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Studies on the New Enhanced Chemiluminescence System of Luminol-H2O2-HRP-BPB and Its Application in Magnetic Enzyme-Linked Immunoassay

Author: BiSai
Tutor: YangXiuYing；ZhangShuSheng
School: Qingdao University of Science and Technology
Course: Analytical Chemistry
Keywords: Chemiluminescence Enhancer Immunoassay Magnetic microspheres Gold Nanoparticles Tumor markers
CLC: Q503
Type: Master's thesis
Year: 2008
Downloads: 265
Quote: 1
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Abstract


The chemiluminescence method due to the equipment is simple, rapid analysis, high sensitivity, wide linear range, more and more extensive attention from scientists. The new enhancer bromophenol blue (BPB) and the enhanced role of Luminol-H 2 O 2 -HRP chemiluminescent reaction Luminol-H 2 O 2 -HRP-BPB Chemiluminescence System. Free HRP and HRP markers were measured under optimal experimental conditions, the linear range and detection limit. This the chemiluminescence enhancement system with magnetic separation technology and enzyme-linked immunoassay combined, carcinoembryonic antigen (CEA), thyroxine (T4) and other tumor markers were measured. In addition, the use of gold nanoparticles as the enzyme-labeled antibody marker the chemiluminescence enhancement system for detection of alpha-fetoprotein (AFP). The method is sensitive, rapid, simple and has broad application prospects in biological analysis. Work mainly in the following aspects: a Luminol-H 2 O 2 -HRP-of the BPB Enhanced Chemiluminescence research study Luminol-H < sub> 2 O 2 -HRP-BPB enhanced chemiluminescence reaction system enhancement. Of free HRP determined the optimal experimental conditions, the linear range of the determination of free HRP is 1.0 × 10-11 ~~ 5.0 × 10-10 g / mL, the detection limit of 1.25 × 10-12 g / mL; and HRP markers were determined, the detection limit of 2 -HRP direct chemiluminescence system 1000 times lower than the luminol to-H 2 O color than tetramethylbenzidine ELISA Act 10 times lower. And Luminol-H 2 O 2 -HRP-the BPB enhanced chemiluminescence system mechanism was discussed. Second, based on the Luminol-H 2 O 2 -HRP-BPB enhanced chemical hair magneto-optical enzyme immune system determination of CEA and T4 carboxyl-modified magnetic microspheres, after EDC / NHS activated magnetic microspheres obtained after the activation treatment, the AFP monoclonal antibody fixed to the activation of the surface of the magnetic microspheres, prepared by immunization of magnetic microspheres having the ability to recognize and capture the corresponding antigen. Magnetic bead separation technology with Luminol-H 2 O 2 -HRP-the BPB enhanced chemiluminescence system combined, carcinoembryonic antigen (CEA) and serum total thyroxine (T4) were measured. Determined by double antibody sandwich method of CEA, CEA measured linear range of 1.0 ~~ 40.0 ng / mL, the detection limit of 1.0 ng / mL, and photometric five times lower than the the tetramethyl benzidine ELISA color; using competition law in the determination of the T4 the measured T4 linear range of from 1.0 ~~ 30.0 ng / mL, the detection limit of 1.0 ng / mL, five times lower than tetramethylbenzidine ELISA spectrophotometry. The method is simple, fast, and on the patient serum samples were measured by the established method, Spectrophotometric and ELISA (ELISA) were both good correlation. Third, based on nanoparticles labeled Luminol-H 2 O 2 -HRP-BPB photomagnetic immune system enhanced chemiluminescence determination of AFP trisodium citrate Restore prepared gold nanoparticles by controlling the added amount of reducing agent is tri-sodium citrate, obtained different particle diameter of the gold nanoparticles. HRP-labeled by the electrostatic interaction of alpha-fetoprotein (AFP) antibody connected to the gold nanoparticles. UV - the visible the optimal concentration of antibodies required for absorption and fluorescence spectra of gold nanoparticles with antibodies connection to 0.6μg/mL. To gold nanoparticles as enzyme markers, magnetic separation technology with Luminol-H 2 O 2 -HRP-the BPB enhanced chemiluminescence system combination, a double antibody sandwich method, AFP were measured, and compared with a method of enzyme-labeled antibody is added directly. The enzyme antibody labeling method for determination of the linear range of the AFP for 1.0 ~ 50.0 ng / mL, the detection limit of 1.0 ng / mL; gold nanoparticles as the enzyme-labeled antibody marker for determination of the AFP the linear range of of 0.1 ~~ 50.0 ng / mL, detection limit of 0.1 ng / mL, spectrophotometry 50 times lower than the the tetramethyl benzidine ELISA color. With the established methods to the patient serum samples were measured with the ELISA method (ELISA) were good correlation.

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CLC: > Biological Sciences > Biochemistry > General issues > Biochemical techniques
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