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..
IMMUNOSENSOR AND IMMUNE REACTION
Biosensor and immunosensor
A biosensor is a device that combines a biological sensing element with a signal transducer to convert biological reaction signals into measurable outputs The biological sensing elements can include oligonucleotides (DNA or RNA), enzymes, proteins, cells, antibodies, or antigens The transducer, typically built on a solid-state substrate, transforms these signals into measurable forms, such as electrical signals Biological reactions can cause changes in pH, electronic or ionic transfer, refraction, luminescence, micro mass, or thermal transfer Immunosensors, a specific type of biosensor, utilize antibodies or antigens, and the four most common types of immunosensors are optical, electrochemical, micro mass, and thermal.
[3] proposed the first concept of the immunosensor in 1985 in which
Immunosensors are innovative devices that transform specific antibody-antigen interactions into measurable signals These sensors utilize either antibodies or antibody-antigen complexes immobilized on a transducer's surface, serving as bio-receptors for detecting target elements, such as other antibodies or antigens.
Immunosensors primarily operate through two mechanisms: biological catalysis and biological affinity Biological catalysts, typically enzymes, facilitate biochemical reactions, whereas biological affinity relies on specific interactions involving proteins, lectins, receptors, live cells, nucleic acids, antibodies, and antigens.
Biosensors and immunosensors are utilized across various fields, including clinical diagnostics, food safety, industrial process control, pollution monitoring, drug discovery, and military applications This growing interest is evident in the surge of related publications, which increased from around 100 in 1985 to 4,500 in 2011, with that year's publications accounting for over 10% of all biosensor articles ever published Additionally, the global biosensor market has expanded significantly, rising from a $2 billion share in 2000 to $13 billion, with forecasts for 2018 estimating it to reach approximately $17 billion.
Electrochemical immunosensors, as defined by IUPAC, are integrated devices that combine an immunochemical recognition element with a transducer element These sensors utilize antibodies or their complementary binding partners, such as antigens or haptens, in conjunction with electrodes or field-effect transistors The advantages of electrochemical immunosensors include low sample consumption, cost-effective instrumentation, and potential for miniaturization, driving their widespread development and application in various fields.
Figure 1.1 The performing principle of electrochemical immunosensor
The core functionality of an electrochemical immunosensor, illustrated in Fig 1.1, consists of three primary components: molecular recognizers such as antibodies or antigens, electrodes that support these recognizers, and the transducer that measures performance.
Based on the measurement method, the several types of transducer employed in electrochemical immunosensors field are listed in the following:
Potentiometric transducers operate on the Nernst equation, which states that potential changes are logarithmically proportional to the specific ion activity at the electrodes These sensors measure the potential difference (voltage) between the working electrode (WE) and the counter electrode (CE) They are commonly employed to determine the analytical concentration of electrically charged components in various analytes.
The transducer principle relies on the accumulation of potential across a sensing membrane, where ion-selective electrodes (ISE) utilize ion-selective membranes to create charge separation between the sample and the sensor surface Additionally, bioreceptors, such as antigens or antibodies, are immobilized on the membrane to bind specific compounds from the solution, resulting in changes to the transmembrane potential Among ISEs, pH measuring electrodes are the most widely used.
This transducer is similar to the transmembrane potential sensor However, an electrode by itself is the surface for the formation of antigen-antibody
11 complexes, changing the electrode potential in relation to the concentration of the analyte
The ion-selective field-effect transistor (ISFET) is a semiconductor device designed to monitor charge accumulation on an electrode's surface, specifically at the metal gate situated between the source and drain electrodes The surface potential of the ISFET varies with the concentration of the analyte, making it a valuable tool in chemical sensing Additionally, this technology shows significant promise for applications in immunosensors, enhancing the detection capabilities in various biochemical analyses.
Amperometric immunosensors are engineered to detect current flow resulting from electrochemical reactions at a constant voltage These sensors rely on the electrical transfer from the redox reactions of biocomponents to the electrode surface Since most protein analytes cannot serve as redox partners in electrochemical reactions, the technique requires the use of electrochemically active labels, such as enzymes or redox labels, to facilitate detection.
Immunosensor transducers detect changes in electrical conductivity within a solution at a constant voltage, driven by biochemical reactions that generate or consume ions These capacitance changes are monitored through an electrochemical system where a bioreceptor is immobilized on noble metal electrodes, such as gold (Au) or platinum (Pt) Specifically, ion-channel conductance immunosensors are employed to accurately capture subtle immune signaling reactions that often go unnoticed due to the high ionic strength of the solution.
Immunoassays, which utilize antibodies or antigens as bioreceptors, represent a significant distinction in the foundational principles of immunosensors compared to other biosensors Typically, conventional electrochemical immunosensors feature immobilized antibody molecules on electrode surfaces that specifically recognize antigens in samples Immunosensors can function through direct or indirect methods, differentiated by the use of non-labeled or labeled antibodies, respectively Direct electrochemical immunosensors detect electrochemical changes during immune complex formation, while indirect immunosensors rely on signal-generating labels attached to antibodies to identify antigens indirectly.
Immunosensors that utilize labeled antibodies commonly employ a sandwich-type immunoassay, which involves two antibodies In this setup, primary antibodies are immobilized on an electrode, creating immune complexes with specific antigens and labeled antibodies, as illustrated in Fig 1.2 The detectable signal generated in this process primarily relies on the labeled signal tags, prompting significant scientific efforts to develop effective labeling methods Among these, enzymes and redox-labels are widely used as electrochemically active labels in indirect electrochemical immunosensors, particularly in amperometric immunosensors, with alkaline phosphatase being one of the most notable enzymes.
[8], horseradish peroxidase , β –galactosidase [9], cholinesterase [10] and glucose oxidase [11], while ferrocene derivatives or In 2+ salts [12], redox polymers (e.g., polymer [PVP-Os(bipyridyl) 2 Cl]) [13] are known as notable redox-labels
Figure 1.2 Direct and Indirect immunosensor Direct munosensor im
Indirect electrochemical immunosensors are known for their high sensitivity due to label activation; however, they have drawbacks such as complex fabrication processes and indirect antigen concentration measurement based on generated signals As a result, label-free antibodies are preferred for immunoassay-based electrochemical immunosensors, particularly for in vitro applications, as they enable real-time measurements without hazardous reagents The first direct electrochemical immunosensor was developed in the 1970s by Janata, who observed a potential change using a PVC membrane-immobilized Concanavalin-A antibody on a potentiometric electrode, allowing real-time monitoring of the binding process without labeling In 1984, Keating advanced this field by modifying an electrode with a dioxin-ionophore antigen conjugate, facilitating the detection of anti-dioxin antibodies.
Immune Reaction
Amperometric immunosensors have traditionally been unsuitable for the direct detection of immune components, requiring the use of enzyme or redox labels However, a groundbreaking study by Hu in 2003 demonstrated that gold nanoparticles modified with anti-paraoxon antibodies could be utilized on a glassy carbon electrode for the direct detection of paraoxon through cyclic voltammetry This method achieved a notable detection limit of 12 μg/L, marking a significant advancement for initial amperometric immunosensors without the need for labels.
