Author Description

This work was done to develop a biosensor system based on fluorescence detection using the evanescent field of a fused silica optical fiber. Intercalating dye was tethered to probe oligonucleotide, and this was assembled by solid-phase oligonucleotide synthesis onto fused silica optical fibers functionalized with linker molecules. The fluorescence signal that was associated with hybridization of target sequences was monitored in real time. This self-contained DNA biosensor system avoided the use of an additional dye to stain dsDNA or target labeling. The approach could also ameliorate false signal generation caused by nonspecific adsorption of labeled targets. Thermal or chemical denaturation of the hybridized strands regenerated the single-stranded DNA (ssDNA), allowing for multiple assay cycles using the same fiber.Computer simulated energy calculations were used to evaluate different tethers for development of suitable ssDNA probes. Due to its high binding affinity to double-stranded DNA, thiazole orange (TO) was chosen and a series of its derivatives were covalently attached to probe oligonucleotides through the 5' terminal phosphate group using polyethylene glycol linkers. The spectroscopic characterization of the tethered probes in solution suggested that up to a 10 fold enhancement of fluorescence could be obtained upon hybridization. Fluorescence titration experiments revealed a 1:1 binding stoichiometry for the tethered oligonucleotides and their complementary sequences.Epoxy silane chemistry was used to activate various silica surfaces, and formed a close-packed monolayer. Hexaethylene glycol was used as a linker. The modified surface was allowed to react with oligonucleotides with amine/aldehyde termini, or was coupled with dimethoxytrityl (DMT) for solid-phase oligonucleotide synthesis. Surface spectroscopy and mass spectrometry data indicated that the immobilized films were continuous and were of monolayer thickness.The fluorescence response from fibers that were coated with TO-tethered sequences was examined by fiber optic spectrofluorimeter using evanescent wave excitation. Ionic strength, tether length and packing density of the probe molecules were examined to determine the influence of environmental parameters on the thermodynamic and kinetic performance of the biosensor. A preliminary application experiment was done to investigate response of the biosensor to a series of PCR products from Erwinia herbicola.