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 H2O2 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.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…A hybridisation efficiency lower than 10% was observed. The 18-mer probes, containing five thiophosphate thymines, were also used by Willner and co-workers (Patolsky et al, 1999;Bardea et al, 1999b;Alfonta et al, 2001). The 18-mer oligonucleotides, modified by a phosphorothioate group either at its 3 or both 3 and 5 terminals, were used by Hianik and co-workers to modify gold electrodes (Hianik et al, 2001).…”

“…The surface-linked, thiol-modified, capture probe 1 was reacted with the complex: target DNA-biotinylated capture probe 2. Ternary complex formation introduced a barrier to interfacial electron-transfer of [Fe(CN) 6 ] 3−/4− redox probe that could be readily detected by faradic impedance spectroscopy (Patolsky et al, 1999) and/or chronopotentiometry (Alfonta et al, 2001). Binding of an avidin-HRP conjugate to the double-stranded superstructure, further increased the electron-transfer resistance.…”

A Novel Type of Nucleic Acid-based Biosensors: the Use of PNA Probes, Associated with Surface Science and Electrochemical Detection Techniques

“…Electrochemical concepts have a l s o p r o v e n t o b e v e r y u s e f u l f o r s e q u e n c e -s p e c i f i c biosensing of DNA, because they provide simple, rapid, lable-free and low-cost detection of nucleic acid sequences [Wang et al, 2000;F. Patolsky et al, 1999].…”

Miniaturized DNA Biosensor for Decentralized Breast-Cancer Screening

“…In this system, the hybridization event resulted in the 'wiring' of the enzyme to the transducer (via an electron-conducting redox hydrogel), hence leading in a continuous hydrogen-peroxide electroreduction current. Willner's group (22) illustrated that multiple amplifications can be achieved by coupling of a peroxidase enzyme label with the surface accumulation of the phenol reaction product.…”