The results suggested the solutions had lower dielectric constants and higher viscosities in the nanometer-sized channels.
  • Yoshikuni Kikutani, Akihide Hibara, Kenji Uchiyama, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 2 (4) 193 - 196 1473-0197 2002/11/01 [Refereed][Not invited]
     
    We made a 'pile-up' microreactor in which ten levels of microchannel circuits were integrated to form a single glass entity. Solutions were distributed to each layer via cylindrical holes with a diameter much larger than that of the microchannel. Fabrication of the pile-up reactor was completed using only conventional photolithography, wet etching and thermal bonding techniques, and no special facilities or instruments were required. An amide formation reaction between amine in aqueous solution and acid chloride in organic solution was carried out using the pile-up reactor. The yield of the amide formation reaction is dependent on the size of the specific surface area between the two solutions, and the small space inside the microchannels is good for acquiring a large specific surface area without any stirring processes. The maximum throughput for the ten-layered pile-up reactor was ten times larger than that of a single-layered one, yet the reaction yield was still high. Productivity of the pile-up reactor for the reaction was as high as on a gram per hour scale. This value suggests that many conventional plants producing fine chemicals can be replaced by microreactors through the numbering-up technology.
  • Yoshikuni Kikutani, Takayuki Horiuchi, Kenji Uchiyama, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 2 (4) 188 - 192 1473-0197 2002/11/01 [Refereed][Not invited]
     
    An integrated multireactor system for 2 × 2 parallel organic synthesis has been developed on a single glass microchip. Three-dimensional channel circuits in the chip were fabricated by laminating three glass plate layers. The fabrication method is a straightforward extension of the conventional one, and topological equivalence for any three-dimensional circuits can be constructed easily with it. 2 × 2 phase-transfer amide formation reactions, which constitute a simple model for combinatorial synthesis, were successfully carried out on the microchip, and the integrity of the three-dimensional circuits was confirmed. Combinatorial chemistry with multi-microreactors, in conjunction with a high-throughput screening method based on μ-TAS technologies, is expected to provide an efficient tool for drug discovery.
  • A Hibara, M Nonaka, H Hisamoto, K Uchiyama, Y Kikutani, M Tokeshi, T Kitamori
    ANALYTICAL CHEMISTRY 74 (7) 1724 - 1728 0003-2700 2002/04 [Refereed][Not invited]
     
    We demonstrated a liquid/liquid and a gas/liquid two-phase crossing flow in glass microchips. A 250-mum-wide microchannel for aqueous-phase flow was fabricated on a top glass plate. Then, as a way to utilize the surface energy difference for stable phase confluence and separation, a 250-mum-wide microchannel for organic-phase (or gas-phase) flow was fabricated on a bottom glass plate and the wall was chemically modified by octadecylsilane (ODS) group. The top and bottom plates were sealed only by pressure. A microchannel pattern was designed so that the two phases made contact at the crossing point of the straight microchannels. The crossing point was observed with an optical microscope. Results showed that the ODS modification of the microchannel wall clearly improved stability of the interface between the two fluids. Pressure difference between fluids was measured and the interface of water and nitrobenzene was stable for the pressure difference from +300 Pa to -200 Pa. The pressure drop in a countercurrent flow configuration was also estimated, and the pressure difference required to realize the countercurrent flow was less than the allowable pressure range. Finally, we discussed the advantages of utilizing this approach.
  • E Tamaki, K Sato, M Tokeshi, K Sato, M Aihara, T Kitamori
    ANALYTICAL CHEMISTRY 74 (7) 1560 - 1564 0003-2700 2002/04 [Refereed][Not invited]
     
    We developed a microsystem for cell experiments consisting of a scanning thermal lens microscope detection system and a cell culture microchip. The microchip system was good for liquid control in microspace, and this results in secure cell stimulation and coincident in vivo observation of the cell responses. The system could detect nonfluorescent biological substances with extremely high sensitivity without any labeling materials and had a high spatial resolution of similar to1 mum. This system was applied to monitoring of cytochrome c distribution in a neuroblastoma-glioma hybrid cell cultured in the microflask (1 mm x 10 mm x 0.1 mm; 1 muL) fabricated in a glass microchip. Cytochrome c release from mitochondria to cytosol during the apoptosis process was successfully monitored with this system. The cytochrome c detected with this system was estimated to be mu10 zmol. We concluded that the system was suitable for measuring the distribution of chemical substances in a single cell because the microchip is good for liquid handling in microspace and the thermal lens microscope has high sensitivity and spatial resolution.
  • M Tokeshi, T Minagawa, K Uchiyama, A Hibara, K Sato, H Hisamoto, T Kitamori
    ANALYTICAL CHEMISTRY 74 (7) 1565 - 1571 0003-2700 2002/04 [Refereed][Not invited]
     
    A new design and construction methodology for integration of complicated chemical processing on a microchip was proposed. This methodology, continuous-flow chemical processing (CFCP), is based on a combination of microunit operations (MUOs) and a multiphase flow network. Chemical operations in microchannels, such as mixing, reaction, and extraction, were classified into several MUOs. The complete procedure for Co(II) wet analysis, including a chelating reaction, solvent extraction, and purification was decomposed into MUOs and reconstructed as CFCP on a microchip. Chemical reaction and molecular transport were realized in and between continuous liquid flows in a multiphase flow network, such as aqueous/aqueous, aqueous/organic, and aqueous/organic/aqueous flows. When the determination of Co(H) in an admixture of Cu(II) was carried out using this methodology, the determination limit (2sigma) was obtained as 18 nM, and the absolute amount of Co chelates detected was 0.13 zmol, that is, 78 chelates. The sample analysis time was faster than that of a conventional processing system. Moreover, troublesome operations such as phase separation and acid and alkali washing, all necessary for the conventional system, were simplified. The CFCP methodology proposed here can be applied to various on-chip applications.
  • K Sato, M Yamanaka, H Takahashi, M Tokeshi, H Kimura, T Kitamori
    ELECTROPHORESIS 23 (5) 734 - 739 0173-0835 2002/03 [Refereed][Not invited]
     
    A bead-bed immunoassay system suitable for simultaneous assay of multiple samples was constructed on a microchip. The chip had branching multichannels and four reaction and detection regions; the constructed system could process four samples at a time with only one pump unit. Interferon gamma was assayed by a 3-step sandwich immunoassay with the system coupled to a thermal lens microscope as a detector. The biases of the signal intensities obtained from each channel were within 10%, and coefficients of variation were almost the same level as the single straight channel assay. The assay time for four samples was 50 min instead of 35 min for one sample in the single-channel assay; hence higher throughput was realized with the branching structure chip.
  • Y Kikutani, A Hibara, K Uchiyama, H Hisamoto, M Tokeshi, T Kitamori
    LAB ON A CHIP 2 (4) 193 - 196 1473-0189 2002 [Refereed][Not invited]
     
    We made a 'pile-up' microreactor in which ten levels of microchannel circuits were integrated to form a single glass entity. Solutions were distributed to each layer via cylindrical holes with a diameter much larger than that of the microchannel. Fabrication of the pile-up reactor was completed using only conventional photolithography, wet etching and thermal bonding techniques, and no special facilities or instruments were required. An amide formation reaction between amine in aqueous solution and acid chloride in organic solution was carried out using the pile-up reactor. The yield of the amide formation reaction is dependent on the size of the specific surface area between the two solutions, and the small space inside the microchannels is good for acquiring a large specific surface area without any stirring processes. The maximum throughput for the ten-layered pile-up reactor was ten times larger than that of a single-layered one, yet the reaction yield was still high. Productivity of the pile-up reactor for the reaction was as high as on a gram per hour scale. This value suggests that many conventional plants producing fine chemicals can be replaced by microreactors through the numbering- up technology.
  • A Hattori, J Yamaguchi, M Tokeshi, T Kitamori
    BIOMEDICAL NANOTECHNOLOGY ARCHITECTURES AND APPLICATIONS 4626 352 - 356 0277-786X 2002 [Refereed][Not invited]
     
    Miniaturization of sensitive detection system is a key factor for integrated chemical systems. Thermal lens microscope can be applied to the ultra-sensitive detection of non-fluorescent molecules in many applications. By using a very small SELFOC(TM) micro lens as an objective, palmtop-sized thermal lens microscope (PTLM) has been constructed. A calibration curve for an aqueous solution in a range of 1.0 x 10(-7)-5.0 x 10(-6) mol/L has been measured in this newly designed PTLM.
  • H Hisamoto, T Saito, M Tokeshi, A Hibara, T Kitamori
    CHEMICAL COMMUNICATIONS (24) 2662 - 2663 1359-7345 2001/12 [Refereed][Not invited]
     
    The large specific interfacial areas and short molecular diffusion distances provided by glass microchips play important roles not only for effective phase-transfer synthetic reaction, but also for avoiding an undesirable side reaction.
  • H Hisamoto, T Horiuchi, K Uchiyama, M Tokeshi, A Hibara, T Kitamori
    ANALYTICAL CHEMISTRY 73 (22) 5551 - 5556 0003-2700 2001/11 [Refereed][Not invited]
     
    A sequential ion-sensing system using a single microchip was successfully realized. The system developed here involves intermittent pumping of plural organic phases into a microchannel, followed by contact with a single aqueous phase to form a stable organic-aqueous two-layer flow inside the microchannel. Because the plural organic phases created by intermittent flow contain the same lipophilic pH indicator dye but different ion-selective neutral ionophores, different ions can be sequentially and selectively extracted into the different organic phases, where they can be determined by thermal lens microscopy (TLM). We used KD-A3 as the lipophilic pH indicator dye and valinomycin and DD16C5 as neutral ionophores to demonstrate sequential ion sensing of potassium and sodium ions by measuring the deprotonated dye caused by the ion extraction. The integrated microfluidic system proposed here allows multi-ion sensing, which is not easily demonstrated by conventional ion sensor technology using a solvent polymeric membrane. The minimum volume of single organic phase needed to obtain an equilibrium response without dilution by cross dispersion of two organic phases was ca. 500 nL in our system, indicating that the required amounts of expensive reagents in one measurement could be reduced to 1.7 ng and 2.8 ng for the dye and ionophore molecules, respectively.
  • Tomoko Minagawa, Manabu Tokeshi, Takehiko Kitamori
    Lab on a Chip 1 (1) 72 - 75 1473-0197 2001/09/01 [Refereed][Not invited]
     
    The integration of a wet analysis system on a glass chip was demonstrated and determination of Co(II) was performed using this system. The Co(II) was extracted into m-xylene from aqueous solution as 2-nitroso-1-naphthol chelates, and colorimetric determination of the: m-xylene phase was applied by a thermal lens microscope. The integration of the chemical operation procedures shown here leads to a considerable reduction in analyzing time. The time for extraction in the integrated system, 10 min, was about tenfold shorter than a conventional system using a separatory funnel and mechanical shaker. Moreover, troublesome operations such as phase separation necessary for the conventional system could be omitted. The determination of Co(II) in the range 2 × 10-7-1 × 10-8 M, which was estimated to be 0.072-1.44 zmol, was achieved.
  • A Hibara, M Tokeshi, T Kitamori
    ELECTROCHEMISTRY 69 (8) 620 - 623 1344-3542 2001/08 [Refereed][Not invited]
  • MN Slyadnev, Y Tanaka, M Tokeshi, T Kitamori
    ANALYTICAL CHEMISTRY 73 (16) 4037 - 4044 0003-2700 2001/08 [Refereed][Not invited]
     
    We have demonstrated that a miniaturized device with IR laser heating of the solvents based on a photothermal effect, is capable of fast and localized control of an enzymatic reaction on a microchip under flow conditions. Using noncontact spectroscopic temperature-sensing techniques, we measured temperature dynamics and spatial distribution and compared the measurements with results of numerical simulation analysis. The device was operated at ultrafast heating and cooling rates of 67 and 53 degreesC/s, respectively, which is 30 times faster than conventional systems and 3-6 times faster than electrothermal miniaturized thermocyclers. The IR laser-mediated heater is characterized by a significantly reduced heated volume of only 5 nL, compared to existing chip-based systems with electrothermal heating. Direct heating of a sample with extremely small heat capacity led us to a fast heating rate, and efficient heat removal through heat transfer to the glass substrate resulted in a fast cooling rate. Reproducible temperature levels with dwell times shorter than 0.5 s were achieved. The enzyme reaction on a chip was successfully controlled with 0.6-s time resolution, using periodic photothermal heating by IR laser. The IR diode laser is compact and thus suits well the miniaturized system design. Our work gives the basis for integration in a chip format of a variety of chemical processes that require fast temperature control.
  • A Hibara, K Sato, H Hisamoto, K Uchiyama, MN Slyadnev, M Tokeshi, T Kitamori
    PROGRESS IN NATURAL SCIENCE 11 S237 - S241 1002-0071 2001/05 [Refereed][Not invited]
     
    Thermal lens microscope (TLM) can be a powerful tool for a detection method for integrated chemistry laboratory (ICL). In spite of great potential of ICL, its application has been restricted due to the lack of highly applicable and sensitive detection methods. TLM developed by our group meets requirements of detection method for ICL, such as high sensitivity, high spatial resolution, and wide applicability. In this review, the principle and characteristics of TLM are introduced and usefulness of TLM in ICL is demonstrated in some analytical applications.
  • M Tokeshi, M Uchida, A Hibara, T Sawada, T Kitamori
    ANALYTICAL CHEMISTRY 73 (9) 2112 - 2116 0003-2700 2001/05 [Refereed][Not invited]
     
