Center for NanoBio Integration- University of Tokyo Speakers

Takuzo Aida
Ung-il Chung
Hiroyuki Fujita
Takashi Funatsu
Murat Gel
Yasuhiro Horiike
Takanori Ichiki
Masaki Ihara
Kazuhiko Ishihara
Takehiko Ishii
Kazunori Kataoka
Takanori Kihara
Takehiko Kitamori
Akira Matsumoto
Yoichiro Matsumoto
Ryosuke Matsuno
Yuji Miyahara
Kanjiro Miyata
Natsuhisa Oka
Seji Okha
Shoji Ohuchi
Takamasa Sakai
Kae Sato
Kiichi Sato
Satoshi Yamaguchi
Toshiyuki Ikoma
Masayuki Takeuchi


Takuzo Aida
Department of Chemistry and Biotechnology, University of Tokyo

"Programmed Self-Assembly toward Functional Nanomaterials"

Abstract:
Understanding and utilization of π-electronic conjugation have become of increasing importance in materials sciences, particularly, for molecular electronics, and a variety of molecules exhibiting interesting optoelectronic properties have been reported. My lecture will highlight some of our recent research activities on this subject.
In 2003, we have reported that grinding single-walled carbon nanotubes in imidazolium ion-based ionic liquids gives 'bucky gels', which serve as highly powerful capacitors and can be applicable to the fabrication of fully plastic actuators by layer-by-layer casting. In 2004, we also succeeded in fabricating graphitic nanotubes by self-assembly of an amphiphilic hexabenzocoronene. They show interesting electronic properties. By chemical, electrochemical, and photochemical stitching approaches, we obtained pseudo-crosslinked versions of this graphitic nanotube with enhanced thermal stability and robustness against solvolysis. Use of a chiral amphiphilic hexabenzocoronene with stereogenic centers results in the formation of graphitic nanotubes with one-handed helical chirality. The most recent achievement includes fabrication of a photoconductive graphitic nanotube with a coaxial architecture.

(1) Science 2003, 300, 2072. (2) Small 2006, 2, 554. (3) Angew, Chem., Int. Ed. 2005, 44, 2410. (4) Chem. Eur. J. (Concepts) 2007, 13, 5048. (5) Chem. Rev. 2007, 107, 718. (6) Science 2004, 304, 1481. (7) JACS 2005, 127, 10020. (8) JACS 2006, 128, 4220. (9) Proc. Natl. Acad. Sci., USA 2005, 102, 10801. (10) Adv. Mater. 2006, 18, 1297. (11) JACS 2007, 129, 719. (12) JACS 2006, 128, 14337. (13) Science 2006, 314, 1761. (14) JACS 2007, 129, in press.



Ung-il Chung/Yuichi Tei
University of Tokyo Graduate Schools of Engineering and Medicine

"Development of high-performance implant devices"

Abstract
Skeletal defects pose grave socio-economical problems with the society aging and the birthrate declining. Our group has been attempting to develop high-performance implantable medical devices that help induce bone and cartilage regeneration. Among the three pillars of tissue engineering/regenerative medicine, i.e., cells, scaffolds and signals, we put special emphasis on scaffolds and signals. First, we have attempted to optimize regenerative signaling factors using cell-based biosensors. These cells are engineered to fluoresce upon osteogenic or chondrogenic differentiation, providing precise, easy and non-invasive tools for detection of bioactive substances. Using these biosensors, genes, proteins and small compounds including their random combinations have been efficiently screened. Second, we have attempted to create ideal 3D scaffolds for skeletal regeneration using inkjet printing technology. Layer molding using inkjet printing of biocompatible and biodegradable calcium phosphate powder enables us to not only precisely regulate external contour but also freely design internal structure. New artificial bone substitutes have been fabricated and safely implanted to patients with craniofacial defects/deformities. Third, we have been attempting to merge the first and second technologies to create high-performance implant devices that release bioactive substances in a controlled manner and act on host cells to induce regeneration. Bioactive substances are printed from the ink-heads at desired positions in desired amounts. These high-performance implant devices are now being tested in large animal experiments.



Hiroyuki Fujita
Professor
The University of Tokyo?Institute of Industrial Science
Center for International Research on MicroMechatronics

"MEMS for Bio Molecular Handling, Characterization and Utilization"

Abstract
Rapid advance in bio MEMS has enabled us not only to observe but also to handle and analyze a very small number of bio molecules, even a single molecule. The presentation covers three topics related to the
issue:

- single molecular handling of DNA by MEMS tweezers
- single molecular analysis of F1-ATPase in a fL chamber
- molecular nano conveyer based on bio motor molecules

I expect that the direct molecular handling by MEMS will enable us to analyze and process a countable number of molecules digitally in the lab-on-a-chip.



Takashi Funatsu
Professor
Graduate School of Pharmaceutical Sciences
The University of Tokyo

"Single-molecule analyses of functions of biomolecules by nano- and micro-devices"

Abstract
The single-molecule imaging technique is a powerful method that allows the observation of the functions and interactions of biomolecules. To analyze weak interactions of protein molecules, we have developed a single molecule imaging technique with zero-mode waveguides (1). Because many biologically relevant events occur at order of ÁM concentrations, the illumination volume of 0.2 fL of TIRFM (Total Internal Reflection Fluorescence Microscopy) should be reduced to less than 1 aL. Arrays of zero-mode waveguides, which are nano-hole arrays lined up on a metal-clad quartz slide, can reduce the illumination volume by three orders of magnitude as compared with TIRFM. We developed a single molecule method for imaging protein-protein interactions of chaperonin utilizing this technique (2).
We are also developing a micro fabricated fluorescence-activated biomolecule sorter (3). Supramacromolecular complexes such as an organelle are structural units that exhibit physiological functions. High purification technologies for separation and purification of bio-molecules have been required. We have developed a micro fabricated fluorescence-activated biomolecule sorter that uses a thermoreversible gelation polymer as a microvalve; opening and closing are controlled by laser-induced heating under epifluorescence microscopy. The flow switching time of 4 ms was achieved. We used this system to demonstrate the sorting of mitochondria labeled with fluorescent MitoTracker.
1) M.J. Levene et al., Science 299: 682-686 (2003)
2) T. Ueno, et al., Molecular Cell, 14: 423-434 (2004)
3) Y. Shirasaki, et al., Anal. Chem. 78: 695-701 (2006)



