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 D NA is described using Faradaic impedance spectroscopy. A thiol-thymine-tagg ed oligonucleotide (1) capable of forming only one double-stranded turn wit h 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 u pon the construction of the double-stranded assembly. This is attributed to the electrostatic repulsion of Fe(CN)(6)(3-) upon formation of the negativ ely charged double-stranded superstructure. Binding of an avidin-HRP conjug ate to the oligonucleotide-DNA assembly further insulates the electrode and increases the interfacial electron-transfer resistance. The HRP-mediated b iocatalyzed oxidation of 4-ckloro-1-naphthol (4) by H2O2 yields a precipita te (5) on the conductive support and stimulates a very high barrier for int erfacial electron transfer, R-et = 14.7 k Omega. Thus, the precipitation of 5 confirms and amplifies the sensing process of the target DNA (2). The an alyte DNA (2) corresponds to the mutated gene fragment characteristic of th e 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 x 10(-9) g.mL(-1). Cyclic voltam metry experiments further confirm the formation of barriers for the interfa cial electron transfer upon the buildup of the double-stranded oligonucleot ide-DNA structure and upon the biocatalytic deposition of 5 on the electrod e surface.