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Electrochemical Detection of Harmful Algae by Means of a Sandwich Hybridization Assay on an Electrode Surface

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Molecular Biological Technologies for Ocean Sensing

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

Abstract

We report on the development of a sandwich hybridization assay on an electrode surface-based sensor device for the detection of toxic algae. This DNA (rRNA)-based sensor uses an electrochemical detection of a target sequence by means of its hybridization to a capture probe, with an attachment label at its 5′ end that binds it to the electrode surface, and a signal probe with a DIG detection label at the 3′ end. The thiol (biotin)-labelled capture probe is immobilized onto gold (carbon, graphite or carbon nanotubes) screen printed electrodes. Synthetic positive control DNA or real rRNA isolated from algal cells is applied to the sensor and allowed to hybridize to the capture probe. A signal probe with a digoxigenin label is then hybridized to this construct, followed by an anti-DIG-antibody coupled to horseradish peroxidase (HRP) and a substrate. The electrical signal obtained from the redox reaction is proportional to the amount of DNA (rRNA) applied to the biosensor, which is in turn proportional to the number of cells harvested when applied to real samples. The optimized hybridization conditions and steps of the fabrication process are presented here. The biosensor has been used to detect different algal species, such as Prymnesium parvum, Gymnodinium catenatum, Pseudo-nitzschia australis, Alexandrium ostenfeldii and Alexandrium minutum, by immobilizing their genetic material over the different transducer platforms.

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References

  1. Maso M, Garces E (2006) Harmful microalgae blooms (HAB); problematic and conditions that induce them. Mar Pollut Bull 53(10–12):620–630

    Article  PubMed  CAS  Google Scholar 

  2. Penna A, Magnani M (1999) Identification of Alexandrium (Dinophyceae) species using PCR and rDNA-targeted probes. J Phycol 35(3):615–621

    Article  CAS  Google Scholar 

  3. Scholin CA, Marin R, Miller PE et al (1999) DNA probes and a receptor-binding assay for detection of Pseudo-nitzschia (Bacillariophyceae) species and domoic acid activity in cultured and natural samples. J Phycol 35(6):1356–1367

    Article  CAS  Google Scholar 

  4. Ayers K, Rhodes LL, Tyrrell J, Gladstone M, Scholin C (2005) International accreditation of sandwich hybridization assay format DNA probes for micro-algae. N Z J Mar Freshw Res 39(6):1225–1231

    Article  CAS  Google Scholar 

  5. Diercks S, Gescher C, Metfies K, Medlin LK (2009) Evaluation of locked nucleic acids for signal enhancement of oligonucleotide probes for microalgae immobilised on solid surfaces. J Appl Phycol 21(6):657–668

    Article  CAS  Google Scholar 

  6. Diercks S, Medlin LK, Metfies K (2008) Colorimetric detection of the toxic dinoflagellate Alexandrium minutum using sandwich hybridization in a microtiter plate assay. Harmful Algae 7(2):137–145

    Article  CAS  Google Scholar 

  7. Gescher C, Metfies K, Medlin LK (2008) The ALEX CHIP - Development of a DNA chip for identification and monitoring of Alexandrium. Harmful Algae 7(4):485–494

    Article  CAS  Google Scholar 

  8. Greenfield DI, Marin R, Jensen S et al (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 

  9. Haywood AJ, Scholin CA, Marin R, Steidinger KA, Heil C, Ray J (2007) Molecular detection of the brevetoxin-producing dinoflagellate Karenia brevis and closely related species using rRNA-targeted probes and a semiautomated sandwich hybridization assay. J Phycol 43(6):1271–1286

    Article  CAS  Google Scholar 

  10. O’Halloran C, Silver MW, Holman TR, Scholin CA (2006) Heterosigma akashiwo in central California waters. Harmful Algae 5(2):124–132

    Article  Google Scholar 

  11. Tyrrell JV, Connell LB, Sholin CA (2002) Monitoring for Heterosigma akashiwo using a sandwich hybridization assay. Harmful Algae 1(2):205–214

    Article  CAS  Google Scholar 

  12. Diercks S, Metfies K, Medlin LK (2008) Development and adaptation of a multiprobe biosensor for the use in a semi-automated device for the detection of toxic algae. Biosens Bioelectron 23(10):1527–1533

