Towards the development of a fibre-optic nucleic acid biosensor: Considerations for the quantitative transduction of hybridization of immobilized DNA

Thermal denaturation profiles were obtained for polythymidylic icosanucleotides, (dT20) hybridized with dA20, d(A9GA 10), d(A9G2A9), d(A18G 2), d(G2A16G2) and (G5A 20G5) in bulk solution by UV-absorption spectrophotometry. Thermal denaturation profiles were also obtained for dT20 covalently immobilized to the surface of fused silica optical fibers via flexible hexaethylene glycol linkers with different packing densities (DNA biosensor configuration based on Total Internal Reflection Fluorescence). The immobilized dT 20 was hybridized with mixtures of fluorescein-labeled and unlabeled dA20 and d(A9GA10). Each thermal denaturation of dA20 and d(A9GA10) at the surface of the optical fibers was accompanied by a 2--3 fold reduction in standard enthalpy change, relative to values determined for denaturation in bulk solution. Additionally, the deviation in observed Tm arising as a result of the presence of a centrally located single base-pair mismatch (SBPM) was significantly larger for thermal denaturation occurring at the surface of the optical fibers (DeltaTm = 6--10°C) relative to that observed in bulk solution (DeltaTm = 3.8--6.1°C). These results suggest that hybridization at an interface occurs in a significantly different physical environment in comparison to hybridization in bulk solution.