Over the past four decades, electrochemical immunosensors have significantly advanced, primarily due to enhancements in key materials like electrodes, immobilized substances, and electrolytes Research has increasingly expanded the range of analytical targets, now encompassing infectious viruses in humans and animals, cancer cell antigens, pathogens, and toxic substances.
Antibodies, or immunoglobulins (Ig), are glycoprotein molecules produced by white blood cells in the immune systems of vertebrates, playing a vital role in the immune response by specifically recognizing harmful substances known as antigens, which include bacteria, fungi, parasites, viruses, and chemicals The interaction between antibodies and antigens is characterized by high specificity and affinity, making it essential to understand the structural and energetic aspects of this binding to comprehend how antibodies precisely identify their corresponding antigens.
The structure of an antibody molecule, specifically immunoglobulin G (IgG), consists of four polypeptide chains interconnected by disulfide bonds (S-S) This structure includes two identical light chains (L chains) and two heavy chains, forming a functional unit essential for immune response.
Immunoglobulins consist of 15 identical heavy chains (H chains) and two light chain isotypes: kappa (κ) and lambda (λ), which are encoded by different genes on mammalian chromosomes There are five classes of immunoglobulins—IgG, IgA, IgM, IgD, and IgE—that vary in amino acid sequences and the number of domains in the constant regions of the heavy chains (CH) Among these, immunoglobulin G (IgG) is the predominant type, constituting approximately 75% of normal serum, and has been the subject of extensive research.
Table 1.1 Properties of immunoglobulin classes [17]
Properties IgG IgM IgA IgD IgE
L-chain type κ or λ κ or λ κ or λ κ or λ κ or λ
H: heavy chain; L: light chain; J: Joining chain; SC: secretory component
Using appropriate enzymes to hydrolyze peptide bonds allows for the cleavage of an immunoglobulin monomer into three fragments Two of these fragments, known as Fab fragments, are identical and retain the ability to bind to antigens The third fragment, called the Fc fragment, contains carbohydrate chains and does not bind to antigens.
The distinctive characteristic of antibody molecules is highlighted through the analysis of amino acid sequences from different immunoglobulin types, revealing that immunoglobulins consist of multiple copies of a folding unit approximately 110 amino acids in length.
Immunoglobulins consist of 16 distinct acids that form a similar structure known as the immunoglobulin fold Each polypeptide's N-terminal domain, found in both heavy and light chains, exhibits high variability, while the other domains maintain constant sequences These domains are categorized into the variable region (V region) and the constant region (C region) Notably, when comparing V region sequences, variability is not evenly spread but is concentrated in three specific areas known as hypervariable regions.
The full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab features a specific structural design, as illustrated in Figure 1.3 This figure includes a schematic representation of the IgG antibody structure, highlighting its domain architecture Research into various antigen-antibody complexes has revealed that the antibody molecule's domain structure resembles a β barrel, underscoring the complexity and functionality of these immunological proteins.
Antibodies consist of nine anti-parallel β strands and seven constant regions, with hypervariable regions clustered at the ends of the variable domain arms The antigen-combining site is primarily formed by six polypeptide segments—three from light variable domains and three from heavy variable domains—exhibiting variability in both sequence and residue count This variability underpins the diverse binding characteristics of antibodies, with the six segments referred to as complementarity-determining regions (CDRs) The specificity of antigen binding is determined by the physical and chemical properties of the surface created by these six CDR loops, while other parts of the variable region, apart from the CDRs, are termed framework regions.
1.2.2 The principle of antibody-antigen interaction
Understanding antigen-antibody interactions, especially with protein antigens, is crucial for the application of antibodies in clinical diagnosis and therapy These interactions involve the formation of specific antibody-antigen bindings during immune responses Richard J Goldberg first described this interaction in 1952, known as "Goldberg's theory." In immune reactions, antibodies bind to specific sites on antigens called antigenic determinants or epitopes, which include various surface configurations and haptenic groups Conversely, the molecular structures within antibodies that interact specifically with these epitopes are referred to as paratopes, which contain framework residues, the amino acid units found in the protein chains of complementarity-determining regions (CDRs).
For better understanding the principle of the antibody-antigen interaction in the immunoreaction, it is necessary to base on two following directions: (1)
The binding between antibodies and antigens exhibits 18 distinct structural features that characterize their complex formation Additionally, the kinetics of antibody-antigen interactions highlight the dynamic properties involved in these specific bindings.
The binding in antibody-antigen complex and structure features
The advent of cell hybridization techniques has enabled the production of monoclonal antibodies with defined specificity, facilitating structural studies of specific antibodies and their interactions with antigens X-ray crystallography has emerged as the preferred method for accurately determining the molecular interaction sites between antibodies and antigens, typically through the crystallization of Fab fragments associated with specific antigens Currently, hundreds of three-dimensional structures of antibody-antigen complexes have been elucidated via X-ray crystallography, contributing valuable data to the immune epitope database Additionally, the crystal structures of highly specific antibody fragments (Fab) in association with protein antigens provide further insights into these interactions.
(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
(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]
The three-dimensional structure of the Fab 1C1/EphA2 complex reveals the heavy and light chains of Fab 1C1 in magenta and beige, respectively, while the human EphA2 ligand binding domain is depicted in cyan Stereographic images illustrate the intermolecular interactions between Fab 1C1 and EphA2, highlighting hydrogen bonds as black dotted lines and showing nitrogen and oxygen atoms in blue and red Notably, Fab 1C1's CDRH3 region penetrates the EphA2 molecule through a hydrophobic tip, with sulfur atoms indicated in yellow The maximum likelihood weighted 2mFo-DFc electron density map further details the area of CDRH3 penetration into EphA2, maintaining the same color coding for clarity.
The interactions forming the antibody-antigen complex, known as "weak interactions," differ from strong covalent bonds, as illustrated in Fig 1.5 The impact of each force on the overall interaction varies based on the specific antibody and antigen involved This highlights a significant distinction in the behavior of antibodies.
FABRICATION OF IMMUNOSENSOR
Antibody Immobilization Approaches
The immobilization of immune proteins, such as antibodies or antigens, is crucial for the effective development of immunosensors In electrochemical immunosensors, antibodies are typically affixed to the solid surface of the sensor's working electrode However, this immobilization often results in a reduced specific binding capacity compared to antibodies in solution, primarily due to the random orientation of the antibodies on the electrode surface To ensure optimal functionality, it is essential that the antibodies maintain their conformations and that their active immune sites remain adequately protected during the immobilization process.
As the previous mention, specific antigen-binding sites are localized in the
Fab tips consist of approximately 110 amino acid residues within six CDR segments on the N-terminal variable domains (Fv) of antibodies like IgG During the immobilization process, active functional groups on the substrate can connect with various moieties in the antibody, including amine groups from lysine residues, thiol groups from cysteine residues, and aldehyde groups from carbohydrate residues in the Fc region This interaction can lead to different orientations of the antibody on the substrate If immobilization occurs at the antigen binding sites, it may impair or completely inhibit the antibody's ability to bind to its antigen.
