▎ 摘　　要

Herein, a novel electrochemical bi-aptasensor using green synthesized reduced graphene-poly(amidoamine)/gold nanocomposite (rGO-PAMAM/Au-nano) as supporting matrix for covalent immobilization of flavin adenine dinucleotide (FAD) and thionine (Th) on the first (W-1) and second (W-2) surfaces of dual working electrode screen printed electrode has proposed. FAD and Th probes were immobilized by glutaraldehyde linker based on a reaction between primary amines (-NH2) of FAD and Th with -NH2 of rGO-PAMAM/Aunano modified dual working electrode for simultaneous determination of cytochrome c (CYC) and vascular endothelial growth factor (VEGF(165)) tumor markers. The principle response of proposed bi-aptasensor was based on the selective interaction of CYC with W-1/rGO-PAMAM-FAD/Au-nano/Anti-ptamer(CYC) and VEGF(165) with W-2/rGO-PAMAM-Th/Au-nano/Anti-ptamer(VEGF165),, respectively. This selective interaction, leads to decrease of response currents of immobilized electrochemical probes of FAD and Th that have different formal potential (E-0'). Electrochemical behavior of bi-aptasensor was studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The liner calibration curve for tumor markers determination was 2.5-320.0 pM. The limit of detections (LOD) for CYC was 1.0 pM and for VEGF(165) was 0.7 pM (3 sigma/S). The biomarker-aptamer dissociation constant (k(d)) was calculated based on the change in current to be 63.9 pM for CYC/Anti-aptamer(CYC) and 38.4 pM for VEGF(165)/Anti-aptamer(VEGF165), respectively. This bi-aptasensor was used for the determination of VEGF(165) and CYC in the human serum sample. (C) 2016 Published by Elsevier B.V.