Murat Gel
Osamu Kurosawa, Hidehiro Oana, Masao Washizu
Department of Mechanical Engineering, University of Tokyo
Hongo, Bunkyoku, 7-3-1, 113-8656, Japan

"Fabrication of Micro Orifice Arrays in PDMS for Electroporation"

Small openings in microstructures can be used to concentrate electric field lines in a microfluidic chip. This concentrated electric field is used for electroporation to introduce material in to cells. The unique advantage of the method lies in the ability to create openings in cell membrane only in the regions where cell is facing to the opening. This keeps the cell viability and increases the efficienty of the electroporation process. Further details of the method can be found elsewhere1. In this poster we report fabrication of orifice array on a thin PDMS membrane integrated with microfluidic channels and characterization of electroporatin efficiency of the method.

Small round shape openings with size less than 10 microns is fabricated on thin suspended PDMS membranes by using photolithography and spin coating of PDMS. The mold which is formed by using a negative resist involves round inverted cones with diameter of 4 microns with 4 microns of thickness. By using extended times of spin coating, PDMS membranes with thickness of less than 5 microns are obtained.

We confirmed the electroporation efficiency of jurkat cells which are immobilized on to such membranes by inserting DNA binding fluorescent dye.

References
1) Kurosawa, O. et al., Meas. Sci. Technol., 2006, 17. 3127.

*Poster Presenter


Yasuhiro Horiike
Advanced Nano Materials Laboratory, National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, 305-0044, Japan

"Development of clinical chips of calorimetric blood analysis and infection disease diagnostic using nano-gap DNA trapping"

Micro TAS or Lab-on-a-Chip is expected as promising POCT techniques which enable us to diagnose our health/disease simply and rapidly from trace amount of specimens. For the goal of the home medical diagnosis, first we developed a painless needle, whose an edge of 150_m diameter SUS tube was cut at 10 degrees for three planes and subsequently sharpened by an electro-polished. A unique sand blast polishing technology made the tube inner surface ultra-smooth, thus allowing us to extract blood with our blood pressure. The clear vain imaging using a polarized NIR light (850 nm) and detection of the vein surface by potential changes between an arm and a vein surface realized the blood collection as seeing a display [1]. A "healthcare chip" [2] employing a calorimetric measurement combined with this electronic blood collection using a painless needle was developed [3]. The chip measures 3 items of triglyceride, total-cholesterol and HDL from whole blood of 6 μL. The centrifugal force is used for a series of process of the separation of plasma from blood, simultaneous metering of plasma/reagents, and mixing of plasma/reagents. Measured values for the 3 items applied for four patients demonstrated almost same for both a commercial blood analyzer and our chip.
A rapid diagnosis chip of infectious diseases was also studied by analyzing gene from viruses in plasma [4]. A virus lyses, DNA purification, DNA trapping and a detection were integrated in a one-chip. Such detection is advantageous in avoiding the infection risk of medical staffs. In the purification process, negatively charged DNA was immobilized on the micro-pillars coated by alumina with positive zeta potential in acid solution and was eluted in alkaline solution, where normally on and off type valves using pH-sensitive hydro-gel [5] were applied. Purified dsDNA was transported to the nano-gap-array, which consisted of 816 triangular channels with minimum width of 50nm. T4 probe dsDNA dyed by YOYO-1 was introduced into the array by electrophoresis. dsDNA was trapped in the nano-gap-array and stretched up to a length of 20μm. However, the fluorescence started to go out around trapping time around 30 min. and the dark area expanded. The initiation time for darkening diminished as applied voltage increased. The joule heating is considered to cause denaturing of dsDNA to ssDNA during trapping in nano-gaps. Indeed, when target ssDNA (50 mer) dyed by YOYO-1 was introduced to the trapping area, the area emitted the fluorescent light , thereby demonstrating hybridization with the trapped prove DNA.
In conclusion, for the home medical diagnosis, the electronic blood collection combined with the painless needle was developed. The calorimetric measurement chip measured 3 items from whole blood of 6 μL. A new infection diagnosis chip which integrated procedures from the virus lyses to the detection in a one-chip was also developed based on finding of denaturing of dsDNA trapped in the nano-gap.

References
[1] H. Ogawa, M. Nagai, J. Kikuchi and Y. Horiike, M. A. Northrup, K. F. Jensen, D. J. Harrison (Eds.) Micro Total Analysis Systems 2003, Kluwer Academic Publishers (2003), pp. 741-744.
[2] A. Oki, M. Takai, H. Ogawa, Y. Takamura, T. Fukazawa, J. Kikuchi, Y. Ito, T. Ichiki and Y. Horiike, Jpn. Appl. Phys., 42 (2003), pp.3722-3727.
[3] Y. Horiike, H. Ogawa, M. Nagai, H. Koda, C.-H.Chang, S. Hashioka, M. Takai and Y. Morimoto, Digest of Technical Papers, Transducers '07, June10-14, Lyon, France, pp. 347-350.
[4]D.J. Beebe, J.S. Moore, J.W. Bauer, Q.Yu, R.H. Liu, C.D. Devadoss and B-H. Jo: Nature, 406, 6, April(2000), pp.588-590.
[5]S. Hashioka, K. Masu and Y. Horiike, Extended Abstracts of the 2007 Intern. Conf. on Solid State Devices and Materials, Tsukuba, 2007, pp.968-969



Takanori Ichiki
Department of Bioengineering, School of Engineering & Center for NanoBio Integration
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 350-8656, Japan
e-mail: ichiki@bioeng.t.u-tokyo.ac.jp