Figure 2.1 Different orientations of the antibody immobilized on the substrate
In recent years, various strategies for antibody immobilization have evolved, ranging from basic adsorption methods to advanced covalent attachments and bio-affinity immobilization The orientation and activity of antibodies on a substrate are crucial for the optimal performance of immunosensors This article will explore the immobilization techniques utilized in electrochemical immunosensors and related applications.
Antibodies can be adsorbed onto solid substrates through intermolecular forces such as hydrophobic, electrostatic, and low energy interactions While physical absorption is one of the simplest immobilization techniques, it has significant drawbacks, including weak attachment and random orientation of antibodies This weak binding can lead to the removal of antibodies by buffers or detergents, and the random orientation can adversely affect the sensitivity of immunosensors in detecting antigens To address these issues, various techniques have been developed, including entrapment in polymer membranes, encapsulation in gels, and the use of gold nanoparticles.
Antibody entrapment on a polyethylene glycol (PEG) surface is a straightforward process utilizing physical absorption, allowing for the direct immobilization of antibodies through their hydrophobic properties Materials like PEG, polydimethylsiloxane (PDMS), and polystyrene are effectively used for antibody entrapment, making them ideal for optical immunosensors due to their optical transparency and low background auto-fluorescence Additionally, conducting polymers such as polypyrrole, polythiophene, and polyaniline are widely employed in electrochemical applications.
28 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
In 1998, sol-gel silicon dioxide processing was introduced for amperometric immunosensors, achieving a low detection limit of 5 ng/mL Agarose gels are frequently utilized on silicon dioxide surfaces to attach antibodies through physical adsorption or covalent linkages Recently, the development of sol-gel thin films has significantly advanced, with their applications in optical immunosensors becoming more prominent than those in electrochemical immunosensors.
In recent years, gold nanoparticles (AuNPs) have become essential in immunosensors for antibody immobilization through physical adsorption, leveraging their unique properties This immobilization occurs via the specific interaction between thiol groups of immunoglobulins and gold atoms Furthermore, AuNPs significantly enhance electron transfer in electrochemical immunosensors due to their high conductivity, leading to improved sensitivity For instance, Wang et al developed an ultrasensitive immunosensor that immobilized antibodies on a glassy carbon electrode modified by electrodeposited AuNPs, enabling the detection of ultralow concentrations of α-1-fetoprotein (AFP) in various 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
Covalent binding is an effective method for immobilizing antibodies onto surfaces, utilizing the accessible functional groups of side-chain-exposed amino acid residues This technique results in irreversible binding and achieves a high degree of surface coverage Functional groups can be introduced either on the substrate or the antibody itself.
The potential for transformable functional groups is crucial for developing surfaces that enable effective coupling chemistries in immunosensors The two primary surfaces utilized are silica (SiO2) on silicon wafers or glass and gold Silica can be surface-modified to introduce functional groups like aldehyde, epoxy, and amine, while gold particles are typically treated with reactive thiol-terminal organic compounds, such as mercaptoundecanoic acid (MUA), via gold-sulfur bonding Additionally, transformable groups on antibodies, including amine, thiol, and hydroxyl residues, serve as functional sites for covalent attachment.
Covalent attachment via amine group
Lysine residues in antibodies are frequently used as anchoring points due to their presence on the antibody's exterior However, the high abundance of lysine can lead to increased binding heterogeneity and limit conformational flexibility.
To prepare silica substrates, the process typically starts with thorough surface cleaning to eliminate contaminants and generate hydroxyl groups These hydroxyl groups serve as attachment points for silane, facilitating covalent bonding Two types of silane are commonly used for this purpose: amine-terminal silane and thiol-terminal silane, which introduce amine and thiol functional groups onto the silica substrate, respectively.
Bifunctional cross-linkers such as glutaraldehyde (GA), succinimidyl-4-N-maleimido-butyrate (GMBS), and N-succinimidyl-4-(maleimido-methyl)-N-cyclohexane-1-carboxylate are commonly used to covalently immobilize car antibodies by facilitating the conjugation between functional groups on a silane layer and amine groups in the antibody Additionally, mixed silanes like APTES and methyltriethoxysilane (MTES) enhance the hydrophobicity of the antibody layer, resulting in more effective immobilization compared to APTES alone.
Gold substrates are typically functionalized to introduce carboxyl groups through a pretreatment with MUA, followed by activation using a combination of EDC and NHS to create NHS esters These esters readily react with primary amine groups (-NH2) found in antibodies, making this method a prevalent choice for covalently immobilizing antibodies on gold surfaces Various antibodies, such as goat anti-E coli, PentaHis, and anti-gIgG, have been successfully immobilized on gold for the development of Surface Plasmon Resonance (SPR) immunosensors Additionally, a bifunctional linker, dithiobis(succinimidyl undecanoate), has been employed as an alternative to the MUA/EDC/NHS method.
Covalent attachment via Thiol group
While coupling through the amine groups in lysine residues of antibodies is a common method for covalent attachment, alternative approaches, such as utilizing thiol groups in cysteine residues, may be more advantageous in certain situations Viitala's study demonstrated the covalent attachment of Fab' fragments from polyclonal anti-human IgG to a polymerizable lipid using maleimide, leading to the formation of internal disulfide bonds This method is less frequent than amine group coupling due to the lower abundance of cysteine compared to lysine To address this limitation, Traut's Reagent (2-Iminothiolane.HCl) is employed for thiolation of primary amine groups in antibodies, resulting in a higher availability of thiol groups for effective coupling.
In Kusnezow's report, it was noted that while the attachment of antibodies via secondary thiol groups may lead to a partial loss of activity, this method offers improved orientation compared to coupling through amine groups.
Figure 2.3 Pre-treated substrate with maleimide and antibody immobilization by thiol groups Covalent attachment via sugar residues
Fabrication of electrochemical sensor based on gold thin film electrodes
This section details the fabrication of an electrochemical sensor utilizing gold thin film electrodes on a silicon substrate, as illustrated in Fig 2.9 The fabrication process on the silicon wafer involves key steps including oxidation, photolithography, etching, and sputtering.
Figure 2.9 Structure of the integrated electrode
The fabrication process utilized a photomask created with CorelDraw software, resulting in a total of 190 electrodes designed on a silicon wafer As illustrated in Fig 2.10, the integrated sensor features three electrodes: the Working Electrode (WE), Reference Electrode (RE), and Counter Electrode (CE), all within a compact dimension of 3.6 x 12 mm², with the Working Electrode occupying an area of 0.785 mm².
This design is optimized for the à-USB configuration, ensuring a reliable connection and seamless interface with the measuring device, thereby eliminating the need for the wire-bonding step in the fabrication process.
