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

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 selfassembled 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 sequenceselective discrimination between perfectly matched and single-base mismatched target oligonucleotides. The DNA microarray approach has become increasingly important in basic research into the genetics of disease and also in the more practical applications of medical diagnosis and treatment. Various methods have been used to detect sequenceselective DNA hybridization, including optical, 1,2 electrochemical, [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] and piezoelectric transduction techniques. 20,21 Of these techniques, the electrochemical method has attracted particular attention because of its high sensitivity, low cost, and compatibility with microfabrication technology. The recent developments of electrochemical DNA biosensor have been reviewed in several reports. [3][4][5][6] Korri-Youssoufi et al. developed a method for direct, label-free, electrical DNA detection. In this approach, the electrical signal is monitored by changes in the conductivity of conducting polymer molecular interfaces, for example, using DNA-substituted or -doped polypyrrole films. 7 Thorp's group has developed a method for the detection of nucleic acids based on the electrochemical oxidation of guanine in target DNA with the mediator Ru(bpy) 3 3+