Fabrication off immunosensor for detection of poultry virus Nghiên cứu chế tạo cảm biến miễn dịch điện hóa để phát hiện virut cúm gia cầm

Fabrication off immunosensor for detection of poultry virus Nghiên cứu chế tạo cảm biến miễn dịch điện hóa để phát hiện virut cúm gia cầm Fabrication off immunosensor for detection of poultry virus Nghiên cứu chế tạo cảm biến miễn dịch điện hóa để phát hiện virut cúm gia cầm luận văn tốt nghiệp thạc sĩ

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

CONTENTS

LIST OF ABBREVIATIONS 3

LIST OF TABLES 4

LIST OF FIGURES 5

Chapter 1 IMMUNOSENSOR AND IMMUNE REACTION 8

1.1 Biosensor and immunosensor 8

1.1.1 Electrochemical immunosensor 9

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 2 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 3 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 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 Ipeak 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) 105, (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 1

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 2 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

The fundamental performance of electrochemical immunosensor is described as shown in Fig 1.1 An electrochemical immunosensor can be classified into three main components, corresponding to the particular roles in its operating principle, namely, antibody or antigen as molecular recognizers, electrodes attached recognizers and performance of a transducer [2].

1.1.1.1 Transducer

Based on the measurement method, the several types of transducer employed

in electrochemical immunosensors field are listed in the following:

+ Potentiometric technique

The fundamental principle of all potentiometric transducers are based on the Nernst equation [7] according to which potential changes are logarithmically proportional to the specific ion activity on the electrodes The signal is measured as the potential difference (voltage) between potentiometric transducer electrodes (working electrodes - WE and counter electrodes - CE) Potentiometric sensors are used to determine the analytical concentration of some components carrying an electrical charge in the analyte

+ Transmembrane potential

This transducer principle is based on the accumulation of a potential across a sensing membrane Ion-selective electrodes (ISE) use ion-selective membranes which generate a charge separation between the sample and the sensor surface Similarly, bioreceptors (antigens or antibodies) immobilized on the membrane binds the corresponding compounds (immune reactions) from the solution at the solid-state surface, which leads to the change the transmembrane potential The electrode measuring pH is the most popular ISE

+ Electrode potential

This transducer is similar to the transmembrane potential sensor However,

an electrode by itself is the surface for the formation of antigen-antibody

complexes, changing the electrode potential in relation to the concentration of the analyte

+ Field-effect transistor (FET)

The FET is a semiconductor device used for monitoring of charges at the surface of an electrode, which have been built up on its metal gate between the so-called source and drain electrodes The surface potential varies with the analyte concentration The integration of an ISE with FET is obtained in the ion-selective field-effect transistor (ISFET) This technique is also highly potential for the applications of immunosensors

+ Conductometric and capacitive technique

These immunosensor transducers measure the alteration of the electrical conductivity in a solution at constant voltage, caused by biochemical reaction (enzymatic activities) which specifically generate or consume ions The capacitance changes are measured using an electrochemical system, in which the bioreceptor is immobilized onto a pair of noble metal electrodes (Au or Pt) For immunosensors,

an ion-channel conductance immunosensor mimicking biological sensory function

is used to record effectively the small signaling immune reactions that are usually lost in the high ionic strength of the solution

1.1.1.2 Bioreceptor

Using antibody or antigen as bioreceptors, also known as immunoassays, is a striking difference in the fundamental basic of immunosensors comparing with other biosensors Generally, conventional electrochemical immunosensors have antibody molecules immobilized on the surface of electrodes, which recognize specific antigen in the sample All types of immunosensor can either operate through direct or indirect way, which are distinguished through using non-labeled antibody or labeled antibody, respectively The direct electrochemical immunosensors are able to detect directly the electrochemical changes during the immune complex formation, while the indirect immunosensors use signal-generating labels attached on antibodies to detect indirectly antigens

