Dual amplification strategy for the fabrication of highly sensitive amperometric immunosensor based on nanocomposite functionalized interface

An effective electrochemical signal amplification strategy based on bilayer enzyme membrane modification and redox probe immobilization was proposed to construct an amperometric immunosensor. At first, porous deposited gold nanocrystals (DpAu) film was performed on bare glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl4) solution and then Prussian blue (PB) was electrodeposited on the surface of DpAu film, which acted not only as redox probe but also as the first amplification membrane layer. Following that, gold colloidal nanoparticles doped chitosan–iron oxide nanocomposite was coated on the surface of PB/DpAu film to avoid the leakage of PB and assemble antibody biomolecules. Finally, horseradish peroxidase (HRP) as the second enzyme membrane was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the excellent electrocatalytic properties of PB and HRP to the reduction of hydrogen peroxide (H2O2), the dual amplification of antigen–antibody interaction and the enhanced sensitivity could be achieved. Under the optimal conditions, the linear range of the proposed immunosensor for the determination of α-1-fetoprotein (AFP) was from 0.05 to 300 ng/mL with a detection limit of 0.02 ng/mL (S/N = 3). Moreover, the immunosensor exhibited good selectivity, stability, reproducibility and regeneration.