Multiplexed electrochemical immunosensing of obesity-related hormones at grafted graphene-modified electrodes

An electrochemical immunosensor was prepared for the simultaneous determination of two hormones, ghrelin (GHRL) and peptide YY (PYY), which play important roles in the regulation of hunger and saciety. Dual screen-printed carbon electrodes modified with reduced graphene oxide (rGO) were used as scaffolds for the immobilization of the corresponding capture antibodies. Grafting of the diazonium salt of 4-aminobenzoic acid (4-ABA) on the modified electrode surfaces allowed covalent immobilization of antibodies. Competitive immunoassays were employed and the affinity reactions were monitored by differential pulse voltammetry upon addition of 1-naphthyl phosphate. Under the optimized working conditions, linear current vs. log [hormone] plots extending between 10−3 and 100 ng/mL GHRL (r2 = 0.990), and 10−4 and 10 ng/mL PYY (r2 = 0.992) were obtained. These ranges are adequate for the determination of both hormones at clinical levels in serum and saliva. An excellent analytical performance in terms of detection limit, reproducibility of the measurements, storage stability and selectivity against other proteins was achieved. The usefulness of the approach was demonstrated by its application to spiked human serum and saliva.