Time-Resolved Fluorescence Detection of Oligonucleotide Hybridization on a Single Microparticle: Covalent Immobilization of Oligonucleotides and Quantitation of a Model System

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Abstract
Several alternative methods have been described for the immobilization of oligodeoxyribonucleotides to uniformly sized glycidyl methacrylate/ethylene dimethacrylate particles. Hybridization of complementary oligodeoxyribonucleotides labeled with photoluminescent europium(III) chelates to these particle-bound oligonucleotide probes was followed by subjecting a single microparticle to a time-resolved fluorescence measurement. The hybridization was further quantified by releasing the europium ion to a fluorescence enhancement solution and determining its concentration against europium(III) chloride standards. Both the efficiency and kinetics of the hybridization were observed to depend markedly on the linker employed to tether the oligonucleotide probes to the particles. These effects and those of the experimental conditions, such as oligonucleotide concentration in solution, oligonucleotide density on particles, and number of particles in a given volume of assay solution, are discussed.