    The photothermal effect of an ultratrace amount of non fluorescent molecules in liquid was determined optimizing the optical arrangement for a thermal lens microscope, The optimized experimental setup could be determined from the evaluation of probing volume and the concentration of the sample solutions even when the expectation of the molecule number in the probing region was less than a single molecule. The minimum expectation, which is explained as being the time average, was 0.4 molecule of Pb(II) octaethylporphyrin (OEP) In benzene. The concentrations in the 9.7 x 10(-11)-7.8 x 10(-10) M region used in this work corresponded to the expected number of 0.4 -3.4 molecules, and the calibration curve in this region showed good linearity. Taking into account the enhancement factor of solvent, the molar absorption coefficient of solute, and the optimization of the optical arrangement, the present result, which was the determination limit of 0.34, was consistent with that previously reported. The relation between molecular behavior in the probing volume and the signal was discussed, The average temperature rise in the probing volume by the photothermal effect for the single OEP molecule was estimated as 3.1 muK and this value nas detectable, based on conventional thermal lens measurements for bulk scale sample.
  • Y Wakabayashi, M Tokeshi, A Hibara, DL Jiang, T Aida, T Kitamori
    JOURNAL OF PHYSICAL CHEMISTRY B 105 (19) 4441 - 4445 1089-5647 2001/05 [Refereed][Not invited]
     
    Radiative and nonradiative relaxation processes after excitation by ultraviolet light were measured for o-, m-, and p-aryl ether dendrimers of fourth generation (o-, m-, and p-Ar(L4)(2)) by using fluorescent and thermal lens spectroscopies. Samples were dissolved in CH2Cl2 to provide concentrations of a constant absorbance at excitation light wavelength (244 nm). Regarding the nonradiative process, we investigated thermal lens signal dependence on the light modulation frequency to determine the nonradiative relaxation rate. When frequency ranged from 4 to 200 Hz, the thermal lens signal became larger in the order p-, o-, m-Ar(L4)(2), while the fluorescent intensity became larger in the order p-, m-, o-Ar(L4)2. We transformed these results into an energy balance of the radiative and nonradiative relaxation processes. Our analysis showed that 50% of the excitation energy was not released from p-Ar(L4)2 for a 100-ms order. Next, the measured fluorescence decay times of the three Ar(L4)2 were obtained as 1.7 ns which revealed that the anomalous properties of dendrimers did not originate in long-lived electronic excitation states, but in long-term storage of internal energy. To explain this phenomenon, a novel mechanism for intramolecular energy storage with nonergodic energy transfer should be considered. Last, we proposed that the nonlinear conjugated oscillator model of Fermi-Pasta-Ulam theory would be suitable for the intramolecular energy storage.
  • H Hisamoto, T Horiuchi, M Tokeshi, A Hibara, T Kitamori
    ANALYTICAL CHEMISTRY 73 (6) 1382 - 1386 0003-2700 2001/03 [Refereed][Not invited]
     
    Integration of a neutral ionophore-based ion pair extraction reaction onto a glass microchip was performed. Since the Liquid microspace provides a short molecular diffusion distance and a large specific interfacial area of the liquid-liquid interface, novel attractive analytical features arise such as extremely fast ion sensing and ultrasmall reagent solution volume. In contrast to the slow response time of a standard ion optode, in which the response time is basically governed by slow diffusion of ionic species in the viscous polymer membrane, that of the on-chip ion-sensing system is clearly faster due to the short molecular diffusion distance and low viscosity of organic solution. In this case, the organic solution containing a neutral ionophore and a lipophilic pH indicator dye and the aqueous solution containing sample ion were independently introduced into microchannel to form an organic-aqueous interface. Then determination of the ion was performed by thermal lens microscopy at the downstream of the organic phase under continuous-now conditions. The response time and minimum required reagent solution volume of the on-chip ion-sensing system are about 8 s and 125 nL, respectively, indicating the advantages of using the liquid microspace. Other advantages of the on-chip ion pair extraction system arising from using the liquid microspace and microfluidic system are also discussed in detail.
  • K Sato, M Tokeshi, H Kimura, T Kitamori
    ANALYTICAL CHEMISTRY 73 (6) 1213 - 1218 0003-2700 2001/03 [Refereed][Not invited]
     
    A bead-bed immunoassay system was structured on a microchip and applied to determine carcinoembryonic antigen (CEA), which is a commonly used marker of colon cancer. Polystyrene beads precoated with anti-CEA antibody were introduced into a microchannel, and then a serum sample containing CEA, the first antibody, and the second antibody conjugated with colloidal gold were reacted successively, The resulting antigen-antibodies complex, fixed on the bead surface, was detected using a thermal lens microscope (TLM). A highly elective and sensitive determination of an ultratrace amount of CEA in human sera was made possible by a sandwich immunoassay system that needs three antibodies for an assay. A detection limit dozens of times lower than the conventional ELISA was achieved. Moreover, when serum samples for 13 patients were assayed with this system, there was a high correlation (r = 0.917) with the conventional ELISA. The integration reduced the time necessary for the antigen-antibody reaction to similar to1%, thus shortening the overall analysis time from 45 h to 35 min. Moreover, troublesome operations required for conventional heterogeneous immunoassays could be much simplified. This microchip-based diagnosis system is the first microchip-based system that is practically useful for clinical diagnoses with short analysis time, high sensitivity, and easy procedures.
  • M. Yamanaka, K. Sato, M. Tokeshi, H. Katou, H. Kimura, T. Kitamori
    2001 International Microprocesses and Nanotechnology Conference, MNC 2001 26 - 27 2001 [Refereed][Not invited]
     
    Immunoassay is known as one of the most important analytical methods and widely used in clinical diagnoses and biochemical studies. The conventional immunoassay, however, requires a long assay time. We developed a bead-bed immunoassay system integrated on a microchip which successfully reduces the assay time from 2 days to 30 minutes using simple operations for assay (Sato et al, 2000 and 2001). Here, aiming at high throughput assay, an integrated immunoassay system using a multichannel microchip was developed for simultaneous analyses of multiple samples.
  • Y. Kikutani, T. Horiuchi, H. Hisamoto, K. Uchiyama, M. Tokeshi, T. Kitamori
    2001 International Microprocesses and Nanotechnology Conference, MNC 2001 18  2001 [Refereed][Not invited]
     
    Summary form only given. Combinatorial chemistry is becoming prevalent methodology in preparing samples for high-throughput drug screening. Here we report a prototype of a combinatorial synthetic system integrated on a single glass microchip. By sandwiching a glass plate having pierced holes between two channel-etched glass surfaces, we made a glass chip in which channels in upper and bottom plates can cross without contacting each other (two-level crossing). To check the 3-D channel network, combinatorial amide syntheses by mixing two acid chlorides with two amines were carried out.
  • M. N. Slyadnev, K. Sato, M. Tokeshi, T. Kitamori
    2001 International Microprocesses and Nanotechnology Conference, MNC 2001 194  2001 [Refereed][Not invited]
     
    DNA melting analysis requires precise temperature measurement. Since heat capacity of the sample inside microchip is extremely small, a measurement device can affect temperature response. In this paper we report a method of temperature monitoring that was implemented on a chip to overcome this difficulties.
  • Yuki Tanaka, Maxim N. Slyadnev, Kiichi Sato, Manabu Tokeshi, Haeng-Boo Kim, Takehiko Kitamori
    Analytical Sciences 17 (7) 809 - 810 0910-6340 2001 [Refereed][Not invited]
  • T Minagawa, M Tokeshi, T Kitamori
    LAB ON A CHIP 1 (1) 72 - 75 1473-0189 2001 [Refereed][Not invited]
     
    The integration of a wet analysis system on a glass chip was demonstrated and determination of Co(II) was performed using this system. The Co(II) was extracted into m-xylene from aqueous solution as 2-nitroso-1-naphthol chelates, and colorimetric determination of the m-xylene phase was applied by a thermal lens microscope. The integration of the chemical operation procedures shown here leads to a considerable reduction in analyzing time. The time for extraction in the integrated system, 10 min, was about tenfold shorter than a conventional system using a separatory funnel and mechanical shaker. Moreover, troublesome operations such as phase separation necessary for the conventional system could be omitted. The determination of Co(II) in the range 2 x 10(-7)-1 x 10(-8) M, which was estimated to be 0.072-1.44 zmol, was achieved.
  • Y Shimizu, H Hisamoto, A Hibara, M Tokeshi, T Kitamori
    MICROPROCESSES AND NANOTECHNOLOGY 2001, DIGEST OF PAPERS 20 - 21 2001 [Refereed][Not invited]
  • MA Proskurnin, M Tokeshi, MN Slyadnev, T Kitamori
    ANALYTICAL SCIENCES 17 S454 - S457 0910-6340 2001 [Refereed][Not invited]
     
    The optical-scheme design for a thermal lens microscope was optimized for the measurements in a microchannel. The actual pathlength, irradiated volume, and the diameter of the thermal lens were estimated The experimental data are in good agreement with the theoretically calculations. The noise sources (laser noises, instrumental flicker noise, convection, and flow noise) were studied, and the possible effects of probe laser on transient and steady-state thermal lens measurements were estimated. The limit of detection of ferroin at 488.0 nm is 1x10(-8) mol dm(-3) (3x10(-21) mol in the detection volume). The linear calibration range is nx 10(-8) - n x 10(-4) mol dm(-3), the repeatability RSD for this range is 3-7%.
  • Y Wakabayashi, M Tokeshi, A Hibara, DL Jiang, T Aida, T Kitamori
    ANALYTICAL SCIENCES 16 (12) 1323 - 1326 0910-6340 2000/12 [Refereed][Not invited]
     
    A specially designed experimental setup including a newly constructed KBr cell, a light source, a monochromator and an IR detection system suitable for strictly calibrated IR absorption measurements was constructed in order to elucidate strange phenomenon associated with cis-trans photo-isomerization at 6.26 mum of the azobenzene core located at the center of an aryl-ether dendrimer (L5AZO). The setup used an ordinary Nichrome source to measure the photon flux, irradiation area, and photon absorption rate as exactly as possible. The photon absorption rate was obtained as 1.2 x 10(-3) photons/s molecule and the absorption cross section of L5AZO molecules was estimated to be 3.1 x 10(-19) cm(2)/molecule. Since these values of the absorption rate and absorption cross section are not abnormal, the occurrence of a simultaneous five-photon absorption is almost impossible. Therefore, the five-photon absorption and photo-isomerization shown by L5AZO suggested that the multiphoton process was not simultaneous absorption, but possibly a sequential absorption and energy storage until the equivalent energy of isomerization was reached.
  • Y Tanaka, MN Slyadnev, A Hibara, M Tokeshi, T Kitamori
    JOURNAL OF CHROMATOGRAPHY A 894 (1-2) 45 - 51 0021-9673 2000/10 [Refereed][Not invited]
     
    Photothermal temperature control of an enzyme-catalyzed reaction in a microchip using a diode laser was demonstrated. A laser beam with energy of 10 mW was used to irradiate an absorbing target placed on top of the microchip cover plate. Theoretical calculations have shown that temperature in the microchannel can be locally increased by 5-7 degrees C during short time intervals, due to heat released by the target. The rate of the enzyme reaction, which was initially inhibited due to cooling of the chip to low temperature, was increased when the target was irradiated. The products were detected by a thermal lens microscope. The product concentration was shown to depend on irradiation time, laser intensity and substrate concentration. Reaction characteristics (rate constant of the reaction) were then derived from these dependencies. The reaction volume and absolute quantity of the reaction product were estimated as 10 nl and 100 fmol, respectively. It was also demonstrated that a direct solvent heating method using infrared radiation could control the reaction in the microchannel. (C) 2000 Elsevier Science B.V. All rights reserved.
  • M Tokeshi, T Minagawa, T Kitamori
    JOURNAL OF CHROMATOGRAPHY A 894 (1-2) 19 - 23 0021-9673 2000/10 [Refereed][Not invited]
     
    A newly designed microchannel for solvent extraction was fabricated in a quartz glass chip and applied to solvent extraction of a Co-2-nitroso-5-dimethylaminophenol complex. The aqueous solution of Co complex and toluene were introduced into the microchannel, and the Co complex extracted in toluene was detected by thermal lens microscopy (TLM). The Co complex was quickly extracted into toluene when the flow was stopped. The observed extraction time, ca. 50 s, was almost equivalent to the value calculated using the diffusion distance and diffusion coefficient. The dependence of the TLM signal on the concentration of the Co complex showed good linearity in the range of 1.10(-7)-1.10(-6) M. (C) 2000 Elsevier Science B.V. All rights reserved.
  • K Sato, M Tokeshi, T Sawada, T Kitamori
    ANALYTICAL SCIENCES 16 (5) 455 - 456 0910-6340 2000/05 [Refereed][Not invited]
  • M Tokeshi, T Minagawa, T Kitamori
    ANALYTICAL CHEMISTRY 72 (7) 1711 - 1714 0003-2700 2000/04 [Refereed][Not invited]
     
