Ali A. Ensafi, Elaheh Khoddami, B. Rezaei,

In this work, a poly-orthophenylene diamine substrate is decorated with gold nanoparticles (GNPs) and then with single-stranded DNA (ss-DNA) aptamer immobilized on the surface of a pencil graphite electrode (PGE) for the detection and determination of plasma insulin. In this procedure, a polymer layer is formed on the surface of the graphite electrode using anodic oxidation of orthophenylene diamine monomers. The parameters affecting the biosensor sensitivity including the potential applied to form the polymer layer, incubation time, concentration of immobilized ss-DNA, and the presence of MgCl2 (as salt) are evaluated. Electrochemical impedance spectroscopy and cyclic voltammetry are then used to characterize the biosensor. The biosensor is employed under optimal conditions to obtain figures of merit, which reveal a linear range and a limit of detection of 1.0–1000.0 nmol L−1 and 0.27 nmol L−1, respectively. Finally, the aptasensor is employed for the determination of insulin in real plasma and urine samples to verify its performance and effectiveness.