T. Fukuba

Japan Agency for Marine-Earth Science Technology, Йокосука, Kanagawa, Japan

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Publications (27)21.01 Total impact

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    ABSTRACT: We developed a new multi-water-sampling system, ANEMONE-11, for autonomous underwater vehicle and remotely operated underwater vehicle exploration. Water samples are continuously collected by the ANEMONE-11 sampler by an in situ water pump at 40 mL/min and are sent to a selection valve unit that consists of 128 valves connected to 40 mL sampling bottles (50 cm in length). Each valve in the unit is selected and opened at preprogrammed intervals. We also discuss the results of observations at a hydrothermal area in the Okinawa Trough.
    Methods in Oceanography 01/2014;
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    ABSTRACT: Underwater hydrothermal vents draw a lot of interest as they are the shelter for unique ecosystems, besides being a possible new opportunity for ore mining. Manganese (Mn) is found at a high concentration in hydrothermal vents, which make it possible to use this metal ion as a tracer to detect and evaluate new hydrothermal sources. Here we present a miniaturized and integrated microfluidic system for the detection of Mn in deep-sea environment, called the integrated in situ analyzer for Mn2+ (IISA-Mn). The detection system is based on the chemiluminescence reaction of Mn contained in the seawater sample with a luminol-based reagent, which offers a high sensitivity. This system is composed of a microdevice for mixing and reaction, a pumping unit, several valving units, and a photomultiplier (PMT) detector. The system is able to detect Mn concentration above 280 nM in seawater, and gives a quite linear response until 500 nM. It is also proven to be able to work continuously during the 8 h of an actual remotely operated vehicle (ROV) dive. This system has led to the discovery of a previously unknown hydrothermal site in the Okinawa Trough.
    IEEE Journal of Oceanic Engineering 01/2013; 38(1):178-185. · 1.16 Impact Factor
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    ABSTRACT: This paper presents a simple method to change the hydrophilic nature of the glass surface in a poly(dimethylsiloxane) (PDMS)-glass hybrid microfluidic device to hydrophobic by an extra-heating step during the fabrication process. Glass substrates bonded to a native or oxygen plasma-treated PDMS chip having microchambers (12.5 mm diameter, 110 µm height) were heated at 200°C for 3 h, and then the hydrophobicity of the glass surfaces on the substrate was evaluated by measuring the contact angle of water. By the extra-heating process, the glass surfaces became hydrophobic, and its contact angle was around 109°, which is nearly the same as native PDMS surfaces. To demonstrate the usefulness of this surface modification method, a PDMS-glass hybrid microfluidic device equipped with microcapillary vent structures for pneumatic manipulation of droplets was fabricated. The feasibility of the microcapillary vent structures on the device with the hydrophobic glass surfaces are confirmed in practical use through leakage tests of the vent structures and liquid handling for the electrophoretic separation of DNA molecules.
    Analytical Sciences 01/2012; 28(1):39-44. · 1.57 Impact Factor
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    ABSTRACT: Pressure and temperature characteristics of in-situ sensors have a big influence on measurements. A pH sensor using ISFET (Ion Sensitive Field Effect Transistor) is regarded as a suitable sensor for measurement pH at deep sea, however, its pressure and temperature characteristics were seldom studied. So, in this paper, we study the pressure and temperature characteristics of ISFET-pH sensor by the structural analysis and by the laboratory experiments, and then consider a new structure which can decrease the pressure effects on ISFET-pH sensor, and investigate these effects on the actual measurement in deep sea. From experimental and actual measurement results, we estimate the applications and efficiency of ISFET-pH sensor. By the results of structural analysis, if the structure of the sensor has sufficient strength for practical use, pressure effects occur. The experimental results shows the pressure and temperature characteristics have large individual difference. But by the results of structural improvement analysis, the pressure effects can be reduced by the change in construction material of the sensor. The actual measurement in deep sea indicates that the temperature characteristics depends on pressure, and show a problem of the present way of calibration that is to calibrate the effects of pressure and temperature, respectively.At the same time, it is also shown that the sensor has high sensitivity, good responsibility, wide application and good efficiency.
    Journal of the Japan Society for Marine Surveys and Technology. 01/2012; 15(2).