    An ion-pair solvent extraction was performed in a microchannel fabricated in a quartz glass chip. The aqueous solution of Fe-bathophenanthrolinedisulfonic acid complex and the chloroform solution of tri-n-octylmethyl-ammonium chloride were introduced into the microchannel, and a parallel two-phase laminar now was formed. The ion-pair product extracted in chloroform was monitored by the thermal lens microscope. The ion-pair product was gradually extracted from aqueous solution into chloroform when the flow was very slow or stopped, while nothing was extracted into chloroform when the flow was fast. The time for extraction in the present 250 mu m microchannel, 45 s, roughly coincided with the molecular diffusion time, and the extraction time was at least 1 order shorter compared with the ordinary extraction time using a separatory funnel and mechanical shaking. The micro-space in the microchannel was characterized by the large specific interface area and short diffusion distance, and these characteristics may contribute to highly efficient extraction without mechanical shaking. The success of this molecular transport may lead to the integration of more complicated separation and chemical operations on a microchip and more applications.
  • Kiichi Sato, Manabu Tokeshi, Tamao Odake, Hiroko Kimura, Takeshi Ooi, Masayuki Nakao, Takehiko Kitamori
    Analytical Chemistry 72 (6) 1144 - 1147 0003-2700 2000/03/15 [Refereed][Not invited]
     
    An immunosorbent assay system was integrated into a glass microchip. Polystyrene beads were introduced into a microchannel, and then hutnan secretory immunoglobulin A (s-IgA) adsorbed on the bead surface was reacted with colloidal gold conjugated anti-s-IgA antibody and detected by a thermal lens microscope. The scale merits of liquid microspace on the molecular behavior remarkably contributed to reduced assay time. The integration cut the time necessary for the antigen-antibody reaction by 1/90, thus shortening the overall analysis time from 24 h to less than 1 h. Moreover, troublesome operations required for conventional immunosorbent assays could be replaced by simple operations.
  • M Tokeshi, T Kitamori
    ELECTROCHEMISTRY 68 (3) 192 - 196 1344-3542 2000/03 [Refereed][Not invited]
  • Manabu Tokeshi, Takehiko Kitamori
    Yosetsu Gakkai Shi/Journal of the Japan Welding Society 69 (6) 514 - 516 0021-4787 2000 [Refereed][Not invited]
  • K Sato, M Tokeshi, T Kitamori, T Sawada
    ANALYTICAL SCIENCES 15 (7) 641 - 645 0910-6340 1999/07 [Refereed][Not invited]
     
    Microchannels having a 150x100 mu m cross section were fabricated in a quartz glass chip as a component in an integrated flow injection analysis (FIA) system. They were put to use for flow, mixing, reaction, and detection. The reaction system was a chelating reaction of divalent iron ion with o-phenanthroline (o-phen), and a photothermal microscope was applied for the ultra-sensitive detection of the non-fluorescent reaction product. Nano liter levels of solutions were introduced and transported by capillary action and mixed by molecular diffusion. Zeptomole levels of the reaction product were detected quantitatively. This was the first demonstration of an on-chip chemical determination device which integrates the primitive FIA system without using electroosmotic liquid control or fluorometric determination.
  • K Sato, H Kawanishi, M Tokeshi, T Kitamori, T Sawada
    ANALYTICAL SCIENCES 15 (6) 525 - 529 0910-6340 1999/06 [Refereed][Not invited]
     
    A thermal-lens microscope which we developed was applied to an ultramicro quantity determination of a dye in an aqueous solution filling a microchannel (150 mu m wide and 100 mu m deep) fabricated in a quartz glass substrate. The detection volume, which corresponded to the confocal volume, was estimated to be 1.3 fl. The detection limit of the dye molecules was 160 ymol, and the calibration line showed good linearity in the sub-zmol-to-zmol region. This detection sensitivity is equivalent to that of laser-induced fluorometry. The thermal-lens signal measured in the microchannel was more stable than that measured in a liquid micro space between a slide glass and a cover glass, which was much wider than the microchannel. This may have been due to a suppression of convection in the microchannel. The thermal lens method can be applied to non-fluorescent chemical species, and is thus very suitable detection method for integrated chemistry systems.
  • Manabu Tokeshi, Marika Uchida, Kenji Uchiyama, Tsuguo Sawada, Takehiko Kitamori
    Journal of Luminescence 83-84 261 - 264 0022-2313 1999 [Refereed][Not invited]
     
    A thermal lens microscope was applied to ultrasensitive detection of non-fluorescent molecule in liquid. The calibration curve showed good linearity in the 5.4-27 molecules region. The minimum number of molecules detected was 5.4 and the detection limit of Pb(II) octaethylporphyrin in benzene was 0.5 molecules. Our thermal lens microscope is a powerful tool for ultrasensitive detection of non-fluorescent molecules at the single-molecule level and offers a great potential for further applications. © 1999 Elsevier Science B.V. All rights reserved.
  • Yuki Wakabayashi, Manabu Tokeshi, Dong-Ling Jiang, Takuzo Aida, Takehiko Kitamori
    Journal of Luminescence 83-84 313 - 315 0022-2313 1999 [Refereed][Not invited]
     
    We have recently reported the anomalous cis-trans isomerization, which induced by 5-photon absorption (5-PA) of a spherical azo-dendrimer (L5AZO) by IR radiation from a nichrom source. In this study, we measured absolute absorption of L5AZO using a calibrated detector in order to investigate these anomalous phenomena. The number of infrared photons absorbed by a L5AZO was estimated as only 10-3 (photons s-1). This result suggests that L5AZO absorbs five photons not simultaneously but sequentially, and suggests the possibility of long-term intramolecular energy storage is anticipated. © 1999 Elsevier Science B.V. All rights reserved.

Books etc

  • Microfluidic Technologies and Platform for Protein Crystallography: Applications of Microfluidic Systems in Biology and Medicine (Manabu Tokeshi, ed.)
    Maeki Masatoshi, Manabu Tokeshi (Joint work)
    Springer 2019/05
  • Applications of Microfluidic Systems in Biology and Medicine
    Manabu Tokeshi (Editor)
    Springer 2019/05
  • Microfabrication and Microfluidic Devices for Drug Delivery: Microfluidics for Pharmaceutical Applications (Hélder A. Santos, Dongfei Liu, Hongbo Zhang, Eds.)
    Niko Kimura, Masatoshi Maeki, Manabu Tokeshi (Joint work)
    Elsevier 2019/01
  • Micro/Nano Devices for Chemical Analysis
    Manabu Tokeshi, Kiichi Sato (Joint editor)
    MDPI 2017
  • 紙を使った分析・診断チップの現状と可能性/機能紙最前線, NPO法人機能紙研究会編集, 第1部進化し続ける機能紙
    真栄城正寿, 渡慶次学 (Joint work)
    加工技術研究会 2017
  • Fabrication and Evaluation of Microfluidic Immunoassay Devices with Antibody-Immobilized Micrbeads Retained in Porous Hydrogel Micropillars: Microchip Diagnostics in Methods in Molecular Biology (Valérie Taly, Jean-Louis Viovy, Stéphanie Descroix eds.)
    Toshihiro Kasama, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    Springer 2017
  • Nanopillars, Nanowires and Nanoballs for DNA and Protein Analysis: Nanofluidcs, 2nd Edition, RSC Nanoscience & Nanotechnology No.41 (Joshua Edel, Aleksandar Ivanov, MinJun Kim, eds.)
    Noritada Kaji, Takao Yasui, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    RSC Publishing 2016
  • 診断・分析機能を集積した免疫分析チップ/バイオチップの基礎と応用
    笠間敏博, 渡慶次学 (Joint work)
    シーエムシー出版 2015
  • Carbon Nanotubes and Modern Nanoagriculture/Nanotechnology and Plant Sciences: Nanoparticles and Their Impact on Plants (Manzer H. Siddiqui, Mohamed H. Al-Whaibi, Mohammad Firoz, Eds.)
    Maged F. Serag, Naritada Kaji, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    Springer 2015
  • Size-Selective Synthesis of Ultrasmall Hydrophilic CdSe Nanoparticles in Aqueous Solution at Room Temperature/Nanoparticles in Biology and Medicine: Methods and Protocols, Methods in Molecular Biology(Mikhail Soloviev, Ed.)
    Yeon-Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    Humana Press 2012
  • ナノピラーデバイス・ナノウォールデバイス/ナノ融合による先進バイオデバイス(民谷栄一監修)
    安井隆雄, 渡慶次学, 馬場嘉信 (Joint work)
    シーエムシー出版 2011
  • マイクロ分析, 光熱変換分析/改訂六版分析化学便覧
    渡慶次 学 (Single work)
    丸善 2011
  • PCR内蔵型DNAチップ/試料分析講座1巻DNA/RNA
    岡本行広, 加地範匡, 渡慶次学, 馬場嘉信 (Joint work)
    丸善 2011
  • DNA前処理/PCR・チップ/バイオチップ実用化ハンドブック
    加地範匡, 岡本行広, 渡慶次学, 馬場嘉信 (Joint work)
    エヌ・ティー・エス 2010
  • 医療に貢献するナノバイオ技術/遺伝子医学「ますます重要になる細胞周辺環境の科学技術-細胞の生存、増殖、機能のコントロールから再生医療まで」
    岡本行広, 加地範匡, 渡慶次学, 馬場嘉信 (Joint work)
    メディカルドゥ 2009
  • 熱レンズ顕微鏡/分光測定入門シリーズ第10巻顕微分光法-ナノ・マイクロの世界をみる分光法(日本分光学会編)
    渡慶次学, 火原彰秀 (Joint work)
    講談社サイエンティフィク 2009
  • 蛍光計測/MEMS/NEMS工学全集
    渡慶次学, 馬場嘉信 (Joint work)
    テクノシステム 2009
  • ナノテクノロジーを利用した超高性能DNA解析手法/マイクロ・ナノ化学チップと医療・環境・バイオ分析, 第5編応用技術
    岡本行広, 加地範匡, 渡慶次学, 馬場嘉信 (Joint work)
    技術教育出版 2009
  • マイクロ化学チップの特性と動作性/マイクロ・ナノ化学チップと医療・環境・バイオ分析, 第2編マイクロ化学チップの動作と原理
    渡慶次学 (Single work)
    技術教育出版 2009
  • Nano-Fabricated Structures for Biomolecule Analysis/Bio-Inspired Nanomaterials and Nanotechnology(Yong Zhou, Ed.)
    Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    Nova Publisher 2009
  • Nanopillars and Nanoballs for DNA Analysis/Nanofluidcs(J. Edel, A. J. deMello, Eds.)
    Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    RSC Publishing 2009
  • ガラスのウェットエッチング-マイクロ化学チップの作製法/ウェットエッチングのメカニズムと処理パラメーターの最適化
    渡慶次学, 菊谷善国 (Joint work)
    サイエンス&テクノロジー 2008
  • Flow Analysis in Microfluidic Devices/Advances in Flow Methods of Analysis(M. Trojanowicz, Ed.)
    Manabu Tokeshi, Takehiko Kitamori (Joint work)
    Wiley-VCH 2008
  • ナノバイオデバイス/ナノバイオ計測の実際
    馬場嘉信, 渡慶次学, 加地範匡 (Joint work)
    講談社 2007
  • ミクロ相分離/ナノバイオ辞典
    渡慶次学 (Single work)
    テクノシステム 2007
  • Solvent Extraction on Chips
    Manabu Tokeshi, Takehiko Kitamori (Joint work)
    CRC Press 2007
  • Nanoscale DNA Analysis
    Laili Mohmoudian, Moham, Reza Mohamadi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba (Joint work)
    CRC Press 2007
  • 化学反応高度分析のためのマイクロ化学チップ~熱レンズ分光分析装置の開発/マイクロリアクターテクノロジー
    渡慶次学 (Single work)
    NTS 2005
  • 分子輸送制御, 化学システム設計/マイクロ化学チップの技術と応用(化学とマイクロ・ナノシステム研究会監修)
    渡慶次学 
    丸善 2004
  • マイクロケミカルテクノロジー/コンビナトリアルテクノロジー:明日を開く‘もの作り’の新世界(鯉沼秀臣, 川崎雅司監修)
    渡慶次学, 北森武彦 (Joint work)
    丸善 2004
  • マイクロチップ化学プロセスの設計, 超高感度検出器, マイクロ分析化学システム/インテグレーテッド・ケミストリー(北森武彦編)
    渡慶次学 
    シーエムシー出版 2004
  • マイクロ流路による微小バイオテクノロジー/バイオエンジニアリングマテリアル(石原一彦編)
    渡慶次学, 佐藤記一, 北森武彦 (Joint work)
    フロンティア出版 2004
  • マイクロ分析チップ/ナノテクノロジーハンドブックIV編 バイオ・化学を使う
    渡慶次学 (Single work)
    オーム社 2003
  • ナノ・マイクロテクノロジー/第6版化学便覧応用化学編I, II編基盤的化学技術, 11章次世代基盤化学技術
    金幸夫, 渡慶次学 (Joint work)
    丸善 2003
  • ヨクト分析-ナノをはるかに超えて/基礎から学ぶナノテクノロジー(平尾一之編)
    渡慶次学, 北森武彦 (Joint work)
    東京化学同人 2003
  • Micro Integrated Chemical Systems for General Use/Lab-On-A-Chip: Miniaturized Systems for (Bio) Chemical Analysis and Synthesis(R.E.Oosterbroek, A.van den Berg, Eds.)
    Yoshikuni Kikutani, Akihide Hibara, Hideaki Hisamoto, Manabu Tokeshi, Takehiko Kitamori (Joint work)
    Elsevier 2003

