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

本研究开发了一种微生物原位分析装置，并利用微加工技术对其进行了评价。因此，IISA（集成原位分析仪）-基因成功开发了紧凑的格式。为了实现该装置，进行了如下开发和评估工作：(1)制作了基于PDMS的微通道芯片和基于玻璃的温控芯片，采用PCR方法对海洋环境中的微生物进行分子分析。(2)采用表面活性剂Tween 20防止蛋白吸附在PDMS表面。有效地提高了反应性能。(3)将朝向试剂（GuSCN）和基于玻璃微珠的DNA纯化功能集成到该装置中。(4)将LED光源和光电检测器（PMT）集成到PCR产物光学检测装置中。(5)在现场作业时，微型泵送系统与微芯片相连，配有泵和阀门。(6)基于微处理器的控制器电路和电源封装在耐压外壳中。IISA-Gene安装在充油外壳中，用于压力均衡。(7)开发并利用高压实验系统对IISA-Gene进行评价。它可以模拟高压（高达60mpa）和低温（低至2℃）。IISA-Gene在真实的深海环境中与RV“NATSUSHIMA”和ROV“HYPERDOLPHIN”（日本JAMSTEC）一起在石垣岛外海作业。日本。结果表明，该系统运行成功，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.