Functionalized electrodes for electrochemical biosensor applications

Abstract

We have developed an enzyme-linked electrochemical method to detect biomolecular recognitions including protein-ligand interaction and DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of biomolecular recognition as well as a building block to immobilize biomolecules. Alkaline phosphatase was acted as a generator of electroactive species for the electrochemical detection. The biospecific interaction of avidin to biotin-functionalized Fc-D layer was detected by the subsequent association with biotinylated alkaline phosphatase, which led to the conversion of electroinactive substrates to electroactive products by the enzymatic reaction. The electroactive products diffuse near the layer of Fc-D. When the ferrocene is electrochemically oxidized, the electrooxidized ferricenium ion accepts electrons from enzymatic products and returns to the ferrocene of the reduced form. In other words, the electrocatalytic reaction occurs, which results in enhancing the electrochemical response of enzymatic product. Using electrochemical quartz crystal microbalance (EQCM), the mass change of enzymatic substrates such as paminophenyl phosphate and a-naphthyl phosphate was simultaneously investigated during each electrochemical oxidation. Detection limit of this method is about 0.3 ㎍/mL of avidin. Furthermore, the alternating multilayered assembly composed of avidin and biotinylated alkaline phosphatase was constructed and investigated with surface plasmon resonance and cyclic voltammetry experiments. In DNA hybridization sensing, thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bi-functional linker. The target DNA was hybridized with the capture probe and an extension in the DNA of target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate th...