Towards the development of a fiber-optic nucleic acid biosensor: An examination of factors affecting selectivity of detection of interfacial nucleic acid hybridization

This work was done to examine the effects of variations in oligonucleotide immobilization chemistry (immobilization density and mechanism of covalent immobilization to silica substrates) on thermal denaturation and non-selective adsorption of nucleic acids in interfacial hybridization experiments using a fiber optic nucleic acid biosensor. Thermal denaturation profiles were obtained by UV-absorption spectrophotometry for dT₂₀ hybridized with complementary and partially complementary targets. Thermal denaturation temperatures (T_ms) were computed for sequences based on SMN1, SMN2, UidA and HrDNA genes as well as for dA₂₀:dT₂₀ via MeltCalc software. Thermal denaturation profiles were obtained using a DNA biosensor configuration based on Total Internal Reflection Fluorescence for dT₂₀, SMN1 and SMN2 sequences covalently immobilized at various densities to the surface of fused silica optical fibers via flexible hexaethylene glycol linkers, immobilized by means of a number of different synthetic routes. The immobilized DNA was hybridized with fluorescein-labeled or Cy5-labeled complementary oligonucleotides. The deviation in T_m arising as result of a centrally-located single base-pair mismatch (SBPM) was as large or larger for thermal denaturation occurring at the surface of the optical fibers (ΔT_m = 6–10°C) relative to that observed in bulk solution (measured or calculated, ΔT_m = 3.8–6.9°C). Selectivity coefficients for hybridization were computed from normalized thermal denaturation profiles. The shapes of thermal denaturation profiles varied as a function of immobilization chemistry and sensor history.; The steady-state fluorescence intensity observed in hybridization assays was dependent on whether target DNA was introduced in the double-stranded (dsDNA) or single-stranded (ssDNA) form, with reductions in fluorescence intensity observed in hybridization assays using symmetric PCR amplicons as targets for immobilized UidA ssDNA oligonucleotide probes, relative to the complementary ssDNA targets. The steady-state fluorescence intensity varied as the position of the target sequence within PCR amplicons was varied. Sonication of genomic DNA provided samples that were detected by the immobilized probes.; In general, it was shown that the selectivity, sensitivity and dynamic range of response of fiber-optic nucleic acid biosensors are sensitive to subtle variations in surface chemistry (immobilization density, mechanism of immobilization), and to the nature of nucleic acid targets (size and molecularity) used.