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    ABSTRACT: In this study, a totally integrated in situ analyzer for microbial gene detection has been developed for oceanography applications. A PDMS–glass microfluidic device that is capable of cell lysis, DNApurification, PCR, and optical detection has been utilized as the core element of the in situ analyzer. Microbial genomic DNA is purified and concentrated on glass beads packed in the microfluidic device. PCR is performed in a flow-through manner, and the amplified products are fluorescently detected using optical fibers. The sensitivity of a completed analyzer with deep-sea operation capabilities has been evaluated in a laboratory setting, and the analyzer has been operated in real deep-sea environments. Field evaluations have shown that the amplification of the eubacterial universal 16S rRNA gene and the recovery of the PCR product to the surface are successfully achieved in deep-sea environments.
    RSC Advances 11/2011; 1(8):1567-1573. · 3.71 Impact Factor
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    ABSTRACT: We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.
    Lab on a Chip 08/2011; 11(20):3508-15. · 5.70 Impact Factor
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    ABSTRACT: Hydrothermal anomalies found in the deep sea are a place where unique ecosystems exist, and may become a possible new resource for metallic ore mining. Elements such as iron and manganese are found in high concentration in hydrothermal vents. Therefore it is possible to use such metal ions as a tracer to detect and evaluate new hydrothermal sources. Here we present a miniaturized and integrated microfluidic system for the detection of manganese in deep-sea environment, called IISA-Mn (In Situ Integrated Analyzer for Mn 2+ ). The detection system is based on the chemiluminescence reaction of manganese contained in the seawater sample with a luminol-based reagent, which offers a high sensitivity. This system is composed of a microdevice for mixing and reaction, a pumping unit, several valving units, and a PMT (photomultiplier) detector. The system is able to detect manganese concentration above 100 nM in seawater, and gives a linear response until 1 µ M. It is also proven to be able to work continuously during the 8 hours of an actual dive. This system has lead to the discovery of a previously unknown hydrothermal site near Okinawa Islands.
    01/2011;
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    ABSTRACT: In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method.
    International Journal of Molecular Sciences 01/2011; 12(7):4271-81. · 2.46 Impact Factor
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    ABSTRACT: Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.
    Electrophoresis 11/2010; 31(22):3719-26. · 3.26 Impact Factor
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    ABSTRACT: Hydrothermal fluids contain high concentration of anoxic chemical species, i.e. methane and hydrogen sulfide, helium-3, and heavy metals derived from the rock-water interaction. During the hydothermal plume spreading, it is known that several chemical species are oxidized which include available energy source for microorganism, however, few results have been reported on the spatial variation of both of chemical and microbiological concentration and species. In the southern Mariana Trough, some site surveys have been conducted with CTD hydrocasts, the manned submersible, and ROVs since 2003. In this field, three hydrothermal vent sites were discovered within the small area, where the chemistry of each hydrothermal fluid was different from each other. These differences of chemistry are prospected to affect the individual plume evolution. In order to discuss the each hydrothermal plume evolution, we conducted high-resolution plume mapping by the AUV "URASHIMA" with some chemical sensors. Additionally, we loaded 24 bottles of water sampler for the geochemical and microbial analysis. During this cruise, we detected hydrothermal plume anomalies derived from each hydrothermal site with the highly precise topographic results. Based on the results, we will discuss the relationships between the spreading of hydrothermal plume (geochemical evolution) and the ecology of plume microbes.
    AGU Fall Meeting Abstracts. 12/2009;
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    ABSTRACT: An integrated in situ analyzer for microbial ATP (IISA-ATP) has been developed with a microfluidic device as its core component to realize a compact and fully integrated system. In the system, a bioluminescence (luciferin---luciferase) reaction is conducted for ATP quantification. The microfluidic device has a coil-shaped microchannel for highly sensitive photo intensity measurement. In this paper, the concept of the IISA-ATP and optimization of the microchannel design to enhance sensitivity are presented. As a result of the optimization, linear correlation of the luminescence intensity with the ATP concentration in the range of 2 to 2 × 104 pM was achieved.
    IEEJ Transactions on Sensors and Micromachines 01/2009; 129(3):73-76.
