In principle, either antibodies or an antibody-antigen complexes immobilized on transducer’s surface play the role as a bio-receptor toward a target element another antibody or antigen..
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 Prof Mai Anh Tuan Dr Nguyen Hien Hanoi – 2016 Tai ngay!!! Ban co the xoa dong chu nay!!! 17051113936031000000 CONTENTS LIST OF ABBREVIATIONS LIST OF TABLES LIST OF FIGURES Chapter IMMUNOSENSOR AND IMMUNE REACTION .8 1.1 Biosensor and immunosensor 1.1.1 Electrochemical immunosensor 1.1.1.1 Transducer 10 1.1.1.2 Bioreceptor 12 1.1.2 Indirect and direct immunosensor 12 1.2 Immune Reaction 14 1.2.1 Structure of antibody 14 1.2.2 The principle of antibody-antigen interaction 17 1.2.3 Monoclonal and polyclonal antibody .23 1.2.4 Immunoglobulin IgG and IgY 23 Chapter FABRICATION OF IMMUNOSENSOR 26 2.1 Antibody Immobilization Approaches 26 2.1.1 Physical adsorption 27 2.1.2 Covalent attachment 28 2.1.3 Bio-affinity .32 2.2 Fabrication of electrochemical sensor based on gold thin film electrodes 35 2.2.1 Photomask design .35 2.2.2 Main processes in the electrochemical sensor fabrication .36 2.2.3 Sensor pretreatment 40 2.3 Antibody Immobilization 41 2.3.1 Antibody Immobilization using PrA/GA approach 42 2.3.2 Antibody Immobilization using SAM/NHS approach .43 2.4 Immunoassay Protocol 46 Chapter DETECTION OF NEWCASTLE DISEASE VIRUS USING ELECTROCHEMICAL IMMUNOSENSOR 47 3.1 Characteristics of electrochemical sensor .47 3.2 Characteristics of PrA-GA immunosensor 50 3.2.1 Cyclic voltammetry characterization of PrA-GA immunosensor 50 3.2.2 Effect of the IgY concentration on the immobilization of PrA-GA immunosensor 53 3.3 Characteristics of SAM-NHS immunosensor 54 3.3.1 Cyclic voltammetry characterization of SAM-NHS immunosensor .55 3.3.2 Effect of the pH value on the immobilization of SAM-NHS immunosensor 58 3.4 Stability of the signal of ND virus immunosensors 59 3.5 Detection of Newcastle disease virus .61 3.4.1 Effect of the immunoreaction time 62 3.5.2 Sensitivity of Newcastle disease virus immunosensor .63 CONCLUSION 68 REFERENCE 69 PUBLICATION 74 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 LIST OF TABLES Table 1.1 Properties of immunoglobulin classes Table 2.1 Sputtering parameters Table 3.1 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3.2 Experimental conditions for the attachment of components Table 3.3 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor Table 3.4 Experimental conditions for the attachment of components Table 3.5 The average and standard deviation of I peak of sensors Table 3.6 The crucial parameters obtained from the calibration Table 3.7 Comparison of analytical properties of different immunosensors for the detection of Avian Influenza LIST OF FIGURES Figure 1.1 The performing principle of electrochemical immunosensor Figure 1.2 Direct and Indirect immunosensor Figure 1.3 (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 1.4 X-ray crystallography of the interactions between Fab of 1C1 antibody and EphA2 antigen Figure 1.5 Non-covalent bonds in the antigen-antibody interaction Figure 1.6 The structural difference between IgG and IgY Figure 2.1 Different orientations of the antibody immobilized on the substrate Figure 2.3 Pre-treated substrate with maleimide and antibody immobilization by thiol groups Figure 2.4 Covalent attachment through carbohydrate residues of antibody Figure 2.5 Biotinylation of antibody by NHS reagent Figure 2.6 Avidin-biotin affinity for immobilization Figure 2.7 Protein A/G-mediated bio-affinity immobilization Figure 2.8 ssDNA-antibody conjugation to form a hydrazone linker Figure 2.9 Structure of the integrated electrode Figure 2.10 Photomask design and detailed structure of electrode sensor Figure 2.11 Main processes for sensor fabrication Figure 2.12 Image of electrochemical sensors on a wafer and a complete sensor Figure 2.13 Electrochemical cleaning and activation of electrodes in sulfuric acid by CV Figure 2.14 The schematic description of the fabrication procedures of PrA-GA immunosensor Figure 2.15 The schematic of antibody immobilization process using SAM-NHS Figure 3.1 CV curves of sensor with commercial Ag/AgCl RE and Ag/AgCl wire Figure 3.2 The uniform of sensors Figure 3.3 The reaction of GA linker with protein A and IgY antibody Figure 3.4 CV characterization of modified electrode recorded on Au electrode Figure 3.5 Effect of the antibody concentration Figure 3.6 The main reactions on the antibody immobilization Figure 3.7 CV characterization of modification of WE Figure 3.8 The schematic description of the CV responses of modified electrode Figure 3.9 Effect of pH value of the immobilization of antibody Figure 3.10 The average and the SD of Ipeak of the bare Au electrode Figure 3.11 The schematic description of the ND virus detection mechanism Figure 3.12 Effect of the immunoreaction time Figure 3.13 (A) The CV curves of PrA-GA immunosensor (a) in buffer solution and after assay with (b) 102, (c) 103, (d) 104, (e) 10 5, (f) 106 EID50/mL ND virus (B) the relationship between ΔIpeak and various ND virus concentrations of PrA-GA immunosensor Figure 3.14 The relationship between ΔIpeak and various ND virus concentrations