Bi-enzyme synergetic catalysis to in situ generate coreactant of peroxydisulfate solution for ultrasensitive electrochemiluminescence immunoassay

A novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of α-1-fetoprotein (AFP) was designed based on the in situ bi-enzymatic reaction to generate coreactant of peroxydisulfate for signal amplification. In this work, AuNPs were electrodeposited on the glassy carbon electrode (GCE) surface, which promoted the electron transfer. Then, L-cysteine and another layer of AuNPs were, respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the ECL signal of peroxydisulfate and immobilizing antibody. At last, glucose oxidase (GOD) and horseradish peroxidase (HRP) were employed to block the nonspecific binding sites. When proper amounts of glucose were added in the detection solution, GOD catalyzed the oxidation of glucose to generate H2O2, which could be further catalyzed by HRP to generate O2 for the signal amplification. The linear range for AFP detection was 0.001–100ngmL−1, with a low detection limit of 3.3×10−4ngmL−1. The novel strategy has the advantages of simplicity, sensitivity, good selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection. ► We developed a novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of α-1-fetoprotein (AFP) based on the in situ bi-enzymatic reaction to generate coreactant of peroxydisulfate for signal amplification. ► L-Cysteine and AuNPs were used to improve the ECL signal and the absorption capacity of antibody. ► GOD and HRP were adopted to in situ generate O2 which is the coreactant of peroxydisulfate system. ► The novel strategy has the advantages of simplicity, sensitivity, good selectivity and reproducibility.