1.1.2 Indirect and direct immunosensor

Indirect immunosensor

For immnosensors using the labeled antibody, the most frequently used sandwich-type immunoassay involves a couple of antibodies As shown in Fig.1.2, primary antibodies are usually immobilized on an electrode, and sandwiched immune-complexes are formed among the immobilized antibodies, specific antigens and labeled antibodies The detectable signal mainly depends on labeled signal tags, thus, a great scientific effort has been devoted for developing effective labels Enzymes and redox-labels are electrochemical active labels which are used widely

in indirect electrochemical immunosensors, especially amperometric immunosensors [2] Several enzymes are prominent such as alkaline phosphatase [8], horseradish peroxidase , β –galactosidase [9], cholinesterase [10] and glucose oxidase [11], while ferrocene derivatives or In2+ salts [12], redox polymers (e.g., polymer [PVP-Os(bipyridyl)2Cl]) [13] are known as notable redox-labels

Figure 1.2 Direct and Indirect immunosensor

Direct immunosensor

Although indirect electrochemical immunosensors are highly sensitive due to the activation of labels, they have also some weak points First of all, their fabrication involves numerous steps, which make experiments more complicated In addition, the antigen concentration is not measured in a direct way, but rather based

on the signal generated by label Therefore, electrochemical immunosensors based

on immunoassays using non-labeled antibodies (label-free antibodies) are preferably chosen for developments in applications To work label free is very

attractive, especially for the development of in vitro immunosensors since in allows

real-time measurement without any additional hazardous reagents In the 1970s, Janata observed a potential change on an immunosensor that was fabricated by covering PVC membrane-immobilized Concanavalin-A antibody (ConA) on a potentiometric electrode [14] This work is referred the first direct electrochemical immunosensor that was possible to follow the binding process directly in real time without any labeling For the detection of antibodies, in 1984, Keating [15] modified an electrode upon a dioxin-ionophore antigen conjugate with a PVC membrane, which was used in the determination of anti-dioxin antibodies

As the mention above, amperometric immunosensors are considered unsuitably for the detection directly immune components, which should be employed with enzyme or redox labels However, in 2003, the report of Hu [16] that gold nanoparticles modifying anti-paraoxon antibodies were used on a glassy carbon electrode to detect directly paraoxon by the cyclic voltammetry measurement This detection limit at 12 μg/L was a quite significant result for the initial amperometric immunosensors without needing any labels

Over forty years, electrochemical immunosensors followed a direct way have developed considerably Most of the studies have focused on the improvement materials in the components of electrochemical immunosensors such as electrodes, immobilized substances and electrolytes Moreover, analytical objects have been extended progressively, which range from infectious viruses in human or animals, antigens on cancer cells, pathogens to toxic substances

1.2 Immune Reaction

1.2.1 Structure of antibody

Antibodies, also known as immunoglobulins (Ig), are glycoprotein molecules produced by white blood cells in the immune system of vertebrates They play an essential role as a critical part of the immune response by specifically recognizing to particular antigens An antigen is any harmful substance that causes the production

of an antibody, such as bacteria, fungi, parasites, viruses and chemicals High specificity and affinity are the most striking characteristics of the antibody-antigen interaction The structural and energetic aspects of antibody-antigen binding are significant to understand how an antibody specifically recognizes its corresponding antigen

Structure of antibody

As shown in Fig 1.3, the typically structure of an antibody molecule (immunoglobulin G - IgG) comprises four polypeptide chains linked together by disulfide bonds (S-S), that divided into two identical light chains (L chains) and two

identical heavy chains (H chains) The light chain has two different isotypes, kappa (κ) chain and lambda (λ) chain, which distributed according to different genes in chromosomes in mammals There are five classes of immunoglobulins (IgG, IgA, IgM, IgD and IgE), which differ in amino acid sequence and number of domains in the constant regions of the heavy chains (CH) In which, immunoglobulin G (IgG) is the major type of immunoglobulins (approximately 75% in all of the normal serum), that is the most extensively investigated

Table 1.1 Properties of immunoglobulin classes [17]

L-chain type κ or λ κ or λ κ or λ κ or λ κ or λ Structure H2L2 (H2L2)5

+J chain

H2L2 and (H2L2)2 + SC

+ J chain

H2L2 H2L2

Molecular weight 150×103 950×103 160×103 180×103 190×103Carbohydrate (%) 3 12 8 10 12

Approximate

concentration in serum

(mg/ml)

