In this work, a novel electrochemical immunoassay system is developed for ultrasensitive detection of cancer biomarker, α-fetoprotein (AFP). This immunosensor is constructed by covalent immobilization of polyamidoamine (PAMAM) dendrimer-encapsulated gold nanoparticles (Au–PAMAM) sensing interface on a Au electrode surface, followed by sequential covalent immobilization of ethyleneamineviologen (Vio) electrochemical redox marker and AFP monoclonal antibody (mAb) on the surface of Au–PAMAM. The Au–PAMAM nanocomposite not only led to increase the electrode surface area and accelerate the electron transfer kinetics, but also it could provide a highly stable matrix for the convenient conjugation of biomolecules. Upon immunorecognition of the immobilized AFP to its antibody, the Vio peak current decreased due to the hindered electron transfer reaction on the electrode surface. Through the differential pulse voltammetry (DPV) experiments, it is found that the proposed method could detect AFP antigen at a wide linear range (0.001–45 ng mL−1) and a detection limit down to 130 fg mL−1. The immunosensor exhibited high specificity for AFP detection, extremely short incubation time (5 min), good stability and acceptable reproducibility. Moreover, the fabricated immunosensor could accurately detect AFP concentration in human serum samples demonstrated by excellent correlations with standard ELISA immunoassay. In addition, electrochemical impedance spectroscopy technique was used as an efficient alternative detection system for AFP measurement with detection limit 0.5 ng mL−1 and concentration range up to 40 ng mL−1. The present protocol is shown to be quite promising for clinical screening of cancer biomarkers and point-of-care diagnostics applications.
