A New Dual Immunoassay for the Determination of α-Fetoprotein and Carcinoembryonic Antigen Based on Chemiluminescence Signal Amplification by Functional Graphite Oxide

A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies of AFP and CEA were immobilized on magnetic mesoporous silica particles (Fe3O4/SiO2) respectively and prepared as the primary probe (AFP-Ab(1)/Fe3O4/SiO2 and CEA-Ab(1)/Fe3O4/SiO2). In order to improve the detection sensitivity, the current work was focused on the signal amplification by using increased amount of horseradish peroxidase (HRP). Graphite oxide (GO) with functioned carboxyl groups were used to immobilize the HRP labeled antibodies to prepare the secondary probes (AFP-HRP-Ab(2)/GO and CEA-HRP-Ab(2)/GO). The enhanced signals were attributed to a high HRP-tag molar ratio on the surface of GO. Two subminiature quartz flow cells were set upon the photomultiplier (PMT) in parallel. As soon as CEA-Ab(1)/Fe3O4/SiO2 and AFP-Ab(1)/Fe3O4/SiO2 were injected into the corresponding flow cell, they were adsorbed on the inside walls by the bar magnet. Then, the mixed solutions of CEA, AFP, AFP-HRP-Ab(2)/GO and CEA-HRP-Ab(2)/GO were injected into the two flow cells and stopped for 30 min. Based on a sandwich immunoassay format, the HRP tags were retained in the flow cells. Finally, the CL substrates of luminol and H2O2 were controlled to the two parallel flow cells, so the sequential determination of two tumor markers was achieved. Due to the increased amount of HRP on the surface of GO and the increased amount of monoclonal antibodies on Fe3O4/SiO2, the signals were largely amplified. The effects on the dual immunoassay, such as the concentrations of luminol and H2O2, the incubation time, and the pH value of the buffer solution, were investigated in the current work. Under the optimal conditions, typical flow injection CL signals have been obtained with the sandwich multiplexed immunoassay for CEA and AFP. AFP could be detected in the linear ranges 0.005 similar to 0.5 and 0.5 similar to 100 ng/mL. CEA could be detected in the linear ranges of 0.0025 similar to 1.0 and 1.0 similar to 80 ng/mL. The detection limits of AFP and CEA were 5.0 and 2.5 pg/mL, respectively. The proposed method was in favor of reducing sample consumption and facilitating the operation.