Biomedical subjects

Shoji Takeuchi

Publications and source records attributed to Shoji Takeuchi.

16 recordsLinked to original sources

PubMed2007

A trap-and-release integrated microfluidic system for dynamic microarray applications.

Dynamic microarrays hold great promise for advancing research in proteomics, diagnostics and drug discovery. However, this potential has yet to be fully realized due to the lack of reliable multifunctional platforms to transport and immobilize particles, infuse reagents, observe the reaction, and retrieve selected particles. We achieved all these functions in a single integrated device through the combination of hydrodynamic and optical approaches. Hydrodynamic forces allow simultaneous transportation and immobilization of large number of particles, whereas optical-based microbubble technique for bead retrieval gives dexterity in handling individual particles without complicated circuitry. Based on the criterion derived in this paper, the device was designed, and fabricated using standard photolithography and soft lithography methods. We examined the dynamics of bubble formation and dissipation in the device, and parametric studies revealed that higher power settings at short intervals were more efficient than low power settings at longer intervals for bead retrieval. We also demonstrated the capabilities of our device and its potential as a tool for screening methods such as the "one-bead-one-compound" (OBOC) combinatorial library method. Although both approaches, hydrodynamic confinement and optical-based microbubbles, are presented in one device, they can also be separately used for other applications in microchip devices.

Journal Article↗

PubMed2006

Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis.

Artificial planar lipid bilayers are a powerful tool for the functional study of membrane proteins, yet they have not been widely used due to their low stability and reproducibility. This paper describes an accessible method to form a planar lipid bilayer, simply by contacting two monolayers assembled at the interface between water and organic solvent in a microfluidic chip. The membrane of an organic solvent containing phospholipids at the interface was confirmed to be a bilayer by the capacitance measurement and by measuring the ion channel signal from reconstituted antibiotic peptides. We present two different designs for bilayer formation. One equips two circular wells connected, in which the water/solvent/water interface was formed by simply injecting a water droplet into each well. Another equips the cross-shaped microfluidic channel. In the latter design, formation of the interface at the sectional area was controlled by external syringe pumps. Both methods are extremely simple and reproducible, especially in microdevices, and will lead to automation and multiple bilayer formation for the high-throughput screening of membrane transport in physiological and pharmaceutical studies.

Automation↗

PubMed2006

Electrophysiological recordings of single ion channels in planar lipid bilayers using a polymethyl methacrylate microfluidic chip.

Planar lipid bilayers are used for functional studies of ion channel proteins using electrophysiological techniques. We have been developing a plastic micro-fluidic device for the reconstitution of planar lipid bilayers and electrophysiological recordings toward a "membrane protein chip" for high-throughput screening. In the previous report [Suzuki, H., Tabata, K.V., Noji, H., Takeuchi, S., 2006. Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip. Langmuir 22 (4), 1937-1942], we presented the method and device in which the reproducibility of planar lipid bilayers reached 90%, and multiple bilayers were formed simultaneously. In this communication, we show that our device has excellent electric properties suitable for ion channel analysis down to single molecular level. Additional aspects on the optical accessibility and controllability on lipid bilayer formation are also presented.

Electrochemistry↗

PubMed2006

Timing controllable electrofusion device for aqueous droplet-based microreactors.

This paper describes an electrofusion device for controlling the precise moment of fusion between droplets by applying an electric field. This device allows (i) accurate determination of the start of chemical/biological reactions, (ii) minimum contact of reactants with channel walls--eliminating surface absorption problems, (iii) easy fabrication and (iv) continuous observation of initiated reaction. We demonstrated the fusion of beta-galactosidase and fluorescein di-beta-D-galactopyranoside (FDG) droplets, and observed the enzymatic reaction using fluorescence microscopy. In addition, sequential fusion of pico-litre droplets was also accomplished.

Alkanes↗

PubMed2006

Highly reproducible method of planar lipid bilayer reconstitution in polymethyl methacrylate microfluidic chip.

We developed a highly reproducible method for planar lipid bilayer reconstitution using a microfluidic system made of a polymethyl methacrylate (PMMA) plastic substrate. Planar lipid bilayers are formed at apertures, 100 microm in diameter, by flowing lipid solution and buffer alternately into an integrated microfluidic channel. Since the amount and distribution of the lipid solution at the aperture determines the state of the lipid bilayer, controlling them precisely is crucial. We designed the geometry of the fluidic system so that a constant amount of lipid solution is distributed at the aperture. Then, the layer of lipid solution was thinned by applying an external pressure and finally became a bilayer when a pressure of 200-400 Pa was applied. The formation process can be simultaneously monitored with optical and electrical recordings. The maximum yield for bilayer formation was 90%. Using this technique, four lipid bilayers are formed simultaneously in a single chip. Finally, a channel current through gramicidin peptide ion channels was recorded to prove the compatibility of the chip with single molecule electrophysiology.