MISC

  • UNDERSTANDING THE LIPID NANOPARTICLES STRUCTURE DYNAMICS USING A TIME-RESOLVED SAXS MEASUREMENT
    Masatoshi Maeki, Niko Kimura, Kazuki Shimizu, Kento Yonezawa, Nobutaka Shimizu, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi  Proc. Micro TAS 2019  1478  -1479  2019/10  [Refereed][Not invited]
  • PORTABLE FLUORESCENCE POLARIZATION ANALYZER FOR ON-SITE MULTISAMPLE IMMUNOASSAY
    Ayano Nakamura, Osamu Wakao, Ken Satou, Mitsutoshi Aoyagi, Kazuhiko Nishimura, Chikaaki Mizokuchi, Ken Sumiyoshi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Koji Shigemura, Akihide Hibara, Manabu Tokeshi  Proc. Micro TAS 2019  1393  -1394  2019/10  [Refereed][Not invited]
  • DEVELOPMENT OF A THREE-DIMENSIONAL MICROMIXER DEVICE FOR PRODUCTION OF VARIOUS LIPID-BASED NUCLEIC ACID NANOCARRIERS
    Niko Kimura, Masatoshi Maeki, Yusuke Sato, Kosuke Sasaki, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi  Proc. Micro TAS 2019  368  -369  2019/10  [Refereed][Not invited]
  • HIGH-THROUGHPUT X-RAY CRYSTALLOGRAPHY BASED ON THE PROTEIN CRYASTAL ARRAY
    Reo Takeda, Masatoshi Maeki, Sho Ito, Go Ueno, Kunio Hirata, Akihiko Ishida, Hirofumi Tani, Masaki Yamamoto, Manabu Tokeshi  Proc. Micro TAS 2019  191  -192  2019/10  [Refereed][Not invited]
  • FLUORESCENCE SIGNAL AMPLIFICATION FOR SENSITIVE ENZYME IMMUNOASSAY UTILIZING AN IMMUNO-WALL
    Keine Nishiyama, Toshihiro Kasama, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi  Proc. Micro TAS 2019  689  -690  2019/10  [Refereed][Not invited]
  • Bioanalysis: Applications of Microfluidic Systems in Biology and Medicine
    Tokeshi Manabu, Daisuke Onoshima, Hiroshi Yukawa, Yoshinobu Baba  Bioanalysis: Applications of Microfluidic Systems in Biology and Medicine  7-  275  -300  2019  [Not refereed][Not invited]
  • Microfluidic stepwise and rapid ethanol dilution for precise size control of lipid nanoparticles
    Niko Kimura, Masatoshi Maeki, Nana Okabe, Yusuke Sato, Akihiko Ishida, Hirofumi Tani, Hideyoshi Harashima, Manabu Tokeshi  Proc. Micro TAS 2018  1404  -1405  2018/11  [Refereed][Not invited]
  • MICROSCOPIC REAL-TIME MEASUREMENT OF PROTEIN CRYSTAL GROWTH IN MICROFLUIDIC DEVICES
    Masatoshi Maeki, Shohei Yamazaki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi  Proc. Micro TAS 2018  2192  -2193  2018/11  [Refereed][Not invited]
  • ナノピラーデバイスを用いたDNAのサイズ分離と無標識検出
    阿尻 大雅, 安井 隆雄, 笠 晴也, 真栄城 正寿, 石田 晃彦, 谷 博文, 西井 準治, 馬場 嘉信, 渡慶次 学  日本分析化学会講演要旨集  67年会-  125  -125  2018/08  [Not refereed][Not invited]
  • Toshihiro Kasama, Yoshinobu Baba, Manabu Tokeshi  Next Generation Point-of-care Biomedical Sensors Technologies for Cancer Diagnosis  305  -322  2017/12/30  [Not refereed][Not invited]
  • A Small-Sized Lipid Nanoparticles Production Method Using Microfluidic Devices with Baffle Structures
    N. Kimura, M. Maeki, Y. Sato, A. Ishida, H. Tani, H. Harashima, M. Tokeshi  Proc. Micro TAS 2017  965  -966  2017/10  [Not refereed][Not invited]
  • High-Throughput Fluorescence Polarization Measurement System Towards Molecular Interaction Analysis
    O. Wakao, M. Maeki, A. Ishida, H. Tani, A. Hibara, M. Tokeshi  Proc. Micro TAS 2017  557  -558  2017/10  [Refereed][Not invited]
  • A Microfluidic-Based Technique for Creating Amyloid Nanostructures and Its Application to Enzyme Reaction
    M. Maeki, S. Sato, A. Ishida, H. Tani, M. Tokeshi  Proc. Micro TAS 2017  1279  -1280  2017/10  [Refereed][Not invited]
  • Noritada Kaji, Takao Yasui, Manabu Tokeshi, Yoshinobu Baba  RSC Nanoscience and Nanotechnology  2017-January-  (41)  76  -98  2017  [Not refereed][Not invited]
     
    © The Royal Society of Chemistry 2017. Recent development of nanofluidic devices using nanopillars, nanowires, and nanoballs for high-performance biomolecules analysis are reviewed in this chapter. Two approaches, "top-down" fabrication techniques and "bottom-up" self-assemble techniques, were applied to construct nanospace inside microchannels, and various biomolecules including DNA, RNA and proteins were successfully separated within a few seconds. These separation techniques enabled high throughput analysis that had never achieved by natural or synthetic polymers and explored a new bioanalytical field based on molecular dynamics in nanospace. Hybrid use of the both approaches might be promising for future home diagnostic devices and clinical applications.
  • Development of a New Technique for Washing Steps in Multistep Assays Using Microfluidic Paper-based Analytical Devices
    Saeed Mohammadi, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi  Proceedings of MicroTAS 2016  970  -971  2016/10  [Refereed][Not invited]
  • Fluorescence Polarization-based Multiple Samples Detection Using Microchamber Array Towards High-throughput Molecular Interaction Analysis
    Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi  Proceedings of MicroTAS 2016  1400  -1401  2016/10  [Refereed][Not invited]
  • Manabu Tokeshi, Kiichi Sato  MICROMACHINES  7-  (9)  2016/09  [Not refereed][Not invited]
  • マイクロ分析チップの新たな可能性
    渡慶次学, 真栄城正寿  JPCA NEWS  576-  8  -13  2016/09  [Not refereed][Invited]
  • がん診断のためのエクソソーム分析法の開発
    阿尻 大雅, 安井 隆雄, 真栄城 正寿, 石田 晃彦, 谷 博文, 馬場 嘉信, 渡慶次 学  日本分析化学会講演要旨集  65年会-  165  -165  2016/08  [Not refereed][Not invited]
  • マイクロ流体デバイスによる脂質ナノ粒子作製とDDSへの応用
    真栄城正寿, 佐藤悠介, 原島秀吉, 渡慶次学  機能材料  36-  15  -21  2016/07  [Not refereed][Invited]
  • T. Kasama, A. Yamamichi, F. Ohka, Y. Kato, H. Suzuki, A. Kato, K. Motomura, M. Hirano, M. Ranjit, L. Chalise, M. Kurimoto, G. Kondo, K. Aoki, N. Kaji, T. Matsubara, H. Suzuki, M. Tokeshi, T. Wakabayashi, A. Natsume, Y. Baba  20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016  651  -652  2016  [Refereed][Not invited]
     
    In the present study, we propose immuno-wall lab-on-a-chip protein analysis devices for ultrarapid detection of R132H mutant isocitrate dehydrogenase 1 (IDH1) that we identified as a specifically and commonly expressed gene in diffusely infiltrative gliomas1. In 9 min immunoassay, we successfully carried out specific detection of R132H mutant IDH1 in a lysate of glioma tissue, which is much faster than intraoperative pathological diagnosis. Moreover, clinical samples from 10 patients with gliomas were analyzed by using the immuno-wall devices. These results were consistent with those of PCR-based genetic mutation detection method. Our devices realize point-of-care tumor testing, resulting in the high precision surgery of gliomas.
  • Y. Fujishima, M. Maeki, Y. Sato, T. Yasui, A. Ishida, H. Tani, Y. Baba, H. Harashima, M. Tokeshi  20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016  1412  -1413  2016  [Refereed][Not invited]
     
    This paper describes lipid nanoparticles (LNPs) formation behavior in microfluidic devices equipped with the staggered herringbone micromixers (SHM) in order to control the LNPs size precisely. Three types of microfluidic devices with different heights of mixer structures were fabricated to examine the effect of mixer structure on the LNPs formation behavior. We found the height of the grooved structures, the lipid concentration, and the ethanol concentration were significant factors for controlling LNPs size and its distribution.
  • 液晶ディスプレイを利用した蛍光偏光イメージング
    若尾摂, 真栄城正寿, 火原彰秀, 渡慶次学  ケミカルエンジニアリング  6-  12  -17  2016/01  [Not refereed][Invited]
  • マイクロ化学チップの最新動向:紙チップとタンパク質結晶構造解析チップ
    真栄城正寿, 渡慶次学  クリーン・テクノロジー  25-  (11)  1  -4  2015/11  [Not refereed][Invited]
  • Fluorescence Polarization Imaging for Multisample Immunoassay
    Osamu Wakao, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi  Proceedings of MicroTAS 2015  1816  -1818  2015/10  [Refereed][Not invited]
  • Tetsunari Hase, Toshihiro Kasama, Nanako Nishiwaki, Naoyuki Yogo, Mitsuo Sato, Noritada Kaji, Masashi Kondo, Manabu Tokeshi, Yoshinobu Baba, Yoshinori Hasegawa  JOURNAL OF THORACIC ONCOLOGY  10-  (9)  S585  -S585  2015/09  [Refereed][Not invited]
  • 紙を利用した分析・診断チップ
    真栄城正寿, 渡慶次学  紙パルプ技術タイムス  58-  37  -42  2015/08  [Not refereed][Invited]
  • T. Kasama, T. Hase, N. Nishiwaki, N. Yogo, M. Sato, M. Kondo, N. Kaji, M. Tokeshi, Y. Hasegawa, Y. Baba  MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences  925  -927  2015  [Refereed][Not invited]
     
    © 15CBMS-0001. 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.
  • Taiga Ajiri, Takao Yasui, Akihiko Ishida, Hirofumi Tani, Yoshinobu Baba, Manabu Tokeshi  MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences  1789  -1791  2015  [Refereed][Not invited]
     
    © 15CBMS-0001. We have developed a label-free detection method of biomolecules using nanostructures [1]. The principle of this method is based on intensity changes of diffracted light derived from the nanostructures. This method is very simple and useful for label-free detection of biomolecules, but further improvement in sensitivity was necessary to apply it to clinical applications. In this paper, we optimized optical system and device design to improve the sensitivity, and applied it to measure extracellular vesicles for cancer diagnosis. These results showed that our method had a potential to be a first screening method for cancer diagnosis.
  • M. Maeki, T. Saito, Y. Node, Y. Sato, T. Yasui, N. Kaji, A. Ishida, H. Tani, Y. Baba, H. Harashima, M. Tokeshi  MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences  838  -840  2015  [Refereed][Not invited]
     
    © 15CBMS-0001. 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.
  • 超解像蛍光顕微鏡の開発
    加地範匡, 渡慶次学, 馬場嘉信  現代化学  (12)  31-34  2014/12/01  [Not refereed][Not invited]
  • LCD-CCD Synchronization Detection for Fluorescece Polarization Immunoassay
    Osamu Wakao, Yusaku Fujii, Akihiko Ishida, Hirofumi Tani, Akihide Hibara, Manabu Tokeshi  Proceedings of the Micro TAS 2014 Symposium  2271  -2273  2014/10  [Refereed][Not invited]
  • Crystal Habit Modification of Protein by Using Microfluidic Chip
    Masatoshi Maeki, Ashtamurthy P. Pawate, Keiichi Watanabe, Manabu Tokeshi, Paul J. Kenis, Masaya Miyazaki  Proceedings of the Micro TAS 2014 Symposium  1083  -1085  2014/10  [Refereed][Not invited]
  • 細胞表層改質による単一細胞抽出法の創成
    岡本 行広, 大川 智生, 小野島 大介, 湯川 博, 渡慶次 学, 馬場 嘉信  日本分析化学会講演要旨集  63年会-  37  -37  2014/09  [Not refereed][Not invited]
  • Daisuke Onoshima, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  BIOPHYSICAL JOURNAL  106-  (2)  699A  -699A  2014/01  [Not refereed][Not invited]
  • Toshihiro Kasama, Yutaka Hasegawa, Haruyasu Kondo, Tsutomu Ozawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014  935  -937  2014  [Refereed][Not invited]
     
    © 14CBMS. 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.
  • Qiong Wu, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba  18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014  233  -235  2014  [Refereed][Not invited]
     