H: heavy chain; L: light chain; J: Joining chain; SC: secretory component

In addition, by using the suitable enzymes for hydrolysis of peptide bonds,

an immunoglobulin monomer is cleaved into three fragments In which, two of the three fragments are identical which retain the ability to bind antigen, that are called

the Fab fragments (fragments of antigen binding) The third fragment called the Fc fragment (fragment crystallizable) contains carbohydrate chains, which does not

bind antigen

The remarkable feature of the antibody molecule is showed by comparison of amino acid sequences from various immunoglobulin molecules This shows that immunoglobulin is composed of various copies of a folding unit of about 110 amino

acids, each of which forms an independent similar structure called the immunoglobulin fold The N-terminal (NH2 terminal) domain of each polypeptide (heavy and light chains) is highly variable, while the remaining domains have constant sequences These domains are called successively the variable region (V region) and the constant region (C region) Furthermore, a comparison of V region sequences shows that variability is not uniformly distributed but concentrated into three areas called the hypervariable regions

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

The investigated structures of various antigen-antibody complexes have demonstrated that the domain structure of the antibody molecule is a β barrel

consisting of nine anti-parallel β strands (V regions) and seven C regions, and that the hypervariable regions are clustered at the end of the variable domain arms, Fig 1.3C The antigen-combining site of antibodies is formed almost entirely by six polypeptide segments, three from light variable domains and three from heavy variable domains These segments show variability in sequence as well as in number of residues which refer single amino acid units making up protein chains This variability provides the basis for the diversity found in the binding characteristics of the different antibodies These six hypervariable segments are often called the complementarity-determining regions or CDRs Thus, the antigen-binding specific is defined by the physical and chemical properties of a binding surface, which formed by six CDR loops in the antibody Moreover, other parts of

the V region, exclusively the CDRs, are known as the framework regions

1.2.2 The principle of antibody-antigen interaction

An understanding of antigen-antibody interactions, particularly those with protein antigens, plays essential roles for the use of antibodies in clinical diagnosis and therapy Antibody-antigen interaction is a chemical one between relevant antibodies and antigens to form specific antibody-antigen bindings during immune reaction Richard J Goldberg proposed the first correct description of the interaction in 1952 [19] and it is now known as “Goldberg’s theory” of antibody-antigen reaction In the typically immune reaction, each antibody binds to several particular sites of antigen called the antigenic determinants (or epitopes) which include surface configurations, haptenic groups, specific areas and so on In contrast, the molecular determinants within the antibody structure that make specific interactions with the epitopes are often termed paratope Antibody paratopes contain “framework” residues that are amino acid units within protein chains of CDRs

For better understanding the principle of the antibody-antigen interaction in the immunoreaction, it is necessary to base on two following directions: (1)

structural features of the binding in the antibody-antigen complex and (2) kinetic of the antibody-antigen interaction These directions are also known as static and dynamic properties in the formation of specific bindings

The binding in antibody-antigen complex and structure features

Structural studies of specific antibodies and their reactions with antigens became possible after the development of the techniques of cell hybridization for the production of monoclonal antibodies of predefined specificity [20] As the example in Fig 1.4, X-ray crystallography appeared as the most effective technique

of choice to determine the precise sites of the molecular interactions of antibodies with their antigens through crystal structures of complexes between them The complex usually is obtained under the crystalline form of the Fab fragments of antibodies associated with specific antigens Nowadays hundreds of the three-dimensional structures of the antibody-antigen complexes are gained by X-ray crystallography to provide for the immune epitope database Furthermore, the crystal structure determinations of highly specific antibody fragments (Fab) associated protein antigens show also [21]:

(1) Both the L and H chains of antibodies make extensive contacts with antigens, although frequently those made by the H chain are more extensive

(2) The specificity of immuno-reaction is determined by the structure of CDRs on

VH and VL part of antibody; in which, the VH CDR3 encoded by the D (diversity) segment of genome makes important contributions to binding

(3) The contacting residues of the antigen are discontinuous in sequence but form a continuous surface (antigenic determinant or epitope)

(4) The contacting surface of the antibody and antigen often show a high degree of complementarity

(5) The contacting surface areas of the antibody-antigen interaction are about 600 to

900 Å2

(6) The formation of multiple bonds by non-covalent interactions as van der Waals forces, hydrogen bonds, electrostatic forces and hydrophobic forces provides stability to antibody-antigen complexes

