Amplified electrochemical immunoassay for 5-methylcytosine using a nanocomposite prepared from graphene oxide, magnetite nanoparticles and β-cyclodextrin

A nanocomposite was prepared from beta-cyclodextrin (beta-CD) functionalized graphene oxide and magnetic nanoparticles (GO/Fe3O4/beta-CD). In parallel, a polyamidoamine dendrimer conjugated to avidinylated alkaline phosphatase (PAMAM-avidin-ALP) was prepared and exploited as a signal amplification unit in a voltammetric immunoassay for 5-methylcytosine (5mC) in genomic DNA. The GO/Fe3O4/beta-CD as a substrate material exhibited good solubility, electrical conductivity and large surface. This is beneficial for the further modification of antibodies (Ab) by host-guest interaction and amide bonds. By taking advantage of three-dimensional structure to capture avidin-ALP by amide linkages, PAMAM was used as a catalytic signal amplification element in this assay. Under the optimized condition and at a typical working potential of 0.94V, the response to 5mC is linear in the 0.01-50nM concentration range with a detection limit of 3.2 pM (atS/N=3). The method is stable, selective and reproducible. It was applied to the determination of5mC in genomic DNA of human tissue.