    © 14CBMS. 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.
  • 湯川 博, 塚本 涼子, 岡本 行広, 水野 正明, 加地 範匡, 渡慶次 学, 馬場 嘉信  Organ Biology  20-  (3)  105  -105  2013/10  [Not refereed][Not invited]
  • ナノワイヤデバイスを用いた細胞破砕
    安井 隆雄, 伊藤 聡, 柳田 剛, 加地 範匡, Yong He, Rahong Sakon, 金井 真樹, 長島 一樹, 渡慶次 学, 川合 知二, 馬場 嘉信  日本分析化学会講演要旨集  62年会-  145  -145  2013/08  [Not refereed][Not invited]
  • Nano Packagingが拓く次世代核酸医療
    松尾保隆, 加地範匡, 畠山浩人, 渡慶次学, 小暮健太朗, 馬場嘉信, 原島秀吉  表面  51-  227-240  2013/05  [Refereed][Not invited]
  • ICP-MS連結マイクロ流路による1細胞レベルでの金属元素検出システムの開発
    宮崎義之, 安井隆雄, 宮下振一, 稲垣和三, 加地範匡, 梅村知也, 渡慶次学, 馬場嘉信  化学とマイクロ・ナノシステム研究会誌,  12-  (1)  16-21  2013/03/29  [Not refereed][Not invited]
  • Qiong Wu, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  2-  1206  -1208  2013  [Refereed][Not invited]
     
    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).
  • Noritada Kaji, Daisuke Shigenaka, Masami Ukawa, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  2-  1170  -1172  2013  [Refereed][Not invited]
     
    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.
  • N. Nishiwaki, T. Kasama, A. Ishida, H. Tani, Y. Baba, M. Tokeshi  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  2-  1164  -1166  2013  [Refereed][Not invited]
     
    We previously developed immuno-pillar devices for rapid and easy-to-use immunoassay [1-3]. However, improvement in the detection sensitivity (nM-pM) has still remained as a problem toward the screening test for disease markers with low cut-off values. We report here a third-generation immuno-pillar device in which the primary antibodies are covalently bounded to polymers on microbeads. By using this device we achieved pM-fM detection sensitivity of C-reactive protein (CRP) spiked in human serum. Moreover, we demonstrated that the device has long-term stability. From these results, it was proved that the third-generation device has capability required for practical use.
  • Takao Yasui, Kensuke Ogawa, Noritada Kaji, Mats Nilsson, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  2-  1123  -1125  2013  [Refereed][Not invited]
     
    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.
  • D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba  17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013  2-  820  -822  2013  [Refereed][Not invited]
     
    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.
  • 連載 超解像顕微鏡の世界:波長の壁を超えて見る(1) 超解像顕微鏡とは:Abbeの法則への挑戦
    加地範匡, 渡慶次学, 馬場嘉信  現代化学  502-  (1)  50-54  2012/12  [Not refereed][Not invited]
  • Portable Liquid Chromatography System Based on Battery-Powered Electroosmotic Pump and Microchip with Packed Column and Electrochemical Detector
    Akihiko Ishida, Takehiro Fujimoto, Satoshi Yokokawa, Hirofumi Tani, Manabu Tokeshi, Ichiro Yanagisawa  Proceedings of the MicroTAS 2012 Symposium  1183  -1185  2012/10  [Refereed][Not invited]
  • On-Chip Bioluminescence Assay of ATP and Kinases Using Immobilized Firefly Luciferase in Three-Dimensional Microfluidic Chip
    Hirofumi Tani, Atsuki Morisaki, Akihiko Ishida, Manabu Tokeshi  Proceedings of the MicroTAS 2012 Symposium  1618  -1620  2012/10  [Refereed][Not invited]
  • エピジェネティクス解析のための一分子DNAメチル化検出技術の開発
    岡本 行広, 佐野 竜輝, 加地 範匡, 渡慶次 学, 馬場 嘉信  日本分析化学会講演要旨集  61年会-  68  -68  2012/09  [Not refereed][Not invited]
  • ナノワイヤデバイスによる生体分子解析
    大塚 康平, 安井 隆雄, Rahong Sakon, 柳田 剛, 加地 範匡, 金井 真樹, 長島 一樹, 渡慶次 学, 谷口 正輝, 川合 知二, 馬場 嘉信  日本分析化学会講演要旨集  61年会-  191  -191  2012/09  [Not refereed][Not invited]
  • マイクロ流体チップによる多機能性エンベロープ型ナノ構造体の作製
    加地 範匡, 北添 雄眞, 重中 大輔, 小暮 健太朗, 秋田 英万, 原島 秀吉, 渡慶次 学, 馬場 嘉信  日本DDS学会学術集会プログラム予稿集  28回-  182  -182  2012/06  [Not refereed][Not invited]
  • Toyohiro Naito, Noritada Kaji, Séverine Le Gac, Manabu Tokeshi, Albert Van Den Berg, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1852  -1854  2012  [Refereed][Not invited]
     
    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.
  • Yukihiro Okamoto, Tatsuki Sano, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1939  -1941  2012  [Refereed][Not invited]
     
    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.
  • Kensuke Ogawa, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Mats Nilsson, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  25  -27  2012  [Refereed][Not invited]
     
    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.
  • Takao Yasui, Sakon Rahong, Takeshi Yanagida, Noritada Kaji, Masaki Kanai, Kentaro Doi, Manabu Tokeshi, Satovuki Kawano, Tomoji Kawai, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  228  -230  2012  [Refereed][Not invited]
     
    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.
  • Jumpei Morikawa, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kazuo Tsubota, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  464  -466  2012  [Refereed][Not invited]
     
    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.
  • Qiong Wu, Koki Motoyama, Takao Yasui, Sakon Rahong, Takeshi Yanagida, Masaki Kanai, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Kazuki Nagashima, Tomoji Kawai, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  599  -601  2012  [Refereed][Not invited]
     
    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.
  • Toyohiro Naito, Rerngchai Arayanarakool, Noritada Kaji, Séverine Le Gac, Manabu Tokeshi, Albert Van Den Berg, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  662  -664  2012  [Refereed][Not invited]
     
    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.
  • Yoshiyuki Miyazaki, Takao Yasui, Kazumi Inagaki, Yukihiro Okamoto, Noritada Kaji, Tomonari Umemura, Manabu Tokeshi, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  767  -769  2012  [Refereed][Not invited]
     
    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.
  • Miaomiao Sun, Toshihiro Kasama, Noritada Kaji, Shin Ichi Akiyama, Yukio Yuzawa, Seiichi Matsuo, Manabu Tokeshi, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  812  -814  2012  [Refereed][Not invited]
     
    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.
  • Yukihiro Okamoto, Yukinori Nakakita, Takahiro Sano, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1108  -1110  2012  [Refereed][Not invited]
     
    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.
  • Kouhei Ootsuka, Yukihiro Okamoto, Tetsunari Hase, Manabu Tokeshi, Noritada Kaji, Yoshinori Hasegawa, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1123  -1125  2012  [Refereed][Not invited]
     
    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.
  • Takao Yasui, Koki Motoyama, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1186  -1188  2012  [Refereed][Not invited]
     
    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.
  • Satoru Ito, Takao Yasui, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1234  -1236  2012  [Refereed][Not invited]
     
    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.
  • Daisuke Shigenaka, Masami Ukawa, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Hidetaka Akita, Hideyoshi Harashima, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1546  -1548  2012  [Refereed][Not invited]
     
    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.
  • Yukihiro Okamoto, Hitoshi Watanabe, Kazutoshi Kubo, Hiroki Kondo, Noritada Kaji, Manabu Tokeshi, Masaru Hori, Yoshinobu Baba  Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012  1633  -1635  2012  [Refereed][Not invited]
     
    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.
  • ナノ構造体による回折現象を利用した無標識検出法
    安井隆雄, 加地範匡, 岡本行広, 渡慶次学, 堀池靖浩, 馬場嘉信  化学とマイクロ・ナノシステム研究会誌,  10-  (2)  22-23  2011/11  [Not refereed][Not invited]
  • Electroosmotic Flow in Nanopillar Chips
    Takao Yasui, Noritada Kaji, M. Reza Mohamadi, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  ACS Nano  5-  (10)  7775-80  2011/10/25  [Refereed][Not invited]
  • 急性肝不全マウスにおける移植幹細胞のIn vivoイメージング
    湯川 博, 渡辺 将生, 宮本 義孝, 岡本 行広, 加地 範匡, 渡慶次 学, 野口 洋文, 馬場 嘉信, 林 衆治  JSMI Report  4-  (2)  57  -57  2011/05  [Not refereed][Not invited]
  • 急性肝不全マウスにおける移植幹細胞のIn vivoイメージング
    湯川 博, 渡辺 将生, 宮本 義孝, 岡本 行広, 加地 範匡, 渡慶次 学, 野口 洋文, 馬場 嘉信, 林 衆治  JSMI Report  4-  (2)  167  -167  2011/05  [Not refereed][Not invited]
  • KUBO KAZUTOSHI, OKAMOTO YUKIHIRO, YAMAMOTO MASAYA, KAJI NORITADA, TOKESHI MANABU, TABATA YASUHIKO, BABA YOSHINOBU  日本化学会講演予稿集  91st-  (3)  716  2011/03/11  [Not refereed][Not invited]
  • Tracking Degradations of Single DNA and Protein Molecules in Fluid
    D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba  Biophysical Society 55th Annual Meeting  100-  (3)  151a-152a  2011/03  [Refereed][Not invited]
  • 量子ドットによる幹細胞のin vivoイメージング
    渡辺将生, 湯川博, 鏡味幸真, 加地範匡, 宮本義孝, 岡本行広, 渡慶次学, 林衆治, 馬場嘉信  生命化学研究レター  35-  38-42  2011/02/01  [Not refereed][Not invited]
  • Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  1-  54  -56  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • K. Iijima, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  2-  1092  -1094  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Koki Motoyama, Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  1-  647  -649  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Tatsuki Sano, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  1-  299  -301  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Yukihiro Okamoto, Yukinori Nakakita, Takahiro Sano, Jumpei Morikawa, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  3-  1909  -1911  2011  [Refereed][Not invited]
     
    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.
  • Toyohiro Naito, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  2-  816  -818  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Toyohiro Naito, Rerngchai Arayanarakool, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Séverine Le Gac, Albert Van Den Berg, Y. Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  1-  248  -250  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  1-  638  -640  2011  [Refereed][Not invited]
     
    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. Copyright © (2011) by the Chemical and Biological Microsystems Society.
  • D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba  15th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2011, MicroTAS 2011  3-  2061  -2063  2011  [Refereed][Not invited]
     
    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.
  • KUBO KAZUTOSHI, OKAMOTO YUKIHIRO, YAMAMOTO MASAYA, KAJI NORITADA, TOKESHI MANABU, TABATA YASUHIKO, BABA YOSHINOBU  化学とマイクロ・ナノシステム研究会講演要旨集  22nd-  31  2010/11/17  [Not refereed][Not invited]
  • 量子ドットによる細胞移植後の幹細胞イメージング
    渡辺将生, 湯川博, 鏡味幸真, 加地範匡, 宮本義孝, 岡本行広, 渡慶次学, 林衆治, 馬場嘉信  ナノ学会会報  9-  (1)  7-12  2010/10  [Not refereed][Not invited]
  • ICP-MSによるマウスに静注した量子ドット標識化脂肪組織由来幹細胞の体内動態の精密測定
    高崎 裕加, 渡辺 将生, 湯川 博, 梅村 知也, 加地 範匡, 岡本 行広, 宮本 義孝, 野口 洋文, 渡慶次 学, 林 衆治, 馬場 嘉信  日本分析化学会講演要旨集  59年会-  153  -153  2010/09  [Not refereed][Not invited]
  • 幹細胞治療に対する量子ドットを用いたin vivoイメージング
    湯川 博, 渡辺 将生, 鏡味 幸真, 宮本 義孝, 加地 範匡, 渡慶次 学, 野口 洋文, 馬場 嘉信, 林 衆治  JSMI Report  3-  (2)  49  -49  2010/05  [Not refereed][Not invited]
  • 高感度脳腫瘍細胞イメージング方法の開発
    岡本 行広, 中尾 早織, 水野 正明, 加地 範匡, 渡慶次 学, 川西 悟基, 榊 裕之, 馬場 嘉信  JSMI Report  3-  (2)  157  -157  2010/05  [Not refereed][Not invited]
  • 幹細胞治療に対する量子ドットを用いたin vivoイメージング
    湯川 博, 渡辺 将生, 鏡味 幸真, 宮本 義孝, 加地 範匡, 渡慶次 学, 野口 洋文, 馬場 嘉信, 林 衆治  JSMI Report  3-  (2)  166  -166  2010/05  [Not refereed][Not invited]
  • OKAMOTO Yukihiro, KAJI Noritada, TOKESHI Manabu, BABA Yoshinobu  電気学会研究会資料. BMS, バイオ・マイクロシステム研究会 = The papers of Technical Meeting on Bio Micro Systems, IEE Japan  2010-  (1)  21  -25  2010/01/29  [Not refereed][Not invited]
  • D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  3-  1439  -1441  2010  [Refereed][Not invited]
     
    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.
  • 国際会議 microTAS2010
    渡慶次学, 馬場嘉信  次世代センサ  20-  15-18  2010  [Not refereed][Not invited]
  • K. Kitazoe, Y. Okamoto, N. Kaji, M. Tokeshi, K. Kogure, H. Harashima, Y. Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  2-  827  -829  2010  [Refereed][Not invited]
     