(7) A large proportion of CDR aromatic residues are appeared in the contacts with antigen

Figure 1.4 X-ray crystallography of the interactions between Fab of 1C1 antibody

and EphA2 antigen [22]

(a) Three-dimensional view of the Fab 1C1/EphA2 complex Fab 1C1 heavy chain and light chain are shown in magenta and beige, respectively Human EphA2 ligand binding domain (LBD) is shown in cyan (b) Stereographic representations of the intermolecular contacts between human EphA2 and Fab 1C1 CDRH3 Fab 1C1 and human EphA2 are shown in magenta and cyan, respectively Nitrogen and oxygen atoms are shown in blue and red, respectively The corresponding interface includes several hydrogen bonds shown as black dotted lines (c) Fab 1C1 CDRH3 penetrates into a channel of the EphA2 molecule via its predominantly hydrophobic tip Sulphur atoms are shown in yellow, whereas the rest of the colour code is identical to that in (b) (d) The maximum likelihood weighted 2mFo-DFc electron density map is shown around the area

of Fab 1C1 CDRH3 penetration into EphA2 Colour code is identical to that in (b)

In addition, the forces joining the antibody-antigen complex which also called “weak interaction” are not strong covalent bonds, as shown in Fig 1.5 The contribution of each of these forces to the overall interaction depends on the particular antibody and antigen involved A striking difference between antibody-

antigen interactions and most other natural protein-protein interactions is that CDRs

of the antibodies contain many aromatic residues, for examples tyrosine or tryptophan, in their binding sites These residues participate manly in van der Waals and hydrophobic forces, and maybe in hydrogen bonds These forces operate over very short ranges and determine the high complementarity of contacting surface In contrast, electrostatic forces and hydrogen bonds linking oxygen and/or nitrogen atoms between charged residues, for example glutamate or lysine, provide specific features and stability in the antibody-antigen complex Moreover, other parts of the

V region do not play directly in contact with the antigen but they provide a stable structural framework for the CDRs and help determine their position and conformation

Figure 1.5 Non-covalent bonds in the antigen-antibody interaction

Thus, structural features of the antibody-antigen complexes have enucleated

by X-ray crystallographic analysis, that the hypervariable loops (CDRs) of immunoglobulin V regions determine the specificity of antibodies The antibody molecule contacts the protein antigen over an area of its surface that is complementary to the surface recognized on the antigen Electrostatic interactions,

hydrogen bonds, van der Waals forces and hydrophobic interactions formed between residues on the antibodies and antigens all contribute to binding In which, the residues in most or all of the CDRs make both the specific and the affinity of the antibody-antigen interaction

Kinetic of the antibody-antigen interaction

In the kinetic consideration, if a monovalent antibody fragment is used for analysis, the equilibrium of antigen-antibody binding is defined as:

At the beginning, a chemical reaction proceeds mostly in one direction, but the reverse rate progressively increases until the forward and reverse speeds are equal At this point, the reaction has reached its equilibrium

According to the law of mass action at equilibrium, the ratio between the concentrations of the product ([complex]) and the reactants ([antigen] and [antibody]) is constant Keq is called the equilibrium constant and is equal to the ratio between the association (ka) and the dissociation (kd) rate constant

or the antibody concentration

It’s known that, the greater the strength of the antibody-antigen binding, the higher its equilibrium constant is This relationship is expressed by the Gibbs’ energy (ΔG) of the formation of an antibody-antigen complex, which is given by:

In the immune technology, it has two terms that are affinity and avidity used widely to describe strengths of the antibody-antigen binding As the mention above, the strength of a single antibody-antigen bond, this obtained from a monovalent antibody fragment with an epitope, is defined the antibody affinity However, each complete monoclonal IgG antibody (or IgY antibody) has two antigen-binding sites

in the Fab fragments and other classes of the monoclonal antibody such as IgM, IgA, IgD, IgE have more than two antigen-binding sites Thus, all complete antibodies are multivalent in their reaction with antigens Similarly, antigens also exhibit a multivalent characteristic because they can bind to more than one

antibody Avidity is a term that refers to the overall strength of that a multivalent antibody binds a multivalent antigen