Figure 2 .10 Photomask design and detailed structure of electrode sensor
2.2.2 Main processes in the electrochemical sensor fabrication
The wafer-cleaning step is crucial in the overall process, as contaminants on the wafer can lead to failed electrodes and negatively impact experimental results This step aims to eliminate all surface contaminants Initially, to remove organic substances, the wafer is immersed in a fresh piranha solution (H2O2:H2SO4, 3:7, v/v) for 5 minutes, followed by rinsing with deionized (DI) water The wafer is then immersed in ethanol and rinsed again with DI water to ensure thorough cleaning.
The wafer undergoes a cleaning process by boiling it in a 65% HNO3 solution for 10 minutes, followed by rinsing in deionized (DI) water to eliminate inorganic contaminants Finally, the wafer is briefly immersed in a 1% HF solution to remove the thin layer of native silicon dioxide After this thorough cleaning, the wafer is ready for subsequent fabrication processes, as illustrated in Fig 2.11.
The objective of this step is to form a silicon dioxide (SiO2) insulator layer, which serves as a substrate for enhancing the performance of metal electrodes To achieve this, a wet oxidation method is utilized to produce a 100 nm SiO2 layer, as illustrated by the accompanying chemical reaction.
Si(solid) + 2H2O(gas)→ SiO2(solid) + 2H2(gas)
Figure 2.11 Main processes for sensor fabrication
Photolithography is a technique used to transfer geometric shapes from a mask onto a silicon wafer's surface coated with a radiation-sensitive material known as photoresist This process relies on the alteration of the photoresist's solubility in a developer solution when exposed to ultraviolet (UV) light In this study, the positive photoresist S1813 is utilized, with a 2% solution of Tetramethylammonium hydroxide serving as the developer The steps of the photolithography process are detailed in Figure 3.
- Spin-coating of the primer in order to clean the surface and to enhance the adhesion of photoresist
- Spin-coating of the photoresist (spinning velocity: 1000 rpm for 5s, slope for 5s, and 4000 rpm for 30s)
- Pre-baking for 10 minutes at 90 o C
- Exposing the coated wafer in UV light for 50 seconds under designed photomask
- Developing of the exposed photoresist by immersing the wafer in developer solution
- Checking by microscope If the patterns are not good, the photeresist should be washed by acetone and the photolithography is performed again
- Post-baking for 30 minutes at 120 o C in order to polymerize the photoresist
Sputtering has emerged as a leading technique for thin film device fabrication, utilizing accelerated ions, typically Ar+ plasma, to bombard a target material This process facilitates energy transfer, allowing atoms from the source material's surface to be deposited onto a substrate, resulting in thin film growth The film's thickness and quality are influenced by various sputtering conditions, including power, pressure, temperature, and sputtering duration.
Process Parameters Lining Ti Au
Base vacuum 2.1e-6 mTorr 2.1e-6 mTorr Sputtering vacuum 5e-3 mTorr 5e-3 mTorr
In this study, a metal film was deposited on a silicon wafer using the sputtering technique To enhance adhesion between the silicon and gold, a 100 nm thick titanium underlayer was sputtered prior to the deposition of the gold film The sputtering conditions are detailed in Table 2.1, and the thickness of the gold film, approximately 200 nm, was measured using an Alpha Step Profilometer.
Thermal annealing is a crucial heat treatment process in planar technology that enhances the properties of materials on wafers Following sputtering, wafers undergo thermal annealing to alleviate stresses, densify the deposited film, repair crystal defects, and improve interface adhesion This study focuses on the thermal annealing of wafers with deposited gold (Au) film in a controlled atmosphere of water vapor and nitrogen.
After depositing a metal layer on the wafer using the sputtering technique, the lift-off method was employed to eliminate the photoresist and excess metals This lift-off process involved the use of acetone and ultrasonic support Upon completion, the device wafer containing the electrodes was produced, as illustrated in Fig 2.12, and is now prepared for cutting into individual integrated electrodes.
Figure 2.12 Image of electrochemical sensors on a wafer and a complete sensor
Each wafer features around 180 sensor structures, which are individually separated using the DAD322 semi-automatic dicing saw at the Nano and Energy Center, Noi University of Science The dicing process is executed with specific parameters tailored for optimal performance.
- Index: size of sensor is 3.6 mm × 12.0 mm
- Feed speed: cutting rate is 3 mm/s
: revolution of dicing blade is 30000 rev/min
- Work thickness: thickness of silicon wafer 0.6 mm
After dicing process, a complete electrochemical sensor is shown in Fig 2.12 and is ready to be treated with cleaning process
Cleaning the electrode surface is a crucial pretreatment process for electrochemical sensors This step aims to eliminate contaminants that can hinder target attachment efficiency and lead to measurement noise.
The cleaning procedure for the gold electrodes involved several steps to ensure optimal preparation Initially, the electrodes were subjected to ultrasonic cleaning in acetone for 15 minutes, followed by rinsing with deionized water They were then immersed in a fresh piranha solution (H2O2:H2SO4 = 3:7) for 5 minutes and rinsed again with deionized water Subsequently, the electrodes underwent ultrasonic treatment in ethanol and deionized water After each cleaning step with deionized water, the electrodes were dried using a stream of nitrogen to complete the process.
Figure 2.13 Electrochemical cleaning and activation of electrodes in sulfuric acid by CV
The sensors underwent electrochemical scanning between -0.5 and 1.0V, using a commercial Ag/AgCl reference electrode at a scan rate of 50 mV/s in 0.5 M H2SO4, until a stable voltammogram was achieved (see Fig 2.13) Following this activation process, the sensors were fully prepared for subsequent electrochemical experiments and attachment procedures.
Antibody Immobilization
This article explores the development of electrochemical immunosensors by immobilizing bio-components on the working electrode (WE) to detect Newcastle disease virus (ND virus) in poultry Specifically, anti-Newcastle disease virus immunoglobulin Y (anti-ND virus IgY), an antibody derived from egg yolk, serves as a targeted immune bioreceptor Two distinct strategies for antibody immobilization are presented: the first, termed P-GA immunosensor, utilizes protein A and glutaraldehyde; the second, known as SAM-NHS immunosensor, employs self-assembled monolayers (SAM) and N-hydroxysuccinimide (NHS) ester to establish covalent bonds between gold surfaces and the antibody.
Protein A, BSA (Bovine Serum Albumin), PBS buffer, K3Fe(CN)6 and
K4Fe(CN)6 were purchased from Sigma-Aldrich Glutaraldehyde 25% solution was bought from Prolabo The supporting chemicals such as KCl, K 2 Cr 2 O 7 : 99 %,
H2SO4 (98%) and N2 (99.9%) were sourced from China, with all chemicals and reagents being of analytical grade and utilized without additional purification Specific IgY antibodies for Newcastle and Gumboro diseases were acquired from Biotech-Vet Co JSc in Hanoi, Vietnam.
2.3.1 Antibody Immobilization using PrA/GA approach
Protein A is a crucial biological reagent for attaching antibodies to solid substrates due to its strong affinity for the Fc fragment of IgG antibodies Its effective adsorption of gold particles enhances the bio-affinity attachment on gold surfaces However, unlike IgG antibodies from mammals, IgY antibodies derived from egg yolk do not bind to Protein A Therefore, when immobilizing IgY antibodies, it is essential to incorporate an appropriate crosslinking agent.
