Enzyme-Linked Amplified Electrochemical Sensing of Oligonucleotide-DNA Interactions by Means of the Precipitation of an Insoluble Product and Using Impedance Spectroscopy

A novel method for the sensitive and specific electrochemical analysis of DNA is described using Faradaic impedance spectroscopy. A thiol-thymine-tagged oligonucleotide (1) capable of forming only one doublestranded turn with the target DNA analyte (2) is assembled on a Au electrode and acts as the sensing interface. The resulting functionalized electrode is reacted with a complex between the target DNA (2) and a biotinylated oligonucleotide (3) to yield a bifunctional double-stranded assembly on the electrode support. The Faradaic impedance spectra, using Fe(CN)6 as redox probe, reveal an increase in the electrontransfer resistance at the electrode surface upon the construction of the double-stranded assembly. This is attributed to the electrostatic repulsion of Fe(CN)6 upon formation of the negatively charged doublestranded superstructure. Binding of an avidin-HRP conjugate to the oligonucleotide-DNA assembly further insulates the electrode and increases the interfacial electron-transfer resistance. The HRP-mediated biocatalyzed oxidation of 4-chloro-1-naphthol (4) by H2O2 yields a precipitate (5) on the conductive support and stimulates a very high barrier for interfacial electron transfer, Ret ) 14.7 kΩ. Thus, the precipitation of 5 confirms and amplifies the sensing process of the target DNA (2). The analyte DNA (2) corresponds to the mutated gene fragment characteristic of the Tay-Sachs genetic disorder. The normal gene (2a) is easily discriminated by the sensing interface. The sensor device enables detection of the target DNA (2) with a sensitivity of at least 20 × 10-9 g‚mL-1. Cyclic voltammetry experiments further confirm the formation of barriers for the interfacial electron transfer upon the buildup of the double-stranded oligonucleotide-DNA structure and upon the biocatalytic deposition of 5 on the electrode surface.