Nanosilver-penetrated polyion graphene complex membrane for mediator-free amperometric immunoassay of alpha-fetoprotein using nanosilver-coated silica nanoparticles

A facile and sensitive mediator-free electrochemical immunosensor for detection of alpha-fetoprotein (AFP) was designed by using nanosilver-coated silica nanoparticles (Ag-SiO2) as bionanolabels. To construct such an electrochemical immunosensor, silver ions/single-stranded DNA/graphene nanosheets were initially immobilized on a gold electrode in turn, then silver ions were in situ reduced to silver nanoparticles with the aid of NaBH4, and anti-AFP antibodies conjugated to silver nanoparticles were used. In the presence of AFP analyte, the sandwiched immunocomplex was formed on the electrode surface by using horseradish peroxidase-anti-AFP conjugate-labeled Ag-SiO2 (HRP-anti-AFP-Ag-SiO2) as secondary antibodies. Compared with pure silver nanoparticles, Ag-SiO2 nanocomposites could provide a large room for the immobilization of HRP-anti-AFP, and improve the electrochemical responses of the immunosensor. Meanwhile, the presence of highly conductive graphene nanosheets and silver nanoparticles provided a good pathway for electron transfer. Under optimal conditions, the immunosensor exhibited good electrochemical responses toward AFP ranging from 0.3 to 200 ng/mL with a detection limit (LOD) of 0.05 ng/mL (at 3σ) in pH 6.0 PBS-H2O2 system. Intra- and inter-assay displayed good precisions with coefficient of variation below 9.5%. In addition, the method was evaluated with 23 clinical serum samples, receiving good correlation with results from commercially available electrochemiluminescent analyzer.

Research highlights
► We fabricate a polyion graphene complex membrane-based immunosensing platform for sensitive electrochemical immunoassay of alpha-fetoprotein.
► Nanosilver-coated silica nanocomposites as bionanolabels.
► Graphene nanosheets, single-stranded DNA and silver nanoparticles as matrices.
► Direct electron transfer without electron mediator.
► Analysis of real samples and method comparison.