This study details the fabrication of an ND virus immunosensor, beginning with the modification of the sensor's working electrode (WE) using protein A through bio-affinity adsorption Subsequently, anti-ND virus IgY antibodies were immobilized on the protein A-modified electrode with glutaraldehyde serving as a cross-linking agent This cross-linker facilitates covalent bonding between protein A and IgY antibodies while ensuring proper antibody orientation, which is crucial for maintaining the antigen-binding sites on the Fab fragments The procedure is illustrated in Fig 2.14.
Figure 2.14 The schematic description of the fabrication procedures of PrA-GA immunosensor
To prepare the protein A-modified electrode, the sensor is incubated in a 1 mg/ml protein A solution in 0.1 M PBS buffer for 3 hours Following this, the electrode is immersed in a 5% glutaraldehyde solution in deionized water for 30 minutes Subsequently, anti-ND virus IgY is immobilized by incubating the electrode in a 60 μg/ml PBS solution for 24 hours at 4°C Finally, to block unspecific sites, a coating of 1% BSA in PBS is applied to the working electrode for 30 minutes.
After each modification step, the working electrode was thoroughly rinsed with PBS to eliminate any unbound molecules Fully modified electrodes were then stored at +4°C in 0.1 M PBS, making them ready for immunoassay protocols The immobilization process is visually represented in Figure 2.14.
2.3.2 Antibody Immobilization using SAM/NHS approach
For the functionalization of the gold electrode surface, many recent works have focused on the production of self-assembled monolayers (SAMs) of organic
Alkanethiols, disulfides, and sulfides exhibit a strong affinity for gold surfaces, enabling the formation of a well-ordered and oriented self-assembled monolayer (SAM) Common functional groups used in SAMs include carboxylic (-COOH), amine (-NH2), and thiols (-SH) The immobilization of antibodies onto the SAM, specifically those with carboxylic groups, is facilitated through a stable ester intermediate, which is generated using reagents such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or N,N'-dicyclohexylcarbodiimide (DCC) in conjunction with N-hydrosuccinimide (NHS).
This study utilized thioglycolic acid (TGA) to create a self-assembled monolayer (SAM) on a gold (Au) surface, featuring oriented COOH functional groups The anti-ND-virus IgY was subsequently attached to the modified electrode through covalent bonding between the COOH terminals of the SAMs and the –NH2 groups of IgY, facilitated by the co-addition of DCC and NHS A schematic diagram illustrating the fabrication of the SAM-NHS immunosensor is provided in Fig 2.15, with the experimental process outlined in detail.
Figure 2.15 The schematic of antibody immobilization process using SAM-
The working electrode (WE) was incubated in a 10 mM thioglycolic acid (TGA) ethanol solution for 24 hours to create a self-assembled monolayer (SAM) After rinsing the SAM-modified Au electrode with ethanol, it was treated with 0.2 M DCC and 0.1 M NHS for one hour at room temperature to convert the terminal carboxylic group into an active NHS ester Following a wash with water and drying, the electrode was incubated with a 60 μg/mL solution of anti-ND-virus antibodies in borate buffer (pH = 8.2) for 12 hours at 4°C Finally, the electrode was rinsed with PBS and incubated for 30 minutes in 1% BSA (in PBS) to block nonspecific sites, before being thoroughly rinsed with PBS and dried with nitrogen.
Immunoassay Protocol
For the preparation of Newcastle disease virus (ND virus) samples, the inactivated Newcastle vaccine type M was sourced from Hanviet Co JSc in Vietnam, with an initial concentration of 10^6 EID50/mL, followed by a series of dilutions.
ND virus was obtained by the dilutions in 0.1 M PBS
The immunoassay procedures were performed as the follows:
A 5 μL drop of ND virus sample was introduced into the working electrode (WE) of the immunosensor, followed by a 60-minute incubation at room temperature After incubation, the immunosensor was rinsed five times with PBS buffer, with each immersion lasting 10 seconds and then dried using a gentle stream of nitrogen The immunoreaction results of the immunosensors developed through these methods are illustrated in Fig 2.16.
Figure 2.16 The schematic of immune ND virus reaction in immunosensor
When the whole immuneassay procedures are completed, the immunosensors is ready for electrochemical measurements
DETECTION OF NEWCASTLE DISEASE VIRUS USING
Characteristics of electrochemical sensor
The sensors utilized in this phase are referred to as bare electrodes, as they have not undergone any treatment or chemical processing Their characteristics were evaluated through cyclic voltammetry (CV) measurements, employing the EC301 Stanford Research System in a three-electrode configuration These sensors consist of a gold thin film deposited on an insulated SiO2/Si substrate An Ag/AgCl wire, produced through a straightforward chemical reaction of silver in an FeCl3 solution, serves as the external reference electrode (RE) in place of the built-in option.
Firstly, a sliver wire (1.0 mm diameter) was cut to 3 cm long, polished and cleaned before immerging it about 2 cm into a solution of 0.5 M FeCl3 solution for
In just 30 seconds, the silver wire changed color to black, indicating the successful formation of an AgCl film Following this reaction, the Ag/AgCl was rinsed with deionized water and dried using a nitrogen stream, making it ready for use as a pseudo reference electrode.
In parallel, a commercial Ag/AgCl RE as a standard RE was also employed to evaluate the performance and quality of the Ag/AgCl wire For CV
48 measurements, potential was cycled from -0.1 V to 0.5 V, with scan rate of 25 mV/s All measurements were conducted in 0.1 M KCl solution containing 0,03 M [Fe(CN) 6 ] 3−/4− (1:1) as a redox probe
The cyclic voltammetry (CV) responses of the redox probe [Fe(CN)6] 3−/4− were analyzed using both commercial Ag/AgCl reference electrodes (RE) and Ag/AgCl wire The results, illustrated in Fig 3.1, demonstrate a reversible cyclic voltammogram for [Fe(CN)6] 3−/4− with the commercial RE, characterized by a notable difference between the anodic and cathodic peak potentials (ΔEpeak = Eanodic - Ecathodic).
The optimal performance range for sensors is indicated by a ΔE peak between 60 mV and 100 mV, as reported by Tang When the ΔE peak exceeds 100 mV, it suggests potential contamination on the sensor's surface, necessitating additional cleaning to restore the peak to normal levels Following tests with a commercial reference electrode (RE), the cyclic voltammetry (CV) of the sensor was recorded using a fabricated Ag/AgCl wire as the reference electrode, revealing a reversible cyclic voltammogram for [Fe(CN)6] 3−/4− with a redox peak difference of 122 mV.
Figure 3.1 CV curves of sensor with commercial Ag/AgCl RE and Ag/AgCl wire
The peak current (Ipeak) derived from the cyclic voltammetry (CV) of sensors serves as the primary signal in measurements, with Ipeak representing the difference between anodic and cathodic currents Consequently, achieving uniformity in peak current values across multiple sensors is a crucial characteristic, playing a vital role in ensuring the accuracy and reliability of the measurements obtained.
