Considerations for the quantitative transduction of hybridization of immobilized DNA

Immobilized single-stranded DNA (ssDNA) can be used as a selective 'reagent ' to bind complementary DNA or RNA for applications such as the detection o f pathogenic organisms, gene therapy agents and genetic mutations. The dens ity of ssDNA on a surface will determine the charge density due to ionizabl e phosphate groups. Such a negatively charged interface will attract positi ve counter-ions from solution, which may result in a local ionic strength, pH and dielectric constant on the surface that is substantially different f rom that in bulk electrolyte solution. It is the local conditions which inf luence the thermodynamics of hybridization, and this can studied by the mel t temperature (T-m) of double-stranded DNA (dsDNA). Experimental work and t heoretical models have been used to examine whether hybridization reactions on a surface can cause dynamic changes in local charge density, and theref ore, changes in selectivity and drift in calibration for quantitative analy sis. Organosilane chemistry has been used to covalently immobilize hexaethy lene glycol linkers and to control the subsequent density of dT(20) that wa s prepared by automated synthesis. Fiber-optic biosensors based on fused si lica that was coated with DNA were used in a total internal reflection fluo rescence instrument to determine T-m from the dissociation of duplexes of f luorescein-labeled dA(20):dT(20) The experimental results suggest that the thermodynamic stability of duplexes that are immobilized on a surface is de pendent on the density of immobilized DNA and on the extent of hybridizatio n of DNA. The experimental results show that the thermodynamic stability of immobilized dsDNA is significantly different than that of dsDNA in bulk so lution, and include observations of the variation of enthalpy at different ionic strengths, asymmetry in the melt curves, and the possibility of a red uced dielectric constant within a DNA layer relative to that in bulk soluti on. (C) 1999 Elsevier Science B.V. All rights reserved.