Development of micro in situ analysis devices for microbiology in oceanic environment

在本研究中，我们利用微加工技术，开发并评估了一种微生物学原位分析装置。因此，成功开发了紧凑格式的IISA（集成原位分析仪）-基因。为了实现该装置，进行了如下开发和评估：（1）制作了基于PDMS的微通道芯片和基于玻璃的温度控制芯片，以使用PCR方法对生活在海洋环境中的微生物进行分子分析。（2）采用表面活性剂吐温20（Tween 20）防止蛋白质吸附到PDMS表面。有效地提高了反应性能。（3）将基于离液剂（GuSCN）和玻璃珠的DNA纯化功能集成到装置中。（4）将LED光源和光电检测器（PMT）集成到装置中，用于PCR产物的光学检测。（5）对于原位操作，具有泵和阀的小型化泵送系统连接到微芯片。（6）基于微处理器的控制器电路和电源被封装在耐压外壳中。将IISA-Gene安装在充油外壳中以进行压力均衡。（7）建立了高压实验系统，并用于ISA-Gene的评价。它可以模拟高压（高达60 MPa）和低温（低至2℃）。IISA-Gene在石垣岛附近的真实的深海环境中与RV“NATSUSHIMA”和探测器“HYPERDOLPHIN”（日本JAMSTEC）一起运行。日本该系统已成功运行，获得了原始数据.

In this research, an in situ analysis device for microbiology is developed and evaluated with microfabrication techniques. As a result, IISA (Integrated In situ Analyzer) -Gene was successfully developed with compact format. To achieve the device, development and evaluation was performed as follows :(1)A PDMS based microchannel chip and a glass based temperature control chip was fabricated to perform molecular analysis of microbes living in oceanic environments with PCR method.(2)Surfactant reagent (Tween 20) was applied to prevent protein adsorption onto PDMS surface. It increased reaction performance effectively.(3)Chaotropic reagent (GuSCN) and glass beads based DNA purification function was integrated to the device.(4)LED light source and photo detector (PMT) was integrated to the device for optical detection of PCR products.(5)For in situ operation, miniaturized pumping system with pumps and valves is connected to the microchip.(6)Microprocessor based controller circuit and power supply are packaged in a pressure resistant housing. IISA-Gene was mounted in oil-filled housing for pressure equalization.(7)High-pressure experiment system was developed and utilized for evaluations of the IISA-Gene. It can simulate high pressure (up to 60 MPa) and low temperature (down to 2℃).The IISA-Gene was operated in real deep-sea environment with RV "NATSUSHIMA" and ROV "HYPERDOLPHIN" (JAMSTEC, Japan) at off Ishigaki Isl. Japan. As a result, the system was operated successfully and the primitive data was obtained.