The cyclic voltammograms (CVs) of five randomly selected sensors were recorded under identical measuring conditions, as illustrated in Fig 3.2A The results indicate a high degree of uniformity in the electrochemical performance of the sensors, evidenced by the similar shapes of the CV curves and the proximity of the peak positions This finding aligns with Tri u Quân's report, which highlights that the discrepancy in current amplitude is minimal.
Figure 3.2 The uniform of sensors (A) CV curves of five 5 sensors (with Ag/AgCl wire RE), (B) The average and standard deviation of Ipeak of five sensors (before and after cleaning)
Fig 3.2B shows the average and error bar of Ipeak of five sensors before and after cleaning process
In this thesis, standard deviation (SD) and relative standard deviation (%RSD) are calculated as the formulas:
%RSD = relative standard deviation x1, , xn = the sample data set x = mean value of the sample data set n = size of the sample data set
The average peak current (Ipeak) of cleaned sensors is significantly higher at 197.11 μA compared to unclean sensors This increase in Ipeak following the cleaning process is attributed to the removal of contaminants that obstruct the electro-transfer of Fe(CN)6 3−/4− at the electrode surface.
The %RSD (n = 5) of the peak current (Ipeak) for cleaned sensors is significantly lower at 1.03% compared to 7.7% for unclean sensors Additionally, the %RSD for unclean sensors is 15% This demonstrates that cleaned sensors exhibit superior uniformity in their performance.
Thus, electrochemical characteristics, that obtained by CV measurements, have shown the stable performance of electrochemical sensors versus Ag/AgCl wire reference electrode.
Characteristics of PrA-GA immunosensor
3.2.1 Cyclic voltammetry characterization of PrA-GA immunosensor
As the experimental procedure presented in chapter 2, the PrA-GA immunosensor formation, based on modification of the sensor’s WE through protein
A and glutaraldehyde (GA), can be described as follows:
First, protein A was directly adsorbed onto the surface of bare gold electrode Then, an amino group (-NH2), that appears in some amino acid residues of protein
A (e.g., lysine), was reacted with one of two aldehyde groups (-CHO) of GA to form a covalent binding After that, the residual aldehyde group of GA was
51 employed for immobilization of IgY antibody, that also constitutes a covalent bond at the same reaction Finally, BSA was used for blocking of unspecific binding sites
The principle of this antibody immobilization, as shown in Fig 3.3, is based on the reaction between aldehyde groups (-CHO) and amine groups (-NH2), which forms imines bonds (C=N bonds)
Figure 3.3 The reaction of GA linker with protein A and IgY antibody
To examine the formation of modified layers on a bare gold (Au) electrode, the modification process was systematically recorded using cyclic voltammetry (CV) The conditions and parameters for the CV measurements were consistent with those used for bare electrodes The resulting CV curves are presented in Figure 3.4, along with essential parameter values listed in Table 3.1.
The bare Au electrode exhibits a reversible redox cyclic voltammogram for the [Fe(CN)6] 3−/4– pairs, with a peak current (Ipeak) of 202.8 μA, indicating efficient electron transfer However, the adsorption of protein A creates a passivating layer that significantly impedes the transfer of Fe(CN)6 3−/4– pairs to the electrode surface, resulting in a notable decrease in Ipeak to 129.2 μA Additionally, the introduction of glutaraldehyde linkers onto the protein A-electrode surface causes a slight further reduction in current.
Ipeak to 110.0 μA (curves c) The immobilization of IgY antibodies, that led to a further decrease of Ipeak to 99.0 μA (curves d), is also observed.
Figure 3.4 CV characterization of modified electrode recorded on Au electrode
(a); PrA-Au electrode (b); GA-PrA-Au electrode (c); IgY-GA-PrA-Au electrode
(d); BSA-IgY-GA-PrA-Au electrode (e)
Table 3.1 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor
Au-PrA-GA-IgY-BSA 85.3 131
The reduction in Ipeak responses with each addition of glutaraldehyde and IgY antibodies to the protein A-Au electrode indicates a successful reaction, highlighting the cross-linking role of glutaraldehyde, as illustrated in Fig 3.2.
3.2.2 Effect of the IgY concentration on the immobilization of PrA-GA immunosensor
The performance of an immunosensor is influenced by several factors, such as the concentration of attached components like protein A and GA linker, the amount of immobilized anti-ND virus IgY, the pH of the liquid substrate, and the temperature This study specifically examines how varying IgY antibody concentrations affect the immobilization process.
Table 3.2 Experimental conditions for the attachment of components
Components Concentrations Incubation time Tem Ref
Protein A 1 mg/ml 3h Room tem [ ] 63
GA linker 5% wt 30 min Room tem [ ] 51
BSA 1% wt 30 min Room tem [ ] 63
ND virus 10 6 EID50/mL - Room tem [ ] 63
In our experiments, we utilized a 0.1 M PBS buffer to maintain a stable pH of 7.4, optimal for the biological properties of immune proteins Additional fixed factors are detailed in Table 3.2.
The procedure was implemented as the follows:
Working electrodes were incubated with 1.0 mg/ml protein A for 3 hours to create a monolayer, followed by immersion in a 5% GA solution for 30 minutes to attach cross-linkers The impact of varying IgY antibody concentrations, ranging from 20 μg/ml to 60 μg/ml, was assessed over 24 hours at 4°C After a blocking step with BSA, all immunosensors were utilized to detect the ND virus at a concentration of 10^6 EID50.
CV measurement was carried out to obtain the peak current of each immunosensor before and after assay with ND virus
The ΔIpeak, expressed in the formula 3.3, is used to evaluate the performance of immunosensors
54 where Ipeak(0) is the Ipeak of each fabricated immunosensor, and the Ipeak(i) is the Ipeak obtained after employed with 10 6 EID50 ND virus concentration
Figure 3.5 Effect of the antibody concentration
The results are exhibited in Fig 3.5 Fig 3.5 shows a rapid increase of ΔIpeak from 20 μg/ml to 60 μg/ml IgY antibody used and a maximum when the concentration of antibody is 60 μg/ml.
Higher concentrations of incubated antibody molecules lead to an increased yield of covalent bond formation, resulting from the reaction between the amino groups of antibodies and the aldehyde groups of the GA layer The commercially available concentration of 60 μg/ml of anti-ND virus IgY is the highest and was selected for antibody immobilization.
Characteristics of SAM-NHS immunosensor
This section discusses the characterization of an immunosensor created using a second approach that involves the formation of a self-assembled monolayer (SAM) and the functionalization of NHS ester groups.
3.3.1 Cyclic voltammetry characterization of SAM-NHS immunosensor
As the experimental procedure in chapter 2, the second approach for the fabrication of immunosensor can be described as the follows:
A self-assembled monolayer (SAM) of thioglycolic acid (TGA) is created on the gold electrode surface via a robust Au-thiolate bond, with the carboxylic group exposed at the interface This monolayer undergoes activation through a sequential process involving the formation and replacement of terminal DCC and NHS, resulting in the creation of an NHS ester Finally, the immobilization of the antibody occurs as the active NHS ester is substituted by the amine groups of the antibody.