"Nanobiodevices: Engine for creation of innovative bioanalytical platforms"

One of the largest barriers faced in conventional cell researches is the difficulty to evaluate single cells invasively and collect them selectively. We aim to create new bioanalytical systems useful for future cell research through the development of highly functional biodevices realized using nano/microfabrication technologies and their combination with microscopic imaging apparatus. In several years, we have proposed and developed some microreactor and/or microfluidic devices so as to enable direct manipulation and precise measurement of individual cells and biomolecules.
Our recent foci lies in the use of electrophoresis as a tool for the quality control in the cell processing. Zeta potential is regulated by the ionization of protein and phospholipids which are produced on the cell membrane. We have developed a microfluidic chip-based cell electrophoresis system and have enabled the highly precise measurement of electrophoretic mobility of individual cells in a minimally invasive manner. Using this system we have revealed that the electrophoretic mobility can be a useful marker for monitoring progress of the cell cycle or the apoptosis, and evaluating damages during cell processing.



Masaki Ihara
Biorecognition Molecular Engineering Laboratory,
Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan
e-mail: ihara@pel.t.u-tokyo.ac.jp

"Nano-biosensor based on open-sandwich system"

We have been developing a novel immunoassay, Open Sandwich immunoassay (OS-IA), which is based on the phenomenon of antigen-dependent stabilization of the antibody variable region (Fv). ELISA based on the OS principle, in which VH fragments bound to immobilized VL in presence of sample are detected, enables non-competitive sandwich assay for monovalent antigens with MW less than 1000 that are not susceptible to conventional sandwich assays. Our midterm goals are (1) to establish a rapid screening method for antibody suitable for OS-IA, (2) to exploit a method to produce antibody fragment available to Open Sandwich from hybridoma, and (3) to integrate our OS-IA with various measurement devices. The final goal is to provide handy diagnostic devices.

References
1) Ueda, H. et al. R. Anal. Chem. 2007, 79, 6193.
2) Ueda, H. et al. Nat. Biotech. 1996, 14, 1714.

*Poster Presenter


Kazuhiko Ishihara
Professor
Graduate School of Engineering
The University of Tokyo

"Nanobiointerfaces with artificial cell membrane structure"

Abstract
A development of new biomaterials was proposed based upon the mimicking of a simple component present on the cell membrane surfaces of the lipid bilayer that forms the matrix of the plasma membranes of cells, namely, the phosphorylcholine (PC) group of phosphatidylcholine. The PC group, an electrically neutral, zwitter ionic head group, which represents the dominant property of the phospholipid head groups present on the external surface of cells, is inert in coagulation assays. From the results, it can be said that these polymerizable phospholipid derivatives are useful for making polymer biomaterials. It was proposed a new concept for making polymer biomaterials that have good stability, process ability and applicability using a methacrylate monomer with a phosphorylcholine group, 2-methacryloyloxyethyl phosphorylcholine (MPC). It was developed finely the synthetic route and purification method of MPC and a sufficient amount of MPC with excellent purity could be obtained. Thus, it became possible to prepare the MPC polymer and their functionality was carefully evaluated. By concerning the nano/micro biodevices, the surface treatment to obtain antibiofolding and bioinert property is extremely important. Nonspecific adsorption and reaction of biomolecules against the materials surface induced significantly. The development of the MPC polymers is one of the triggers for opening surface modifications of the substrate with phospholipid derivatives and these surfaces also demonstrated the usefulness of the improvement of protein adsorption and cell adhesion resistance.



Takehiko Ishii
Department of Bioengineering, Graduate School of Engineering,
Center for NanoBio Integration, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
e-mail: tishii@bmw.t.u-tokyo.ac.jp

"Surface Design of PEGylated Nanocarrier for Active targeting"

Ligand installation on the surface of PEGylated nanoparticles is one of the promising approaches to achieve a successful active targeting for drug delivery. In some cases, however, the reactivity of the PEGylated nanoparticles to the target surface was not as dramatic as we expected. In this study, we reconsider the surface design of PEGylated nanoparticles possessing a ligand molecule at the PEG free end to give more highly recognition ability. PEGylated gold nanoparticle was employed as a model nanoparticle due to its well defined size. To increase the degree of freedom to the ligand molecules at the end of PEG, the mixture of a-lactosyl-w-mercapto-PEG (Lactose-PEG-SH) and shorter PEG-SH with some given ratio were co-immobilized on the surface of gold nanoparticles. Then the reactivity of the gold nanoparticles having different PEG chain lengths and different ligand densities on their surface to lectin-immoblized surface was evaluated by SPR.

References
1) Uchida, K. et al. Anal. Chem. 2005, 77, 1075.
2) Takae, S. et al. Biomacromolecules 2005, 6, 818.
3) Takae, S. et al. Bioconjugate Chem. 2007, 18, 1214.

*Poster Presenter


Kazunori Kataoka
Center for NanoBio Integration
Department of Materials Engineering and Center for Disease Biology and Integrative Medicine
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
E-mail: kataoka@bmw.t.u-tokyo.ac.jp

"Supramolecular nanocarriers assembled from biocompatible block copolymers for gene and drug delivery"

Polymeric micelle, the self-assembly of block copolymers with core-shell architecture, is a promising nanocarrier for drug and gene delivery. There are several relevant properties in polymeric micelle as nanocarrier systems, such as longevity in blood circulation, tissue-penetrating ability, spatial and temporal controlled drug release, and reduced inherent toxicity. Also, engineering of the block copolymer structure allows the preparation of polymeric micelles with integrated smart functions, such as targetability as well as stimuli-sensitivity. This presentation overviews the recent achievements as well as the future perspectives of polymeric micelles as smart nanocarriers for drug and nucleic acid delivery. Notable anti-tumor efficacy against hypovascular cancer, including pancreatic cancer and diffused-type stomach cancer, of the doxorubicin-incorporated polymeric micelles with pH-responding property will be demonstrated to emphasize a promising utility of the nanocarrier-modulated chemotherapy for the treatment of intractable cancers. Then, the focus of the talk will be placed to the application of gene-loaded polymeric micelles as non-viral vectors in the field of regenerative medicine, particularly bone regeneration. The result using micellar nanovector systems will be demonstrated for the successful generation of new bone in experimental animals by transducting genes encoding differentiation factors. Further, the future perspective of supramolecular nanodevices, including polymeric micelles, polymer vesicles, and photosensitive dendrimer assemblies, will be featured in the last part of this presentation, directing to the new medical paradigm of smart nanotheranostic systems controlled by external physical stimuli, particularly, photoillumination (nano-photomedicine).



