Enzyme-Amplified Electrochemical Detection of DNA Using Electrocatalysis of Ferrocenyl-Tethered Dendrimer

Abstract

We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.

Single-step electronic detection of femtomolar DNA by target-induced strand displacement in an electrode-bound duplex

“…A wide range of electronic DNA detection schemes have been described to date (3)(4)(5), the best of which achieve limits of detection (LOD) ranging from picomolar to femtomolar. Among promising recent examples are approaches based on conductive links produced through the catalytic deposition of silver by nanoparticle-linked secondary probes (LOD 500 fM) (6), the electrocatalytic oxidation of modified bases by Os(bpy) 3 3ϩ (LOD 400 fM) (7), the electrochemistry of water-soluble, ferrocene-functionalized cationic polythiophenes (LOD 500 pM) (8) or ferrocene-linked triblock copolymer-DNA hybrids (LOD 100 pM) (9), the electron transfer of ferrocenyl-tethered poly(amido-amine)dendrimer in a sandwich-type enzyme-linked DNA sensor (LOD 100 pM) (10), charge transport from electroactive DNA intercalators (with magnetic sample concentration) (LOD 2 pM) (11), the chronopotentiometric detection of micrometer-long indium rod tracer in a DNA sandwich hybridization assay (with magnetic sample concentration) (LOD 2.6 pM) (12), and the anodic stripping voltammetry of silver nanoparticles deposited in a multistep reduction process initiated by a labeled secondary probe (LOD 0.1 fM) (13).…”

Where organometallics and dendrimers merge: the incorporation of organometallic species into dendritic molecules

“…Most of the uses for these ferrocenyl systems exploit their wellstudied redox behavior and some speciality devices have been fashioned in which electrodes with dendrimer coatings for redox sensing of analytes have been built. Some recent reports have included the electrochemical sensing of bio-relevant molecules, such as biotin, DNA, nucleic acids and carbohydrates using electrodes incorporating dendrimers of ferrocene along with a specific analyte binding function [21,22].…”

Label-Free Electrochemical DNA Detection Based on Electrostatic Interaction between DNA and Ferrocene Dendrimers

“…Because a dendrimer partially contains ferrocenes by covalent bonding, an Fc-Den can interact with the electrode via a redox reaction of ferrocenes. [21][22][23] Ferrocenes in a dendrimer can amplify a signal whereas ferrocene molecules attached to oligonucleotide probes act as a redox tag because a single dendrimer contains multiple ferrocene molecules. 17 Under a physiological condition where the pH is 7.4, terminal protonated amine groups in a dendrimer have a positive charge due to the dendrimer's high pKa (of approximately 9.5).…”