    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.
  • K. Sugiura, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  2-  806  -808  2010  [Refereed][Not invited]
     
    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).
  • H. Suzuki, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  3-  1880  -1882  2010  [Refereed][Not invited]
     
    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.
  • J. Wang, M. Aki, D. Onoshima, K. Arinaga, N. Kaji, M. Tokeshi, S. Fujita, N. Yokoyama, Y. Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  3-  1514  -1516  2010  [Refereed][Not invited]
     
    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.
  • Kazutoshi Kubo, Yukihiro Okamoto, Masaya Yamamoto, Noritada Kaji, Manabu Tokeshi, Yasuhiko Tabata, Yoshinobu Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  1-  181  -183  2010  [Refereed][Not invited]
     
    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.
  • Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010  1-  485  -487  2010  [Refereed][Not invited]
     
    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.
  • BIOINDUSTRY センシングバイオロジー-生命科学そして医療を支えるセンシング技術 量子ドットを用いた脂肪組織由来幹細胞のバイオイメージング
    鏡味幸真, 渡辺将生, 湯川 博, 加地範匡, 岡本行広, 渡慶次学, 林 衆治, 馬場嘉信  BIOINDUSTRY センシングバイオロジー-生命科学そして医療を支えるセンシング技術 量子ドットを用いた脂肪組織由来幹細胞のバイオイメージング  26-  (12)  14  -18  2009/12  [Not refereed][Not invited]
  • 量子ドットを用いた脂肪組織由来幹細胞のバイオイメージング
    鏡味幸真, 渡辺将生, 湯川博, 加地範匡, 岡本行広, 渡慶次学, 林衆治, 馬場嘉信  Bioindustry  309-  ((5))  14-18  2009/12  [Not refereed][Not invited]
  • マイクロチップを利用した簡易細胞アッセイ技術の開発
    岡本 行広, 杉浦 佳奈子, 加地 範匡, 渡慶次 学, 馬場 嘉信  日本バイオマテリアル学会大会予稿集  31回-  316  -316  2009/11  [Not refereed][Not invited]
  • 臨床応用が始まるチップ疾患診断
    渡慶次学, 舘知也, 馬場嘉信  現代化学  464-  ((11))  34-38  2009/10  [Not refereed][Not invited]
  • 非標識型タンパク質検出手法の迅速化と高感度化 毒素タンパク質検出への応用
    安藝 理彦, 汪 俊, 小野島 大介, 吉瀬 智康, 金 万春, 藤原 健志, 有永 健児, 加地 範匡, 渡慶次 学, 山田 景子, 藤田 省三, 馬場 嘉信, 太田 美智男, 横山 直樹  日本分析化学会講演要旨集  58年会-  52  -52  2009/09  [Not refereed][Not invited]
  • Noritada Kaji, Y. Okamoto, Manabu Tokeshi, Yoshinobu Baba  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  238-  .  2009/08  [Refereed][Not invited]
  • 【ますます重要になる細胞周辺環境(細胞ニッチ)の最新科学技術 細胞の生存、増殖、機能のコントロールから創薬研究、再生医療まで】細胞周辺環境のための材料加工・利用技術 医療に貢献するナノバイオ技術
    岡本 行広, 加地 範匡, 渡慶次 学, 馬場 嘉信  遺伝子医学MOOK  別冊-  (ますます重要になる細胞周辺環境(細胞ニッチ)の最新科学技術)  161  -168  2009/08  [Not refereed][Not invited]
     
    大きさや形状を厳密に制御した様々なナノ構造体の作製が可能となっており,近年これを生体試料へ適用する試みが行われている。なかでもナノピラー構造体を用いてDNAあるいは細胞の分離,細胞培養を行うと従来法をしのぐ性能を得ることが可能である。一方,ナノ材料の中でナノファイバーは生体試料の高性能分離媒体として利用可能であり,量子ドットは微量な生体試料の高感度検出試薬に加えて,癌細胞のみにアポトーシス誘起可能な治療材料としての利用も期待される。このようにナノテクノロジーを用いて細胞あるいは分子周辺環境を適切に整備すると,従来では見受けられない現象が観測されるとともに,従来法をしのぐ機能を生み出し,医療への多大な貢献が期待される。(著者抄録)
  • 舘 知也, 加地 範匡, 渡慶次 学, 馬場 嘉信  TDM研究  26-  (3)  s134  -s134  2009/06  [Not refereed][Not invited]
  • Yousuke Inoue, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  BUNSEKI KAGAKU  58-  (6)  517  -521  2009/06  [Not refereed][Not invited]
     
    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 trite 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 rim, while CSCNTs Suspension did not. The application of CSCNTs for MCE enabled its 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.
  • Yong Zhang, Jun Wang, Yukihiro Okamoto, Manabu Tokeshi, Noritada Kaji, Yoshinobu Baba  Analytical Chemistry  81-  (7)  2745  -2750  2009/04  [Refereed][Not invited]
     
    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. © 2009 American Chemical Society.
  • ナノ構造体を用いたDNA解析
    安井隆雄, 加地範匡, 岡本行広, 渡慶次学, 馬場嘉信  NEW GLASS  24-  ((1))  22-27  2009/03  [Not refereed][Not invited]
  • 金 万春, 山田 景子, 岡本 陽, 太田 美智男, 伊神 舞, 渡慶次 学, 加地 範匡, 馬場 嘉信, 木下 圭司, 水谷 誠, 村井 篤嗣, 並河 鷹夫  日本細菌学雑誌  64-  (1)  106  -106  2009/02  [Not refereed][Not invited]
  • Nanopillars and Nanoballs for DNA Analysis
    Kaji Noritada, Tokeshi Manabu, Baba Yoshinobu  NANOFLUIDICS: NANOSCIENCE AND NANOTECHNOLOGY  (6)  179-191  2009  [Refereed][Not invited]
  • Toyohiro Naito, Ai Yatsuhashi, Noritada Kaji, Taeko Ando, Kazuo Sato, Hisao Moriya, Hiroaki Kitano, Yukihiro Okamoto, Manabu Tokeshi, Yosinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  627  -629  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Jun Wang, Yong Zhang, Yeon Su Park, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  791  -793  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Takao Yasui, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  1198  -1200  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Kanako Sugiura, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  1243  -1245  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Mitsuru Shibata, Yukihiro Okamoto, Kaji Noritada, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  624  -626  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Yukihiro Okamoto, Yousuke Inoue, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  794  -796  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Tomoya Tachi, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  427  -429  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Ayako Kawakami, Mai Ikami, Yukihiro Okamoto, Noritada Kaji, Jin Wanchun, Keiko Yamada, Michio Ohata, Hidekatsu Tazawa, Tomohiko Ebata, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  436  -438  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Hiroshi Suzuki, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  827  -829  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Katsuma Kitazoe, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Kentaro Kogure, Hideyoshi Harashima, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  1267  -1269  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Yong Zhang, Jun Wang, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  788  -790  2009  [Not refereed][Not invited]
     
    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. © 2009 CBMS.
  • Daisuke Onoshima, Jun Wang, Michihiko Aki, Kenji Arinaga, Noritada Kaji, Manabu Tokeshi, Shozo Fujita, Naoki Yokoyama, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  1587  -1589  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • Kazuki Iijima, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba  Proceedings of Conference, MicroTAS 2009 - The 13th International Conference on Miniaturized Systems for Chemistry and Life Sciences  854  -856  2009  [Refereed][Not invited]
     
    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. © 2009 CBMS.
  • A versatile method for the surface modification of the microchannels in polymer microchips
    Yukihiro Okamoto, Yeon-Su Park, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2008  892-894  2008/10  [Refereed][Not invited]
  • Yeon-Su Park, Yukihiro Okamoto, Jun Wang, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  236-  .  2008/08  [Refereed][Not invited]
  • 治療薬物モニタリングのためのマイクロ化学システムの開発
    渡慶次 学, 舘 知也, 加地 範匡, 馬場 嘉信  日本分析化学会講演要旨集  57年会-  63  -63  2008/08  [Not refereed][Not invited]
  • 量子ドットを用いた非ウイルスベクターの細胞内トラフィッキングの可視化
    水船 祥吾, 加地 範匡, 渡慶次 学, 馬場 嘉信  日本分析化学会講演要旨集  57年会-  98  -98  2008/08  [Not refereed][Not invited]
  • TOKESHI Manabu, KAJI Noritada, BABA Yoshinobu  電気学会研究会資料. BMS, バイオ・マイクロシステム研究会 = The papers of Technical Meeting on Bio Micro Systems, IEE Japan  2008-  (1)  21  -24  2008/02/12  [Not refereed][Not invited]
  • YASUI Takao, KAJI Noritada, TOKESHI Manabu, BABA Yoshinobu  電気学会研究会資料. OQD, 光・量子デバイス研究会  2008-  (1)  13  -16  2008/02/08  [Not refereed][Not invited]
  • K. Kitazoe, N. Kaji, Y. Okamoto, M. Tokeshi, K. Kogure, H. Harashima, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  1891  -1893  2008  [Refereed][Not invited]
     
    In this paper, a new method to construct a multifunctional envelope-type nano device (MEND) on a chip is described. MEND is an extremely efficient non-viral vector for a gene delivery system, which is composed of a condensed DNA and a lipid envelope. Using the microfluidic device, MEND we constructed had stable size distribution for gene therapy. We also could make the volume of accessory product lower. © 2008CBMS.
  • Kaji Noritada, Okamoto Yukihiro, Tokeshi Manabu, Baba Yoshinobu  SEIBUTSU BUTSURI KAGAKU  52-  (3)  95  -99  2008  [Not refereed][Not invited]
     
    Various kinds of functional polymers have been developed for capillary and microchip electrophoresis. A versatile alternative to entangled and random-coiled polymers, Pluronic F127, is a typical functional polymer 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. In this review, we will describe different type of viscosity-tunable polymers as well as our recent work using Pluronic F127.
  • M. R. Mohamadi, T. Yasui, N. Kaji, M. Tokeshi, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  495  -497  2008  [Refereed][Not invited]
     
    In the current research we studied coating efficiency of series of water soluble polymers with different hydrophilicity on microchip made of Poly(methyl methacrylate) (PMMA). Various characteristics of the dynamic coatings such as the efficiency for suppression of protein adsorption and electroosmotic flow (EOF), improving hydrophilicity of the microchannel surface and resolution for separation of protein samples were studied. The results give a better understanding of the interaction of protein samples to the PMMA surface. © 2008 CBMS.
  • Maged Fouad, Noritada Kaji, Mohammad Jabasini, Manabu Tokeshi, Yoshinobu Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  227  -229  2008  [Refereed][Not invited]
     
    Carbon nanotubes (CNT) can intracellulary traffic through different cellular barriers and deliver biomolecules into living cells. However, their use in plants is limited by the cellulosic wall surrounding the plant cell. Here we show that CNT with immobilized cellulase can serve as an efficient DNA delivery system for plant cells. Tracking the cellular fate of nanotubes revealed two novel phenomena: (1)A possible nuclear localization and (2)When the transfected cell decides to differentiate into tracheary cell (water conducting cell), nanotubes were observed to incorporate into cellular structure. Our work aims at methodological development that paves the way toward on-chip-nanoscale-gene delivery applications. © 2008 CBMS.
  • Mitsuru Shibata, Yukihiro Okamoto, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  1426  -1428  2008  [Refereed][Not invited]
     
    In this paper, a new method to form liposomes in microfluidic device is presented. We demonstrated a novel formation method of liposomes by using counter-current flows in an asymmetric microfluidic device. We revealed that the counter-current flows enabled the liposome formation, which weren't based on diffusive mechanism of organic solvent and the control of size and size distribution of liposomes. We investigated the formation efficiency of liposomes of counter-current flows and co-current flows.
  • K. Kondo, N. Kaji, S. Toita, K. Akiyoshi, M. Tokeshi, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  1132  -1134  2008  [Refereed][Not invited]
     
    This paper describes a novel DNA separation matrix, nanogels, for microchip electrophoresis of DNA. This small (~30 nm in diameter) but multi-functional nanogels enabled easy loading into microchannels and provided excellent resolutions especially in the relatively small DNA fragments. © 2008 CBMS.
  • K. Fujiyoshi, N. Kaji, M. Tokeshi, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  441  -443  2008  [Refereed][Not invited]
     
    In this paper, we successfully observed dynamics of conformational transition of DNA induced by ethanol and histone using microfluidic devices. Real-time monitoring of DNA contour length gave different contraction profiles in ethanol and histone solution at a single DNA molecule level. © 2008 CBMS.
  • Yousuke Inoue, N. Kaji, Y. Okamoto, M. Tokeshi, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  1456  -1458  2008  [Refereed][Not invited]
     
    We developed an inkjet-based DNA injector for microchip electrophoresis system based on a straight microchannel. The inkjet-based DNA injector enables us to precisely control injection volume of DNA and to use a simple straight microchannel for chip electrophoresis, whereas we need cross channel injector or T injector as well as complicated voltage programming for conventional chip electrophoresis. In this paper, microchip electorophoresis with inkjet injection (MCE-wii) for DNA separation was demonstrated. MCE-wii could reduce the sample volume by 0.1 % compared to that of the conventional cross injection.
  • Mai Ikami, Manabu Tokeshi, Yukihiro Okamoto, Noritada Kaji, Yoshinobu Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  1111  -1113  2008  [Refereed][Not invited]
     
