Studies On The New Enhanced Chemiluminescence System Of Luminol-H₂O₂-HRP-BPB And Its Application In Magnetic Enzyme-Linked Immunoassay

Chemiluminescence (CL) has become a more and more popular analytical technique owing to its simple instrumentation, high sensitivity, wide dynamic range, reproducibility, simplicity and rapidity. This thesis studied a novel CL enhancer, bromophenol blue (BPB), in the system of Luminol-H₂O₂-HRP, and proposed a new enhanced CL system of Luminol-H₂O₂-HRP-BPB. Under the optimum conditions, the linear ranges and detection limits of free horseradish peroxidase (HRP) and labeled HRP were determined. This established enhanced CL system was also used to detect carcino enbryonic antigen (CEA) and T4 which integrates this method with magnetic separation technique. Furthermore, this new enhanced CL system was also applied to detect aloha-fetal protein (AFP) using Au nanoparticles (AuNPs) as the HRP-antibody (HRP-Ab) labels. The results showed that this method is of high sensitivity, rapidity and simplicity, and could be widely used in the field of biological analysis.The main jobs of this thesis can be concluded as follows:1. The studies on the new enhanced CL system of Luminol-H₂O₂-HRP-BPBThis thesis studies a new enhanced CL system of Luminol-H₂O₂-HRP-BPB. Under the optimum conditions, the free HRP was detected with a linear range of 1.0×10-11～5.0×10-10 g/mL and a detection limit of 1.25×10-12 g/mL. The labeled HRP was also detected with the detection limit 1000 folds lower than that of direct CL system of Luminol-H₂O₂-HRP and 10 folds of the traditional ELISA absorptionmetric method using 3,3',5,5'-Tetramethylbenzidine (TMB) as the chromogenic agent. The mechanism of this new enhanced CL system of Luminol-H₂O₂-HRP-BPB was discussed preliminarily.2. The detection of CEA and T4 based on the system of Luminol-H₂O₂-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassayCarboxyl-modified magnetic beads were activated by EDC/NHS, and then conjugated monocloning antibody which were endowed with the capacity to capture and detect target antigens. CEA was detected by double antibody sandwish immunoassay method by integrating the magnetic separation technique with this new enhanced CL system of Luminol-H₂O₂-HRP-BPB with the linear range of 1.0～40.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. T4 was detected by competitive immunoassay method with the linear range of 1.0～30.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. Under the optimum conditions, the human serum samples were detected, and the results showed good corresponding relationship with those of ELISA absorptionmetric method.3. The detection of AFP based on the system of Luminol-H₂O₂-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassay using AuNPs as HRP labelsUniformly and excellent dispersivity AuNPs were prepared by water phase synthetical method. Different preparation conditions were chosen, and different size AuNPs were gotten. HRP-labeled AFP antibody were bound on the surface of AuNPs by electrostatic at special pH condition. The optimum concentration of HRP-labeled antibody was 0.6μg/mL based on UV-Vis and fluorescence spectrum. Using AuNPs as HRP labels, AFP was detected by integrating the system of Luminol-H₂O₂-HRP-BPB with magnetic separation. Based on the double antibody sandwich immunoassay method, the linear range of AFP was 0.1～50.0 ng/mL and the detection limit was 0.1 ng/mL, which was 10 folds lower than that of the system without using AuNPs as the labels and 50 folds of ELISA.