This paper discussed the novel and sensitive chemiluminescence immunoassay biosensors integrating with the chemiluminescence technique and nanotechnique. The CL reaction conditions and the performance of the biosensors were studied by using chemiluminescence assay, spectral analysis, and electrochemical methods. The novel and sensitive chemiluminescence immunoassay (CLIA) has been developed by employing the new chemiluminescence (CL) enhancers based on colloidal gold nanoparticles (AuNPs) modified with HRP-labeled antibody (or antibody) for further signal amplification. In summary, ease of fabrication, a low cost and high sensitivity make the proposed biosensors in this study may provide an interesting alternative tool for detection protein in clinical laboratory.The main jobs of this thesis can be concluded as follows:1. The study on the new enhanced CL system of luminol-H2O2-horseradish peroxidase -4-(1,2,4-triazol-1-yl)phenol and its applicationsThis study described the employment of a novel p-phenol derivative 4-(1,2,4-triazol-1-yl)phenol (TRP) as a highly potent signal enhancer of the luminol-H2O2-HRP CL system. The CL reaction conditions were optimized and its enhancement characteristics were compared with that of p-iodophenol (PIP). Under the optimized conditions, the dynamic range of HRP by using TRP as an enhancer was 8.0×10-10 g/mL-5.0×10-8 g/mL, with the detection limit of 5.0×10-10 g/mL, compared with 4-iodophenol (PIP). TRP is a more potent enhancer for the luminol chemiluminescence. TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of TRP has been discussed. The developed system was then applied to the determination of H2O2 with immobilized HRP using magnetic beads as a solid support. The linear range for H2O2 was 2.0×10-6 mol/L-1.0×10-3 mol/L. The detection limit for H2O2 was 2.0×10-6 mol/L.2. The studies on the new proenhanced CL system of luminol-H2O2-HRP- p-[1,2,4]-triazole-phenol phosphate monoesterIn this work, a novel proenhancer, p-[1,2,4]-triazole-phenol phosphate monoester (TAPM) was synthesized by the PClO3 method. Under the enzymatic hydrolysis of the alkaline phosphatase (ALP), TAPM produced a strong proenhancement on the luminol-H2O2-HRP chemiluminescence system. The reaction conditions of luminol-H2O2-HRP-TAPM-ALP chemiluminescence system were optimized, and the system was applied to the determination of ALP. The chemiluminescence intensity was linear to the concentration of ALP in the range of 1.0μg/L-100μg/L, with a detection limit of 1.0μg/L (S/N=3).3. The study on the new enhanced CL system of luminol-H2O2-HRP-4-(4′-iodo)- phenyl-phenol and its applicationsIn this work, a novel enhancer, 4-(4′-iodo)phenylphenol (IPP) was employed in the luminol-H2O2-HRP CL system. The optimized CL reaction conditions and the evaluation of its enhancing capabilities in the luminol-H2O2-HRP CL system were described. Under the optimized conditions, the dynamic range of HRP by using PIP as an enhancer was 5.0×10-11 g/mL-4.0×10-8 g/mL, with the detection limit of 9.0 pg /mL. The results showed that IPP would be a more potent enhancer than PIP, which was the most widely used enhancer. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of IPP has been discussed. AFP was chosen to prove the novel CL immunoassay as a typical model. The new CL immunoassay involved a binding event between carboxylic acid coated MBs and anti-AFP, and the formation of sandwich immunocomplexes between the MBs and anti-AFP-HRP. The concentration of AFP was monitored based on the HRP label activity toward the oxidation of luminol, which was quantified by CL method. The corresponding calibration plot of relative CL intensity versus the concentration of human AFP was linear over the range from 0.5 ng/mL to 5.0 ng/mL, and the detection limit was 0.5 ng/mL, which was lower than that in the ELISA. The feasibility of the immunoassay system for clinical applications was investigated by analyzing several real samples, in comparison with the ELISA method. The calibration curve indicated that there was no significant difference between the results given by two methods. 4. The study on the new enhanced CL system of luminol-H2O2-HRP-bromophenol blue and its applicationsIn this work, a novel enhancer, bromophenol blue (BPB) was employed in the luminol-H2O2-HRP CL system. The optimized CL reaction conditions and the evaluation of its enhancing capabilities in the luminol-H2O2-HRP CL system were described. Under the optimized conditions, the dynamic range of HRP by using BPB as an enhancer was 5.0×10-11 g/mL-1.0×10-9 g/mL, with the detection limit of 1.0×10-11 g/mL. The results showed that BPB would be a more potent enhancer than PIP, which was the most widely used enhancer. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of BPB has been studied. After optimizing the CL reaction conditions, this new luminol-H2O2-HRP-BPB CL system was applied to a sandwich-type CLIA based on the magnetic separation. A linear range was obtained when the concentrations of AFP were from 1.0 ng/mL to 50.0 ng/mL, with the detection limit of 0.2 ng/mL. The present method was successfully applied to the determination of AFP in human serum samples. The results indicated that this proposed protocol could be quite promising for the application in immunoassay.5. The studies on the enhanced chemiluminescence immunoassay biosensor by using gold nanoparticles as solid supportsIn this work, we described a sensitive and promising analytical mehtod employing new CL enhancers, BPB or IPP, based on MBs separation and gold nanoparticles (AuNPs) labeling technique. HRP-labeled AFP antibody was first bound on the surface of AuNPs by electrostatic, then the new luminol-H2O2-HRP-BPB (or IPP) CL systems were applied to a sandwich-type CLIA for the determination of AFP based on the magnetic separation. This novel strategy takes advantage of easy magnetic separation by MBs and amplification feature of colloidal gold label along with CL intensity enhanced by BPB (or IPP) for luminol-H2O2-HRP system. A linear ranges of AFP standard solution were 0.1 ng/mL-5.0 ng/mL and 0.008 ng/mL-0.3 ng/mL,respectively. And the detection limits of AFP were 0.01 ng/mL and 5.0 pg/mL, respectivrly. An enhanced chemiluminescence immunoassay method for the sensitive and rapid determination of CEA was proposed in this paper. Firstly, luminol and anti-CEA-antibody were simultaneously bound on the surface of AuNPs by covalence. Secondly, the new luminol-H2O2-HRP-IPP CL systems were applied to a sandwich-type CLIA for the determination of CEA based on the magnetic separation. A linear range of CEA standard solution was 0.5 ng/mL-50.0 ng/mL, with detection limit of 0.5 ng/mL.The proposed methods were also used to determine AFP (or CEA) in human serum samples, and the results were well corresponding to those of conventional ELISA.
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