Nanogold-enhanced graphene nanosheets as multienzyme assembly for sensitive detection of low-abundance proteins

A new electrochemical immunosensing protocol was designed for detection of carcinoembryonic antigen (CEA, as a model protein) by using graphene-carried poly(o-phenylenediamine)/gold hybrid nanosheets (GNPGs) as signal tags on the hierarchical dendritic gold microstructures (HDGMs)-modified glassy carbon electrode. To prepare the signal tags, poly(o-phenylenediamine) molecules were initially immobilized on the surface of graphene nanosheets via the π-stacking interaction. Then gold nanoparticles were assembled onto the poly(o-phenylenediamine)-modified graphene nanosheets, which were used for the labeling of anti-CEA detection antibodies and horseradish peroxidase (HRP). The as-prepared GNPGs were characterized by using atomic force microscopy (AFM), transmission electron microscopy (TEM) and UV-vis absorption spectroscopy. The assay was carried out with a sandwich-type immunoassay format in pH 5.5 acetic acid-buffered saline solutions containing 2.5 mmol L(-1) H2O2. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of 0.005-80 ng mL(-1) toward CEA standards with a low detection limit of 5.0 pg mL(-1). Intra- and inter-assay coefficients of variation were less than 11.5%. No significant difference at the 0.05 significance level was encountered in the analysis of 6 clinical serum specimens and 6 spiked blank new born cattle serum specimens between the developed immunoassay and commercially available electrochemiluminescent (ECL) method for the detection of CEA.