Takatoki Yamamoto

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane‐glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)‐glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS‐glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS‐glass hybrid microchip with 500 bp target of λDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS‐glass microchip. The chip could be considered as an inexpensive single‐use apparatus compared to glass or silicon‐made microchips for the same purpose.

PDMS–glass hybrid microreactor array with embedded temperature control device. Application to cell-free protein synthesis

A microreactor array was developed which enables high-throughput cell-free protein synthesis. The microreactor array is composed of a temperature control chip and a reaction chamber chip. The temperature control chip is a glass-made chip on which temperature control devices, heaters and temperature sensors, are fabricated with an ITO (indium tin oxide) resistive material. The reaction chamber chip is fabricated by micromolding of PDMS (polydimethylsiloxane), and is designed to have an array of reaction chambers and flow channels for liquid introduction. The microreactor array is assembled by placing the reaction chamber chip on the temperature control chip. The small thermal mass of the reaction chamber resulted in a short thermal time constant of 170 ms for heating and 3 s for cooling. The performance of the microreactor array was examined through the experiments of cell-free protein synthesis. By measuring the fluorescence emission from the products, it was confirmed that GFP (Green Fluorescent Protein) and BFP (Blue Fluorescent Protein) were successfully synthesized using Escherichia coli extract.

Molecular surgery of DNA based on electrostatic micromanipulation

A novel method for the space-resolved dissection (molecular surgery) of DNA using electrostatic molecular manipulation is proposed and demonstrated. In conventional biochemistry, DNA cutting enzymes and DNA are mixed in water, so the cutting reactions occur only by stochastic chances. In contrast, the present method is based upon a physical manipulation and enables the deterministic cutting of DNA at arbitrary position on a DNA molecule. In order to realize this space-resolved cutting, the target DNA is stretched straight by electrostatic orientation, and anchored onto a solid surface by dielectrophoresis, using a high intensity (/spl ges/1 MV/m) high frequency (/spl ap/1 MHz) field generated in microfabricated electrodes. The molecular surgery presented in this paper is expected to realize space-resolved molecular operations, not only limited to dissections, but also chemical modifications, or even insertion of genes may become possible in the future.

Electroactive Microwell Arrays for Highly Efficient Single‐Cell Trapping and Analysis

We present a novel method, implemented in the form of a microfluidic device, for arraying and analyzing large populations of single cells. The device contains a large array of electroactive microwells where manipulation and analysis of large population of cells are carried out. On the device, single cells can be actively trapped in the microwells by dielectrophoresis (DEP) and then lysed by electroporation (EP) for subsequent analysis of the confined cell lysates. The DEP force in the selected dimensions of the microwells could achieve efficient trapping in nearly all the microwells (95%) in less than three minutes. Moreover, the positions of the cells in the microwells are maintained even when unstable flow of liquid is applied. This makes it possible to exchange the DEP buffer to a solution that will be subsequently used for stimulating or analyzing the trapped cells. After closing the microwells, EP is conducted to lyse the trapped cells by applying short electric pulses. Tight enclosure is critical to prevent dilution, diffusion and cross contamination of the cell lysates. We demonstrated the feasibility of our approach with an enzymatic assay measuring the intracellular-galactosidase activity. The use of this method should greatly help analysis of large populations of cells at the single-cell level. Furthermore, the method offers rapidity in the trapping and analysis of multiple cell types in physiological conditions that will be important to ensure the relevance of single cell analyses.

Development of microfluidic device for electrical/physical characterization of single cell

A novel device with microchannels for flowing cells and twin microcantilever arrays for measuring the electrical impedance of a single cell is proposed. The fabrication process is demonstrated and the twin microcantilever arrays have been successfully fabricated. In our research, we measured the electrical impedance for normal and abnormal red blood cell over the frequency range from 1 Hz to 10 MHz. From the electrical impedance experiment of normal and abnormal red blood cell, it was examined that the electrical impedance between normal and abnormal red blood cells was significantly different in magnitude and phase shift. In this paper, we show that the normal cell can be taken apart from the abnormal cell by electrical impedance measurement. Therefore, it is expected that the applicability of this technology can be used in cellular studies such as cell sorting, counting or membrane biophysical characterization.

A microfluidic in situ analyzer for ATP quantification in ocean environments

We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.