INTRODUCTION Newcastle disease (ND) is one of the most popular infection diseases in poultry that widely spreads in Southern East Asian countries, including Vietnam Its most notable effect is that causes severe economic losses in domestic poultry due to its highly contagion, especially in chicken Over the past years, the conventional qualitative methods (haemagglutination inhibition, agar gel precipitation test and Latex agglutination test) as well as semi-quantitative analysis (enzyme-linked immunosorbent assay and immunofluorescence test) were introduced for clinical diagnosis of ND Although these methods allow effective determinations ND virus in infective samples, which require rather complicated procedures for sample preparations and sophisticated instruments for assays Thus, it is necessary to develop methods that offer a simple, rapid, cost-effective analytical strategy, which can be easily used for applications in contamination studies of ND To investigate infection diseases, the fabrication and application of electrochemical immunosensor have been considerably developed However, most of the works have used monoclonal immunoglobulin G (antibody IgG) from mammalian blood Egg yolk immunoglobulin (IgY) from chickens can be employed as an alternate IgG in immunoassay, which offers some advantages with respect to animal care, high productivity and special suitability in the source of antibodies In our work, electrochemical immunosensor using IgY as receptors in configuration has been developed to detect ND virus This thesis is organized into three chapters: In the first chapter, the basic concepts about immunosensor and fundamental theory of immune reaction will be introduced In the second chapter, the fabrication of electrochemical immunosensor will be described in detail In the last chapter, the characterization of immunosensor carried out with ND virus will be discussed Chapter IMMUNOSENSOR AND IMMUNE REACTION 1.1 Biosensor and immunosensor A biosensor is an analyte device consisting of a biological sensing element attached a signal transducer, which converts signals of the biological reactions into measurable signals [1] The biological sensing element ranges from oligonucleotides (DNA or RAN) to enzymes, proteins, cells, antibodies or antigens Transducer designed on a solid-state substrate that plays a role converting the signals recorded from biological sensing element into measurable signals like the electric signals Biological reactions are able to lead to that include the changing of pH value, electronic or ionic transfer, refraction, luminescence, micro mass or thermal transfer… The biosensors based on antibodies or antigens are known as immunosensors Thus, the four most common kind of immunosensors based on the signal of biological reactions are optical, electrochemical, micro mass and thermal [2] North [3] proposed the first concept of the immunosensor in 1985 in which the bioelement was antibody Recently, the term immnosensors were described as the ones that can convert the specific antibody-antigen interactions into measurable signals In principle, either antibodies or an antibody-antigen complexes immobilized on transducer’s surface play the role as a bio-receptor toward a target element (another antibody or antigen) Most of the immunosensors are designed that based on the two mechanisms such as biological catalysis and biological affinity The biological catalysts are usually enzymes catalyzing for biochemical reactions, while the biological affinity bases on the specific interaction of proteins, lectins, receptors, live cells, nucleic acids, antibodies and antigens [2] The applications of the biosensor and immunosensor comprise a wide range of tasks, ranging from clinical diagnostics, food safety, industrial processes control, pollution monitoring, drug discovery, to military and security applications [4] The interest in the fields of biosensors is reflected directly in its fast rise in the number of publications In 1985, there were approximately 100 papers on this subject and this number rose to 4500 in 2011 Furthermore, the papers published in 2011 alone represented more than 10% of all articles ever published concerning the biosensors This upward trend can also be seen in the global market for biosensors which increased from billion US dollars market share in 2000 to 13 billion dollars and predictions for 2018 show figures around 17 billion dollar mark [5] 1.1.1 Electrochemical immunosensor According to the IUPAC suggestion of definition for electrochemical biosensors [6], an immunosensor is an integrated device consisting of an immunochemical recognition element in direct spatial contact with a transducer element Electrochemical immunosensors employ either antibodies or their complementary binding partners, i.e antigens or haptens as biological recognition elements in combination with electrodes or field-effect transistors Advantage of this kind of immunosensor ranges from low sample consumption, reasonable cost of instrumentations to miniaturization possibility, which are the main reasons for extensive development of electrochemical immunosensors Figure 1.1 The performing principle of electrochemical immunosensor