A high sensitive single luminophore ratiometric electrochemiluminescence immunosensor in combined with anodic stripping voltammetry

Herein, we report a high sensitive ratiometric electrochemiluminescence (ECL) method for carcinoembryonic antigen (CEA) determination. Porous g-C3N4 nanosheets were immobilized on the surface of conductive polymer film doped with carbon nanotubes on glassy carbon electrode and used as ECL luminophore. Signal amplification was achieved by using CuS nanoparticles conjugated second antibody. After the immune recognition, the tag of CuS nanoparticles was dissolved and Cu(II) was electrochemically reduced to Cu on the surface of modified electrode at the potential of - 0.3 V (vs. Ag/AgCl), enhancing greatly the sensitivity. The cathodic ECL of g-C3N4 generated with K2S2O8 as the co-reactant was firstly measured and used as the reference signal in the ratiometric measurement. Afterwards, the anodic ECL of g-C3N4 was measured as the sensing signal with triethanolamine as the co-reactant. During the anodic ECL process, the pre-deposited Cu was stripped to Cu(II), which can quench the ECL signal with higher efficiency due to the short distance of g-C3N4 and Cu(II) within the Helmholtz layer on the electrode surface. In this ratiometric method, the amount of Cu(II) was determined to quantify the concentration of CEA in the range of 1 fg mL−1 to 0.1 ng mL−1, with the limit of detection of 0.3 fg mL−1, which is lower than that of 3 fg mL−1 in signal-on ECL immunesensor using g-C3N4 as the tag. The combination of biometallization and anodic stripping voltammetry with ratiometric ECL can enhance significantly the sensitivity and reliability in immunosensors.