Application of Microfluidics to Oceanography

■Microfluidics technology is one of the key technologies for automating various analytical operations that previously had to be performed by human researchers. By applying semiconductor processing technology, micro-machine fabrication technology, and more recently, 3D printing technology, it has become possible to realize extremely compact analyzers. This technology is being applied mainly in the fields of medical diagnosis and biotechnology, and there are many examples that have already led to commercialization. Here, we are attempting to apply this technology to underwater in situ measurements. In particular, there have been attempts to realize ultra-compact and highly functional in situ flow analyzers by applying both matured and cutting-edge microfluidic technologies.

■So far, we have developed an "in-situ microbial gene analyzer," "in-situ manganese ion quantitative analyzer," and "in-situ ATP quantitative analyzer" using the microfluidic technology, and have conducted sea trials in actual underwater environments.

■The in situ microbial gene analyzer is an ambitious device that aims to perform gene extraction and purification from microorganisms on-site in the sea, including deep-sea, and detection of targeted microorganisms by PCR. Although this is our first effort and it was the most complicated device developed so far, we have succeeded in evaluating the prototype device at deep-sea environment using ROV. It is now being applied to the automated detection of environmental DNA (eDNA) in combination with multiscale fluidics technology for sample preparation before PCR.

■For the determination of manganese ions, the challenge was to construct a simple and robust Luminol chemiluminescence reaction system suitable for use in the underwater environment and to integrate advanced microfluidic technologies such as microvalves, flow- regulators, and micro-mixer structures on single PDMS microfluidic device that can be operated in a deep-sea environment automatically. In 2010, we succeeded in discovering a new hydrothermal site in the Okinawa Trough by conducting survey operation using an ROV with the Mn analyzer and other miniaturized sensor suites.

■Currently we are working on the development of the in situ ATP quantitative analyzer based on our motivation to improve the problems identified during the development of the genetic analyzers and the manganese analyzers and to systematize the technology by applying a well-established fluidic technology. ATP (adenosine triphosphate) is an indicator of microbial biomass, and highly sensitive quantitative ATP assays based on the Luciferin-Luciferase bioluminescence method are in practical use in the field of hygiene testing. By bringing the ATP analyses underwater, we aim to visualize "where and how many microorganisms are present," which no one has ever seen before.In-situ ATP quantification may be useful for underwater resource exploration, since microbial biomass is known to be anomalous around seafloor hydrothermal areas. ATP quantification by the Luciferin-Luciferase method is an assay that uses an enzymatic bioluminescence reaction, so fundamental technologies such as highly sensitive optical measurement and temperature control are essential. In addition, the miniaturization and integration of the fluidics is also important for continuous measurement while using expensive reagents.Therefore, we are developing the quantitative ATP analyzer with accumulating the know-how and establishing the fundamental technology for applying the microfluidic technology in the sea.

＜Related Publications＞

T. Fukuba, T. Fujii “Lab-on-a-chip technology for in situ combined observations in oceanography”, Lab on a Chip, 21(1), pp. 55-74. (Critical Review), DOI: 10.1039/D0LC00871K, 2020

T. Fukuba T. Noguchi, K. Okamura, and T. Fujii, “Adenosine Triphosphate Measurement in Deep Sea Using a Microfluidic Device” Micromachines, vol. 9, 370, 2018, DOI 10.3390/mi9080370

T. Fukuba, T. Noguchi and T. Fujii “The Yoron Hole: The Shallowest Hydrothermal System in the Okinawa Trough” J. Ishibashi, K. Okino, and M. Sunamura eds., Subseafloor Biosphere Linked to Hydrothermal Systems TAIGA Concept, Springer, 2015 DOI: 10.1007/978-4-431-54865-2_38, pp. 489-492, 2015

C. Provin, T. Fukuba, K. Okamura, and T. Fujii, “An Integrated Microfluidic System for Manganese Anomaly Detection based on Chemiluminescence: Description and Practical Use to Discover Hydrothermal Plumes Near the Okinawa Trough” IEEE Journal of Ocean Engineering, 38(1), pp. 178-185, DOI:10.1109/JOE.2012.2208849, 2012

T. Fukuba, A. Miyaji, T. Okamoto, T. Yamamoto, S. Kaneda and T. Fujii, “Integrated in situ Genetic Analyzer for Microbiology in Extreme Environments” RSC Advances, 1, pp. 1567-1573, DOI:10.1039/C1RA00490E, 2011

Y. Aoki, T. Fukuba, T. Yamamoto, and T. Fujii, “Design Optimization and Evaluation of a Bioluminescence Detection Part on a Microfluidic Device for in situ ATP Quantification” IEEJ Transactions on Sensors and Micromachines, 129 (3), pp. 73-76, DOI: 10.1541/ieejsmas.129.73, 2009

T. Fukuba, C. Provin, K. Okamura, and T. Fujii, “Development and Evaluation of Microfluidic Device for Mn Ion Quantification in Ocean Environments” IEEJ Transactions on Sensors and Micromachines, 129(3), pp. 69-72, DOI:10.1541/ieejsmas.129.69, 2009

福場 辰洋， 山本 貴富喜， 長沼 毅， 藤井 輝夫，「極限環境微生物学のための現場型微生物遺伝子解析装置の開発」海の研究, 14(2), pp. 361-368, DOI:10.5928/kaiyou.14.361, 2005

T. Fukuba, T. Yamamoto, T. Naganuma, and T. Fujii, “Microfabricated Flow-Through Device for DNA Amplification - Towards in situ Gene Analysis” Chemical Engineering Journal, 101(1-3), pp. 151-156, DOI:10.1016/j.cej.2003.11.016, 2004