# Simple Label-Free Electrochemical Immunosensor in a Microchamber for Detecting Newcastle Disease Virus

Authors: Luyen Thi Tran; Thinh Quang Tran; Ha Phu Ho; Xuan Thi Chu; Tuan Anh Mai
Journal: Journal of Nanomaterials
 (2019)

---

## Abstract

In this study, a simple, label-free, electrochemical immunosensor system, including a three-electrode transducer and a microchamber, was designed, fabricated, and integrated with focus toward the detection of Newcastle disease virus (NDV). The chicken egg yolk antibodies (IgY) against NDV were used as the biological recognition element, replacing purified IgG antibodies that require a complex extraction process and time-consuming. The IgY against NDV was immobilized on the sensor surface using PrA/GA and SAM/NHS approaches. The immunosensor showed high sensitivity with NDV concentrations ranging from 106 to 102 EID50/mL with good specificity, repeatability, and small standard deviations. Compared to traditional methods, the immunosensor with advantages such as simple fabrication, quick response, direct detection, and possibility for miniaturization by integrating the immunosensor with the microchamber is potential for applications in contamination studies and field measurements.


---

## Body

## 1. Introduction

Newcastle disease (ND) is one of the most contagious diseases in poultry that has widely spread in South East Asian countries including Vietnam and causes severe economic losses [1, 2]. So far, the conventional qualitative methods such as hemagglutination inhibition (HI) [2], agar gel precipitation [3], and Latex agglutination tests [4] have been introduced for clinical diagnosis of ND. In addition, enzyme-linked immunosorbent assay (ELISA) [2], polymerase chain reaction (PCR) [5, 6], and immunofluorescence test [7] have also been used for the semiquantitative analysis. Although these methods effectively determine NDV in infective samples, they require complicated procedures for sample preparations, sophisticated instruments for assays, and large periods of time for the completion of the assays. Therefore, alternative methods that offer a simple, rapid, and cost-effective analytical strategy and possible on-site and in-field measurements are essential.Label-free electrochemical immunosensor detects antigens using specific antibodies immobilized on a surface of metallic electrodes [8–11]. This method has been developed with rapid analysis, high sensitivity, and portability purposes [12–15]. In this work, the three-electrode setup including the integrated Au electrodes consisted of a working electrode (WE), a counter electrode (CE) in a chip, and a quasireference Ag/AgCl electrode (qRE) was designed and fabricated. The polydimethylsiloxane- (PDMS-) based microchamber was bonded with the integrated three-electrode setup to facilitate miniaturization of the analytical system, and thus, the volume of the consumed biological sample is reduced. Moreover, this structure also simplifies the microelectrode fabrication process in a clean room and, on the other hand, is convenient for electrical connection with a measuring circuit.In immunosensor development, most of the studies, using monoclonal immunoglobulin G (IgG) antibody from mammalian blood as the biological recognition element, have been focused on antibody immobilization methods to improve the sensitivity, response time, and detection limit [16–18]. However, IgG antibody production and purification require modern techniques, expensive biologicals, long time, and complicated procedures [19, 20]. In our previous work [21], chicken egg yolk immunoglobulin (IgY) antibody was used as an alternative to IgG in immunosensor fabrication. The immobilization using IgY antibody offers advantages with respect to cost-effective and rapid detection of virus in infectious disease outbreaks. In addition, it is less dependent on purified IgG sources with shorter lease time and reduced costs. In this work, chicken egg yolk antibodies (IgY) against ND virus were immobilized on the Au electrodes (WE) using two different methods: (1) the combination protein A/glutaraldehyde (PrA/GA) and (2) the self-assembly modification of thioglycolic acid with the activation by N-succinimidyl ester (SAM/NHS). The fabricated immunosensors were used to detect ND virus. The effect of virus incubation time on the output signal of the immunosensors and specificity and sensitivity of the ND virus immunosensors was also investigated. Both immunosensors showed advantages over conventional ones such as simple process, quick response, direct detection, high specificity and sensitivity, and potential miniaturization by integrating with micro reactors.