Active immobilization of biomolecules on a hybrid three-dimensional nanoelectrode by dielectrophoresis for single-biomolecule study

We propose and experimentally demonstrate a method of active immobilization for biomolecules on a three-dimensional nanometre-scale electrode (3D nanoelectrode) using dielectrophoresis to immobilize the biomolecules at predetermined locations for single-biomolecule study. We have developed a novel two-step fabrication process for obtaining a 3D nanoelectrode having a sharp top, which is necessary for immobilizing a single biomolecule at a single point. The first step is to fabricate the backbone structure, which is rigid and defines the shape of the 3D nanoelectrode. It was fabricated with diamond-like carbon (DLC) obtained using focused ion beam assisted chemical vapour deposition followed by post-plasma etching, which reshapes the DLC structure. The second step coats the DLC structure with a thin layer of aluminium, which supplies electrical conductivity to the DLC structure. By applying a high frequency (of the order of megahertz) and high intensity (greater than or equal to a few megavolts per metre) electric field using the 3D nanoelectrodes, the generated dielectrophoresis attracted and then immobilized target biomolecules onto the tops of 3D nanoelectrodes, as a demonstration of active immobilization of biomolecules.

On-Chip Single Embryo Coculture With Microporous-Membrane-Supported Endometrial Cells

In vitro culture (IVC) of the mammalian embryo is an essential technique in reproductive technology and other related life science disciplines. Although embryos are usually cultured in groups, a single embryo culture has been highly desired for IVC to investigate developmental processes. In this study, we proposed and developed the first single embryo coculture device, which allows making an array of a single embryo coculture with endometrial cells by controlling the culture environment in a microfluidic device. To realize this concept, we investigated three key issues: selection of a culture medium for the embryo coculture with endometrial cells using a mouse embryo and endometrial cells, evaluation of an on-microporous-membrane coculture of endometrial cells and an embryo to control the polarization of endometrial cells on the membrane, and evaluation of the coculture of endometrial cells and the embryo in the microfluidic device. We successfully obtained an array of a single coculture of embryo with endometrial cells in a microfluidic device. This concept will open and enhance the management of an individual embryo for assisted reproductive technology, livestock breeding, and fundamental stage research by further development.

Nanometer-level high-accuracy molding using a photo-curable silicone elastomer by suppressing thermal shrinkage

Although the so-called “labs-on-a-chip” or micro total analysis systems (micro TAS) fields hold high promise for applications in many fields, conventional fabrication processes based on the semiconductor industry such as photolithography have limitations in terms of productivity. Silicone elastomers are widely used for micromodeling and offer biocompatibility and chemical stability, but they are generally thermosetting and undergo unacceptable levels of shape deformation during curing. In this study, a photocurable silicone elastomer that has recently become commercially available was examined, and its basic optical, mechanical, and other related characteristics, along with its shape transfer capabilities, particularly its nanostructure replication characteristics, were measured in comparison with those of a representative existing thermosetting silicone elastomer. As a result, the photo-cured elastomer was shown to be superior to existing heat-cured silicone elastomers, having mechanical strength approximately three times greater, and was shown to have the same optical transmittance, extending from the near-IR to the near-UV regions. In addition, it was shown that the elastomer is sensitive to light in a wide range of wavelengths, from 254 to 600 nm, with no large difference in its curing characteristics, indicating that curing can be performed under a variety of common forms of illumination. Most importantly, the photocured elastomer provided extremely high replication accuracy due to its thermal shrinkage of less than 0.02%, compared to 2.91% in the heat-cured elastomer.

Evaporative pumping of liquid in nanochannel for electrical measurement of a single biomolecule in nanofluidic format

We present a solution to obtain planar micro-electrodes self-aligned around a nanochannel drilled using focused ion beam (FIB) machining . The inter-electrode spacing is exactly that of the width of the nanochannel (100 to 600 nm ). The system is sealed with a PDMS (polydimethylsiloxane) thin foil that includes microfluidic channels. We are aiming at manipulation and characterization of single biomolecules by taking advantages of electrical measurements at nanoscale. As a result of pumping in the nanochannel, it has been observed that high-speed motion of DNA molecules due to evaporation-coupled capillary action. Electrical detection and measurements are currently being conducted.