Glucose oxidase as a blocking agent-based signal amplification strategy for the fabrication of label-free amperometric immunosensors

An effective electrochemical signal amplification strategy based on enzyme membrane modification and redox probe immobilization was proposed to construct an amperometric immunosensor. L-cysteine@ferrocene functionalized chitosan, which possessed not only efficient redox-activity but also excellent film-forming ability, was coated on the bare glass carbon electrode. Moreover, the thiol groups (-SH) in the ferrocenyl compound were used for gold nanoparticles immobilization via the strong bonding interaction, which could further be utilized for the immobilization of antibody biomolecules with well-retained bioactivities. Finally, glucose oxidase (GOD) as the enzyme membrane was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the excellent electrocatalytic properties of GOD towards glucose, the 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 carcinoembryonic antigen (CEA) was from 0.05 to 100 ng/mL with a detection limit of 0.02 ng/mL (S/N = 3). Moreover, the immunosensor exhibited good selectivity, stability and reproducibility, which provided a promising potential for clinical immunoassay.