Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes

A novel immunoassay protocol for simultaneous electrochemical determination of alpha-fetoprotein (AFP), carcinoembryonic (CEA) and streptococcus suis serotype 2 (SS2) was designed. As standard sandwich-type immunoassay format, three primary antibodies (Ab1), anti-CEA and anti-AFP and anti-SS2, were immobilized via protein A (PA) adsorbed by Nafion modified electrodes, and functionalized graphene sheets (GS), containing abundant gold nanoparticles (AuNPs) and carboxyl group, were used to immobilize secondary antibody@redox-probe so as to act as tracer. Concentration of each analyte was quantitatively related to the reduction peak current of corresponding redox-probe in differential pulse voltammetry (DPV) scan. The resulting immunosensor exhibited high selectivity and sensitivity in simultaneous determination of three analytes. The linear range was from 0.016 to 50 ng/mL for AFP with a detection limit of 5.4 pg/mL, 0.010 to 50 ng/mL for CEA with a detection limit of 2.8 pg/mL and 0.012 to 50 ng/mL for SS2 with a detection limit of 4.2 pg/mL (S/N=3).

► Immunosensor for simultaneous determination of three antigens on identical interface. ► Using PA/Nafion to immobilize primary antibodies. ► Preparing PTCA/Au/GS/redox-probe@Ab2 as tracer. ► The immunosensor exhibited excellent sensitivity and selectivity.