Skip to main content
SpringerLink
Log in

Microfabricated Flow-Through Device for In Situ Gene Analysis

  • Protocol
  • First Online:
Molecular Biological Technologies for Ocean Sensing

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 696 Accesses

Abstract

A miniaturized and self-contained in situ genetic analyzer Integrated In Situ Analyzer-Gene (IISA-Gene) was developed to conduct PCR-based targeted sequence detection using microfluidic technology. The core element of the in situ analyzer is a microfluidic device that conducts cell lysis, DNA purification, PCR, and optical detection of amplified DNA fragments. In this device, a standard three-step SYBR green PCR amplification with a pair of oligonucleotide primers is conducted using environmental microbial DNA templates eluted from glass beads packed in a microchannel. The microfluidic device is integrated with pumping components and control electronics for real-field deployments. Here, the performance of the microfluidic device and results of in situ operation of the IISA-Gene in deep-sea hydrothermal areas are presented.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cavanaugh CM, Gardiner SL, Jones ML, Jannasch HW, Waterbury JB (1981) Prokaryotic cells in hydrothermal vent tube worm Riftia pachyptila Jones: possible chemoautotrophic symbionts. Science 213(4505):340–342

    Article  PubMed  CAS  Google Scholar 

  2. Karl DM, Wirsen CO, Jannasch HW (1980) Deep-sea primary production at the Galapagos hydrothermal vents. Science 207(4437):1345–1347

    CAS  Google Scholar 

  3. Gold T (1992) The deep, hot biosphere. Proc Natl Acad Sci U S A 89(13):6045–6049

    Article  PubMed  CAS  Google Scholar 

  4. Stetter K (1999) Extremophiles and their adaptation to hot environments. FEBS Lett 452(1–2):22–25

    Article  PubMed  CAS  Google Scholar 

  5. McDonald I, Murrell J (1997) The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs. FEMS Microbiol Lett 156(2):205–210

    Article  PubMed  CAS  Google Scholar 

  6. Gulledge J, Ahmad A, Steudler P, Pomerantz W, Cavanaugh C (2001) Family-and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria. Appl Environ Microbiol 67(10): 4726–4733

    Article  PubMed  CAS  Google Scholar 

  7. Amann R, Binder B, Olson R, Chisholm S, Devereux R, Stahl D (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56(6):1919–1925

    PubMed  CAS  Google Scholar 

  8. Karner M, DeLong E, Karl D (2001) Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409(6819):507–510

    Article  PubMed  CAS  Google Scholar 

  9. Sloan E (2003) Fundamental principles and applications of natural gas hydrates. Nature 426(6964):353–363

    Article  PubMed  CAS  Google Scholar 

  10. Halbach P, Nakamura K, Wahsner M, Lange J, Sakai H, Käselitz L, Hansen RD, Yamano M, Post J, Prause B, Seifert R, Michaelis W, Teichmann F, Kinoshita M, Märten A, Ishibashi J, Czerwinski S, Blum N (1989) Probable modern analogue of Kuroko-type massive sulphide deposits in the Okinawa Trough back-arc basin. Nature 338(6215):496–499

    Article  CAS  Google Scholar 

  11. Zierenberg RA, Fouquet Y, Miller DJ, Bahr JM, Baker PA, Bjerkgar T, Brunner CA, Duckworth RC, Gable R, Gieskes J, Goodfellow WD, Groschel-Becker HM, Guerin G, Ishibashi J, Iturrino G, James RH, Lackschewitz KS, Marquez LL, Nehlig P, Peter JM, Rigsby CA, Schultheiss P, Shanks WC, Simoneit BRT, Summit M, Teagle DAH, Urbat M, Zuffa GG (1998) The deep structure of a sea-floor hydrothermal deposit. Nature 392(6675): 485–488

    Article  CAS  Google Scholar 

  12. Zhang C, Pancost R, Sassen R, Qian Y, Macko S (2003) Archaeal lipid biomarkers and isotopic evidence of anaerobic methane oxidation associated with gas hydrates in the Gulf of Mexico. Org Geochem 34(6):827–836