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    ABSTRACT: This paper presents design, fabrication and evaluation of a polydimethylsiloxane (PDMS) ---glass microfluidic device for quantitative determination of manganese ion (Mn(II)) in oceanic environments. The microfluidic device can perform luminol-hydrogen peroxide chemiluminescence (CL) reaction in a flow-through manner for highly sensitive Mn (II) quantification. A chelate resin column was integrated in the microfluidic device for removal of coexisting metal ion such as Fe(II). The developed device could quantify Mn(II) in nM level under coexistence of muM level of Fe(II). The microfluidic device developed here will be used as a core element of a totally integrated in situ chemical analyzer that we have been developing for oceanography applications.
    IEEJ Transactions on Sensors and Micromachines 01/2009; 129(3):69-72.
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    ABSTRACT: In this study, "Integrated in situ analyzer-ATP (IISA-ATP)" which is to estimate microbial activities in deep-sea environments has been developed. On the system, a microfluidic device is used as a core element to perform a bioluminescent (luciferin-luciferase) reaction for ATP quantification. The microfluidic device has a coil-shaped microchannel for photo intensity measurement. In this paper, concept of the IISA-ATP and optimization process of the microchannel design are presented. As a result of the microchannel design and flow rate optimization, 2 pM to 2 x 10<sup>4</sup> pM (20 nM) of ATP was successfully quantified.
    OCEANS 2008 - MTS/IEEE Kobe Techno-Ocean; 05/2008
  • C. Provin, T. Fukuba, T. Fujii
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    ABSTRACT: Many metal ions that can be found at trace or ultratrace level in ocean are coming from deep-sea hydrothermal vents. In particular, manganese (Mn<sup>2+</sup>) can be assimilated by phytoplankton and so can be found in all the food chain. It is then of interest to be able to detect manganese to determine its spatial distribution and/or to find new hydrothermal sources. Usually, determination of manganese concentration is done by sampling seawater in bottles, followed by analysis in lab onboard a ship or on the ground. Major drawbacks of that method are that only a few samples can be taken during each dive and that there is a risk of contamination of the sample. The use of microfabrication techniques to produce an in-situ microanalyzer that can be installed in an autonomous underwater vehicles (AUVs) or remotely operated vehicles (ROVs) can allow a higher sampling rate, with experiments over a longer time, a reduced risk of contamination. We propose here a microdevice designed for in situ detection of manganese ion in deep-sea environment. Detection is based on chemiluminescence of luminol triggered by the H<sub>2</sub>O<sub>2</sub> decomposition catalyzed by manganese ion. The set-up is composed of pressure-proof micropumps for handling of the reagents, a 6-way valve for samples introduction, a PDMS (poly(dimethylsiloxane)) microchip made by soft lithography, and a photomultiplier for photon detection. The limit of detection of this system is about 10 nanomolar at the moment.
    OCEANS 2008 - MTS/IEEE Kobe Techno-Ocean; 05/2008
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    ABSTRACT: This paper describes the study of the DNA markers of uncultured microorganisms in order to develop the technology for detecting methane leakage in the deep sea. Methane oxidizing bacteria called methanotrophs consume methane as their sole energy and carbon source and are distributed at methane producing sites. Aerobic methanotrophic bacteria use soluble methane monooxygenases (sMMO) or particulate methane monooxygenases (pMMO) to convert methane to methanol. The anaerobic oxidation of methane mediated by anaerobic methanotrophs (ANME) and sulphate-reducing bacteria plays a critical role in anoxic sediments of cold seeps. Methanotrophs are ubiquitous in soil, fresh water and the open ocean but have not been well characterized in deep-sea hydrocarbon seeps and gas hydrates, where methane is unusually abundant. In the last few decades, cultivation-independent molecular methods have been applied widely to investigate microbial diversity and quantify predominant organisms in natural microbial community. Methanotroph diversity has been studied in different environments using the polymerase chain reaction (PCR). Our goal in this study was to compare the composition of bacterial and archaeal communities associated with gas hydrates with those of bacterial and archaeal communities in normal marine sediments in the Nankai Trough of Japan in order to identify specific DNA markers (16S RNA and pmoA genes) for methane leakage in the deep sea. The development of in-situ detection of DNA markers associated with methane leakage is now ongoing with the application of a totally integrated and automated system for in-situ gene analysis of microbes (IISA-gene system).
