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 proc
Trang 1
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
Trang 21.1 Biosensor and immunosensor et eas saat _ 8
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
3.3.1 Cyclic voltammetry characterization of SAM-NHS mmmunosensor SŠ
Trang 33.3.2 Effect of the pH value on the immobilization of SAM-NHS
3.4 Stability of the signal of ND virus immunosensors 59 3.5, Detection of Noweastle discase ViTu8 cuoi .ỐT
3.5.2 Sensitivity of Neweastle disease virus immunosensor 63
Trang 4The 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
Trang 5UIDs 50 Percent Embryo Infectious Dose
Trang 6The 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
Trang 7LIST 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
Trang 8INTRODUCTION
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.
Trang 9LIST 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
Trang 10LIST 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
Trang 11The 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
Trang 12The 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
Trang 13INTRODUCTION
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.
Trang 14INTRODUCTION
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.
Trang 15INTRODUCTION
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.
Trang 16UIDs 50 Percent Embryo Infectious Dose
Trang 17UIDs 50 Percent Embryo Infectious Dose
Trang 18LIST 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
Trang 19LIST 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
Trang 20UIDs 50 Percent Embryo Infectious Dose
Trang 21Figure 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
Trang 22UIDs 50 Percent Embryo Infectious Dose
Trang 23LIST 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
Trang 24LIST 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
Trang 25Chapter 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
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]
Trang 26LIST 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
Trang 27Figure 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
Trang 28LIST 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
Trang 29INTRODUCTION
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.
Trang 30The 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
Trang 31Chapter 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
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]
Trang 32LIST 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
Trang 33Chapter 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
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]
Trang 34LIST 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
Trang 35Chapter 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
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]
Trang 36The 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
Trang 37LIST 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