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Analytical and Bioanalytical Chemistry

, Volume 380, Issue 1, pp 46–53 | Cite as

Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus

  • Natalya V. Zaytseva
  • Richard A. Montagna
  • Eun Mi Lee
  • Antje J. Baeumner
Original Paper

Abstract

A multi-analyte biosensor based on nucleic acid hybridization and liposome signal amplification was developed for the rapid serotype-specific detection of Dengue virus. After RNA amplification, detection of Dengue virus specific serotypes can be accomplished using a single analysis within 25 min. The multi-analyte biosensor is based on single-analyte assays (see Baeumner et al (2002) Anal Chem 74:1442–1448) developed earlier in which four analyses were required for specific serotype identification of Dengue virus samples. The multi-analyte biosensor employs generic and serotype-specific DNA probes, which hybridize with Dengue RNA that is amplified by the isothermal nucleic acid sequence based amplification (NASBA) reaction. The generic probe (reporter probe) is coupled to dye-entrapping liposomes and can hybridize to all four Dengue serotypes, while the serotype-specific probes (capture probes) are immobilized through biotin–streptavidin interaction on the surface of a polyethersulfone membrane strip in separate locations. A mixture of amplified Dengue virus RNA sequences and liposomes is applied to the membrane and allowed to migrate up along the test strip. After the liposome-target sequence complexes hybridize to the specific probes immobilized in the capture zones of the membrane strip, the Dengue serotype present in the sample can be determined. The amount of liposomes immobilized in the various capture zones directly correlates to the amount of viral RNA in the sample and can be quantified by a portable reflectometer. The specific arrangement of the capture zones and the use of unlabeled oligonucleotides (cold probes) enabled us to dramatically reduce the cross-reactivity of Dengue virus serotypes. Therefore, a single biosensor can be used to detect the exact Dengue serotype present in the sample. In addition, the biosensor can simultaneously detect two serotypes and so it is useful for the identification of possible concurrent infections found in clinical samples. The various biosensor components have been optimized with respect to specificity and sensitivity, and the system has been ultimately tested using blind coded samples. The biosensor demonstrated 92% reliability in Dengue serotype determination. Following isothermal amplification of the target sequences, the biosensor had a detection limit of 50 RNA molecules for serotype 2, 500 RNA molecules for serotypes 3 and 4, and 50,000 molecules for serotype 1. The multi-analyte biosensor is portable, inexpensive, and very easy to use and represents an alternative to current detection methods coupled with nucleic acid amplification reactions such as electrochemiluminescence, or those based on more expensive and time consuming methods such as ELISA or tissue culture.

Keywords

Dengue virus Biosensor Multi-analyte Liposome Serotype-specific Rapid 

Notes

Acknowledgements

The authors acknowledge financial support for this project from Innovative Biotechnologies International, Inc., from the National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA, from the New York State CAT, Biotechnology Program at Cornell University, and from the Cooperative State Research, Education and Extension Services (NYC-123314). We thank M.G. Sarngadharan and Roxanne N. Shurtliff from Advanced BioScience Laboratories, Inc., for their many discussions and help in moving the project along. We also want to thank Sutee Yoksan from Mahidol University, Thailand and Shuenn-Jue Wu from Naval Medical Research Center, USA, for providing us with Dengue virus samples that were used in this publication.

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Natalya V. Zaytseva
    • 1
  • Richard A. Montagna
    • 2
  • Eun Mi Lee
    • 3
  • Antje J. Baeumner
    • 1
  1. 1.Department of Biological and Environmental EngineeringCornell UniversityIthacaUSA
  2. 2.Innovative Biotechnologies International Inc.Grand IslandUSA
  3. 3.Advanced BioScience Laboratories Inc.KensingtonUSA

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