Incubation-free electrochemical immunoassay for diethylstilbestrol in milk using gold nanoparticle-antibody conjugates for signal amplification

We report on a novel enzyme-enhanced label for the electrochemical determination of diethylstilbestrol (DES). The label was obtained by orientation-controlled immobilization of a multiplex horseradisch peroxidase (HRP) conjugated polymer on gold nanoparticles (AuNPs) using the Envision reagent (EV) which is an enzyme-polymer complex that contains HRP and anti-IgG antibody in a polydextrin amine skeleton. The AuNPs were modified with Concanavalin A (Con A) and served as a carrier for immobilization of the EV?DES antibody composite. This resulted in a bioconjugate of the type AuNP?Con A?EV?DES Ab which was employed as the label. On exposure to samples containing DES, a sandwich immunocomplex is formed between antibody against DES (which was immobilized on a glassy carbon electrode and is acting as a capture probe), DES (the analyte), and the above label as the signal tracer. Hemin was used as an electronic mediator in the reaction of HRP. The HRP on the label catalyzes the oxidative formation of hydrogen peroxide at pH 7.0, and this induces an increased reductive current in the presence of hemin as an electron mediator. Under optimal conditions, the current increases linearly with increasing concentrations of DES in the range from 5 to 500 pg?·?mL^?1, with a detection limit as low as 2 pg?·?mL^?1 (at an S/N of 3). The method exhibits high selectivity and good stability. It works without incubation so that the time for an assay is shortened to 5 min. The assays was successfully applied to the determination of DES in milk samples.