DETECTION OF OLIGONUCLEOTIDE HYBRIDIZATION ON A SINGLE MICROPARTICLE BY TIME-RESOLVED FLUOROMETRY - HYBRIDIZATION ASSAYS ON POLYMER PARTICLES OBTAINED BY DIRECT SOLID-PHASE ASSEMBLY OF THE OLIGONUCLEOTIDE PROBES

Oligodeoxyribonucleotides were assembled by conventional phosphoramidi te chemistry on uniformly sized (50 mu m) porous glycidyl methacrylate /ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatiz ed with DMTrO-acetyl groups. The linker was completely resistant towar d ammonolytic deprotection of the base moieties. The quality of oligon ucleotides was assessed by repeating the synthesis on the same particl es derivatized with a cleavable ester linker. The ability of the oligo nucleotide-coated particles to bind complementary sequences via hybrid ization was examined by following the attachment of oligonucleotides b earing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hyb ridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentrat ion of the target oligonucleotide in solution, length of the hybridizi ng sequence, presence of noncomplementary sequences, and ionic strengt h. fluorescence signal measured on a single particle after hybridizati on was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.