ND virus IgY Finally, BSA is used to block unspecific binding sites
Figure 3.6 The main reactions on the antibody immobilization
The immobilization method in chemical principles relies on three key reactions: the formation of Au-thiolate bonds through the interaction of Au atoms with thiol groups (S-H), the activation of carboxylic groups to produce NHS esters, and the reaction of NHS ester terminals with the primary amine of IgY antibodies These processes are illustrated in Fig 3.6.
The formation of modified layers on gold electrodes was monitored through cyclic voltammetry (CV) measurements taken before and after each treatment, with results illustrated in Fig 3.7 Additionally, the I peak and ΔEpeak values from the CV curves are summarized in Table 3.3.
Figure 3.7 CV characterization of modification of WE (a) Au electrode, (b) TGA-Au electrode, (c) active NHS-TGA-Au electrode, (d) IgY-NHS-TGA-Au electrode, (e) BSA-IgY-NHS-TGA-Au electrode
The bare Au electrode exhibited a reversible cyclic voltammogram, indicating a clean surface In contrast, the formation of the TGA-monolayer on the Au electrode was characterized by a lack of peak currents and redox peaks, demonstrating that the TGA-monolayer creates a highly insulating surface that effectively blocks faradic currents This phenomenon occurs because the negatively charged terminal carboxylic groups of TGA, formed through deprotonation in aqueous solution, significantly impede the transfer of the negative Fe(CN)63- /4 probe to the electrode surface.
Table 3.3 The crucial parameters obtained from experimental CV data for fabrication procedures of immunosensor
Au-TGA-DCC+NHS-IgY 124.75 300
Au-TGA-DCC+NHS-IgY-BSA 116.70 350
The co-addition of DCC and NHS activates the formation of NHS ester functional groups on the TGA monolayer, evidenced by an increased current response in curve c This peak current increase indicates that the negatively charged carboxylic groups of TGA are replaced by NHS ester, which, due to electrostatic interactions, allows for the unhindered transfer of negative redox probes to the electrode surface Consequently, the TGA monolayer modified with NHS ester exhibits reduced insulating properties The changes observed in the CV characterizations of the Au electrode modified by TGA monolayer and NHS ester are illustrated in Fig 3.8.
Figure 3.8 The schematic description of the CV responses of modified electrode
(a) bare Au electrode, (b) TGA-electrode, (c) active NHS-Au electrode
The immobilization of IgY macromolecules on the active NHS ester significantly hinders the access of Fe(CN)63- /4 pairs to the electrode surface, resulting in a notable decrease in current response Furthermore, the blocking of nonspecific binding sites on the antibody by BSA leads to an additional slight reduction in peak current, as observed in the electrochemical response.
Thus, CV characterizations provided useful information on the changes of the electrode behavior after each modifying step
3.3.2 Effect of the pH value on the immobilization of SAM-NHS immunosensor
This section examines the pH factors that influence the performance of the SAM-NHS immunosensor, while other fixed variables are detailed in Table 3.4.
Table 3.4 Experimental conditions for the attachment of components
Components Concentration Incubated time Tem Media (pH) Ref
SAM (TGA) 10 mL 24 h Room Tem Ethanol [ ] 54
DCC/NHS 0.2/0.1 M 1 h Room Tem DMF [ ] 54
BSA 1% wt 30 min Room Tem PBS (7.4) [ ] 63
ND virus 10 2 EID 50 /mL - Room Tem PBS (7.4) [ ] 63
In the experiment, IgY antibody samples were prepared at the same concentration using various buffers: Citrate (pH = 5.0), PBS (pH = 7.4), and Borate (pH = 8.4) All immunosensors were fabricated under consistent conditions as outlined in Table 3.4, aside from the differing buffers The impact of pH on antibody immobilization was assessed by measuring the ΔIpeak of immunosensors using a 10² EID50 ND virus concentration in PBS, as illustrated in Fig 3.9.
Figure 3.9 Effect of pH value of the immobilization of antibody
At pH values of 8.2 and 9.4, there is a significant increase in ΔIpeak, peaking at pH 8.2, indicating optimal antibody immobilization on the SAM-modified electrode with a slight alkaline buffer (Borate buffer) The attachment of IgY antibodies occurs through the aminolysis reaction, where NHS esters on the electrode surface react with the primary -NH2 groups of the antibodies to form stable amide bonds Research shows that the efficiency of this reaction is reduced at lower pH values, resulting in a decreased density of antibody molecules on the immunosensor, as evidenced by lower ΔIpeak readings at pH levels such as 5.0 and 7.4.
Therefore, Borate buffer providing the pH value 8.2 would be used to immobilize IgY antibody in the fabrication of SAM-NHS immunosensor.
Stability of the signal of ND virus immunosensors
The peak current value of the immunosensor, represented as Ipeak(0), is crucial in the formula for ΔIpeak, measured after the sensor's complete fabrication To ensure effective performance, immunosensors must exhibit signal stability, which is achieved through high uniformity in their construction.
The Ipeak(0) value is crucial for effective virus detection, as a higher Ipeak(0) enhances the sensitivity of the immunosensor, allowing for the identification of viruses at low concentrations In this context, Ipeak(0) refers to the initial peak value of the immunosensor, while Ipeak(i) represents the peak value measured after the immunoreaction with the ND virus.
This study examines the stability of signal in two types of immunosensors by analyzing the average and standard deviation (SD) of Ipeak(0) with n = 5 Figure 3.10 illustrates the comparison of Ipeak(0) between the two immunosensor types, while also displaying the Ipeak of a bare Au electrode to highlight the signal changes following modifications.
Figure 3.10 The average and the SD of I peak of the bare Au electrode (A), PrA-GA immunosensor (B), and SAM-NHS immunosensor (C)
The analysis of Fig 3.10 and Table 3.5 reveals that both types of immunosensors demonstrate high uniformity, evidenced by their low standard deviation (SD) values Notably, the initial peak current (Ipeak(0)) of the SAM-NHS immunosensor significantly surpasses that of the PrA-GA immunosensor.
Table 3.5 The average and standard deviation of I peak of sensors (n=5)
Both types of immunosensors demonstrate strong signal stability However, the SAM-NHS immunosensor may excel in virus detection, owing to its higher peak current (Ipeak(0)).
Detection of Newcastle disease virus
This study focuses on the development of an electrochemical immunosensor for the detection of the inactivated Newcastle Disease (ND) virus By attaching anti-ND virus antibodies and supporting substances to the sensor's working electrode (WE), the sensor transforms into an effective detection device The detection method employs Fe(CN)63- /4 as an electroactive marker, allowing for direct virus detection without the need for additional labeling substances.
Figure 3.11 The schematic description of the ND virus detection mechanism
The detection of the ND virus involves measuring CV before and after treatment with the immunosensor The change in peak currents, represented by ΔIpeak and calculated using formula 3.3, is essential for assessing the immunosensor's performance The mechanism of ND virus detection utilizing a label-free electrochemical immunosensor is illustrated in Fig 3.11.
