Hidenori Nagai

High-throughput PCR in silicon based microchamber array.

Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

Carbon nanotube–liposome supramolecular nanotrains for intelligent molecular-transport systems

Biological network systems, such as inter- and intra-cellular signalling systems, are handled in a sophisticated manner by the transport of molecular information. Over the past few decades, there has been a growing interest in the development of synthetic molecular-transport systems. However, several key technologies have not been sufficiently realized to achieve optimum performance of transportation methods. Here we show that a new type of supramolecular system comprising of carbon nanotubes and liposomes enables the directional transport and controlled release of carrier molecules, and allows an enzymatic reaction at a desired area. The study highlights important progress that has been made towards the development of biomimetic molecular-transport systems and various lab-on-a-chip applications, such as medical diagnosis, sensors, bionic computers and artificial biological networks.

Development of a novel running buffer for the simultaneous determination of nitrate and nitrite in human serum by capillary zone electrophoresis.

In order to improve NO2- peak height and obtain a convenient buffer system for the assay of nitrogen monooxide metabolites, we developed a novel running buffer for the simultaneous determination of nitrite and nitrate in human serum by capillary electrophoresis. The addition of cetyltrimethylammonium chloride to the running buffer resulted in high-speed separation using reverse electroosmotic flow. Highly sensitive determination was also achieved using stacking with 10-fold diluted sample solutions. The samples were injected hydrodynamically for 100 s into a 50 cm x 75 microm I.D. capillary. The separation voltage was 10 kV (negative polarity). UV detection was performed at 214 nm. We obtained complete separation of nitrite and nitrate in deproteinized human serum within 6 min with optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and peak area were obtained with standard addition method. The limits of detection obtained at a signal-to-noise ratio of 3 for nitrite and nitrate were 4.1 and 2.0 microM, while the values of relative standard deviation of peak height were 2.4 and 2.6%, respectively.

Ultra-rapid flow-through polymerase chain reaction microfluidics using vapor pressure.

A novel flow-through polymerase chain reaction (PCR) microfluidic system using vapor pressure was developed that can achieve ultra-rapid, small-volume DNA amplification on a chip. The 40-cycle amplification can be completed in as little as 120 s, making this device the fastest PCR system in the world. The chip device is made of a pressure-sensitive polyolefin (PSP) film and cyclo-olefin polymer (COP) substrate which was processed by cutting-work to fabricate the microchannel. The enclosed structure of the microchannel was fabricated solely by weighing the PSP film on the COP substrate, resulting in superior practical application. The vapor pressure in the denaturation zone of the destabilizing flow source was applied to the flow force, and ultra-rapid, efficient amplification was accomplished with a minimal amount of PCR reagents for detection. The flowing rhythm created by vapor pressure minimized the residual PCR products, leading to highly efficient amplification. For field test analysis, airborne dust was collected from a public place and tested for the presence of anthrax. The PCR chip had sufficient sensitivity for anthrax identification. The fastest time from aerosol sampling to detection was theoretically estimated as 8 min.

High-throughput characterization for organic pollutants in environmental waters using a capillary electrophoresis chip.

To evaluate organic pollution in water, we did preliminarily studies on high‐throughput characterization of organic pollution in water using microchip‐based capillary electrophoresis (CE) with laser‐induced fluorescence (LIF) detection. The applied voltage was investigated to control the gated valve injection and CE separation for conventional cross type microchips using a self‐made personal computer (PC)‐based controller as the voltage supply. We obtained high‐throughput data for the reproducible separation of fluorescein isothiocyanate (FITC)‐labeled river‐water samples using a zwitter‐ion based buffer solution to avoid adsorption of the labeled sample onto the channel of a microchip made from quartz glass. We used real samples from the Hino River that flows into Lake Biwa, from ten sampling points and obtained several reproducible peaks in different separation patterns for each sample within 2 min. We successfully demonstrated high‐throughput characterization of dissolved organic carbon (DOC) in environmental water using the microchip.

Living cells captured on a bio-microsystem devoted to DNA injection

Abstract This paper deals with the realization of a microsystem for living cell manipulation. The aim of this research is to make a high-efficiency DNA injection microsystem, thus providing a powerful tool for genetic therapy. Our microsystem is multifunctional: it is expected to catch ill cells, range them as an array, insert the therapeutic DNA as well as a control gene, detect nontransfected cells for their lysis, and liberate transfected cells for their culture and reinsertion into the body of the patient. For the cell trapping specificity, we use antibodies. For the spatial selectivity of the cell trapping (the cell has to be localized precisely on specified areas of the microsystem), two methods are presented in the paper to pattern the surfaces covered with antibodies. To make such a microsystem, we have to integrate two types of technologies: the technology of micromachining to realize the mechanical part of the microsystem (for instance, microcapillaries to lead the gene up to the cell), and the technology that is more related to biochemistry and biology for the cells linking part.