    We developed a new rapid and easy-to-use immunoassay platform to simultaneously evaluate multiple biomarkers in a single sample. As proof of principle, we demonstrated a multiplex assay measuring three biomarkers: α-fetoprotein (AFP), C-reactive protein (CRP), and prostate-specific antigen (PSA). The assay was completed within 12 min, and the limit of detections (LODs) were several tenths of pg/ml for the three biomarkers. © 2008 CBMS.
  • Takao Yasui, Noritada Kaji, Mohamad Reza Mohamadi, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  432  -434  2008  [Refereed][Not invited]
     
    In this paper, EOF mesurement and biomolecular separation (21-340 kDa in proteins, 1-48.5 kbp in DNA) were carefully studied by using nanopillar chips arranged in tilted array pattern with 100-nm spacing. We revealed that the nanopillar chips had an intrinstic function to suppress electro-osmotic flow (EOF), and as a result, DNA and SDS-protein complexes could be separated without any sieving matrices and surface coatings. © 2008 CBMS.
  • D. Onoshima, N. Kaji, M. Tokeshi, Y. Baba  12th International Conference on Miniaturized Systems for Chemistry and Life Sciences - The Proceedings of MicroTAS 2008 Conference  18  -20  2008  [Refereed][Not invited]
     
    A microfluidic analytical system, which uses simple technique to pick reacting DNA molecules out of a crowd of molecules during enzymatic reaction, was developed on the basis of single-molecule imaging. Restriction enzymes EcoR I and Not I with their target and non-target DNA were analyzed. With this system, we measured the duration difference of restriction-site-searching by these enzymes at the single-molecule level. © 2008 CBMS.
  • Eiki Maeda, Masatoshi Kataoka, Mami Hino, Kazuaki Kajimoto, Noritada Kaji, Manabu Tokeshi, Jun-Ichi Kido, Yasuo Shinohara, Yoshinobu Baba  Electrophoresis  28-  (16)  2927  -33  2007/08  [Refereed][Not invited]
     
    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 microM of glucose (r(2) = 0.9963; p <0.01), and the detection limit was 0.92 microM. 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.
  • Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  BUNSEKI KAGAKU  56-  (7)  521  -534  2007/07  [Not refereed][Not invited]
     
    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.
  • Laili Mahmoudian, Jonas Melin, Mohamad Reza Mohamadi, Keiko Yamada, Michio Ohta, Noritada Kaji, Manabu Tokeshi, Mats Nilsson, Yoshinobu Baba  Chemistry Letters  36-  (3)  396  -397  2007/03/05  [Refereed][Not invited]
     
    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. Copyright © 2007 The Chemical Society of Japan.
  • Yoshitake Akiyama, Keisuke Morishima, Atsuna Kogi, Yoshikuni Kikutani, Manabu Tokeshi, Takehiko Kitamori  ELECTROPHORESIS  28-  (6)  994  -1001  2007/03  [Not refereed][Not invited]
     
    A newly developed vacuum hot press system has been specially designed for the thermal bonding of glass substrates in the fabrication process of Pyrex glass microchemical chips. This system includes a vacuum chamber equipped with a high-pressure piston cylinder and carbon plate heaters. A temperature of up to 900 degrees C and a force of as much as 9800 N could be applied to the substrates in a vacuum atmosphere. The Pyrex substrates bonded with this system under different temperatures, pressures, and heating times were evaluated by tensile strength tests, by measurements of thickness, and by observations of the cross-sectional shapes of the microchannels. The optimal bonding conditions of the Pyrex glass substrates were 570 degrees C for 10 min under 4.7 N/mm(2) of applied pressure. Whereas more than 16 h is required for thermal bonding with a conventional furnace, the new system could complete the whole bonding processes within just 79 min, including heating and cooling periods. Such improvements should considerably enhance the production rate of Pyrex glass microchemical chips. Whereas flat and dust-free surfaces are required for conventional thermal bonding, especially without long and repeated heating periods, our hot press system could press a fine dust into glass substrates so that even the areas around the dust were bonded. Using this capability, we were able to successfully integrate Pt/Ti thin film electrodes into a Pyrex glass microchip.
  • 渡慶次 学, 馬場 嘉信  化学とマイクロ・ナノシステム  6-  (1)  5  -7  2007/03  [Not refereed][Not invited]
  • Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Electrophoresis  28-  (5)  830  -6  2007/03  [Refereed][Not invited]
     
    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%).
  • KAJI Noritada, TOKESHI Manabu, BABA Yoshinobu  電気学会研究会資料. BMS, バイオ・マイクロシステム研究会 = The papers of Technical Meeting on Bio Micro Systems, IEE Japan  2007-  (1)  25  -28  2007/01/30  [Not refereed][Not invited]
  • Lihua Zhang, Toshikatsu Shinka, Yutaka Nakahori, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Sensors and Actuators, B: Chemical  121-  (1)  124  -128  2007/01/30  [Refereed][Not invited]
     
    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. © 2006 Elsevier B.V. All rights reserved.
  • A New On-Chip Platform For Rapid And Easy-To-Use Immunoassay
    Mai Ikami, Manabu Tokeshi, Noritada Kaji, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  655-657  2007  [Not refereed][Not invited]
  • Development of new microfluidic immunoassay chips
    Tokeshi Manabu, Ikami Mai, Kaji Noritada, Baba Yoshinobu  PROCEEDINGS OF THE FIRST SHENYANG INTERNATIONAL COLLOQUIUM ON MICROFLUIDICS  199-200  2007  [Refereed][Not invited]
  • A novel high efficient and speed construction method of a multi functional envelope-type nano device on microdevice
    Hiroshi Kuramoto, Noritada Kaji, Kentarou Kogure, Manabu Tokeshi, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  781-783  2007  [Not refereed][Not invited]
  • Cell Sorting Of Live And Dead Cells By Laser Radiation Pressure And Sheath Flow In Microchannel
    Masaya Murata, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  116-118  2007  [Refereed][Not invited]
  • Molecular Crowding Effect On Enzymatic Reaction In A Fl-Microchamber To Mimic Crowded Intracellular Enviromnent
    Hisashi Murahara, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  1003-1005  2007  [Not refereed][Not invited]
  • Dna Separation By Square Patterned Nanopillar Chips
    Takao Yasui, Noritada Kaji, Ryo Ogawa, Shingi Hashioka, Manabu Tokeshi, Yasuhiro Horiike, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  1207-1209  2007  [Not refereed][Not invited]
  • Viscosity-Tunable Polymer For Microchip Electrophoresis
    Daisuke Kuroda, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  841-843  2007  [Not refereed][Not invited]
  • Quantum-Dot-Connected Dna For Single-Molecular Nanobiodevices
    Daisuke Onoshima, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  1619-1621  2007  [Not refereed][Not invited]
  • 800 Fold Signal Enhancements By Transient Isotachophoresis In Linear Polymer Solution For Immunoassay Of Hsa On Standard Cross Channel Microchips
    Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  1402-1404  2007  [Not refereed][Not invited]
  • Dynamics Measurement Of Structural Change Of Helical Polymer Using Thermal Lens Microscopy And Microfluidic Technique
    Keiko Osato, Manabu Tokeshi, Noritada Kaji, Kazuma Mawatari, Takehiko Kitamori, Eiji Yashima, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  1565-1567  2007  [Not refereed][Not invited]
  • Micromixer Based On Baker'S Transformation
    Keiko Osato, Manabu Tokeshi, Noritada Kaji, Yusuke Omoto, Yasuhiko Sakai, Eiji Shamoto, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  309-311  2007  [Not refereed][Not invited]
  • Fast Differentiative Quantitation Of Salivary And Pancreatic Amylase Activities In Human Blood By Microchip Electrophoretic Separation Of Substrate/Hydorlysate Coupled With Immunoinhibition
    Eiki Maeda, Masatoshi Kataoka, Yasuo Shinohara, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2007  2007-  65-67  2007  [Not refereed][Not invited]
  • マイクロチップイムノアッセイ
    舘知也, 加地範匡, 渡慶次学, 馬場嘉信  分析化学  56-  (7)  521-534  2007  [Not refereed][Invited]
  • Nanotechnology in Proteome Analysis
    Y. Zhang, N. Kaji, M. Tokeshi, Y. Baba  Expert Review of Proteomics  4-  (4)  565-572  2007  [Refereed][Not invited]
  • Noritada Kaji, Akio Oki, Ryo Ogawa, Yuzuru Takamura, Takahiro Nishimoto, Hiroaki Nakanishi, Yasuhiro Horiike, Manabu Tokeshi, Yoshinobu Baba  Israel Journal of Chemistry  47-  (2)  161  -169  2007  [Refereed][Not invited]
     
    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.
  • マイクロリアクターを用いた高効率化学合成
    渡慶次学, 菊谷善国  バイオインダストリー  24-  (2)  53-58  2007  [Not refereed][Invited]
  • 渡慶次 学, 加地 範匡, 馬場 嘉信  臨床検査  50-  (12)  1567  -1575  2006/11  [Not refereed][Not invited]
  • 加地 範匡, 渡慶次 学, 馬場 嘉信  現代化学  (425)  31  -38  2006/08  [Not refereed][Invited]
  • Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Hiroshi Chuman, Yoshinobu Baba  Nano Today  1-  (1)  38  -45  2006/02  [Refereed][Not invited]
     
    This review, though not comprehensive, looks at recent developments in nanodevices for genomics and proteomics and some of the new applications in biomedicine. © 2006 Elsevier Ltd. All rights reserved.
  • M Tokeshi, T Kitamori  PROGRESS IN NUCLEAR ENERGY  48-  (2)  182  -182  2006  [Not refereed][Not invited]
  • Hiroshi Kuramoto, Noritada Kaji, Kentarou Kogure, Manabu Tokeshi, Yasuo Shinohara, Hideyoshi Harashima, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  1005  -1007  2006  [Refereed][Not invited]
     
    We developed a simple microdevice to construct a multifunctional envelope-type nano device (MEND) on a chip. MEND is an extremely efficient technology for a non-viral gene delivery system, however, the existing method to construct it is time-consuming, labor intensive, and tedious task. In the simple microdevice developed here, just by changing flow rate and concentrations of materials in microchip, we succeeded to construct MEND efficiently and easily. © 2006 Society for Chemistry and Micro-Nano Systems.
  • Kentaro Fujiyoshi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  1396  -1398  2006  [Refereed][Not invited]
     
    This paper presents the development of a direct monitoring system of conformational transition from B- to A-DNA in microfluidics devices. Well-controlled laminar flows produced elongated large single DNA molecules onto chemically modified surfaces, and furthermore, ease of liquid-exchange operation of microfluidics devices enabled dynamic observation of B- to A-DNA conversion. © 2006 Society for Chemistry and Micro-Nano Systems.
  • ナノバイオデバイスのバイオ応用
    渡慶次 学, 加地範匡  細胞工学  25-  873-877  2006  [Not refereed][Invited]
  • Sonoko Inoue, Masatoshi Kataoka, Yasuo Shinohara, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  903  -905  2006  [Not refereed][Not invited]
     
    Microchip electrophoretic mobility shift assay (μEMSA) has been developed for DNA-protein interaction study, and finally, we succeed in detecting the interaction of DNA and Nuclear Factor κB (NFκB) within only 200 s. Since the method provides quantitative data simultaneously, kinetics data analysis of DNA-protein interaction was also conducted. © 2006 Society for Chemistry and Micro-Nano Systems.
  • Tomoya Tachi, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  816  -818  2006  [Not refereed][Not invited]
     
    A simple and rapid immunoassay on a microchip has been developed by using fluorescence polarization measurement. We here report a microchip-based quantitative analysis of a kind of bronchodilator, theophylline, which is important for the treatment of asthma. © 2006 Society for Chemistry and Micro-Nano Systems.
  • Eiki Maeda, Masatoshi Kataoka, Yasuo Shinohara, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  813  -815  2006  [Not refereed][Not invited]
     
    In this study, high-performance monitoring system for blood sugar was developed by using microchip electrophoresis with a plastic chip. A combination of a reductive amination as sugar labeling and sugar-borate complex formation realized highly selective detection of blood sugar even in a blood sample. This paper demonstrates that this system enables us to determine blood sugar with high sensitivity and high accuracy, and it will be applicable to clinical diagnosis. © 2006 Society for Chemistry and Micro-Nano Systems.
  • Hiroki Okada, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  401  -403  2006  [Not refereed][Not invited]
     
    For protein analysis, polymeric microchip was extremely incompatible with protein samples. To overcome the incompatibility, we developed a novel physically adsorbed polymer coating, which was a new approach and different from conventional coating methods, on PMMA microchip using cellulose derivatives as the coating reagents. The coating method remarkably progressed analytical performance and we could successfully separate 5 proteins dependent of their own sizes with high reproducibility (RSD; within 3 %, n=12) on coated PMMA microchip with 12 channels array. © 2006 Society for Chemistry and Micro-Nano Systems.
  • APPLICATION OF 1,4-DIOXANE TO ENHANCING SELECTIVITY IN ANALYSIS OF GLYCOSAMINOGLYCAN DISACCHARIDES BY MICROCHIP ELECTROPHORESIS
    Yong Zhang, Guichen Ping, Bingmei Zhu, Hiroshi Chuman, Noritada Kaji, Manabu Tokeshi, Yoshinobu Baba  Micro Total Analysis System 2006  μTAS2006-  2006  [Not refereed][Not invited]
  • Mohamad Reza Mohamadi, Laili Mahmoudian, Noritada Kaji, Manabu Tokeshi, Hiroshi Chuman, Yoshinobu Baba  Micro Total Analysis Systems - Proceedings of MicroTAS 2006 Conference: 10th International Conference on Miniaturized Systems for Chemistry and Life Sciences  μTAS2006-  353  -355  2006  [Not refereed][Not invited]
     
