DETECTION OF OLIGONUCLEOTIDE HYBRIDIZATION ON A SINGLE MICROPARTICLE BY TIME-RESOLVED FLUOROMETRY - QUANTITATION AND OPTIMIZATION OF A SANDWICH-TYPE ASSAY

Uniformly sized (50 mu m) porous glycidyl methacrylate/ethylene dimeth acrylate particles (SINTEF) were used as the solid phase in a sandwich type mixed-phase hybridization assay based on time-resolved fluoresce nce detection on a single particle. These particles were coated with o ligodeoxyribonucleotide probes by conventional phosphoramidite chain a ssembly. An oligodeoxyribonucleotide bearing a photoluminescent europi um(III) chelate, {2,2',2 ichloro-1,3,5-triazin-2-yl)amino]phenyl}-2,2' :6',2 ''-terpyridine-6,6 ethylenenitrolo)}tetrakis(acetato)}europium-( III), was hybridized to a complementary sequence of the target oligonu cleotide, and the resulting duplex was further hybridized to the parti cle-bound probes. The latter binding was quantified by time-resolved m easurement of the emission signal of a single particle. Kinetics of hy bridization and the effect of the concentration of the target oligomer and the fluorescently tagged probe on the efficiency of hybridization were studied. The intensity of the emission signal was Linearly relat ed to the concentration of the target oligomer over a range of 5 order s of magnitude. The length of the complementary region between the tar get oligomer and the particle-bound probe was varied, and the effect o f point mutations and deletions on the hybridization efficiency was de termined in each case. The maximal selectivity was observed with 10-16 -base pair complementary sequences, the optimal length depending on th e oligonucleotide loading on the particle. Discrimination between the complete matches and point mismatches was unequivocal, a single point mutation and/or deletion decreasing the efficiency of hybridization by more than 2 orders of magnitude.