Rotatable reagent cartridge for high-performance microvalve system on a centrifugal microfluidic device.

Recently, microfluidic lab-on-a-CD (LabCD) has attracted attentions of researchers for its potential for pumpless, compact, and chip-inclusive on-site bioassay. To control the fluids in the LabCD, microvalves such as capillary, hydrophobic, siphon, and sacrificial valves have been employed. However, no microvalve can regulate more than one channel. In a complicated bioassay with many sequential mixing, washing, and wasting steps, thus, an intricate fluidic network with many microchannels, microvalves, and reservoirs is required, which increases assay costs in terms of both system development and chip preparation. To address this issue, we developed a rotatable reagent cartridge (RRC), which was a column-shaped tank and has several rooms to store different reagents. By embedding and rotating the RRC in the LabCD with a simple mechanical force, only the reagent in the room connected to the following channel was injected. By regulating the angle of the RRC to the LabCD, conservation and ejection of each reagent could be switched. Our developed RRC had no air vent hole, which was achieved by the gas-permeable gap between the bottle and cap parts of the RRC. The RRC could inject 230 nL-10 μL of reagents with good recoveries more than 96%. Finally, an enzymatic assay of L-lactate was demonstrated, where the number of valves and reservoirs were well minimized, significantly simplifying the fluidic system and increasing the channel integratability. Well quantitative analyses of 0-100 μM L-lactate could easily be carried out with R(2) > 0.999, indicating the practical utility of the RRC for microfluidic bioanalysis.

Application of micromachine techniques to biotechnological research

Abstract Novel methods to construct a multianalyte biosensing chip are described. A method is two-step immobilization in which materials are not immobilized directly but indirectly via small support materials. Biomaterials are immobilized on a certain support particle in the first step. The particles are placed or stuck on a chip in the second step. The other method is random fluidic self-assembly of the microsupports for reducing the complication in the second step on the chip. The combination of the methods enables us to immobilize various kinds of biomaterials densely on a chip. The ways to avoid possible problems due to random distribution are discussed. Several examples are also described.

Application of a partial filling technique to electrophoretic analysis on microchip with T-cross channel configuration

To achieve a high performance sample injection, we designed microchips with both T- and cross-type channel geometry (T-cross chip), which enables two sample solutions to be introduced into a separation channel simultaneously with desired volumes by a combination of gated and pinched injection techniques. The developed microchips were applied to microchip micellar electrokinetic chromatography (MCMEKC) using a partial filling (PF) technique. In the PF technique, to suppress the increase in the background noise due to the presence of a pseudostationary phase (PSP), e.g., ionic surfactant micelles in MCMEKC with mass spectrometric (MS) detection, in a background solution (BGS), the separation channel is partially filled with a PSP while the rest of the channel including the detection point is filled with the BGS without a PSP. As a result, both sodium dodecyl sulfate (SDS) micellar and sample solutions were simultaneously injected into the separation channel on the T-cross chip with a good repeatability of the injection lengths by applying programmed voltages, and a baseline separation of rhodamine derivatives was achieved within 40 s by MCMEKC, in which the micellar zone was partially injected with the PF technique. It was confirmed that the detection could be performed without any influence of SDS by the fluorescence imaging measurements. These results demonstrated that the developed T-cross chips will allow a combination of the MCMEKC separation with the MS detection by employing the PF technique for high performance analysis of biogenic compounds. In the PF?MCMEKC analysis, furthermore, the resolution of two rhodamines clearly increased from 1.30 to 1.73 with the increase of the injection length of PSP from 0.16 to 0.89 mm, whereas at the injection length of 1.47 mm the resolution decreased. Therefore, the selection of an optimal injection length on the T-cross chip is very important and the effects of the injection length on the separation processes in PF?MCMEKC were discussed on the basis of theoretical calculations.

Quantification of Ag(I) and kinetic analysis using ion-pair extraction across a liquid/liquid interface in a laminar flow by fluorescence microscopy

To analyze the kinetics of complicate ion-pair extraction, we have utilized a microfluidic approach and fluorescence detection. We have already developed a Ag(I)-specific thia-crown ether as an ion-association reagent. Furthermore, a fluorescent anion was added to detect the generated complex of Ag(I), ion-association reagent, and the counteranion in the ion-pair extraction system. A two-phase laminar flow consisting of an aqueous liquid and an organic liquid in a microchannel was formed, and the relationship between the initial conditions and reaction rate was examined. The microfluidic device could realize a spatiotemporal approach to solvent extraction, because the traveling length along the interface corresponded to the reaction time. The rate-determining step was estimated according to ion-pair formation behavior. Furthermore, due to the miniaturized reaction volume in the microchannel, rapid extraction of Ag(I) was achieved. The microchannel width was optimized to carry out the rapid extraction of A...