Enzyme-linked amplified electrochemical sensing of oligonucleotide-DNA interactions by means of the precipitation of an insoluble product and using impedance spectroscopy

Fernando Patolsky, Eugenii Katz, Amos Bardea, Itamar Willner

Research output: Contribution to journalArticlepeer-review

178 Scopus citations

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)63- 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)63- 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 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.

Original languageEnglish
Pages (from-to)3703-3706
Number of pages4
JournalLangmuir
Volume15
Issue number11
DOIs
StatePublished - 25 May 1999
Externally publishedYes

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