## 2. Materials and Methods

### 2.1. Chemicals

Polydimethylsiloxane (PDMS) Sylgard 184 was obtained from Dow Corning. Staphylococcal protein A (PrA), bovine serum albumin (BSA, 98% purity), phosphate buffer solution (PBS, 0.01 M, pH 7.4), potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)6·3H2O, 99.5% purity), potassium ferricyanide(III) (K3Fe(CN)6, 97% purity), potassium chloride (KCl, 99.0% purity), glutaraldehyde solution (25 wt. % in H2O), thioglycolic acid (TGA, 98.0% purity), N,N′-dicyclohexylcarbodiimide (DCC, 99.0% purity), N-hydroxysuccinimide (NHS, 97% purity), and N,N-dimethylformamide (DMF, 99.8% purity) were purchased from Sigma-Aldrich. All other reagents were of analytical grade and used without further purification.Chicken egg yolk antibodies against Newcastle disease virus (IgY, 60μg/mL) were obtained from Biotech-Vet Co. (Hanoi, Vietnam). Newcastle disease vaccine type M (inactivated NDV, 106 embryo infectious dose 50% (EID50)/mL) was purchased from Hanvet Co. JSc. (Hanoi, Vietnam). Avian influenza virus vaccines including H5N1 subtype (inactivated AIV/H5N1 virus) and H5N2 subtype (inactivated AIV/H5N2 virus) were supplied by Joint Stock Company Central Veterinary (Hanoi, Vietnam).


### 2.2. Measuring Setup

The integrated Au electrodes consisted of a 1 mm in diameter working electrode (WE) and a counter electrode (CE) in an8.5×12.2mm2 chip. These electrodes were deposited on a SiO2/Si substrate by cathode sputtering technique. The detailed fabrication process was previously discussed [22]. The (100 μm in diameter) Ag/AgCl wire, used as a quasireference electrode (qRE), was prepared by oxidizing the silver wire [23]. The three-electrode system (including qRE) is integrated with a PDMS-based microchamber in which the qRE is immersed (independently) in a 100 μL open chamber (Figure 1(a)). This structure, on the one hand, reduces the microelectrode fabrication process in a clean room and, on the other hand, is convenient for electrical connection with a measuring circuit (Figure 1(b)). The system is wired with EC301 from Stanford Research Systems.Figure 1
(a) Schematic illustration of the measuring setup and (b) equivalent circuit of the measuring system.
(a)
(b)


### 2.3. IgY Antibody Immobilization

To prepare a clean surface with ready functional groups before immobilizing IgY antibody, the integrated Au electrodes were pretreated in acetone, Piranha solution (H2O2 : H2SO4, 3 : 7), and ethanol. The electrodes were then rinsed with deionized water and nitrogen dried. After that, the Au electrodes were electrochemically activated in 0.5 M H2SO4 solution by sweeping the voltage from -0.5 V to +1 V vs. Ag/AgCl (in sat. 3 M KCl) at 50 mV/s until the cyclic voltammetry characteristics were stable.Right after the surface treatment, chicken egg yolk antibodies (IgY) against ND virus were immobilized on the Au electrodes (WE) by two approaches, namely, protein A/glutaraldehyde (PrA/GA) and self-assembly modification/N-succinimidyl active ester (SAM/NHS).


#### 2.3.1. The Scheme of Antibody Immobilization Process Using PrA/GA

The scheme of antibody immobilization process using PrA/GA is shown in Figures2(a)–2(e). Firstly, the Au working electrode (WE) was incubated with a solution of protein A (1 mg/mL in PBS) for 3 hours. The PrA-modified electrode was then soaked in a solution of GA (5% wt. in deionized water) for 30 minutes. Then, IgY antibodies (60 μg/mL in PBS) were immobilized on the Au WE for 3 hours at 4°C. Lastly, BSA (1% wt./vol. in PBS) was deposited on the WE for 30 minutes to block unbound sites. After each modified step, the WE was rinsed with PBS to remove unbound molecules. The immunosensor was stored in a refrigerator at 4°C for further use.Figure 2
The scheme of antibody immobilization process using PrA/GA.