    OCEANS 2008 - MTS/IEEE Kobe Techno-Ocean; 05/2008
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    ABSTRACT: An integrated and miniaturized device for in situ quantitative analysis of microbial ATP was developed in this study. The device can perform extracellular ATP elimination, cell lysis and ATP quantification in a microfluidic chip. A pattern of a microchannel has been optimized to measure luminescence caused by luciferin-luciferase reaction. As a result of evaluation of the developed microfluidic device, 10 nM to 100 muM of ATP was successfully quantified. Environmental samples and bacterial culture media were also analyzed by the microfluidic device. Towards in situ calibration of the ATP quantitative analysis device, a prototype of caged ATP based online calibration component was also fabricated and evaluated.
    Underwater Technology and Workshop on Scientific Use of Submarine Cables and Related Technologies, 2007. Symposium on; 05/2007
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    T. Fujii, T. Fukuba
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    ABSTRACT: Microfluidics is one of the emerging technologies to achieve advanced biological and chemical sensing. A project to develop a series of microfluidics-based in situ measurement systems, named IISA (Integrated In Situ Analyzer)' is underway in author's group at IIS, University of Tokyo. In this talk, a basic concept and technologies of microfluidics will be presented along with some examples of USA devices and systems. The possibilities and future perspectives of this new technology towards integrated and real-time measurement will be discussed.
    Underwater Technology and Workshop on Scientific Use of Submarine Cables and Related Technologies, 2007. Symposium on; 05/2007
  • C. Provin, T. Fukuba, T. Fujii
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    ABSTRACT: Manganese is a metal ion that can be found at ultra trace level in the ocean. Mainly, manganese comes into the ocean through hydrothermal vents. Usually, determination of manganese concentration is done by sampling seawater in bottles and measuring afterward onboard a ship or in a lab on the ground. Major drawbacks of that method are that only a few samples can be taken at a time and that there is always a risk of contaminations. The use of microfabrication techniques to produce an in situ microanalyzer that can be installed in Autonomous Underwater Vehicles (AUVs) or Remotely Operated Vehicles (ROVs) can overcome these issues. Miniaturization is an asset for mounting an analyzer in AUVs or ROVs due to low total volume (less reagents) and electrical consumption, and for increasing spatial and temporal resolution (higher rate of sampling) to make 3D manganese cartography possible. Here we show the development of an integrated in-situ analyzer for manganese based on chemiluminescence detection. Manganese catalyzes the decomposition of H<sub>2</sub>O<sub>2</sub> into radicals OHldr that react with luminol to produce light, which intensity is proportional to Mn<sup>2+</sup> concentration. Reagents are brought by syringe pumps or pressure-proof mieropumps to a reaction chamber made of polydimethylsiloxane (PDMS) material.
    Underwater Technology and Workshop on Scientific Use of Submarine Cables and Related Technologies, 2007. Symposium on; 05/2007
  • N. Fukuzawa, T. Fukuba, T. Fujii
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    ABSTRACT: An integrated in situ analyzer for ATP concentration (IISA-ATP) has been developed in this study. Concept of IISA-ATP that has function for cell lysis, extracellular ATP elimination, ATP quantification with luciferin-luciferase reaction and in situ calibration is proposed in this study. A simplified prototype of PDMS based microfluidic chip that has a coiled microchannel pattern for optical detection is developed with micro fabrication technique. As a result of evaluation of the prototype, more than 10-10 M range of ATP was successfully detected
    01/2006;
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    ABSTRACT: Various miniaturized and integrated in situ devices for biological and chemical oceanography have been developed in our research group. The devices targeted to analyze microbial gene, heavy metal ion (manganese: M<sup>2+</sup>) concentration and pH of seawater are evaluated and presented here. Flow-through PCR polymerase chain reaction) can be performed with the microbial gene analysis device with integrated heating and pumping system. Mn<sup>2+</sup> ion concentration is quantified by applying chemiluminescence reaction by the heavy metal ion analysis device. Micropumps are integrated to develop well-integrated pH measurement device. All devices are based on PDMS (polydimethylsiloxane) microchannel chip fabricated with conventional molding method.
    Microtechnology in Medicine and Biology, 2005. 3rd IEEE/EMBS Special Topic Conference on; 06/2005