1.2.3 Monoclonal and polyclonal antibody

Monoclonal and polyclonal IgG antibodies purified from mammalian bloods are used widely in the immunoassays Both of them have different advantages that make them useful for individual applications Polyclonal antibodies are relatively quick and inexpensive to produce compare to monoclonal antibodies However, monoclonal antibodies are higher specific to detect only one epitope on the antigen than those of polyclonal antibodies [23] Traditionally, bigger animals such as horses, sheep, pigs and rabbits, were used for the production of polyclonal antibodies, while rats were used as a source of production of monoclonal antibodies Nowadays, most frequently chosen mammals for polyclonal and monoclonal antibody production are rabbits and mice, respectively A quite significant disadvantage of the production of antibodies from mammalian bloods is that are the painful collecting and final sacrificing of animals Thus, the interest in developing alternative methods for the traditional production of antibodies is enhanced increasingly in the sense of animal protection

1.2.4 Immunoglobulin IgG and IgY

Initially, immunoglobulins that found from avian serum or egg yolk were classified as IgG-like immunoglobulins In 1969 Leslie [24] showed experimental data demonstrating great differences in their structure and proposed the name IgY (immunoglobulin from egg yolk) Now IgY is recognized as a typical low-molecular weight antibody of birds, reptiles, amphibian, and as an evolutionary ancestor of IgG and IgE antibodies that are found in mammals General structure of IgY molecule is the same as of IgG with two heavy chains and two light chains As shown in Fig 1.6., the major difference between IgG and IgY is the number of constant regions in heavy chains (CH), which consists of two structural forms of IgY: full-length and truncated IgY The full-length structural IgY found on ducks,

that has four C regions (CH1 to CH4) The truncated structural IgY found on chickens, lacking the two terminal domains of the constant region, CH3 and CH4, of the H chains, thus named as IgY(ΔFc) In addition, both IgY forms are much less flexible than IgG due to the absence of the hinge between CH1 and CH2, which is a unique mammalian feature

The major structure difference between IgG and IgY is reflected in typical properties of IgY, of which IgY do not bind to protein A or G as well as do not bind

to mammalian antibodies [25]

Figure 1.6 The structural difference between IgG and IgY

Among all sources of avian animals, chicken IgY from egg yolk is most frequently produced It provides an excellent substitution for IgG from mammalian blood in many applications (as a research, diagnostic, detection of antigens…) IgY was also demonstrated to work in practically all tested immunological methods that were traditionally developed for IgG, for examples immunofluorescence, immune-enzyme techniques and immunosensors

In this work, the antibody-antigen interaction between anti-ND virus IgY and

ND virus occurs on a working electrode surface in triple electrode configuration This interaction will be investigated using CV measurement that is an important and widely used electroanalytical technique in many areas of chemistry From obtained

characteristic of CV measurements, we will develop the electrochemical immunosensor towards quantitative detection of ND virus

Chapter 2

FABRICATION OF IMMUNOSENSOR

This chapter presents the development of electrochemical immunosensor including the fabrication of a planar triple electrode configuration and the attachment techniques of anti-ND virus IgY (specific immunoglobulin Y for Newcastle virus) onto the gold electrode In addition, immunoassay protocol, which

is the experimental immune reaction of immunosensor with Newcastle disease virus, will also be discussed and reported

2.1 Antibody Immobilization Approaches

The immobilization steps of the immune proteins, antibodies or antigens are essential in the development of the immunosensors In the electrochemical immunosensors, antibodies are usually attached to a surface of a solid surface that is chiefly employed as a working electrode of the sensor Generally, when antibodies are immobilized, their specific binding capacity is usually less than that of antibody solutions [26] One of the main reasons for this reduction is attributed to the random orientation of the antibodies on the surface of electrode Thus, to achieve full functionality, the conformations of the antibodies should not be altered and their active immune sites should not be fully exposed to the binding agent during immobilization