Takanori Kihara
Takanori Kihara1,*, Chikashi Nakamura2, Jun Miyake1,2 1Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan 2Research Institute of Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan e-mail: tkihara@will.dpc.u-tokyo.ac.jp

"Development of nanoprobe for inner cell function"

We have developed new molecular delivery system to target single living cell by using atomic force microscope (AFM) and ultlathin needle, named nanoneedle. This system delivers molecules into living cell by attaching them to surface of nanoneedle. Thus, the molecules can be integrated on the surface by choosing appropriate binding method and delivered to particular space of single living cell. By using this molecular delivery system and sensor molecules, it is able to analyze function and amounts of the inner cell molecules in situ. However, to analyze that precisely, it is necessary to develop high sensitive nanoprobe. To realize development of nanoprobe for inner cell molecules, we focus on two points of it. One is regulation of surface environment of nanoneedle and the other is production of sensor molecules appropriate for attaching nanoneedle. In this presentation, we introduce our current study to develop nanoprobe for inner cell function.
*Poster Presenter



Takehiko Kitamori
Department of Applied Chemistry, School of Engineering
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

"Extended-Nano chemical systems on chip for bio and clinical application"

Abstract
We have developed general methods for micro integration of chemical systems built around a concept similar to that in electronics. In place of resistors, capacitors, and transistores in an IC, micro unit operations (MUOs) for mixing, extraction, phase separation, etc. have been developed to be integrated components of a microchemical system. The MUOs can be combined to each other freely, and a continuous flow chemical process (CFCP) can thus be constructed like an electronic circuit. The microchemical chip functions as a chemical CPU in the miniaturized chemical or biological system.
One further direction is to develop novel research tools for micro and nano scale sciences. In particular, the extended nanoscale, 101?102 nm; larger than macro and supra molecular space, but smaller than is possible to construct with conventional microfabrication technologies, is of great scientific interest, but experimental tools are lacking. We have applied our fabrication technologies to glass substrates, and succeeded in preparing microchips with channel widths down to 20 nm:s. Here, the very properties of liquid appear to change. NMR studies conducted in our lab have shown unique characteristics in the extended nanospace, ranging from 20 nm to 800 nm, which were consistent with results obtained from measurements of capillary motion and time resolved LIF. From these results we proposed a three-layer model; adsorbed water, high proton mobility later, and bulk water.
We intend to apply our nanotechnology to biological analysis in the near future.



Akira Matsumoto1,
Naoko Sato1, Chiho Kataoka2, Toshiya Sakata1, Kazunori Kataoka1, Yuji Miyahara1,2
1 Center for NanoBio Integration, The University of Tokyo,
2 National Institute for Materials Science,
Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8656, Japan
e-mail: amatsumoto@bmw.t.u-tokyo.ac.jp

"Polmer-gate Field Effect Transistor for Noninvasive and Quantitative Detection of Carbohydrate Chains"

Development of Field Effect Transistor (FET) devices capable of detecting cell membrane dynamics in a noninvasive manner is being attempted. Phenylborate derivative is used as a functional molecule exhibiting specific bindings with carbohydrate chains, particularly targeting to sialic acid, a carbohydrate existing with the largest population among all others on the cell membrane. Modification of the transistor gate with phenylborate moiety leads to a promoted cell adhesion. By detecting negative charges on the sialic acid molecules, construction of "cell transistor" may be possible through which cell membrane dynamics can be continuously and quantitatively analyzed.

The introduction of phenylborate moiety onto the gate surface was accomplished by forming a copolymer gel: poly(N-isopropylacrylamide-co-3-acrylamidophenylboronic acid) (9:1 in molar ratio = NB10 gel) covalently attached onto the gate. For the assessment of sugar recognizing ability of the prepared transistor, it was tested for its glucose sensitivity. Change in the threshold voltage (VT) was monitored for various glucose concentrations using an FET realtime analyzer. Pronounced glucose-dependent changes in the VT value were achieved with good reversibility. Detailed detection principle and a technical rationale for the application of the presented transistor device to carbohydrate chain sensors will also be discussed.

*Poster Presenter


Yoichiro Matsumoto
Professor
Dept. of Mechanical Engineering, The University of Tokyo

"Development of a microbubble-enhanced ultrasound gene transfer system"

Abstract
Ultrasound has a potential to deliver some therapeutic materials, such as genes, drugs or proteins, into cells. This method is called "sonoporation." Moreover, it is shown that microbubble contrast agents can enhance sonoporation efficiency. However, the mechanism of sonoporation has not been clarified and the efficiency is still insufficient. In this study, we try to deliver GFP plasmid into fibroblast cells. We established experimental apparatus and protocol, and investigated the relationships between the number of transfected cells and the several experimental conditions (ultrasound intensity, with and without microbubbles). Cell viability was also measured under some experimental conditions.
Midterm Goal: Establish experimental apparatus for parametric study, and investigate the efficiency of gene transfer with various conditions of the ultrasound and microbubbles.
Final Goal: Clarify the mechanism of gene transfer with the ultrasound and microbubbles, and develop efficient method for the transfection based on the knowledge.