Lipid Bilayers↗

PubMed2005

Utilization of cell-sized lipid containers for nanostructure and macromolecule handling in microfabricated devices.

We propose an original approach to handle submicrometer-sized biological or inorganic materials in microfabricated devices for micro total analysis applications. Cell-sized liposomes were utilized as containers for nanoparticles, green fluorescent proteins, or DNA and handled within a microfluidic chip. Due to the micrometer size of these liposomes, their detection could be achieved by conventional optical systems. Moreover, liposomes are hardly sensitive to Brownian motion; their trapping or transportation is thereby made easy with electrostatic-based techniques, for instance, developed the past few years for cells and particles. Encapsulated materials were confined for long durations with respect to the diffusive scale time, and the liposome membrane provided excellent protection from the outside environment, inhibiting undesirable interactions. A microfluidic device consisting of a flow cell covering an array of asymmetric electrodes allowed us to convey readily liposomes by the AC electroosmosis effect. We also assessed the electrofusion of liposomes between micromachined electrodes, opening up controlled initiation of reaction inside these containers; it was exemplified by fusing differently colored liposomes. We observed that a large fraction of the liposomes fused for electric field intensity around 6 kV/cm. Applications ranging from ultrasmall biomimetic reactors to large-scale drug delivery or cell labeling can be envisaged.

Capsules↗

PubMed2005

Parylene flexible neural probes integrated with microfluidic channels.

The fluidic channel in the flexible probe has three functions: (i) to inject chemicals into the tissues, (ii) to measure the neural activities from the tissues, and (iii) to improve the mechanical stiffness of the probe by filling the channel with a solid material. A 10-microm-thick microfluidic channel was embedded into the probe by using sacrificial photoresist patterns. Polyethylene glycol (PEG), which is solid at room temperature and dissolves when in contact with water, was used to fill the channel and increase the stiffness of the probe before insertion into the tissue. The impedance of the electrode inside the fluidic channel was around 100 kOmega at 1 kHz when the channel was filled with saline solution. We were able to insert the probe into a rat's brain and measure the neural signals with the electrode.

Animals↗

PubMed2005

Microfabricated arrays of femtoliter chambers allow single molecule enzymology.

Precise understanding of biological functions requires tools comparable in size to the basic components of life. Single molecule studies have revealed molecular behaviors usually hidden in the ensemble- and time-averaging of bulk experiments. Although most such approaches rely on sophisticated optical strategies to limit the detection volume, another attractive approach is to perform the assay inside very small containers. We have developed a silicone device presenting a large array of micrometer-sized cavities. We used it to tightly enclose volumes of solution, as low as femtoliters, over long periods of time. The microchip insures that the chambers are uniform and precisely positioned. We demonstrated the feasibility of our approach by measuring the activity of single molecules of beta-galactosidase and horseradish peroxidase. The approach should be of interest for many ultrasensitive bioassays at the single-molecule level.

Biological Assay↗

PubMed2005

Highly coupled ATP synthesis by F1-ATPase single molecules.

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.

Adenosine Triphosphate↗

PubMed2005

Controlling the shape of filamentous cells of Escherichia coli.

This paper describes a technique for growing filamentous cells of Escherichia coli with defined shapes, including crescents, zigzags, sinusoids, and spirals. The procedure begins with the fabrication of embossed microchambers in agarose. Cells are trapped in the chambers by placing a flat, flexible "ceiling", either a slab of agarose or poly(dimethylsiloxane), against an agarose mold on which a suspension of cells has been added; the use of agarose keeps cells hydrated and allows nutrients to diffuse into the chambers. Cells grown in microchambers in the presence of cephalexin grow into a multinucleate, nonseptate, filamentous phenotype and adopt the shape of the microchambers. The resulting cells are motile and can be released by removing the "ceiling" from the agarose microchambers and rinsing the cells into solution.

Cells, Immobilized↗

PubMed2004

Planar lipid bilayer reconstitution with a micro-fluidic system.

A planar lipid bilayer which is widely used for the electrophysiological study of membrane proteins in laboratories is reconstituted using a micro-fluidic system, in a manner that is suitable for automated processing. We fabricated micro-channels on both sides of the substrate, which are connected through a 100-200 microm aperture, and showed that the bilayer can be formed at the aperture by flowing the lipid solution and buffer, alternately. Parylene coating is found to be suitable for both bilayer formation and electric noise reduction. Future applications include a high-sensitivity ion sensor chip and a high-throughput drug screening device.

