Effects of oligonucleotide immobilization density on selectivity of quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective "reagent" to bind complementary nucleic acids for applications including detection of pathogenic organisms and genetic mutations. The density of ssDNA on a surface will determine nearest neighbor interactions, surface interactions, and charge density due to ionizable phosphate groups. This may result in a local ionic strength, pH, and dielectric constant at the surface that is substantially different from that in bulk electrolyte solution. It is the local conditions that influence the thermodynamics of hybridization, and this can be studied by the melt temperature (T-m) of double-stranded DNA (dsDNA). Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT(20) that was prepared by automated synthesis. Fiber-optic biosensors based on fused silica optical fibers that were coated with DNA were used in a total internal reflection fluorescence instrument to determine T-m from the dissociation of duplexes of mixtures of fluorescein-labeled and unlabeled dA(20) and d(A(9)GA(10)). Each thermal denaturation of dsDNA 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. The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is dependent on the density of immobilized DNA. Additionally, the deviation in T-m, arising as a result of the presence of a centrally located single base-pair mismatch was significantly larger for thermal denaturation occurring at the surface of the optical fibers (Delta T-m = 6-10 degrees C) relative to that observed in bulk solution (Delta T-m = 3.8-6.1 degrees C). These results suggest that hybridization at an interface occurs in a significantly different physical environment in comparison to hybridization in bulk solution, and that surface density can be tuned to design analytical figures of merit.