#### 2.3.2. The Scheme of Antibody Immobilization Process Using SAM/NHS

The scheme of antibody immobilization process using SAM/NHS is shown in Figures3(a)–3(e). The Au WE was incubated with ethanol solution of 10 mM TGA for 24 hours for self-assembly modification (SAM) to occur. The excess TGA molecules on the modified WE were removed by rinsing with ethanol. The SAM-modified Au WE was treated with a DMF solution of 0.2 M DCC and 0.1 M NHS for 1 h at room temperature to convert the terminal carboxylic group to the active NHS ester. After rinsing with deionized water, the WE was incubated with 60 μg/mL solution (borate buffer, pH=8.2) of IgY antibodies for 12 hours at 4°C. The excess antibodies were then washed off with PBS. BSA (1% wt./vol. in PBS) was also used to block unbound sites. Finally, the WE was rinsed with PBS, nitrogen dried, and stored at 4°C.Figure 3
The scheme of antibody immobilization process using SAM/NHS.


### 2.4. Newcastle Disease Virus Detection

In the three-electrode system, the Au (WE) was prepared with IgY antibodies and the fabricated Ag/AgCl qRE was wired with the microchamber (Figure1). ND virus was detected using the label-free electrochemical immunosensor. 20 μL NDV sample (with varied concentrations of the NDV in 0.01 M PBS, pH 7.4) was injected into the microchamber. After the microchamber was incubated for 1 hour at 25°C, it was rinsed five times with PBS buffer. Electrolyte, composed of 0.1 M KCl and 0.03 M K3Fe(CN)6/K4Fe(CN)6 (1 : 1) as a redox couple, was pumped into the microchamber with the rate of 100 μL/min. Cyclic voltammetry (CV) measurements were performed using EC301 Stanford Research Systems; the potential was cycled from -0.2 to 0.5 V with scan rate 25 mV/s.


## 2.1. Chemicals

Polydimethylsiloxane (PDMS) Sylgard 184 was obtained from Dow Corning. Staphylococcal protein A (PrA), bovine serum albumin (BSA, 98% purity), phosphate buffer solution (PBS, 0.01 M, pH 7.4), potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)6·3H2O, 99.5% purity), potassium ferricyanide(III) (K3Fe(CN)6, 97% purity), potassium chloride (KCl, 99.0% purity), glutaraldehyde solution (25 wt. % in H2O), thioglycolic acid (TGA, 98.0% purity), N,N′-dicyclohexylcarbodiimide (DCC, 99.0% purity), N-hydroxysuccinimide (NHS, 97% purity), and N,N-dimethylformamide (DMF, 99.8% purity) were purchased from Sigma-Aldrich. All other reagents were of analytical grade and used without further purification.Chicken egg yolk antibodies against Newcastle disease virus (IgY, 60μg/mL) were obtained from Biotech-Vet Co. (Hanoi, Vietnam). Newcastle disease vaccine type M (inactivated NDV, 106 embryo infectious dose 50% (EID50)/mL) was purchased from Hanvet Co. JSc. (Hanoi, Vietnam). Avian influenza virus vaccines including H5N1 subtype (inactivated AIV/H5N1 virus) and H5N2 subtype (inactivated AIV/H5N2 virus) were supplied by Joint Stock Company Central Veterinary (Hanoi, Vietnam).