T.Ikeda, et al., Ultrasound Med. Biol., 32:1383-1397, 2006
Y.Matsumoto, et al., Exp. Thermal and Fluid Science, 29, 255-265, 2005
Y.Kaneko, et al., European Radiology, 15, 1415-1420, 2005
Y.Matsumoto and S.Yoshizawa, Int. J. Numerical Methods in Fluids, 47, 591-601, 2005



Ryosuke Matsuno
Yusuke Goto, Tomohiro Konno, Madoka Takai, and Kazuhiko Ishihara
Center for NanoBio Integration and Department of Material Engineering, School of Engineering, The University of Tokyo,
Hongo, Bunkyo-ku, Tokyo 113-8586, Japan.
e-mail: matsuno@mpc.t.u-tokyo.ac.jp

"Preparation of bioaffinity nanoparticles with well-defined structure for molecular diagnosis"

Quantum dot (QD) has advantage for fluorescent label in biological research. QDs were widely used for in vitro application and in vivo application. Obviously, the QDs surface needs to be coated by biomolecules to disperse into aqueous or biocompatible polymer to use in biological environment. In the process of the preparation of QDs, QDs surface was generally covered by hydrophobic ligand, trioctylphosphine oxide (TOPO). In order to disperse into aqueous solution, the QDs surface ligands were exchanged from TOPO to the other ligands or coated by other molecules throw hydrophobic interaction. However, nonspecific binding of biomolecules may reduce the detection sensitivity of QDs. To prevent nonspecific binding, commercial available QDs surface was covered by polyethylene glycol with thiol end group. On other biocompatible polymer coating by using radical polymerization, it has not been reported except two papers as possible as we know, because the fluorescence peak change and disappear with degreasing QD core by radical.
We have proposed three type modification methods to dissolve above problem. (1) polymer coating using poly lactic acid (PLA) as a first layer and PMBN copolymer that consisted of poly 2-methacryloyloxyethylphosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenyloxycarbonyl polyethylene glycol methacrylate (MEONP) as second layer, (2) Polymer grafting from atom transfer radical polymerization (ATRP) initiator fixed on QD surfaces using activators are continuously regenerated by electron transfer (ARGET) ATRP, and (3) polymer grafting over QD surfaces using reversible addition-fragmentation chain transfer (RAFT) polymerization in water aqueous solution. It is well known that PMPC has been not affected for reduction of nonspecific binding, salt concentrations, pH and temperature. Until now, (1) and (3) method has been succeeded. In the case of (1), PLA concentration was important for dispersion in water. Furthermore, other molecules with amine could be combined throw active ester located in NEONP. Fluorescence resonance energy transfer (FRET) was confirmed between QD core (Em 580 nm) and Alexa Fluor 568 IgG fixed via active ester. In the case of (3), we successfully synthesized MPC polymer over QD core using new strategy with multifunctional RAFT agent, 2-Dodecylsulfanylthiocarbonylsulfanyl-2-methyl propionic Acid (DMP) that poses both surface activity and RAFT polymerization ability. The fluorescence peak of the MPC polymer covered QD in distilled water and PBS buffer solution did not change compared with before polymerization. For molecular diagnosis, the polymer covered QD could also conjugate with other molecular with amine group via carboxyl group located end group of polymers. This research was partially supported by a Grant-in-Aid for Scientific Research and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.



Yuji Miyahara
Bioelectronics Group, Biomaterials Center, National Institute for Materials Science /
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo

"Detection of Biomolecular recognition based on intrinsic molecular charges"

Abstract
We have been investigating a new approach to detect biomolecular recognition without labeling materials. We have proposed the novel concept of a genetic field effect transistor (FET) which is based on the direct transduction of charge density change of biomolecules into electrical signal by the field effect. Potentiometric measurements of allele specific oligonucleotide hybridization, intercalation and primer extension at the surface of the gate insulator have been demonstrated using a genetic field effect transistor. Since DNA molecules are negatively charged in an aqueous solution, hybridization event at the gate surface leads to charge density change in the channel of the FET and can be transduced into electrical signal directly without any labeling for target DNA molecules. One of the unique features of our method is to utilize a single-base extension reaction on the gate for DNA sequencing. Single-base mismatch of the target DNA as well as DNA sequencing could be successfully demonstrated with the use of the genetic FETs. By combining cell-processing technology with nano/micro fabrication technologies, we will develop bio-sensing devices for cell functional analysis. As one of the examples, we have been investigating cell-based field effect transistors (FETs) to realize non-invasive analysis of cell membrane dynamics. We also report the recent progress of the cell-based field effect devices. Since bio-sensing devices using field effect transistors are easily integrated and arrayed, they are useful for simple, inexpensive and high throughput systems for SNP typing and DNA sequencing as well as drug development.



Kanjiro Miyata
Makoto Oba, Takehiko Ishii, Nobuhiro Nishiyama, Kazunori Kataoka
Center for Nano-Bio Integration, School of Engineering, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

"Preparation and characterization of a series of cationic polyasparagine derivatives as a gene carrier"

Successful transfection with polycation-based gene vectors (polyplexes) significantly depends on the chemical structure of the incorporated polycations. Indeed, polyethylenimine (PEI) and its derivatives showing the protonation of amino groups over a wide pH range are well-known polycations as effective vehicles, which exhibit high transfection efficiency through endosomal escape owing to the proton sponge hypothesis [1]. However, there has been great concern for human gene therapy with PEI-based vectors mainly due to their problematic toxicity. Thus, the rational design of a cationic structure alternative to PEI should be crucial for success in in vivo gene delivery.
In this study, to screen the cationic structure equipped with both efficient transfection and low cytotoxicity, a series of cationic polyasparagine derivatives were prepared from a single-platform polymer through aminolysis reaction. This reaction allows a benzyl group in the side chain of poly(benzyl-L-aspartate) (PBLA) to be quantitatively converted to a cationic moiety under the restricted condition (Scheme 1). The obtained polymers were used for polyplex formation with plasmid DNA and characterized as a gene vector. Among them, a cationic polyasparagine with a ethylene diamine moiety (PAsp(DET)) showed appreciably high transfection efficiency without marked cytotoxicity in vivo as well as in vitro conditions [2-5]

 

