MINISTRY OF EDUCATION AND TRAINING HANOI UNOVERSITY OF TECHNOLOGY AND SCIENCE INTERNATIONAL TRAINING INSTITUTE FOR MATERIALS SCIENCE --------------------------------------- TRAN QUANG THINH FABRICATION OF IMMUNOSENSOR FOR DETECTION OF POULTRY VIRUS MASTER THESIS OF MATERIALS SCIENCE Batch ITIMS-2014 SUPERVISOR Assoc. Mai Anh Tuan Dr. Nguyen Hien Hanoi – 2016 17051113936031000000 CONTENTS LIST OF ABBREVIATIONS. 3 LIST OF TABLES.
4 LIST OF FIGURES. IMMUNOSENSOR AND IMMUNE REACTION. Biosensor and immunosensor. Indirect and direct immunosensor.
Structure of antibody. The principle of antibody-antigen interaction. Monoclonal and polyclonal antibody. Immunoglobulin IgG and IgY.
FABRICATION OF IMMUNOSENSOR. Antibody Immobilization Approaches. Fabrication of electrochemical sensor based on gold thin film electrodes. Main processes in the electrochemical sensor fabrication.
Antibody Immobilization using PrA/GA approach. Antibody Immobilization using SAM/NHS approach. DETECTION OF NEWCASTLE DISEASE VIRUS USING ELECTROCHEMICAL IMMUNOSENSOR. Characteristics of electrochemical sensor.
Characteristics of PrA-GA immunosensor. Cyclic voltammetry characterization of PrA-GA immunosensor. Effect of the IgY concentration on the immobilization of PrA-GA immunosensor. Characteristics of SAM-NHS immunosensor.
Cyclic voltammetry characterization of SAM-NHS immunosensor. Effect of the pH value on the immobilization of SAM-NHS immunosensor. Stability of the signal of ND virus immunosensors. Detection of Newcastle disease virus.
Effect of the immunoreaction time. Sensitivity of Newcastle disease virus immunosensor. 74 2 LIST OF ABBREVIATIONS BSA Bovine serum albumin CDR Complementarity-determining region CE Counter electrode CV Cyclic Voltammetry DCC N,N'-Dicyclohexylcarbodiimide DNA Deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EID50 50 Percent Embryo Infectious Dose EIS Electrochemical Impedance Spectroscopy GA Glutaraldehyde IgG Immunoglobulin G IgY Immunoglobulin Y LOD Limit of detection LOQ Limit of quantification ND Newcastle Disease NDV Newcastle Disease virus NHS N-hydroxysuccinimide PCR Polymerase chain reaction PrA Protein A RE Reference electrode TGA Thioglycolic acid SAM Self-assembled monolayer SD Standard deviation WE Working electrode 3 LIST OF TABLES Table 1.1 Properties of immunoglobulin classes Table 2. Sputtering parameters Table 3.
The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3. Experimental conditions for the attachment of components Table 3. The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3. Experimental conditions for the attachment of components Table 3.
The average and standard deviation of I peak of sensors Table 3. The crucial parameters obtained from the calibration Table 3. Comparison of analytical properties of different immunosensors for the detection of Avian Influenza 4 LIST OF FIGURES Figure 1. The performing principle of electrochemical immunosensor Figure 1.
Direct and Indirect immunosensor Figure 1. (A) Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab [18], (B) The schematic description of the structure of an IgG antibody, (C) The domain structure of an IgG antibody Figure 1. X-ray crystallography of the interactions between Fab of 1C1 antibody and EphA2 antigen Figure 1. Non-covalent bonds in the antigen-antibody interaction Figure 1.
The structural difference between IgG and IgY Figure 2. Different orientations of the antibody immobilized on the substrate Figure 2. Pre-treated substrate with maleimide and antibody immobilization by thiol groups Figure 2. Covalent attachment through carbohydrate residues of antibody Figure 2.
Biotinylation of antibody by NHS reagent Figure 2. Avidin-biotin affinity for immobilization Figure 2. Protein A/G-mediated bio-affinity immobilization Figure 2. ssDNA-antibody conjugation to form a hydrazone linker Figure 2.
Structure of the integrated electrode Figure 2. Photomask design and detailed structure of electrode sensor Figure 2. Main processes for sensor fabrication Figure 2. Image of electrochemical sensors on a wafer and a complete sensor Figure 2.
Electrochemical cleaning and activation of electrodes in sulfuric acid by CV Figure 2. The schematic description of the fabrication procedures of PrA-GA immunosensor 5 Figure 2. The schematic of antibody immobilization process using SAM-NHS Figure 3. CV curves of sensor with commercial Ag/AgCl RE and Ag/AgCl wire Figure 3.
The uniform of sensors Figure 3. The reaction of GA linker with protein A and IgY antibody Figure 3. CV characterization of modified electrode recorded on Au electrode Figure 3. Effect of the antibody concentration Figure 3.
The main reactions on the antibody immobilization Figure 3. CV characterization of modification of WE Figure 3.