## 2.2. Measuring Setup

The integrated Au electrodes consisted of a 1 mm in diameter working electrode (WE) and a counter electrode (CE) in an8.5×12.2mm2 chip. These electrodes were deposited on a SiO2/Si substrate by cathode sputtering technique. The detailed fabrication process was previously discussed [22]. The (100 μm in diameter) Ag/AgCl wire, used as a quasireference electrode (qRE), was prepared by oxidizing the silver wire [23]. The three-electrode system (including qRE) is integrated with a PDMS-based microchamber in which the qRE is immersed (independently) in a 100 μL open chamber (Figure 1(a)). This structure, on the one hand, reduces the microelectrode fabrication process in a clean room and, on the other hand, is convenient for electrical connection with a measuring circuit (Figure 1(b)). The system is wired with EC301 from Stanford Research Systems.Figure 1
(a) Schematic illustration of the measuring setup and (b) equivalent circuit of the measuring system.
(a)
(b)


## 2.3. IgY Antibody Immobilization

To prepare a clean surface with ready functional groups before immobilizing IgY antibody, the integrated Au electrodes were pretreated in acetone, Piranha solution (H2O2 : H2SO4, 3 : 7), and ethanol. The electrodes were then rinsed with deionized water and nitrogen dried. After that, the Au electrodes were electrochemically activated in 0.5 M H2SO4 solution by sweeping the voltage from -0.5 V to +1 V vs. Ag/AgCl (in sat. 3 M KCl) at 50 mV/s until the cyclic voltammetry characteristics were stable.Right after the surface treatment, chicken egg yolk antibodies (IgY) against ND virus were immobilized on the Au electrodes (WE) by two approaches, namely, protein A/glutaraldehyde (PrA/GA) and self-assembly modification/N-succinimidyl active ester (SAM/NHS).


### 2.3.1. The Scheme of Antibody Immobilization Process Using PrA/GA

The scheme of antibody immobilization process using PrA/GA is shown in Figures2(a)–2(e). Firstly, the Au working electrode (WE) was incubated with a solution of protein A (1 mg/mL in PBS) for 3 hours. The PrA-modified electrode was then soaked in a solution of GA (5% wt. in deionized water) for 30 minutes. Then, IgY antibodies (60 μg/mL in PBS) were immobilized on the Au WE for 3 hours at 4°C. Lastly, BSA (1% wt./vol. in PBS) was deposited on the WE for 30 minutes to block unbound sites. After each modified step, the WE was rinsed with PBS to remove unbound molecules. The immunosensor was stored in a refrigerator at 4°C for further use.Figure 2
The scheme of antibody immobilization process using PrA/GA.


### 2.3.2. The Scheme of Antibody Immobilization Process Using SAM/NHS

The scheme of antibody immobilization process using SAM/NHS is shown in Figures3(a)–3(e). The Au WE was incubated with ethanol solution of 10 mM TGA for 24 hours for self-assembly modification (SAM) to occur. The excess TGA molecules on the modified WE were removed by rinsing with ethanol. The SAM-modified Au WE was treated with a DMF solution of 0.2 M DCC and 0.1 M NHS for 1 h at room temperature to convert the terminal carboxylic group to the active NHS ester. After rinsing with deionized water, the WE was incubated with 60 μg/mL solution (borate buffer, pH=8.2) of IgY antibodies for 12 hours at 4°C. The excess antibodies were then washed off with PBS. BSA (1% wt./vol. in PBS) was also used to block unbound sites. Finally, the WE was rinsed with PBS, nitrogen dried, and stored at 4°C.Figure 3
The scheme of antibody immobilization process using SAM/NHS.


## 2.3.1. The Scheme of Antibody Immobilization Process Using PrA/GA

The scheme of antibody immobilization process using PrA/GA is shown in Figures2(a)–2(e). Firstly, the Au working electrode (WE) was incubated with a solution of protein A (1 mg/mL in PBS) for 3 hours. The PrA-modified electrode was then soaked in a solution of GA (5% wt. in deionized water) for 30 minutes. Then, IgY antibodies (60 μg/mL in PBS) were immobilized on the Au WE for 3 hours at 4°C. Lastly, BSA (1% wt./vol. in PBS) was deposited on the WE for 30 minutes to block unbound sites. After each modified step, the WE was rinsed with PBS to remove unbound molecules. The immunosensor was stored in a refrigerator at 4°C for further use.Figure 2
The scheme of antibody immobilization process using PrA/GA.


