ABSTRACT
Neutron reflection has been used in combination with electrochemical and quartz microbalance techniques to characterize a mixed monolayer of thiolated hairpin ss-DNA and 4-mercaptobutan-1-ol monolayer self-assembled on gold before and after hybridization with a cDNA target. Neutron reflection has revealed the opening of the stem-loop configuration of the probe associated with helix formation. This change in conformation correlates to the modification of the electron-transfer resistance associated with the [Fe(CN)6](3-/4-) redox marker present in solution.
Subject(s)
DNA Probes/chemistry , Gold/chemistry , Inverted Repeat Sequences , Base Sequence , DNA Probes/genetics , Electric Impedance , Gold/pharmacology , Models, Molecular , Neutron Diffraction , Nucleic Acid Conformation/drug effects , Nucleic Acid Hybridization , Quartz/chemistry , Surface PropertiesABSTRACT
An electrochemical DNA hybridization detection method based on the electrostatic interactions of [Ru(NH3)6]3+ cations with the anionic phosphate backbone of DNA is proposed. PNA molecules are immobilized as capture probes on the gold substrate. The cationic ruthenium complexes do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after hybridization, [Ru(NH3)6]3+ is adsorbed on the DNA backbone, giving a clear hybridization detection signal in ac voltammetry. The analytical parameters (sensitivity, selectivity and reproducibility) are evaluated. Very good discrimination against the single-base mismatch A2143G, internal to the 23S rRNA gene of Helicobacter pylori, is observed. Moreover the system is successfully applied to the detection of complementary PCR amplicons.
