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

Abstract

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.