Study on electrochemical biosensor for immunoassay

Abstract

We report here an enzyme-amplified, sandwich-type immunosensor for detecting the biospecific interaction between an antibody and antigen using an electrocatalytic reaction. We employed biotin/anti-biotin IgG as a model immunosensing pair. Partially ferrocenyl-tethered dendrimer (Fc-D), whose ferrocene moiety acts as an electrocatalyst, was immobilized to the electrode surface by covalently binding between the dendrimer amines and the carboxylic acids of a self-assembled monolayer. The unreacted amines of the immobilized Fc-D were modified with biotin groups to allow the specific binding of goat anti-biotin IgG. Rabbit anti-goat IgG-conjugated alkaline phosphatase was bound to goat anti-biotin IgG to catalyze conversion of p-aminophenyl phosphate monohydrate to p-aminophenol. This product is oxidizedto quinoimide by the reduction of ferrocenium back to ferrocene, producing an electrocatalytic anodic current.Cyclic voltammograms and surface plasmon resonance experiments showed that the binding of nonspecific proteins is not significant on the biotinylated Fc-D surface. We also examined the change in peak current according to the concentration of anti-biotin IgG and found that the detection range of this immunosensing scheme is between 0.1 and 30 μg/mL. Signal amplification is the most important strategy in lowering detection limits of immunosensors, and labels that can generate many signaling molecules per biospecifically bound target have been widely used.To achieve higher signal amplification in electrochemical immunosensors, the enzymatic amplification step has been combined with a further amplification step, like the redox cycling of enzymatically amplified electroactive species. Redox cycling of enzymatically amplified electroactive species has been widely employed for high signal amplification in electrochemical biosensors. However, gold (Au) electrodes are not generally suitable for redox cycling using a reducing (or oxidizing) agent because of the hi...