Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of α-fetoprotein

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with γ-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The α-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-100 ng mL⁻¹. The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.