When ND virus binds specifically to the antibody layer, it obstructs the electro-transfer of Fe(CN)63- /4, resulting in a reduced peak current on the CV curve.
3.4.1 Effect of the immunoreaction time
Immunoreaction time, influenced by the incubation duration of the ND virus, is a crucial factor impacting the efficacy of immunosensors in virus detection This section will explore the optimal experimental immunoreaction times for various types of fabricated immunosensors, specifically focusing on the PrA-GA immunosensor.
PrA-GA immunosensors were incubated with ND virus samples at the same
In a study examining the immunoreaction time at a concentration of 10^6 EID50/ml, results indicated that the optimal virus incubation time for achieving maximum immunoreactivity was 60 minutes, as evidenced by the peak ΔIpeak observed in the data presented in Fig 3.12B.
ND viruses on the immunosensor This is referred to the complete specific binding of ND virus with anti-ND virus antibody.
Figure 3.12 Effect of the immunoreaction time
SAM-NHS immunosensors were incubated with ND virus samples at a consistent concentration of 10^2 EID 50/ml, with incubation times ranging from 15 to 90 minutes The results indicated that the peak change in current (ΔIpeak) was maximized at an incubation time of 60 minutes, as illustrated in Fig 3.12B.
When the incubation time exceeds 60 minutes, both types of immunosensors exhibit a slight decrease in the ΔI peak This phenomenon occurs because the immunoreaction is reversible and reaches equilibrium at around 60 minutes, where the maximum number of immune complexes is formed In an open system, external factors significantly influence this equilibrium Beyond 60 minutes, the equilibrium gradually shifts towards the reverse reaction, leading to the dissociation of immune complexes As a result, there is a reduction in the number of viruses bound to the antibodies on the immunosensor, causing an increase in Ipeak(i) and a corresponding decrease in ΔI peak.
Therefore, 60 min is chosen as an effective incubation time of ND virus samples, which will be used for both kinds of immunosensor
3.5.2 Sensitivity of Newcastle disease virus immunosensor
Herein, quantitative assessment of sensitivity of two kinds of immunosensor is performed using inactivated ND virus solution in PBS buffer from 1×10 0 to 1×10 6 EID50/mL at room temperature for 1 h
The correlation between Ipeak and ΔIpeak was analyzed through CV curves and ΔIpeak values in an immunoassay involving the PrA-GA immunosensor and varying concentrations of ND virus, as illustrated in Fig 3.12 As shown in Fig 3.12A, the peak currents progressively decrease with increasing ND virus concentrations, highlighting the inhibitory effects of the virus on the electron transfer between Fe(CN)63-/4 and the PrA-GA immunosensor Additionally, Fig 3.12B further supports these findings.
64 increase of ΔIpeak is exhibited by a nearly linear relationship between ΔIpeak and the logarithm of the concentration of ND virus
The CV curves of the PrA-GA immunosensor demonstrate its performance in a buffer solution and after exposure to varying concentrations of ND virus, specifically at 10², 10³, 10⁴, 10⁵, and 10⁶ EID50/mL Additionally, there is a clear relationship between the change in peak current (ΔIpeak) and the different concentrations of the ND virus detected by the PrA-GA immunosensor.
The sensitivity of the two types of immunosensors was assessed through calibration curves that illustrate the relationship between ΔIpeak and the logarithm of ND virus concentration, as shown in Fig 3.14 These curves, analyzed using Origin software, represent the average of five measurements, with error bars indicating the standard deviation for each data point Key parameters derived from the calibration process are detailed in Table 3.6.
Table 3.6 The crucial parameters obtained from the calibration
Parameter PrA- GA SAM-NHS
Linear range of concentration 10 2 -10 6 EID50/ml 10 2 -10 6 EID50/ml
Standard deviation of the slope (SD S ) 0.00188 0.00286 y-intercept (y i ) -0.0099 0.2606
LOD 0.95 (9 EID 50 /ml) 0.67 (5 EID 50 /ml)
LOQ 2.88 (10 3 EID 50 /ml) 2.03 (10 2 EID 50 /ml)
Both types of immunosensors demonstrate a nearly linear relationship between ΔIpeak and lgC NDvirus within the concentration range of 10² EID50/ml to 10⁶ EID50/ml The linear regression equation for the PrA-GA immunosensor is ΔIpeak = 0.0289lgCNDvirus + 0.0099, with an R² value of 0.983 In contrast, the SAM-NHS immunosensor has a linear regression equation of ΔIpeak = 0.0588lgCNDvirus + 0.261.
Figure 3.14 The relationship between ΔIpeakand various ND virus concentrations
The SAM-NHS immunosensor demonstrates superior performance compared to the PrA-GA immunosensor, exhibiting a higher slope value on the calibration curve and increased ΔIpeak values at each concentration level According to Michael Swartz's report, the limits of detection (LOD) and limits of quantification (LOQ) can be calculated using specific equations.
66 where σ is the standard deviation of the response y-intercepts of the regression lines and S is the slope of the calibration curve
In our study, the PrA-GA immunosensor demonstrated limit of detection (LOD) and limit of quantification (LOQ) values of 10^0.95 EID50/ml and 10^3 EID50/ml, respectively Conversely, the SAM-NHS immunosensor exhibited superior sensitivity, with LOD and LOQ values of 10^0.67 EID50/ml and 10^2 EID50/ml These findings highlight that the SAM-NHS immunosensor outperforms the PrA-GA immunosensor in terms of sensitivity.
Table 3.7 Comparison of analytical properties of different immunosensors for the detection of Avian Influenza
Technique Immobilization Type Antibody Virus Detection limit Ref
Optic APTES/CDI Indirect Goat monoclonal
IgY Purified NDV 2 ng/ml [53]
RRT-PCR NDV RNA 10 1 EID 50 /ml [60]
CV PrA/GA Direct IgY from egg yolk Inactivated
CV SAM/DCC/NHS Direct IgY from egg yolk Inactivated
Cyclic voltammetry (CV) is a key technique used in various studies involving Newcastle disease virus (NDV) and Avian influenza (AI) Important methodologies include carbonyldiimidazole (CDI) for chemical reactions and the use of Flinders Technology Associates (FTA) cards for sample preservation In virology, terms like Embryo Infectious Dose (EID) and Embryo Lethal Dose (ELD) are critical for assessing viral impacts Additionally, reverse transcriptase-polymerase chain reaction (RT-R) and real-time reverse-transcription PCR (RRT-PCR) are essential tools for detecting and quantifying viral RNA in red blood cells (RBCs), facilitating research and diagnostics in infectious diseases.
In the same conditions of measurement as well as of immune reaction (temperature, pH, time), a high sensitivity of immunosensor is closely related to a
The SAM-NHS immunosensor demonstrates a significantly higher sensitivity due to the dense immobilization of specifically active antibodies on the working electrode (WE) This indicates that utilizing SAM and NHS for antibody immobilization enhances the performance of the ND virus immunosensor.