    Article  CAS  Google Scholar 

  13. Awashima Y, Saito H, Hoaki T, Sawada S, Fukasawa T, Sukizaki S, Ishihara Y, Kano K, Arata N (2008) Development of monitoring system on methane hydrate production. OCEANS 2008 - MTS/IEEE Kobe Techno-Ocean, Kobe, Japan, pp 1–7

    Google Scholar 

  14. Niehaus F, Bertoldo C, Kähler M, Antranikian G (1999) Extremophiles as a source of novel enzymes for industrial application. Appl Microbiol Biotechnol 51(6):711–729

    Article  PubMed  CAS  Google Scholar 

  15. Jones W, Preston C, Martin R III, Scholin CA, Vrijenhoek R (2008) A robotic molecular method for in situ detection of marine invertebrate larvae. Mol Ecol Resour 8(3):540–550

    Article  PubMed  CAS  Google Scholar 

  16. Greenfield D, Marin R III, Jensen S, Massion E, Roman B, Feldman J, Scholin C (2006) Application of Environmental Sample Processor (ESP) methodology for quantifying Pseudo-nitzschia australis using ribosomal RNA-targeted probes in sandwich and fluorescent in situ hybridization formats. Limnol Oceanogr Methods 4:426–435

    Article  Google Scholar 

  17. Fries D, Paul J, Smith M, Farmer A, Casper E, Wilson J (2007) The Autonomous microbial genosensor, an in situ sensor for marine microbe detection. Microsc Microanal 13(S02):514–515

    Article  Google Scholar 

  18. Scholin C, Doucette G, Jensen S, Roman B, Pargett D, Marin R III, Preston C, Jones W, Feldman J, Everlove C, Harris A, Alvarado N, Massion E, Birch J, Greenfield D, Vrijenhoek R, Mikulski C, Jones K (2009) Remote detection of marine microbes, small invertebrates, harmful algae, and biotoxins using the environmental sample processor (ESP). Oceanography 22(2):158–161

    Article  Google Scholar 

  19. Chapin T, Jannasch H, Johnson K (2002) In situ osmotic analyzer for the year-long continuous determination of Fe in hydrothermal systems. Anal Chim Acta 463(2):265–274

    Article  CAS  Google Scholar 

  20. Jannasch H, Johnson K, Sakamoto C (1994) Submersible. Osmotically pumped analyzer for continuous determination of nitrate in situ. Anal Chem 66(20):3352–3361

    Article  CAS  Google Scholar 

  21. Thouron D, Vuillemin R, Philippon X, Lourenço A, Provost C, Cruzado A, Garçon V (2003) An autonomous nutrient analyzer for oceanic long-term in situ biogeochemical monitoring. Anal Chem 75(11):2601–2609

    Article  PubMed  CAS  Google Scholar 

  22. Fukuba T, Provin C, Okamura K, Fujii T (2009) Development and evaluation of microfluidic device for Mn ion quantification in ocean environments. IEEJ Trans SM 129(3): 69–72

    Article  Google Scholar 

  23. Aoki Y, Fukuba T, Yamamoto T, Fujii T (2009) Design optimization and evaluation of a bioluminescence detection part on a microfluidic device for in situ ATP quantification. IEEJ Trans SM 129(3):73–76

    Article  Google Scholar 

  24. Fukuba T, Yamamoto T, Naganuma T, Fujii T (2004) Microfabricated flow-through device for DNA amplification -towards in situ gene analysis. Chem Eng J 101(1–3):151–156

    Article  CAS  Google Scholar 

  25. Kemp B, Smith D (2005) Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int 154(1):53–61

    Article  PubMed  CAS  Google Scholar 

  26. Fukuba T, Miyaji A, Okamoto T, Yamamoto T, Kaneda S, Fujii T (2011) Integrated in situ genetinc analyzer for microbiology in extreme environments. RSC Adv 1:1567–1573

    Article  CAS  Google Scholar 

  27. Fujii T (2002) PDMS-based microfluidic devices for biomedical applications. Microelec Eng 61–62:907–914

    Article  Google Scholar 

  28. Nakano H, Matsuda K, Yohda M, Nagamune T, Endo I, Yamane T (1994) High speed polymerase chain reaction in constant flow. Biosci Biotech Biochem 58(2):349–352

