FIBEROPTIC EVANESCENT-WAVE BIOSENSOR FOR THE DETECTION OF OLIGONUCLEOTIDES

An automated optical biosensor system based on fluorescence excitation and detection in the evanescent held of a quartz fiber was used to de tect 16-mer oligonucleotides in DNA hybridization assays. A biotinylat ed capture probe was immobilized on the fiber surface via avidin or st reptavidin, The hybridization with fluorescein-labeled complementary s trands was monitored in real time by fluorescence detection, The doubl e strands formed by hybridization could be dissociated by chemical or thermal regeneration, allowing one to perform hundreds of assay cycles with the same fiber. The signal loss during longtime measurements, i. e., consecutive hybridization assays, can be described by a single-exp onential function. Over more than 200 cycles, the net signal decreased by 50% with a signal variation of 2.4% after correction for this sign al loss. By binding the capture probe with the 5'-end to the optical f iber surface, and by using a 50% (W/w) aqueous urea solution for chemi cal regeneration, the duration of an assay cycle could be reduced to 3 min, By applying longer assay cycles, the detection limit for the hyb ridization with a complementary fluorescein-labeled oligonucleotide wa s 2.0 x 10(-13) M (24 fmol), To detect an unlabeled complementary le-m er oligonucleotide, competitive hybridization assays were performed, r esulting in a detection limit of 1.1 x 10(-9) M (132 pmol). Poly(acryl ic acid) 5100 sodium salt and Tween 20 were used in the hybridization buffer to prevent nonspecific binding caused by ionic or hydrophobic i nteraction. The amount of nonspecific binding of noncomplementary olig onucleotides was in the range of 1-2%, compared with the specific bind ing in the different hybridization assays.