Electrochemistry↗

PubMed2004

Ultra-smooth glass channels for bioassay with motor proteins.

Optically-flat glass channels were fabricated for the non-fluorescent observation of bio-molecules. Two bioassays of motor proteins were successfully performed, in which nanoscale beads and microtubules were clearly observed by a differential interference contrast (DIC) microscope and a dark-field microscope, respectively. The concentration of hydrofluoric acid (HF) was optimized to obtain an optically flat surface which was evaluated by AFM, SEM and dark-field microscopy. A glass channel was etched using a poly(dimethyl siloxane)(PDMS) microfluidic channel as a mask and sealed with a PDMS-coated coverslip. The channel volume of 2-3 microl realized the drastic reduction of proteins required per assay compared with a conventional flow cell method requiring 20 microl.

Animals↗

PubMed2004

Micro patterning of active proteins with perforated PDMS sheets (PDMS sieve).

We propose a novel technique for patterning active proteins on a glass substrate using a perforated polydimethylsiloxane (PDMS) sheet-sieve. The sieve, which has tapering holes, is fabricated by spin-coating PDMS on a pyramidal-shaped mold. By means of this sieve, FITC (fluorescent isothiocyanate, bovine)-albumin was successfully spotted in a 5 x 5 microm(2) area in an array. The patterned spots were perfectly isolated, which eliminates the problem of non-specific binding of proteins to undesired areas. To show that proteins maintained their activity after the patterning, we used F(1)-ATPase biomolecular motors; their activity can easily be verified by observing their rotary motion after patterning. Selective patterning with three kinds of fluorescent micro beads indicated the possibility of patterning of different proteins on the same substrate by using the sieve.

Adenosine Triphosphatases↗

PubMed2004

A concept for controlling singlet oxygen (1 Delta g) yields using nitroxide radicals: phthalocyaninatosilicon covalently linked to nitroxide radicals.

In this study, we have investigated the singlet oxygen ((1)Delta(g)) generation mechanism using phthalocyaninatosilicon (SiPc) covalently linked to nitroxide radicals (NRs), and we succeeded in increasing the singlet oxygen quantum yield (Phi(Delta)) by linking the NRs. This originates from both an increase in the triplet quantum yield and excited-state lifetimes long enough to utilize photochemical reactions. Because the electron exchange interactions with paramagnetic species were known to result only in very fast excited-state relaxation, leading to a decrease in photochemical reaction yields, this increase in Phi(Delta) is an unusual and precious example for increasing photochemical reaction yields by electron exchange interactions with paramagnetic species. In addition, our experiments and theoretical analyses show that the spin-selective energy transfer rate constant is not influenced by linking the NRs and can be evaluated by the product of spin-statistical factors and matrix elements between the initial and final states.

Indoles↗

PubMed2004

A microfluidic device for electrofusion of biological vesicles.

This paper reports a microfabricated device with high aspect-ratio electrodes and low power consumption for the electrofusion of liposomes and cells. The applications may range from gene transfection or cell tracking to biophysical studies of membrane proteins. The device consists of 250 microm thick silicon electrodes bonded to a glass substrate and covered by a PDMS-coated glass slide. Liposomes were first aligned by AC voltage at 300 kHz and then fused with short DC pulses. The fusion yield can reach 75% and is globally better for liposome diameters larger than 10 microm. The encapsulation of microbeads inside liposomes has also been demonstrated and opens up the route towards fusion-based delivery of artificial microstructures into cells.

Cell Membrane↗

PubMed2004

A radio-telemetry system with a shape memory alloy microelectrode for neural recording of freely moving insects.

A radio frequency (RF) telemetry system with a shape memory alloy microelectrode was designed and fabricated. The total size and weight are 15 mm x 8 mm and 0.1 g, respectively. Since the telemeter is small and light enough to be loaded on a small animal such as an insect, the system can be used for the neural recording of a freely moving insect. The RF-telemeter can transmit signals by frequency modulation transmission at 80-90 MHz. The transmitted signals can be received up to about 16 meters away from the telemeter with a high signal-to-noise ratio. The neural activity can be detected without attenuation by using an instrumentation amplifier with its input impedance set to 2 Mohms at 1 kHz. The telemeter was loaded on a cockroach and the neural activity during a free-walk was successfully measured through this telemetry system.

Alloys↗