    Article  CAS  Google Scholar 

  29. Kopp M, Mello A, Manz A (1998) Chemical amplification: continuous-flow PCR on a chip. Science 280(5366):1046–1048

    Article  PubMed  CAS  Google Scholar 

  30. Cheng Y, Halsey J, Fode K, Remsen C, Collins M (1999) Detection of methanotrophs in groundwater by PCR. Appl Environ Microbiol 65(2):648

    PubMed  CAS  Google Scholar 

  31. Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, Ludwig W, Backhaus H (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J Bacteriol 178(19):5636–5643

    PubMed  CAS  Google Scholar 

  32. Boom R, Sol C, Salimans M, Jansen C, Wertheim-van Dillen P (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28(3):495–503

    PubMed  CAS  Google Scholar 

  33. Kato C, Li L, Tamaoka J, Horikoshi K (1997) Molecular analyses of the sediment of the 11000-m deep Mariana Trench. Extremophiles 1(3):117–123

    Article  PubMed  CAS  Google Scholar 

  34. Tsuchida S, Watanabe K, Ishibashi J, Miyake H, Watabe H, Yamaguchi T, Kitajima T, Nakano A, Matsumura M, Watanabe H (2000) Preliminary report of a biological, geological, and geochemical survey on hydrothermalism at the Hatoma and Minna Knolls. JAMSTEC J Deep Sea Res 17:34–42

    Google Scholar 

  35. Takai K, Inagaki F, Nakagawa S, Hirayama H, Nunoura T, Sako Y, Nealson KH, Horikoshi K (2003) Isolation and phylogenetic diversity of members of previously uncultivated ε-Proteobacteria in deep sea hydrothermal fields. FEMS Microbiol Lett 218(1):167–174

    PubMed  CAS  Google Scholar 

  36. Holmes AJ, Costello A, Lidstrom ME, Murrell JC (1995) Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett 132(3):203–208

    Article  PubMed  CAS  Google Scholar 

  37. Kinoshita H, Atsumi T, Fukuba T, Fujii T (2010) Active micro flow-rate reguration technique based on soft membrane deformation using miniaturized electroosmotic pumps. 14th international conference on miniaturized systems for chemistry and life sciences, Groningen, The Netherlands. pp 390–392

    Google Scholar 

  38. Fukuba T, Hiraga M, Takamatsu A, Provin C, Yamamoto T, Fujii T (2008) Simple method for quantitative PCR using flow-through PCR device. 12th international conference on ­miniaturized systems for chemistry and life ­sciences, San Diego, USA, pp 1473–1475

    Google Scholar 

Download references

Acknowledgements

The authors are grateful to the crew of the R/V NATSUSHIMA and the operating team of the ROV HYPER-DOLPHIN (JAMSTEC) for their helpful assistance during scientific cruise NT08-11. This work was supported by a grant-in-aid for scientific research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT). The Engineering Advancement Association of Japan (ENAA) also supported this study.

Author information

Authors and Affiliations

  1. Department of Mechanical and Biofunctional Systems, Institute of Industrial Science, University of Tokyo, Tokyo, Japan

    Tatsuhiro Fukuba

  2. Marine Technology Development Department, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Marine technology and Engineering Center, Natsushima-cho 2-15, Yokosuka-city, Kanagawa, Japan

    Tatsuhiro Fukuba

  3. Center for International Research on MicronanoMechatronics (CIRMM), Institute of Industrial Science, University of Tokyo, Tokyo, Japan

    Teruo Fujii

Authors
  1. Tatsuhiro Fukuba
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Teruo Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tatsuhiro Fukuba .

Editor information

Editors and Affiliations

  1. 4901 Evergreen Road, Dearborn, 48128, USA

    Sonia M. Tiquia-Arashiro

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Fukuba, T., Fujii, T. (2012). Microfabricated Flow-Through Device for In Situ Gene Analysis. In: Tiquia-Arashiro, S. (eds) Molecular Biological Technologies for Ocean Sensing. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-915-0_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-915-0_3

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-914-3

  • Online ISBN: 978-1-61779-915-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation