Luận văn 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

Pre-treated substrate with maleimide and antibody immobilization by thiol groups Figure 2.4, Covalent attachment through carbolrvdrate residues of antibody Figure 2.5.. In principle, ei

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

1.2.2 The principle of antibody-antigen interaction

1.2.3 Monoclonal and polyclonal antibody

1.2.4 Immunoglobulin IgG and Ig

Chapter 2 FABRICATION OF IMMUNOSENSOR

2.1 Antibody Immobilization Approaches

2.3.1 Antibody Immobilization using ¿ PrA/GA approach

2.3.2 Antibody Immobilization using SAM/NHS approach

3.22 Effect of the IgY concentration on the immobilization of PrA-GA

3.3.1 Cyclic voltammetry characterization of SAM-NHS mmmunosensor SŠ

3.3.2 Effect of the pH value on the immobilization of SAM-NHS

TNTUIOS€TSOT à cọc tnhớnh HH hueerrerdirradieroore TẾ

3.4.1 Liffect of the immunoreaction time eee seieeereenre OD

TIS Trlsetrochemical Impedanse Speciroscopy

LIST OF TABLES

Table 1.4 Properties of trunmoglobulin classcs

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

fabncalion procedures of immumosensor

Table 3.4 Experimental conditions for the atlachmen! of components

Table 3.5 he average and standard deviation of lpear of sensors

Table 3.6 The crucial parameters obtained from the calibration

Table 3.7 Comparison of analytical properties of different immurtosensors for the detvclion of Avian Influenza

LIST OF FIGURES

Figure 1-4 The porforming principle of cloctrochemical immuntosensor

Figure 1.2 Direct and Indirect immunosensor

Figure 1.3 (A) Structure of full-length human anti-PD1 therapeutic IgG 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 1Cl antibody and Epha2 antigen

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

Figure 1.6 The structural difference betwoen 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 carbolrvdrate 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 ss)NA-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 Tmage of electrochemical sensors on a wafer and a complete senser

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 rcaction of GA linkor with protein A and Ig¥ antibody

Figure 3.4 CV characterization of modified elecirode recorded on Au electrode Figure 3.5 Effect of the antibody concentration

Figure 3.6 The main reactions ơn 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 Elect of pH valup of the inumobilization of antibody

Figure 3.10 The average and the STD of Tyeak of the bare Au electrode

Vigure 3.11 ‘the schematic description of the ND virus detection mechanism

Figure 3.12 Efoct of the immunoreaction time

Figure 3.13 (A) The CV curves of PrA-GA immiunoxensor (a) in buffer solution and aller assay with (b) 10, (c) 10%, (đ) 10%, (e) 10%, (1) 10 BLDse'mL ND virus (B) the relationship between Alpen and various ND virus concentrations of PrA-GA

immunosensor

Ligure 3.14, ‘he relationship between Alpes and various ND virus concentrations

INTRODUCTION Newcastle disease (ND) is one of the most popular infection diseases in

poultry that widely spreads in Southem Last 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 (haemagghutination inhibition, agar gel precipitation test and

Latex agglutination test) as well as semi-quantitalive analysis (envyme-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 complcaled procedures for sample preparations and sophisticated mstruments for assays Thus, it is necessary to develop methods that offer a simple, rapid, cost-effective analytical strategy, which gan be easily used for applications in contamination studies of ND

To investigate infection diseases, the fabrication and application of electrochemical immunoscnsor have bean considerably developed However, most

of the works have used monoctonal immunoglobulin G (antibody IgG) from mammalian blood Egg yolk immunoglobulin (Ig Y) from chickens can be employed

as an altemate TgG in wmnuncassay, which offers some advantages with respect to anunal care, high productivity and special suitability in the source of antibodies

Tn our work, clectrochemical immnunosensor using TgY as receptors in configuration has been developed to detect ND virus ‘Ihis 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

4n 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, ie 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

The fundamental performance of clectrochemical immunosensor is desoribed as shown in “ig 1.1, An electrochemical immunosensor can be classified into three

qnain componens, corresponding to the particular roles im 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:

1 Potentiometric technique

‘the fundamental principle of all potentiometric transducers are based on the Nerst equation [7] according to which polential changes arc logarithmically propartional to the specific 10n activity on the electrodes ‘he 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 Ton-seleclive elecirodes (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) [rom the solution al the solid- state surface, which leads to the change the transmembrane potential ‘Lhe electrode measuring pll is the most popular 1SE

+ Electrode potential

This transducer is similar to the transmembrane potential sensor Llowever,

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

complexes, changing the clectrode potential in relation to the concentration of the

analyte

+ Hield-effect transistor (M1)

‘The FLT 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 he surface potential varies with the analyte concentration The integration of an ISL with FET is obtained in the ion-selective field-effect transistor (ISFET) This technique is also highly potential for the applications of immunosensors

+ Amperometric technique

This immunoscnsors are designed to measure a current flow generated by an

electrochemical reaction at constant voltage Amperometric immunosensors depend

on (he electrical transfer from redox reaction of biocompononis to the surface electrode, Because most protein analytes are not intrinsically able to act as redox partners in an electrochemical reaction, this technique needs using

electrachemically active labels such as enzyme or redox labels

| Conductometric and capacitive technique

These immunosensor transducers measure the alteration of the electrical

conductivily in a soluliơn al constant vollage, caused by biochemical reaction (enzymatic activities) which specifically generate or consume ions The capacitance

changes are measured using an eleclrochesnical system, in which the bioreceplor is

tnmobilized onto a pair of noble metal clectrodes (Au or Pt) For ummunosensors,

an ion-channel conductance immunosensor mimicking biological sensory function

is used lo record effectively the small signaling immune reactions thal are usually

Jost in the high ionic strength of the solution

1.1.1.2 Rioreceptor

Using antibody or antigen as bioreeeptors, 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 swface 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 diect 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 indircetly 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 Kig.1.2, primary antibodies are usually immobilized on an electrode, and sandwiched immune-complexes are formed among the immobilized antibodies, specific antigens and labeled antibodics The detectable signal mainly depends on labeled signal tags, thus, a reat scientific effort has been devoted for developing effective labels Favymes and redox-labels are electrochemival active labels which are used widely

immunosensors [2] Several enzymes are prominent such as alkaline phosphatase 18], horseradish peroxidase , J galactosidase |9], cholinesterase [10] and glucose oxidase [11], while ferrocene derivatives or In’ salts [12], redox polymers (e.g., polymer [PVP-Os(bipyridyl):Cl}) [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 tho mention above, amperometic immunoscnsors are considered unsuitably for the detection directly immune components, which should be employed wath 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 al 12 pg/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 im the components of electrochemical immunosensors such as electrodes,

immobilized substances and clectrolytes Morcover, analytical objects have been

extended progressively, which range from infectious viruses in human or animals, antigens on cancer cells, palhogens lo loxie substimees

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 # critical part of the immune response by specilically 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 characleristics of the antibody-mtigen interaction ‘The structural and energetic aspects of antibody-antigen binding are significant lo understand low an antibody specifically recognizes ils corresponding

antigen

Structare 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 («) chain and lambda (4) chain, which distributed according to different genes in chromosomes in mammals There are five classes of immunoglobulins (IgG, TgA, TgM, IgD and Igh), which differ in amino acid sequence and number of domains in the constant regions of the heavy chains (Cu) In which, immunoglobulin G (ig@) is the major type of immunoglobulins (approximately 75% in all of the normal serum),

that is the mosl extensively investigaied

Table 1.1 Properties of sumunoglobulin classes [17]

TH: heavy chain; 1: 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 Fe fragment Yfragment crystallizable) contains carbohydrate chains, which docs not bind antigen

The remarkable feature of the antibody molecule is showed by comparison of amino acid sequences from various immunoglobulin mutecules This shows thal 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

IgG

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 B barrel

16

consisting of nine anti-parallel strands (V regions) and seven C regions, and that the hypervariable regions are clustered at the end of the variable domain arms, lig 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 amine 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 dwing immune

reaction Ricl

d 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 tramnmore: ulion, il is necessary to base on two followmg dircclisns: (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 proportics in the formation of specific bindings

The binding in antibody-antigen camplex 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 mosl effective technique

of choice to determine the precise sites of the molecular isteractions of antibedics with their antigens through crystal structures of complexes between them The

complex usually is oblained under the erystalline 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 la provide for the itumme epitope database Furthermore, the crystal structure determinations of highly specific antibody fragments (Kab)

associated protein antigens show also [221]:

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

(2) The specificily of tmmuno-reaction is determined by the structure of CDRs on

Vu and Vi, part of antibody, in which, the Va CDR3 encoded by the 1D (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 (anligenic determinant or epitope)

(4) The contacting surface of the antibody anc antigen ollen show a high degree of

complementarity

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

900 A.

(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 1C] 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) (@) The maximum likelihood weighted 2mFo-DFe 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

complementary to the surface recognized on the antigen Electrostatic interactions,

hydrogen bonds, van der Waals forecs 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

According to the law of mass action at equilibrium, the ratio betweon the concentrations of the product ([complex]) and the reactants ({antigen] and lantibody|) is constant Kyg is called the equilibrium constant and is equal to the ratio between the association (ks) and the dissociation (ka) rate constant

This can be obtained in two ways: increasing either the equilibrium constant

or the antibody concentration

It’s known that, the greater the strength of the antibody-antigen binding, the higher ils equilibrium constant is, This relationstrip is expressed by the Gibbs’

energy (AG) of the formation of an antibody-antigen complex, which is given by:

21

other hand, entropy is gained when water molecules are displaced from the surfaces

that become the new interface, which is in the structures observed by Xzay analysis This latter effect is quite significant and it appears that water molecules are

abnost totally excluded from the tmterface by the close conlact between antibodies

and antigens or the non-covalent bonds in the antibody-antigen interaction,

because the formation of hydrogen bonds is exothermal, they are mostly formed by

the enthalpy (actor In contrast, hydrophobic bonds are olien driven by the entropy

factor due to their endothermic property

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 cbiained from a monovalent antibody fragment with an epitope, is defined the antibody affinity However, each complete monoclonal IgG antibody (or Ig¥ antibody) has two antigen-binding sites

in the Fab fragments and other classes of the monoclonal antibody such as IgM, IgA, IgD, Igli have more than two antigen-binding sites Ihus, 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 tan those of polyclorat 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, mos frequently chosen mammals (or polyclonal and monoclonal antibody production are rabbits and mice, respectively A quite significant disadvantage of the production of amibodies from mammalian bloods is that are the painful collecting and (inal savrificing 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, Immunoglobutin IgG and Ig

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 siructure and proposed the name TgY (immunoglobulin from egg yolk) Now Ig is recognized as a typical low- molecular weight antibody of birds, repliles, amphibian, and as an evolutionary ancestor of IgG and IgE antibodies that arc fod in mammals General structure of IgY molecule is the same as of [g( with two heavy chains and two light chains As shown in Fig 1.6., the major difference between IgG and IY is the number of constant regions in heavy chains (Cx), which consists of two structural fonns of IgY: full-length and truncated IgY The full-length structural IgY found on ducks,

that has four C regions (Cal to Cad) The truncated structural Ig¥ found on chickens, lacking the two terminal domains of the constant region, Cu3 and Cu, of

the H chains, thus named as Ig¥(AFc) In addition, both Tg¥ forms are much tess

flexible than lzG due to the absence of the hinge between Cul and Cx2, which is a unique mammalian feature

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

Figure 1.6 The structural difference between TgG and TgY

Among all sources of avian animals, chicken IgY from egg yolk is most frequently produced It provides an excellent substitution for 1g( from mammalian blood in many applications (as a research, diagnostic, detection of antigens ) IZ¥

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 ocews on a working cloetrode surlace in Inple clectrode 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

F, tips that comprise around 110 amino acid residues at six CDR segments on the

N-terminal variable domains (Fy) (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 Fe 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

26

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

2?

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 a-J-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

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 lwo 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, ¢.g., aldehyde-, epoxy-, and amine-, while gold particles are usually incubaled with reactive thiolterminal organic compounds (e.g, mereaptoundecanoic acid - MUA) through gold-sulfur bonding Tor antibodies, the transformable proups of exposed residues, such as amine, thiol,

hydroxyl, are performed as functional ones for covalent, allachmert,

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

hoteroganeily binding and restricts conformational flexibility

For silica substrales, generally, the procedure begins with surlace cleaning to remove contaminations and to create hydroxyl groups ‘Ihe presence of hydroxyl proups provides sites for the covalent attachment of silane Two kinds of silane available for covalent attachment namely amine-lerminal silane and thiol-terminal silane oth produce amine or thiol functional groups, respectively, on the silica

Bifunctional cross-linkers such as glutaraldehyde (GA), N-succinimidyl-4-

maleimido-butyrate (GMBS), and M-sucemimidyl-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, eg, 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 (-NH;) 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-glgG [39] have all been immobilized on a

gold surface to fabricate Surface plasmon resonance (SPR) ummunosensors

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

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secondary thiol groups for immobilization, as shown in Fig 22 [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]

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 Fe 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 NalO4-oxidation of carbohydrate residues

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/O, 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

Biotin Modified biotin Biotin-labeled antibody

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 (Ka =

10 MĐ) [47]

— Biotinylted antibody

Biotin

Avidin

—unker

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

v 6

oriented antibody immobilization

gi vê

Q, ° undecanoate)

Dithiobis(succinimidyl- Š ©

Protein WG

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

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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]:

ny now

Hydrazone

ssDNA-antibody conjugate

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

3

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 electrodes

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

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

ona 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 mm? and WE’s area is 0,785 mm?

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This design is compatible with the 1-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% HNO; 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

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