TIME-RESOLVED FLUORESCENCE DETECTION OF OLIGONUCLEOTIDE HYBRIDIZATIONON A SINGLE MICROPARTICLE - COVALENT IMMOBILIZATION OF OLIGONUCLEOTIDES AND QUANTITATION OF A MODEL SYSTEM

Several alternative methods have been described for the immobilization of oligodeoxyribonucleotides to uniformly sized glycidyl methacrylate /ethylene dimethacrylate particles. Hybridization of complementary oli godeoxyribonucleotides labeled with photoluminescent europium(III) che lates to these particle-bound oligonucleotide probes was followed by s ubjecting a single microparticle to a time-resolved fluorescence measu rement. The hybridization was further quantified by releasing the euro pium ion to a fluorescence enhancement solution and determining its co ncentration against europium(III) chloride standards. Both the efficie ncy and kinetics of the hybridization were observed to depend markedly on the Linker employed to tether the oligonucleotide probes to the pa rticles. 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 solut ion, are discussed.