Direct, label-free and highly sensitive detection of DNA hybridization is important for the development of DNA chips potentially avoiding initial PCR amplifi­cation. Electrochemical impedance spectroscopy (EIS) was already successfully used for the visualization of DNA hybridization. In EIS, the binding of the target strands to surface-immobilized capture probes is indicated by a shift in the impedance spectrum of the modified electrode [1]. In addition, label-free detection of DNA can be obtained by interaction of DNA with electrochemically active ligands [2]. Actinomycin D is an antitumor antibiotic that contains a 2-aminophenoxazin-3-one chromophore and two cyclic pentapeptide lactones. The chromophore is intercalating with high affinity constant into double-stranded DNA preferentially at GC-Watson-Crick base pairs. In this work, the interactions between ActD and single as well as double stranded DNA is studied by means of EIS. For this purpose, monolayers of thiol-modified ss-DNA in combination with different types of alkanethiols with variable chain length were assembled on gold electrode. The influence of the modified surface on the electron-transfer properties of suitable reversible redox mediators such as Fe(CN)₆^3-/4- or Ru(NH₃)₆^2+/3+ was investigated by means of EIS. The sequence of monolayer formation using thiol-modified single-stranded DNA capture probes, additional surface modification with a thiol derivative, and hybridization with the complementary strand were followed in-situ by means of EIS. Previously, it has been reported [3] that ActD is weakly interacting with ss-DNA, whereas preserves a high binding affinity with immobilised ds-DNA. Intercalation of Actinomycin D into the formed double-stranded DNA could be monitored using EIS, however, a weak non-covalent adsorption onto bare gold or thiolated-gold electrodes was demonstrated. Parameters, such as the nature of the electrolyte, pH value, concentration of the redox mediator, type of thiol used for gold modification were evaluated with respect to their influence on the detection of DNA-hybridization by means of EIS. Based on these results, a new strategy for the detection of DNA hybridization will be proposed.

References:

[1] Eugenii Katz, Itamar Willner, Electroanalysis 2003, 15, 913-947

[2] Wang, S.; Peng, T. Z.; Yang, C. F. J. Electroanal. Chem. 2003, 544, 87-92

[3] Reich E., Goldberg H. I., Prog. Nucleic Acid Res. Mol. Biol. 1964, 3, 183-234

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