Effects of Oligonucleotide Immobilization Density on Selectivity of Quantitative Transduction of Hybridization of Immobilized DNA

Abstract

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 (Tm) of double-stranded DNA (dsDNA). Organosilane chemistry has been used to covalently immobilize hexaethylene glycol linkers and to control the subsequent density of dT20 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 Tm from the dissociation of duplexes of mixtures of fluorescein-labeled and unlabeled dA20 and d(A9GA10). 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 Tm 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 (∆Tm ) 6-10 °C) relative to that observed in bulk solution (∆Tm ) 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, and that surface density can be tuned to design analytical figures of merit.

Controlled Orientation of DNA in a Binary SAM as a Key for the Successful Determination of DNA Hybridization by Means of Electrochemical Impedance Spectroscopy

“…Among others, the most important parameters are i) the surface coverage of the immobilized ssDNA, which determines the DNA orientation at the interface [22] and thus the efficiency of the hybridization process; [23,24] ii) the properties of the shortchain thiol derivatives which are co-assembled with the thioltethered capture probe or immobilized at the transducer surface after the grafting of the ssDNA. These thiol derivatives fill the gaps between the individual surface-bound DNA molecules, remove non-specifically bound DNA strands and ensure a better orientation of the immobilized DNA at the interface.…”

DNA surface hybridization regimes

“…A variety of aids to enhance hybridization performance is used, including surfactants and blocking agents to control nonspecific adsorption, washing procedures to develop contrast between fully and partially complementary sequences, and variation of assay parameters such as ionic strength or temperature. Physical studies (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) aim to distill down this complexity into scenarios where specific features of surface hybridization are brought out; for example, the impact of probe surface coverage on target hybridization (17,20,22,23,28).…”

A Nucleic Acid Biosensor for Detection of Hepatitis C Virus Genotype 1a Using Poly(l-Glutamic Acid)-Modified Electrode

“…It was reported in the literature that the concentration of immobilized DNA can affect the hybridization thermodynamics and the biosensor selectivity [30]. In this work, we used a series of concentration of the probe solution so as to find an optimal probe density for hybridization efficiency and results are given in Fig 5a. Optimum probe concentration was determined by comparing c/nc ratio [31].…”