    Article  PubMed  CAS  Google Scholar 

  13. Metfies K, Huljic S, Lange M, Medlin LK (2005) Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor. Biosens Bioelectron 20(7):1349–1357

    Article  PubMed  CAS  Google Scholar 

  14. Liao JC, Mastali M, Li Y et al (2007) Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection. J Mol Diagn 9(2):158–168

    Article  PubMed  CAS  Google Scholar 

  15. Drummond TG, Hill MG, Barton JK (2003) Electrochemical DNA sensors. Nat Biotechnol 21(10):1192–1199

    Article  PubMed  CAS  Google Scholar 

  16. Wang J (2006) Electrochemical biosensors: towards point-of-care cancer diagnostics. Biosens Bioelectron 21(10):1887–1892

    Article  PubMed  CAS  Google Scholar 

  17. Rautio J, Barken KB, Lahdenpera J, Breinstein A, Molin S, Neubaure P (2003) Sandwich hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. Microb Cell Fact 2(1):4

    Article  PubMed  Google Scholar 

  18. Zammatteo N, Moris P, Alexandre I, Vaira D, Piette J, Remacle J (1995) DNA-probe hybridization in microwells using a new bioluminiscent system for the detection of PCR-amplified HIV-1 proviral DNA. J Virol Methods 55(2):185–197

    Article  PubMed  CAS  Google Scholar 

  19. Ronkainen-Matsuno NJ, Thomas JH, Halsall HB, Heinemann WR (2002) Electrochemical immunoassay moving into the fast lane. Trac-Trends Anal Chem 21(4):213–225

    Article  CAS  Google Scholar 

  20. Anderson DM, Kulis D, Erdner D, Ahn S, Walt D (2006) Fibre optic microarrays for the detection and enumeration of harmful algal bloom species. Afr J Mar Sci 28(2):231–235

    Article  Google Scholar 

  21. Diercks S, Metfies K, Medlin LK (2008) Molecular probe sets for the detection of toxic algae for use in sandwich hybridization formats. J Plankton Res 30(4):439–448

    Article  CAS  Google Scholar 

  22. Orozco J, Medlin L (2010) Electrochemical performance of a DNA-based sensor device for detecting toxic algae. Sens Actuators B Chem 153:71–77

    Article  Google Scholar 

  23. Orozco J, Baudart J, Medlin L (2011) Evaluation of probe orientation and effect of the digoxigenin-enzymatic label in a sandwich hybridization format to develop toxic algae biosensors. Harmful Algae 10:489–494

    Article  Google Scholar 

  24. Groben R, John U, Eller G, Lange M, Medlin LK (2004) Using fluorescently labelled rRNA probes for hierarchical estimation of phytoplankton diversity. Nova Hedw 79:313–320

    Article  Google Scholar 

  25. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar, Buchner A, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer K-H (2004) ARB, a software environment for sequence data. Nucl Acids Res;32:1363–1371

    Google Scholar 

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Acknowledgments

We acknowledge to Dr. Julia Baudart and Sebastien Peuchet from the Observatoire Oceanologique de Banyuls Sur Mer for their help in the discussion of the results and to the latter for the results reported in Fig. 6.

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Authors and Affiliations

  1. Department of NanoEngineering, University of California, San Diego (UCSD), La Jolla, CA, USA

    Jahir Orozco & Linda K. Medlin

  2. UPMC, Univ Paris 06, UMR 7621, LOMIC, Banyuls/mer, France

    Jahir Orozco & Linda K. Medlin

  3. CNRS, UMR 7621, LOMIC, Banyuls/mer, France

    Jahir Orozco & Linda K. Medlin

Authors
  1. Jahir Orozco
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  2. Linda K. Medlin
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Corresponding author

Correspondence to Jahir Orozco .

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Editors and Affiliations

  1. 4901 Evergreen Road, Dearborn, 48128, USA

    Sonia M. Tiquia-Arashiro

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© 2012 Springer Science+Business Media New York

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Orozco, J., Medlin, L.K. (2012). Electrochemical Detection of Harmful Algae by Means of a Sandwich Hybridization Assay on an Electrode Surface. 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_12

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  • DOI: https://doi.org/10.1007/978-1-61779-915-0_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-914-3

  • Online ISBN: 978-1-61779-915-0

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