Microarrays pp 385-400 | Cite as

Quantification of Mixed-Phase Hybridization on Polymer Microparticles by Europium (III) Ion Fluorescence

  • Kaisa Ketomäki
  • Harri Lönnberg
Part of the Methods in Molecular Biology™ book series (MIMB, volume 381)


A protocol for quantification of oligonucleotide hybridization on polymer microparticles by europium(III) ion fluorescence is described. The procedure involves modification of commercially available amino-functionalized microparticles in such a manner that oligonucleotide probes may be assembled in situ on these particles or, alternatively, they may be immobilized postsynthetically. The oligonucleotide-coated particles obtained are then used as the solid phase in a mixed-phase hybridization assay. The efficiency of hybridization is quantified with the aid of oligonucleotides tagged with a europium(III) chelate. Either, the fluorescently tagged probe is hybridized directly to a complementary particle-anchored oligonucleotide, or a sandwich-type assay set up, where a third oligonucleotide complementary both to the tagged and particle-bound probe mediates the attachment to the particles, is exploited. The number of europium(III) ions attached to the solid-phase is determined by the DELFIA® protocol, involving release of the europium(III) ions in solution and development of the fluorescence by addition of an enhancement solution. Alternatively, the fluorescence intensity of the photoluminescent chelate may be measured directly from a single particle.

Key Words

DELFIA® immobilization microparticles mixed-phase hybridization oligonucleotides time resolved fluorometry 


  1. 1.
    O’Donnell-Maloney, M. J., Smith, C. L., and Cantor, C. R. (1996) The development of microfabricated arrays for DNA sequencing and analysis. Tibtechnol. 14, 401–407.Google Scholar
  2. 2.
    Niemeyer, C. M. and Blohm, D. (1999) DNA Microarrays. Angew. Chem. Int. Ed. 38, 2865–2869.CrossRefGoogle Scholar
  3. 3.
    Epstein, C. B. and Butow, R. A. (2000) Microarray technology-enhanced versatility, persistent challenge. Curr. Opin. Biotechnol. 11, 36–41.CrossRefGoogle Scholar
  4. 4.
    Jung, A. (2002) DNA chip technology. Anal. Bioanal. Chem. 372, 41–42.CrossRefGoogle Scholar
  5. 5.
    Gerhold, D. L., Jensen, R. V., and Gullans, S. R. (2002) Better therapeutics through microarrays. Nature Genet. Suppl. 32, 547–552.CrossRefGoogle Scholar
  6. 6.
    Lockhart, D. J., Dong, H., Byrne, M. C., et al. (1996) Expression monitoring by hybridization to high-density oligonucleotide arrays. Nature Biotechnol. 14, 1675–1680.CrossRefGoogle Scholar
  7. 7.
    Czarnik, A. (1998) Illuminating the SNP genomic code. Modern Drug Discov. 1, 49–55.Google Scholar
  8. 8.
    Southern, E., Mir, K., and Shchepinov, M. (1999) Molecular interactions on microarrays. Nat. Genet. 21, 5–9.CrossRefGoogle Scholar
  9. 9.
    Fahy, E., Davis, G. R., DiMichele, L. J., and Ghosh, S. S. (1993) Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics. Nucleic Acids Res. 21, 1819–1826.CrossRefGoogle Scholar
  10. 10.
    Hakala, H. and Lönnberg, H. (1997) Time-resolved fluorescence detection of oligonucleotide hybridizations on a single microparticle: covalent immobilization of oligonucleotides and quantification of a model system. Bioconjugate Chem. 8, 232–237.CrossRefGoogle Scholar
  11. 11.
    Hakala, H., Heinonen, P., Iitiä, A., and Lönnberg, H. (1997) Detection of olionucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes. Bioconjugate Chem. 8, 378–384.CrossRefGoogle Scholar
  12. 12.
    Hakala, H., Mäki, E., and Lönnberg, H. (1998) Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: quantification and optimization of a sandwich type assay. Bioconjugate Chem. 9, 316–321.CrossRefGoogle Scholar
  13. 13.
    Ketomäki, K., Hakala, H., and Lönnberg, H. (2002) Mixed-phase hybridization of short oligodeoxyribonucleotides on microscopic polymer particles: Effect of one-base mismatches on duplex stability. Bioconjugate Chem. 13, 542–547.CrossRefGoogle Scholar
  14. 14.
    Ketomäki, K., Hakala, H., Kuronen, O., and Lönnberg, H. (2003) Hybridizations properties of support-bound oligonucleotides: the effect of the site of immobilization on the duplex stability and selectivity of duplex formation. Bioconjugate Chem. 14, 811–816.CrossRefGoogle Scholar
  15. 15.
    Fulton, R. J., McDade, R. L., Smith, P. L., Kienker, L. J., and Kettman, J. R., Jr. (1997) Advanced multiplexed analysis with the FlowMetrix? system. Clin. Chem. 43, 1749–1756.Google Scholar
  16. 16.
    Hakala, H., Virta, P., Salo, H., and Lönnberg, H. (1998) Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay. Nucleic Acids Res. 26, 5581–5588.CrossRefGoogle Scholar
  17. 17.
    Lövgren, T., Heinonen, P., Lehtinen, P., et al. (1997) Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry. Clin. Chem. 43, 1937–1943.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Kaisa Ketomäki
    • 1
  • Harri Lönnberg
    • 1
  1. 1.Department of ChemistryUniversity of TurkuTurkuFinland

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