As the previous mention, specific antigen-binding sites are localized in the

Fab tips that comprise around 110 amino acid residues at six CDR segments on the N-terminal variable domains (Fv) (e.g., in IgG) Thus, during the immobilization step, active functional groups on the substrate can be coupled with random moieties

in the antibody, such as amine groups in lysine residues, thiol groups in cysteine residues, and the aldehyde groups in carbohydrate residues in the Fc This may result in different orientations of the antibody on the substrate When immobilization occurs through the antigen binding sites, the ability of that antibody

to bind antigen may be impaired or eliminated entirely

Figure 2.1 Different orientations of the antibody immobilized on the substrate

In the past years, numerous strategies for antibody immobilization have been developed from simpler adsorption processes to better covalent attachments, and to bio-affinity immobilization in which the orientation and activity of antibodies on a substrate play a particularly important role for the effective performance of immunosensors The following sections will describe the immobilization techniques that are applied for electrochemical immunosensors and so on

2.1.1 Physical adsorption

Antibodies can be physically adsorbed onto a solid surface of a substrate via intermolecular forces, containing hydrophobic, electrostatic, and low energy interactions [27] Among the immobilization techniques, physical absorption is the simplest process However, it has considerable weak points that antibodies are uncontrollable, weak attachment, and randomly oriented Antibodies attached weakly on a substrate can be removed by buffers or detergents, while the random orientation definitely decreases sensitivity of immunosensors detecting antigens To improve this, some techniques like entrapment on polymer membrane, encapsulation in gels, and gold nanoparticles have been performed

Antibody entrapment on a polyethylene glycol (PEG) surface is a simple process based on physical absorption [28], in which antibodies can be immobilized directly via their hydrophobic properties PEG, polydimethylsiloxane (PDMS), polystyrene is effectively employed as antibody entrapments, which are suited for optical immunosensors due to they are optically transparent with a low back-ground auto-fluorescence [27] While conducting polymers such as polypyrrole, polythiophene, and polyaniline [29] are used widely to in electrochemical

immunosensors Besides, porous gel matrices offer advantages in physical absorption as well because they provide a large surface area, forming denser active sites to which the antibodies can bind

Sol-gel silicon dioxide processing was first prepared in 1998 for amperometric immunosensors that resulted in a low detection limit of 5 ng/mL [30] Agarose gels are commonly used on silicon dioxide surfaces to attach antibodies by physical adsorption or covalent linkages [27] In recent years, sol-gel thin films have been developed considerably, their applications in optical immunosensors more prominent than that of electrochemical immunosensors [31]

In the past few years, gold nanoparticles (AuNPs) have commonly used in immunosensors to trap antibodies by physical adsorption due to their specially properties The immobilization is carried out through the particular interaction between the thiol groups of immunoglobulin and gold atoms Moreover, in electrochemical immunosensors, AuNPs plays important role for the promotion of electron transfer due to their high conductivity, which increase significantly the sensitivity of immunosensors As for example, the report of Wang et al., ultrasensitive immunosensor was developed in which antibodies was immobilized

on the glassy carbon electrode surface modified by electrodepositing AuNPs [32]

The immunosensor could detect ultralow concentration of α-1-fetoprotein (AFP) in

the media

The conjugation of antibody to AuNPs has also been reported in recent years[33] [34] [35] according to which the sensitivity and response time was improved

2.1.2 Covalent attachment

Covalent binding is the preferred techniques for immobilizing antibody to a surface by the accessible functional groups of side-chain-exposed amino acid residues, which leads to irreversible binding and gives a high degree of surface coverage [36] The functional groups can be created on the substrate or antibody, or

both under chemical treatments The possibility of a transformable functional group

is a significant concept that would provide a starting surface for using suitable coupling chemistries Generally, there are two most common surfaces used in immunosensors such as silica (SiO2 on the silicon wafers or glass) and gold Silica

is surface-modified to create functional groups, e.g., aldehyde-, epoxy-, and amine-, while gold particles are usually incubated with reactive thiol-terminal organic compounds (e.g., mercaptoundecanoic acid - MUA) through gold-sulfur bonding For antibodies, the transformable groups of exposed residues, such as amine, thiol, hydroxyl, are performed as functional ones for covalent attachment

Covalent attachment via amine group

The amine groups in the lysine residues of the antibody are the most commonly utilized anchoring points because they are typically present on the exterior of the antibody However, lysine abundance in antibody can also increase heterogeneity binding and restricts conformational flexibility