Reference
[1] Pack, D.W.; Hoffman, A.S.; Pun, S.; Stayton, P.S. Nature Rev. Drug Discovery 2005, 4, 581-593.
[2] Kanayama, N.; Fukushima, S.; Nishiyama, N.; Itaka, K.; Jang, W.D.; Miyata, K.; Yamasaki, Y.; Chung, U.; Kataoka, K. ChemMedChem 2006, 1, 439-444.
[3] Akagi, D.; Oba, M.; Koyama, H.; Nishiyama, N.; Fukushima, S.; Miyata, T.; Nagawa, H.; Kataoka, K. Gene Ther. 2007,
[4] Han, M.; Bae, Y.; Nishiyama, N.; Miyata, K.; Oba, M.; Kataoka, K. J. Control. Release, in press.
[5] Itaka, K.; Ohba, S.; Miyata, K.; Kawaguchi, H.; Nakamura, K.; Takato, T.; Chung, U.; Kataoka, K. Mol. Ther., in press.

*Poster Presenter


Seii Ohka
Department of Microbiology, Graduate School of Medicine,
The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

"Real-time imaging of poliovirus in motor neurons"

Poliovirus (PV) is a causative agent of paralytic poliomyelitis which occurs as a result of the destruction of neurons in the central nervous system, especially motor neurons (MNs), induced by the lytic replication of PV. In humans, a neural pathway is thought to be one of the main dissemination routes for PV. This pathway also exists in PV-sensitive transgenic (Tg) mice carrying the human PV receptor (hPVR / CD155) gene. Using the Tg mice, we demonstrated that PV inoculated into the calf was incorporated into vesicles in the sciatic nerve and retrogradely transported in MNs through a fast retrograde axonal transport pathway, crucial for viral dissemination2,4. Moreover, we showed that the pathogenesis of PV infection via the neural pathway was inhibited by the anti-hPVR monoclonal antibody, which was able to block the infection4. We also demonstrated that a dynein subunit interacts directly with the cytoplasmic domain of hPVR. We observed PV transport in rat primary MNs expressing GFP-tagged hPVRa (hPVRa-GFP) or GFP-tagged mutant hPVRa (hPVRMa-GFP) that shows a reduced ability to bind Tctex-12. Fluorescent-labeled PV1 was transported retrogradely with hPVRa-GFP as well as hPVRMa-GFP. However, in hPVRMa-GFP expressing cells, the fast transport kinetic component of carriers containing both hPVRMa-GFP and PV was greatly reduced compared to that observed in the case of hPVRa-GFP containing organelles. This result suggests that cytoplasmic dynein contributes to the fast directional transport of axonal carriers containing both hPVR and PV in rat primary MNs3. Moreover, this investigation provides the first demonstration of PV axonal transport via direct PV imaging.

References
1) Ohka S., et al. (2007) J. Virol., 81:7902-7912.
2) Ohka S., et al. (2004) J. Virol., 78:7186-7198.
3) Ohka S. and Nomoto A. (2001) Trends Microbiol., 9:501-506.
4) Ohka S., et al. (1998) Virology, 250(1): 67-75.

*Poster Presenter


Shoji Ohuchi
Department of Basic Medical Sciences, Institute of Medical Science,
The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

"Aptamers against live cells: Toward the development of a novel 3D-culture method"

Aptamers are artificial molecules isolated from large combinatorial nucleic acid libraries by their high affinity to a target molecule. Due to a variety of desired properties, aptamers are attractive alternatives to antibodies in molecular biology and medical applications. Aptamers are isolated via an iterative selection-amplification process known as SELEX (Systematic Evolution of Ligands by EXponential enrichment). Although SELEX is typically carried out using purified target molecules, the resulting aptamers are not always reactive to the native targets in the native conditions, such as receptor proteins displayed on cell surface. Recently, several groups have succeeded in the isolation of aptamers using live cells as targets1-4 . Such aptamers, reactive with native targets on live cells, have large advantages compared with canonical aptamers isolated against purified targets. Herein, I report my trial to develop a novel three-dimensional (3D)-culture method employing aptamers against live cells as follows: (1) A universal method to isolate target cell-specific aptamers without knowledge of the target cell-specific molecules. (2) A cell-adhesion method to 3D-culture scaffolds using bi-functional aptamers. (3) A cell-recovery method from 3D-cultue scaffolds based on antisense hybridization to inactivate aptamers.

References
1) Pestourie, C. et al. Biochimie, 2005, 87, 921. 
2) Chu, T. et al. Curr. Opin. Mol. Ther., 2007, 9, 137. (Erratum in: Curr. Opin. Mol. Ther., 2007, 9, 305)
3) Ohuchi, S.P. et al. Nucl. Acids Symp. Ser., 2005, 49, 351.
4) Ohuchi, S.P. et al. Biochimie, 2006, 88, 897.

*Poster Presenter


Takamasa Sakai
Center for NanoBio Integration, Graduate School of Engineering,
The University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-8656, Japan
e-mail: sakai@cnbi.t.u-tokyo.ac.jp

High-strength biocompatible hydrogel with highly homogeneous diamond- like structure

Hydrogels are defined as the 3D polymer networks in which aqueous solution exists as a solvent. Indeed, most of the hydrogels are composed of 90% water. Initially, taking advantage of their high water absorption and retention properties, hydrogels were applied to diapers, contact lenses, drug reservoirs, etc. After the phase transition behavior was discovered by Tanaka et al. in 1978, a variety of functional materials such as sensors and actuators were developed based on their stimulus-responsiveness. Despite these unique characteristics, practical applications of hydrogels are restricted because of low mechanical strength, which is originated from the micro-inhomogeneities of polymeric topological structure created by cross-linking. The inhomogeneities are categorized into spatial, topological, connectivity, and motility inhomogeneities. As the networks cannot behave co-operatively due to these inhomogeneities, they begin to break from the weakest link, reducing the whole mechanical strength.

