Kentaro Kawai

Cyanobacterial cell lineage analysis of the spatiotemporal hetR expression profile during heterocyst pattern formation in Anabaena sp. PCC 7120

Diazotrophic heterocyst formation in the filamentous cyanobacterium, Anabaena sp. PCC 7120, is one of the simplest pattern formations known to occur in cell differentiation. Most previous studies on heterocyst patterning were based on statistical analysis using cells collected or observed at different times from a liquid culture, which would mask stochastic fluctuations affecting the process of pattern formation dynamics in a single bacterial filament. In order to analyze the spatiotemporal dynamics of heterocyst formation at the single filament level, here we developed a culture system to monitor simultaneously bacterial development, gene expression, and phycobilisome fluorescence. We also developed micro-liquid chamber arrays to analyze multiple Anabaena filaments at the same time. Cell lineage analyses demonstrated that the initial distributions of hetR::gfp and phycobilisome fluorescence signals at nitrogen step-down were not correlated with the resulting distribution of developed heterocysts. Time-lapse observations also revealed a dynamic hetR expression profile at the single-filament level, including transient upregulation accompanying cell division, which did not always lead to heterocyst development. In addition, some cells differentiated into heterocysts without cell division after nitrogen step-down, suggesting that cell division in the mother cells is not an essential requirement for heterocyst differentiation.

100 picoliter droplet handling using 256 (2 8 ) microvalve array with 18 multiplexed control lines

100 picoliter droplet handling with 256 (28) microvalves is demonstrated. The 256 microvalves are positioned on the 256 cross points of 16 vertical fluidic channels and 16 horizontal fluidic channels. 256 microvalves are individually controlled by multiplexed pneumatic circuit having only 18 (2*8+2) pneumatic control lines. The 100 picoliter order droplet of sample is handled in inert liquid to avoid evaporation. The droplets about 100 pl are successfully transferred to any directions by 256 microvalves.

Microfluidic valve array control system integrating a fluid demultiplexer circuit

This paper proposes an efficient control method for the large-scale integration of microvalves in microfluidic systems. The proposed method can control 2n individual microvalves with 2n + 2 control lines (where n is an integer). The on-chip valves are closed by applying pressure to a control line, similar to conventional pneumatic microvalves. Another control line closes gate valves between the control line to the on-chip valves and the on-chip valves themselves, to preserve the state of the on-chip valves. The remaining control lines select an activated gate valve. While the addressed gate valve is selected by the other control lines, the corresponding on-chip valve is actuated by applying input pressure to the control line to the on-chip valves. Using this method would substantially reduce the number of world-to-chip connectors and off-chip valve controllers. Experiments conducted using a fabricated 28 microvalve array device, comprising 256 individual on-chip valves controlled with 18 (2 × 8 + 2) control lines, yielded switching speeds for the selected on-chip valve under 90 ms.

Spatially Focused Reagent Injection System for Cell Analysis using 3-D Sheath Flow Scanner

We developed a spatially focused reagent injection system using 3-D sheath flow scanner. The proposed 3-D sheath flow scanner enables to introduce reagent specifically to target cell colonies cultivated in a microchamber. SiO2 mesh strainer is formed in the microwell to improve spatial resolution of the reagent flow. This enables selective delivery of the reagent to target cell colonies cultivated in the microwell. The specific reagent flow is shifted by eight buffer flows which are controlled by four electro- osmotic flow (EOF) pumps. High performance positioning is realized by PC control with a joystick. Observation of confocal fluorescent image confirmed that the reagent flow is focused to small spot of about 100mumPhi on the mesh strainer where biological cells are cultivated.

Pit Formation, Patterning and Flattening of Ge Surfaces in O2-Containing Water by Metal-Assisted Chemical Etching

Metal-assisted chemical etching is a novel method of etching a Ge surface in contact with a noble metal in water. Its basic mechanism involves the catalytic activity of metals to reduce dissolved O2 molecules in water, which accompanies the formation of a soluble oxide (GeO2) on the Ge surface around the metal. Here, we apply this electroless etching to the pit formation, nanoscale patterning and surface flattening of Ge. The fundamental etching properties for these three processes are also presented.

Pricise Titration Microfluidic System using Sub-Picoliter Droplet Injection

Precise titration using sub-picoliter droplet injection microfluidic system is proposed. This system can to measure less than 1 picoliter of reagent and to deliver to the micro chamber without diffusion which becomes major concern when the reagent volume is smaller. A prototype device can generate a droplet of 0.8 picoliter (800 femtoliter) in the metering channel of 40 ¿m × 5 ¿m × 4 ¿m, and step-by-step titration by precise reagent injection per two hundred thousand (2.0 × 10¿5) in volume of the micro chamber (157 nanoliter: 1 mm in diameter × 200 ¿m in height).

Cells and Biomolecules Handling Micro/Nanofluidic Systems

Micro Electro Mechanical Systems (MEMS) technologies and top down nano-technologies have been applied for the biological cell analysis. Micro/nano fluidic devices and systems for biological cells and biomolecules handling are described.

Improved passive cell distributing method to micro cellular diagnostic well array

We developed a novel cell injection method to the microwell array, which can distribute cells evenly to each microwell only by introducing cell suspension from the inlet. This method facilitates simultaneous cell injection into a number of diagnostic well with simple channel configuration. In order to enhance the uniformity of injected cell numbers in each microwell, we propose a new concept of the flow interchange, which can suppress deviation of the cell concentration in the cell loading channels. The uniformity of the injected fluorescent beads in each microwell was evaluated in prototype devices of 8 microwell array. It was confirmed that the cross interchange structure is effective to improve the uniformity compared to the previous microchannel. The coefficient of variation between the 8 microwells was 20.7%.