For silica substrates, generally, the procedure begins with surface cleaning to remove contaminations and to create hydroxyl groups The presence of hydroxyl groups provides sites for the covalent attachment of silane Two kinds of silane available for covalent attachment namely amine-terminal silane and thiol-terminal silane Both produce amine or thiol functional groups, respectively, on the silica substrate

Bifunctional cross-linkers such as glutaraldehyde (GA),

N-succinimidyl-4-maleimido-butyrate (GMBS), and

N-succinimidyl-4-(N-maleimido-methyl)-cyclohexane-1-car-boxylate, are usually employed to covalently immobilize antibodies, which make a conjugation between functional groups on a silane layer and amine groups in antibody Mixed silanes, e.g., APTES and methyltriethoxysilane (MTES), are also used to improve the hydrophobicity of antibodies layer, that showed more effective immobilization than that of APTES alone [37]

For gold substrates, surfaces are usually functionalized to create carboxyl groups by a pretreatment process with MUA Then carboxyl groups are activated by mixed reagents containing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxyl sucinimide (NHS) to form NHS esters, which can react easily with primary amine groups (-NH2) of antibody This approach is the most common

covalent immobilization for antibody on gold surfaces Goat anti-E coli

antibody[38], PentaHis antibody, and anti-gIgG [39] have all been immobilized on a gold surface to fabricate Surface plasmon resonance (SPR) immunosensors Moreover, a bifunctional linker as dithiobis(succinimidyl undecanoate) was also used instead of MUA/EDC/NHS combination [40]

Covalent attachment via Thiol group

Although coupling through the amine groups in lysine residues of antibody is a popular approach in covalent attachment However, in certain circumstances, alternative approaches may be preferable, for example, thiol groups in cysteine residue In Viitala’s study [41], Fab’ fragments of polyclonal anti-human IgG were covalently attached onto a polymerizable lipid by using maleimide which is described as Fig 2.3 The process could create internal disulfide bonds, that is less likely than the process obtained by amine groups due to cysteine is not as abundant

as lysine To solve this problem, Traut's Reagent (2-Iminothiolane.HCl) is used for thiolation of primary amine groups in an antibody, which create more abundant

secondary thiol groups for immobilization, as shown in Fig 2.2 [42] In Kusnezow’s report, antibodies attached through thiol group may result in the partial loss of activity, but they have better orientation than coupling by the amine groups [43]

Figure 2.2 Traut's Reagent

Figure 2.3 Pre-treated substrate with maleimide and antibody immobilization by

thiol groups

Covalent attachment via sugar residues

The immobilization by hydroxyl groups in carbohydrate residues antibody is not a common method due to the inert chemical properties of hydroxyl groups In advantage, activity of antibody is not affected after attachment, because carbohydrate residues are only present at Fc fragment of antibody This approach was used to attach IgG antibodies on an APTES-modified surface [44] In which, reactions were performed between amines in the silane and aldehydes (-CHO) which produced by the NaIO4-oxidation of carbohydrate residues

Figure 2.4 Covalent attachment through carbohydrate residues of antibody

Plasma-assisted covalent technique

In the recent years, surface modification by using plasma treatment has opened up a novel avenue for antibody immobilization A striking advantage of the technique is that offer high-density bonding sites for APTES Functional groups can

be controlled onto the surface by plasma technique depending on plasma parameters such as power, used gases, pressure, and treating time Hydroxyls (-OH) and amino groups (-NH2) are two most popular kinds of functional group, that are introduced onto a surface by using Ar/O2 plasma treatment [45] and ammonia plasma treatment [46], respectively Therefore, plasma-assisted technique provides an efficient solution for surface pre-treatment instead of wet chemical techniques

2.1.3 Bio-affinity

Biochemical affinity reactions offer a good oriented, homogenous immobilization of antibodies on the substrate surface Most of bio-affinity immobilization techniques are based on the reactions: avidin-biotin system, his-tag system, protein A/protein G-mediated attachment, and DNA-mediated attachment

+ Avidin-biotin reaction

The interaction between avidin and biotin has been exploited to conjugate antibodies on a solid surface This approach has been widely used in the immobilization process

Figure 2.5 Biotinylation of antibody by NHS reagent

Biotinylated antibodies or biotin-labeled, react with avidin to generate a biocompatible layer on a surface by one of the strongest non-covalent bonds (Kd =