In order to create homogeneous crosslinking network structure, we have designed a novel homogeneous hydrogel (Tetra-PEG gel) constructed by two different tetrahedral polyethyleneglycol (PEG) solutions with mutually reactive terminal groups. Because the nano-structural unit of the gel network was defined by the length of the tetrahedral PEG arm, the gel was expected to have a homogeneous structure and resultant high mechanical strength. Dynamic light scattering indicated the presence of a single gel mode with the blob size corresponding to the length of two PEG arms. Small angle neutron scattering and optical transmittance indicated the absence of inhomogeneities. These results demonstrate that Tetra-PEG gel has a homogeneous network structure, achieving high mechanical strength. Furthermore, Tetra-PEG gel can be easily fabricated in situ by simply mixing two biocompatible component solutions, offering the possibilities for various biomedical applications.

*Poster Presenter


Kae Sato
Atsuki Tachihara, Mats Nilsson, Takehiko Kitamori
Department of Applied Chemistry, School of Engineering, The University of Tokyo
7-3-1, Hongo, Bunkyo, Tokyo, 113-8656, JAPAN

"Single DNA Molecule Detection by on-Bead Rolling Circle Amplification in a Microchip"

This paper reports a single DNA molecule detection by on-bead rolling circle amplification (RCA) in a microchip. Single molecule counting system based on padlock probes and RCA was reported [1]. To count the individual RCA products, microscale chamber was necessary, because the RCA product had a diameter of approximately 1 Ám, and it is difficult to analyze it in a bulk scale. A microchip-based RCA system was developed, in which on-bead DNA enrichment and the solid-phase amplification in a microspace was realized. The microchannels had a dam structure with 10 Ám. Beads could be retained one side of the dam region. The beads were pre-immobilized with primer. Sample DNA solution was introduced from one inlet into the channel with the packed beads. Next, ligase was injected into the microchannel, and padlock primer was circularized. Extension of the primer hybridized to the DNA circle, i.e. RCA, was performed by DNA polymerase in the presence of excess dNTPs in a solution. The RCA products were labeled by adding fluorescence DNA probe. The enzymatic reactions were realized on the beads successfully. Individual RCA products on beads were visible with a fluorescence microscope as a bright object, and they could be easily counted. System optimization and detection of pathogenic bacteria using this system are under investigation.

[1] "Digital quantification using amplified single-molecule detection." J. Jarvius et al, Nat Methods. 3, 725 (2006).

*Poster Presenter


Kiichi Sato
Department of Applied Biological Chemistry
The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657 Japan

"Development of an integrated micro total bioassay system for oral medicine"

A bioassay using cultured cells is one of the most important analytical methods in a search for new drugs, a safety evaluation of foods and chemical compounds, and basic biochemical studies. We have reported integration of several bioassay systems into a microchip to realize reduction of assay time, cell and reagent consumption, and troublesome manual operations [1-3]. In this study, a micro total bioassay system for an oral anticancer agent was developed, and overall activity of an anticancer agent, i.e. intestinal absorption, hepatic metabolism, and anticancer activity, can be assayed with simple operation.
The microchip was composed of a slide glass and two PDMS sheets. Human intestinal model cells were cultured on the membrane in the microchip. Human hepatocellular carcinoma cells were cultured on the microcarrier beads, and then packed in the microchannel. Human breast carcinoma cells were cultured on the fibronectin-coated glass surface of the microchannel as target cells. An anticancer agent for breast cancer, cyclophosphamide (CPA), which is known as an oral prodrug, was used as a model drug. CPA shows its anticancer activity after intestinal absorption and activation at liver. By using the system, overall characters of an anticancer agent could be assayed with simple operation, and the system realized reduction of assay time and cell consumption.

[1] "Microbioassay System for an Anti-Cancer Agent Test using Animal Cells on a Microfluidic Gradient Mixer" S. Fujii et al., Anal. Sci. 22, 87 (2006).
[2] "Microbioassay System for Antiallergic Drug Screening using Suspension Cells Retaining in a Poly(dimethylsiloxane) Microfluidic Device" T. Tokuyama et al., Anal. Chem. 77, 3309 (2005).
[3] "Development of a Microchip-based Bioassay System using Cultured Cells" M. Goto et al., Anal. Chem. 77, 2125 (2005).

*Poster Presenter


Natsuhisa Oka
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo

"Stereocontrolled synthesis and properties of backbone-modified DNA and RNA"

In recent years, a great deal of attention has been focused on small DNA and RNA molecules as therapeutic reagents for selective inhibition of gene expression. Oligonucleotide therapeutic reagents, such as antisense DNAs, ribozymes, and siRNAs act on a target mRNA to arrest the protein synthesis. In order to stabilize these molecules in cells, a chemical modification of internucleotidic phosphodiester bonds is quite effective. Phosphorothioate DNA is one of the most widely used backbone-modified antisense DNAs to date. However, the currently used phosphorothioate DNAs are random mixturese of diastereomers since the chirality of the phosphorous atom cannot be controlled by use of the current synthetic methods. Because the properties of phosphorothioate DNAs, such as hybridization abilities with mRNA, affinities to proteins, and tolerances against nucleases are considered to be affected by the chirality of the phosphorous atoms, it is an important subject to develop an efficient method for obtaining stereoregulated phosphorothioate DNAs. Quite recently, we have developed a novel approach for the stereocontrolled synthesis of phosphorothioate DNAs and RNAs by the use of nucleoside 3'-O-oxazaphospholidine derivatives as monomers and N-(cyanomethyl)ammonium salts as activators (oxazaphospholidine approach).1-5 In this presentation, I wish to describe a recent progress of stereocontrolled synthesis of P-chiral DNA and RNA analogs, which are useful as oligonucleotide therapeutic reagents, by the oxazaphospholidine approach.

