A novel experimental methodology based on the unique characteristics of chitosan-branched ferrocene (CS-Fc) and gold nanoparticles (Au NPs) was developed to design a label-free amperometric immunosensor for the sensitive detection of hepatitis B surface antigen (HBsAg) as a model protein. The controllable electrodeposition of CS-Fc solution formed a three-dimensional robust film with good biocompatibility and large surface area for the assembly of Au NPs and further immobilization of hepatitis B surface antibody (HBsAb) on an electrode. The morphologies and electrochemistry of the formed nanocomposite biofilm were investigated by using scanning electron microscopy and electrochemical techniques including cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The HBsAg concentration was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody. The decreased differential pulse voltametric values were proportional to the HBsAg concentration in the range of 0.05-305ngmL(-1) with a detection limit 0.016ng. This would provide an approach for the application of mediator in immunoassays.
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