1015 M-1) [47]

Figure 2.6 Avidin-biotin affinity for immobilization

Therefore, this technique allows the use of harsh conditions during immunoassays Other proteins, including streptavidin, neutravidin, tamavidin, and captavidin, known as biotin-binding proteins, can also be used for the same purpose Avidin can be attached to substrates by absorption or covalent binding

+ Protein A/G-mediated bio-affinity immobilization

Protein A and protein G, which are derived from group G Streptococcus and

Staphylococcus aureus, respectively, have been used as mediated agents for

oriented antibody immobilization

Figure 2.7 Protein A/G-mediated bio-affinity immobilization

Both antibody-binding proteins have a high affinity for the Fc region of many IgG subclasses, and thus they are employed without interfering with the antigen-combining sites located on the Fab tips Protein A/G can be attached on the gold surface by direct adsorption [48] or using a covalent coupling reagent such as dithiobis succinimidyl undecanoate

+ DNA-directed immobilization

DNA-direct immobilization (DDI), in which DNA is used as a linker molecule, has been developed recently with considerable success for immunosensors This technique is based on the hybridization interaction of complementary single-stranded DNA (ssDNA) sequences Firstly, primary antibodies are labeled with ssDNA oligomers to produce ssDNA-antibody conjugates The labeling of antibodies is usually performed in three steps that are showed in the following figure [49]:

Figure 2.8 ssDNA-antibody conjugation to form a hydrazone linker

(A) The S-HyNic linker is used to modify the antibody, and the S-4FB for modification of the amino-ssDNA (B) Modified components are combined in the presence of an aniline

catalyst and react to form (C) a hydrazone-conjugated ssDNA-antibody

In addition, the complementary ssDNA oligomers are attached on an amine surface by the covalent coupling technique, for example, using NHS active esters

Finally, ssDNA-antibody conjugates are immobilized on the ssDNA-surface through specific DNA hybridization

In conclusion, a brief overview of this section has presented various techniques for antibody immobilization, which have been recently used to fabricate immunosensors Basing on fundamentally difference of bindings, attaching techniques can be classified into three main types, containing physical adsorption, covalent binding, and bio-affinity Indeed, no generally applicable immobilization technique predominated because of wide variations in the properties of materials Adsorption on the polymer matrixes or gels is a simple, easy approach Covalent coupling is the most universal and reliable approach Bio-affinity agents are advantageously used for oriented antibody attachment

2.2 Fabrication of electrochemical sensor based on gold thin film electrode s

In this section, we present fabrication of electrochemical sensor based on Au thin film electrodes on silicon substrate, which has a structural description as shown

in Fig 2.9 The planar process on silicon wafer, containing main steps such as oxidation, photolithography, etching, sputtering, is employed for the fabrication

Figure 2.9 Structure of the integrated electrode 2.2.1 Photomask design

Only a photomask, designed with CorelDraw software, was used in the fabrication process As shown in Fig 2.10, the total number of electrodes designed

on a silicon wafer is 190 The three electrodes (Working Electrode - WE, Reference Electrode - RE and Counter Electrode - CE) are all integrated on one sensor of which the dimension is 3.6 x 12 mm2 and WE’s area is 0,785 mm2

This design is compatible with the µ-USB configuration This will make sure a good connection and interface with the measuring device and the wire-bonding step will be no longer used within the fabrication process

Figure 2.10 Photomask design and detailed structure of electrode sensor

2.2.2 Main processes in the electrochemical sensor fabrication

Wafer-cleaning

Wafer-cleaning step plays an important role in the whole process and directly affects the experimental results because contaminants appearing on the wafer cause unsuccessful electrodes This step purposes to remove all contaminations on the surface of the wafer Firstly, in order to remove the organic substances, the wafer is immersed into fresh piranha’s solution (H2O2:H2SO4, 3:7, v/v) for 5 min, and rinsed with DI water, further immersed in ethanol and rinsed with DI water respectively

Then the wafer is boiled in a 65% HNO3 solution for 10 min and is rinsed in DI water again This step is to remove inorganic contaminations Lastly, wafer is immersed into 1% HF solution for some seconds to remove thin native silicon dioxide After the wafer-cleaning step, wafer will be employed with other fabrication processes, that is shown in Fig 2.11