References
1) Oka, N., Wada, T., Saigo. K. J. Am. Chem. Soc. 2002, 124, 4962-4963.
2) Oka, N., Wada, T., Saigo. K. J. Am. Chem. Soc. 2003, 125, 8307-8317.
3) Wada, T.; Fujiwara, S.; Sato, T.; Oka, N.; Saigo, K. Nucleic Acids Symp. Ser. 2004, 48, 57-58.
4) Iwamoto, N.; Sato, T.; Oka, N.; Wada, T. Nucleic Acids Symp. Ser. 2006, 50, 159-160.
5) Wada, T.; Maizuru, Y.; Oka, N.; Saigo, K. Bioorg. Med. Chem. Lett. 2006, 16, 3111-3114.

*Poster Presenter


Satoshi Yamaguchi
Center for NanoBio Integration (CNBI) & School of Engineering, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan

"A Photocleavable Linker for Temporal Control of Gene Expression"

Transfected cell microarrays allow us to analyze thousands of genes at one time, and are promising tools for a high-throughput analysis of the gene networks that are associated with cancer and other severe diseases. However, unlike the analysis of one target gene, it is difficult to clarify the gene networks because the effects of the timing and the temporal order of multiple gene expressions need to be analyzed. Thus, a new technology for temporal regulation of gene expression on cell-based microarrays is required.

Recently, various "caged" biomolecules have been reported as a molecular tool for controlling biological phenomena spatially and temporally. In the strategy based on "cage" chemisty, the biomolecules are inactivated by the covalent attachment of a photocleavable protecting group, a process known as caging, and then are reactivated by photo-illumination (uncaging) at desired space and time. Therefore, we decided to apply the caged chemistry to transfected cell microarrays. Our ultimate goal of this study is to develop a photo-regulated cell microarray.

Until now, the photo-mediated gene expression using randomly caged DNA vector in living cells were reported by other groups. However, in their reports, inactivation by caging was not complete, and the efficiency of reactivation by photo-uncaging was extremely low. Therefore, to enhance the efficiencies in the blockage and photo-recovery of gene expression, we synthesized a new photocleavable linker that can link a plasmid vector and a bulky protecting group. The 6-bromo-7-hydroxylcoumarin-4-ylmethyl (Bhc) group was employed as a photocleavable group because of its high degradability. This photocleavable linker carries two reactive groups, the diazo group and the bromoacetyl group, separated by Bhc moiety. These reaction groups can be used to couple polynucleotide and thiol-containing species. In this poster presentation, the experimental result of the caged DNA modified with cationic peptides will be reported.

*Poster Presenter
Toshiyuki Ikoma
Biosystem and Biomolecule Control Group, Biomaterials Center,
National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki, 305-0044, Japan

"Calcium Phosphate Biomaterials and Its Related Applications based on Nanotechnology"

Abstract
Calcium Phosphate has been known to be a biocompatible material, which is found in our bone and tooth tissues. The nano-scaled hydroxyapatite (HAp) crystal is much more useful for the bone tissue engineering and drug delivery carriers than the conventional HAp ceramics sintered at high temperature with low solubility product. The nanocrystals are dissolved in our body and the dissolution rate is easily controlled with the sintering temperature. We have developed a HAp /collagen nanocomposite with unidirectional porous structure by control of ice crystal growth1, and injectable HAp drug delivery carriers2,3 with high specific surface area and high porosity for sustained release of proteins, human growth hormone, and osteoclast inhibitory factor (OCIF), etc. The novel HAp sensor was developed by using quartz crystal micro- balance method to elucidate the protein adsorption mechanism4. The HAp functions to osteoblast-like cell have been analyzed by comprehensive gene analyses with cell culture 5.
We have developed the zeolite and apatite composites for a long term assurance material for the disposal of radioactive waste, focused on the low solubility product of conventional HAp ceramic6. The target waste is iodine-129 produced by the fission of uranium atoms due to its long half-life time of 15.7Î106. We have constructed the unique composite structure; the target iodine was adsorbed to zeolite crystals of which surface were covered with HAp nanocrystals as nanolayer, and the particles were dispersed and sintered into the matrix of fluro-hydroxyapatite.

References
1) Yunoki S, Ikoma T, et al., J. Biomed. Mater. Res., 80B 166-173 2007
2) Ikoma T, et al., J. Nanosci. Nanotech., 7 822-827 2007
3) Mizushima Y, Ikoma T, et al., J. Control. Rel., 110 (2): 260-265 2006
4) Monkawa A, Ikoma T, et al., Biomaterials, 27 5748-5754 2006
5) Hanagata N, et al., Biochem. Biophy. Res. Comm., 344 (4): 1234-1240 2006
6) Watanabe Y, Ikoma T, et al., Phosphorous Res. Bull., 20 89-100 2006

Masayuki Takeuchi
Macromolecules Group, Organic Nanomaterials Center,
National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki, 305-0047, Japan

"Supramolecular Approach toward the Alignment of Conjugated Polymers"

Abstract
Exploring new methods for controlling the orientation and electronic state of p-conjugated oligomers and polymers is of importance for the production of materials having optimized properties and for their ultimate assembly into molecular circuitry. Unlike synthetic macromolecular systems, the bundling proteins found in animal cells bind one-dimensional (1D) actin filaments in high affinity to elicit the formation of actin bundles.?The bundling proteins posses two interactive modules for cross-linking actin filaments; their distinct properties determine the types of assemblies. If one can reconstruct such modules interacting with 1D materials in a supramolecular manner, not only would such systems provide a new means of aligning these materials but also they would create complex mesoscopic structures and networks akin to those found in nature. We report herein a new concept for aligning and assembling conjugated polymers (CPs) through the action of supramolecular bundling ("aligner") molecules1-3 and the use of twining polymers4 ("twimers") acting as helical hosts that would twine around and include within a single conjugated polymer.

References
1) Wakabayashi, R. et al. R. Chem. Commun. 2005, 5742.
2) Kubo, Y. et al. Angew. Chem. Int. Ed. 2006, 45, 1548.
3) Wakabayashi, R. et al. J. Am. Chem. Soc. 2006, 128, 8744.
4) Takeuchi, M. et al. Angew. Chem. Int. Ed. 2006, 45, 5494.

*Poster Presenter