    An efficient dynamic coating using methylcellulose and a non-ionic detergent (Tween-20) was developed which controlled protein adsorption onto the surface of microchannels on a microchip made of poly (methyl methacrylate), PMMA. Highly stacking microchip electrophoresis under non-denaturing condition was used for analyzing high molecular weight protein complexes on a plastic microchip. The method has the efficiency of 6*10 6 plate/m for a complex of mAb-HSA with MW of ∼220kDa and with the detection limit of few pM. © 2006 Society for Chemistry and Micro-Nano Systems.
  • 田中有希, 西中正弘, 馬渡和真, 渡慶次学, 渡慶次学, 北森武彦, 北森武彦  分析化学討論会講演要旨集  66th-  16  2005/04/30  [Not refereed][Not invited]
  • 渡慶次学, 馬渡和真, 火原彰秀, 北森武彦  応用物理  73-  (6)  741  -748  2004  [Not refereed][Not invited]
  • High Throughput Bonding Technique of Pyrex Chip using Hot Pressing
    Keisuke Morishima, Yoshitake Akiyama, Manabu Tokeshi, Takehiko Kitamori  Proceedings of the Micro Total Analysis Systems, Malmö, Sweden, (2004).  410  -412  2004  [Not refereed][Not invited]
  • M Tokeshi, Y Kikutani, A Hibara, K Sato, H Hisamoto, T Kitamori  ELECTROPHORESIS  24-  (21)  3583  -3594  2003/11  [Not refereed][Not invited]
     
    This review describes our recent research on miniaturization of chemical systems. We have developed a miniaturization methodology based on pressure-driven multiphase laminar flow and a highly sensitive detection tool, the thermal lens microscope. Some representative applications of the methodology in the fields of analysis, synthesis, and bioassay are described.
  • K Sato, A Hibara, M Tokeshi, H Hisamoto, T Kitamori  ADVANCED DRUG DELIVERY REVIEWS  55-  (3)  379  -391  2003/02  [Not refereed][Not invited]
     
    This review focuses on chemical and biochemical analysis systems using pressure-driven microfluidic devices or microchips. Liquid microspace in a microchip has several characteristic features, for example, short diffusion distances, high specific interfacial area and small heat capacity. These characteristics are the key to controlling micro unit operations and constructing new integrated chemical systems. By combining multiphase laminar flow and the micro unit operations, such as mixing, reaction, extraction and separation, continuous flow chemical processing systems are realized in the microchip format. By applying these concepts, several different analysis systems were successfully integrated on a microchip. In this paper, we introduce the microchip-based chemical systems for wet analysis of cobalt ion, multi-ion sensors, immunoassay, and cellular analysis. (C) 2002 Elsevier Science B.V. All rights reserved.
  • K Sato, A Hibara, M Tokeshi, H Hisamoto, T Kitamori  ANALYTICAL SCIENCES  19-  (1)  15  -22  2003/01  [Not refereed][Not invited]
     
    This review focuses on the integration of chemical and biochemical analysis systems into glass microchips for general use. By combining multiphase laminar flow driven by pressure and micro unit operations, such as mixing, reaction, extraction and separation, continuous-flow chemical processing systems can be realized in the microchip format, while the application of electrophoresis-based chip technology is limited. The performances of several analysis systems were greatly improved by microchip integration because of some characteristics of microspace, i.e., a large specific interface area, a short molecular diffusion time, a small heat capacity and so on. By applying these concepts, several different analysis systems, i.e., wet analysis of cobalt ion, multi-ion sensor, immunoassay, and cellular analysis, were successfully integrated on a microchip. These microchip technologies are promising for meeting the future demands of high-throughput chemical processing.
  • Multichannel Micro ELISA System
    Kiichi Sato, Maho Yamanaka, Manabu Tokeshi, Keisuke Morishima, Takehiko Kitamori  Proceedings of the Micro Total Analysis Systems,Squaw Valley, USA, (2003).  781  -784  2003  [Not refereed][Not invited]
  • Development of Integration ELISA System Using a Multichannel Microchip
    Maho Yamanaka, Kiichi Sato, Manabu Tokeshi, Keisuke Morishima, Hajime Katou, Takehiko Kitamori  25th International Symposium on Capillary Chromatography, Riva del Garda, Italy, (2002)  --  (-)  2002  [Not refereed][Not invited]
  • Microchip-Based Enzyme-Linked Immunosorbent Assay (ELISA) System
    Kiichi Sato, Maho Yamanaka, Manabu Tokeshi, Keisuke Morishima, Takehiko Kitamori  Proceedings of the Micro Total Analysis Systems,Nara, Japan, (2002).  311  -312  2002  [Not refereed][Not invited]
  • A Hibara, M Tokeshi, K Uchiyama, H Hisamoto, T Kitamori  ANALYTICAL SCIENCES  17-  (1)  89  -93  2001/01  [Not refereed][Not invited]
     
    We utilized microchip technology and found that the multilayer flow of liquids can be formed in microchannels. Liquid/liquid interfaces were formed parallel to the side wall of the microchannels, because the surface tension and friction force are stronger than the force of gravity. A water/ethylacetate/water interface was formed in a 70-mum-wide and 30-mum-deep channel. The interface was observed to be quite stable and to be maintained for a distance of more than 18 cm. As an example of a multilayer flow application, we demonstrated the liquid/liquid extraction of Co-dimethylaminophenol complex in a microchannel. The solvent-extraction process of the complex into m-xylene in the multilayer flow was found to reach equilibrium in 4 s, while it took 60 s in a simple two-phase extraction.

Awards & Honors

  • 2021/02 北海道大学 教育研究総長表彰
     
    受賞者: 渡慶次学
  • 2019/04 Royal Society of Chemistry Fellow(FRSC)
     
    受賞者: TOKESHI Manabu
  • 2018/09 日本分析化学会 学会賞
     
    受賞者: 渡慶次 学
  • 2011/10 堀場製作所 堀場雅夫賞
     
    受賞者: 渡慶次 学
  • 2007/10 Pioneers of Miniaturisation Prize
  • 2007/07 可視化情報学会学会賞(論文賞)
  • 2007/05 CHEMINAS奨励賞

Research Grants & Projects

  • オンサイト蛍光偏光イムノアッセイ装置の開発
    JST:先端計測分析技術・機器開発プログラム
    Date (from‐to) : 2016/10 -2021/03 
    Author : 渡慶次 学
  • 小型蛍光偏光測定デバイスの開発
    JSPS:科学研究費補助金挑戦的萌芽研究
    Date (from‐to) : 2012 -2015/03 
    Author : 渡慶次 学
  • 次世代診断チップの開発とその臨床診断への応用
    JSPS:科学研究費補助金基盤研究A
    Date (from‐to) : 2012 -2015/03 
    Author : 渡慶次 学
  • 食品中の抗生物質検出のためのマイクロチップ蛍光偏光免疫分析法の開発
    JSPS:二国間交流事業
    Date (from‐to) : 2012 -2014/03 
    Author : 渡慶次 学
  • 超高性能マイクロチップ電気泳動による糖たんぱく質のアイソフォーム解析
    JSPS:二国間交流事業
    Date (from‐to) : 2011 -2013/03 
    Author : 渡慶次 学
  • 細胞アッセイ用マイクロデバイスの開発
    JSPS:科学研究費補助金基盤研究B
    Date (from‐to) : 2008 -2011/03 
    Author : 渡慶次 学
  • 流路型免疫分析チップの開発
    NEDO:産業技術研究助成事業費助成金
    Date (from‐to) : 2007 -2011/03 
    Author : 渡慶次 学
  • 実診断を目指したマイクロチップ免疫分析システムの研究
    JSPS:科学研究費補助金基盤研究B
    Date (from‐to) : 2006 -2009/03 
    Author : 渡慶次 学
  • マイクロ・ナノ反応場を利用した革新的アクチノイド分離法の開発
    文部科学省:原子力システム開発事業
    Date (from‐to) : 2005 -2008/03 
    Author : 渡慶次 学
  • ファンクショナル熱レンズ顕微鏡
    JST:先端計測分析技術・機器開発事業
    Date (from‐to) : 2005 -2008/03 
    Author : 渡慶次 学
  • マイクロ空間における新規免疫化学反応場の創出
    川崎市:川崎市新技術・新製品開発等支援事業補助金
    Date (from‐to) : 2006 -2007/03 
    Author : 渡慶次 学
  • 心筋細胞の駆動力を電気エネルギーに変換するナノファイバー型バイオ発電素子の開発
    JSPS:科学研究費補助金萌芽研究
    Date (from‐to) : 2004 -2006/03 
    Author : 渡慶次 学
  • マイクロチップを用いた再処理工場用分析装置の開発
    経済産業省:革新的実用原子力技術開発費補助金
    Date (from‐to) : 2003 -2005/03 
    Author : 渡慶次 学
  • 簡易型マイクロチップの実用化研究
    NEDO:基盤技術研究促進事業
    Date (from‐to) : 2001 -2004/03 
    Author : 渡慶次 学

Educational Activities

Teaching Experience

  • 総合化学特別研究
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 総合化学院
  • Applied Biochemistry A (Analytical Biochemistry)
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : 分子認識,酵素反応,免疫反応,生体分子間相互作用,生物計測化学,マイクロ分析・診断デバイス
  • 総合化学実験指導法
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 総合化学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : マイクロ化学,ナノ化学,微細加工,化学チップ,バイオチップ,マイクロリアクター,単一原子・分子操作
  • 総合化学実験研究法
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 総合化学院
  • Micro-Nanochemistry
    開講年度 : 2020
    課程区分 : 修士課程
    開講学部 : 総合化学院
    キーワード : マイクロ化学,ナノ化学,微細加工,化学チップ,バイオチップ,マイクロリアクター,単一原子・分子操作
  • 総合化学特別研究第一
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
  • Analytical Chemistry Ⅰ
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 化学量論,溶液内化学平衡,反応速度,分析試薬,分析反応場,滴定,沈殿,溶媒抽出,吸光・蛍光光度法,クロマトグラフィー,電気泳動,酵素分析法,免疫測定法,誤差,相関分析,回帰分析
  • 総合化学研究・指導法
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 総合化学院
  • Internship
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : インターンシップ,就業体験
  • 卒業論文
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
  • Internship Ⅰ
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 国内外インターンシップ,企業インターンシップ,就業体験
  • Internship Ⅱ
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 企業インターンシップ
  • Internship Ⅱ
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 国内外インターンシップ,企業インターンシップ,就業体験
  • Exercise in Creative Engineering Design
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 社会的ニーズ,調査研究,研究発表,既存知識,課題解決法
  • Theory of Chemical Bonding
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 元素、原子軌道、周期律表、シュレディンガー方程式、波動関数
  • Exercise in Scientific English
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 英語,読解,作文,英会話,プレゼンテーション,ディスカッション
  • Applied Chemistry and Biochemistry
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 工学部
    キーワード : 有機合成,有機材料,化学プロセス,反応器設計,生体材料,高分子材料,分子機能,無機材料,複合材料,電子材料,光機能材料

Committee Membership

  • 2021/04 - Today   The Chemical Society of Japan   Hokkaido branch secretary
  • 2020/05 - Today   Society for Chemistry and Micro-Nano Systems   Auditor
  • 2017/05 - Today   Japan Science and Technology Agency   PRESTO Research Area Advisor
  • 2007 -2009   International Conference on Miniaturized Systems for Chemistry and Life Sciences (µTAS)   Technical Program Committee Member   The Japan Society for Analytical Chemistry

Social Contribution

Social Contribution

Social Contribution

  • Editorial Advisory Board
    Date (from-to) : 2018/01-Today
    Role : Advisor
    Sponser, Organizer, Publisher  : Elsevier
    Event, Program, Title : Sensors and Actuators B
  • Editorial Advisory Board
    Date (from-to) : 2016/01/01-2018/12/31
    Role : Advisor
    Sponser, Organizer, Publisher  : American Chemical Society
    Event, Program, Title : Analytical Chemistry
  • Editorial Advisory Board
    Date (from-to) : 2018/04
    Role : Advisor
    Sponser, Organizer, Publisher  : Wiley
    Event, Program, Title : Separation Science Plus

Media Coverage

  • Associate Editor
    Date : 2018/01
    Publisher, broadcasting station: Royal Society of Chemistry
    Program, newspaper magazine: Lab on a Chip
    Paper
  • Editorial Board
    Date : 2015/11
    Publisher, broadcasting station: MDPI
    Program, newspaper magazine: Micromachines
    Paper
  • Editor
    Date : 2013/04
    Publisher, broadcasting station: Elsevier
    Program, newspaper magazine: Sensors and Actuators B
    Paper


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