A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-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-Ab₁/Fe₃O₄/SiO₂ and CEA-Ab₁/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-Ab₂/GO and CEA-HRP-Ab₂/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-Ab₁/Fe₃O₄/SiO₂ and AFP-Ab₁/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-Ab₂/GO and CEA-HRP-Ab₂/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 0.005～0.5 and 0.5～100 ng/mL. CEA could be detected in the linear ranges of 0.0025～1.0 and 1.0～80 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.

Key words: chemiluminescent immunoassay, flow injection analysis, graphite oxide, horseradish peroxidase, signal amplification