Bioelectrocatalytic signaling from immunosensors with back-filling immobilization of glucose oxidase on biorecognition surfaces

We report a novel method of electrochemical signaling from antigen–antibody interactions at immunoelectrodes with bioelectrocatalyzed enzymatic signal amplification. For the immunosensing surface construction, a poly(amidoamine) G4‐dendrimer was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization. As a model biorecognition reaction, the dinitrophenyl (DNP) antigen‐functionalized electrode was fabricated and an anti‐DNP antibody was used. Glucose oxidase (GOX) was chosen to amplify electrochemical signal by enzymatic catalysis. The signal amplification strategy introduced in this study is based on the back‐filling immobilization of biocatalytic enzyme to the immunosensor surface, circumventing the use of an enzyme‐labeled antibody. The non‐labeled native antibody was biospecifically bound to the immobilized ligand, and the activated enzyme (periodate‐treated GOX) reacted and “back‐filled” the remaining surface amine groups on the dendrimer layer by an imine formation reaction. From the bioelectrocatalyzed signal registration with the immobilized GOX, the surface density of biospecifically bound antibody could be estimated. The DNP functionalization reaction was optimized to facilitate the antibody recognition and signaling reactions, and ≈6% displacement of surface amine to DNP was found to be an optimum. From quartz crystal microbalance measurement, immunosensing reaction timing and the surface inertness to the nonspecific biomolecular binding were tested. By changing the surface functionalization level of DNP in the calibration experiments, immunosensors exhibited different dynamic detection ranges and limits of detection, supporting the capability of parameters modulation for the immunosensors. For the anti‐DNP antibody assay, the fabricated immunosensor having 65% functionalization ratio exhibited the linear detection range of 10−4 to 0.1 g/L protein and a limit of detection around 2 × 10−5 g/L. © 2005 Wiley Periodicals, Inc.