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

Abstract

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 double-stranded 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 3 - as redox probe, reveal an increase in the electron-transfer resistance at the electrode surface upon the construction of the double-stranded assembly. This is attributed to the electrostatic repulsion of Fe(CN) 6 3 - upon formation of the negatively charged double-stranded 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 H 2 O 2 yields a precipitate ( 5 ) on the conductive support and stimulates a very high barrier for interfacial electron transfer, R et = 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.