## 2.3.2. The Scheme of Antibody Immobilization Process Using SAM/NHS

The scheme of antibody immobilization process using SAM/NHS is shown in Figures3(a)–3(e). The Au WE was incubated with ethanol solution of 10 mM TGA for 24 hours for self-assembly modification (SAM) to occur. The excess TGA molecules on the modified WE were removed by rinsing with ethanol. The SAM-modified Au WE was treated with a DMF solution of 0.2 M DCC and 0.1 M NHS for 1 h at room temperature to convert the terminal carboxylic group to the active NHS ester. After rinsing with deionized water, the WE was incubated with 60 μg/mL solution (borate buffer, pH=8.2) of IgY antibodies for 12 hours at 4°C. The excess antibodies were then washed off with PBS. BSA (1% wt./vol. in PBS) was also used to block unbound sites. Finally, the WE was rinsed with PBS, nitrogen dried, and stored at 4°C.Figure 3
The scheme of antibody immobilization process using SAM/NHS.


## 2.4. Newcastle Disease Virus Detection

In the three-electrode system, the Au (WE) was prepared with IgY antibodies and the fabricated Ag/AgCl qRE was wired with the microchamber (Figure1). ND virus was detected using the label-free electrochemical immunosensor. 20 μL NDV sample (with varied concentrations of the NDV in 0.01 M PBS, pH 7.4) was injected into the microchamber. After the microchamber was incubated for 1 hour at 25°C, it was rinsed five times with PBS buffer. Electrolyte, composed of 0.1 M KCl and 0.03 M K3Fe(CN)6/K4Fe(CN)6 (1 : 1) as a redox couple, was pumped into the microchamber with the rate of 100 μL/min. Cyclic voltammetry (CV) measurements were performed using EC301 Stanford Research Systems; the potential was cycled from -0.2 to 0.5 V with scan rate 25 mV/s.


## 3. Results and Discussion

A cyclic voltammetry (CV) test was conducted using conventional reference electrode and the fabricated Ag/AgCl qRE (located inside the microchamber). In both cases, two peaks at 0.2 V/0.1 V and 0.27 V/0.17 V, representing the oxidation of Fe(CN)64– and reduction of Fe(CN)63–, were observed on the CV curves. The measured peak current (Ianodic and Icathodic) and the difference between the anodic and cathodic peak potentials, ΔE, stayed almost the same after 20 CV cycles (detail was described in supplemental data).


### 3.1. Immobilization of the IgY Antibody from Chicken Egg Yolk on the Au Electrode

Figure4 shows CV characteristics of the WE measured in Fe(CN)63−/4– solution with different IgY antibody immobilization step for PrA/GA immunosensor (Figure 4(a)) and SAM/NHS immunosensor (Figure 4(b)). The difference in the current peak (Ipeak=Ianodic−Icathodic) was used to demonstrate the formation of the layers of the immunosensor during stepwise modification procedures (see Table S1 in supplemental information).Figure 4
CV characterization of modified electrodes measured in the microchamber. (a) PrA/GA immunosensor: CV curve of (A) Au electrode, (B) PrA-Au electrode, (C) GA-PrA-Au electrode, (D) IgY-GA-PrA-Au electrode, and (E) BSA-IgY-GA-PrA-Au electrode and (F) after immunoreaction of 104 EID50/mL NDV solution with PrA/GA immunosensor. (b) SAM/NHS immunosensor: (A) Au electrode, (B) TGA-Au electrode, (C) NHS-TGA-Au electrode, (D) IgY-NHS-TGA-Au electrode, and (E) BSA-IgY-NHS-TGA-Au electrode and (F) after immunoreaction of 104 EID50/mL NDV solution with SAM/NHS immunosensor.
(a)
(b)


#### 3.1.1. Antibody Immobilization Using PrA/GA Approach

As observed in Figure4(a), the highest Ipeak (202.8 μA) on the CV curve obtained with the bare Au electrode due to high conductivity and low barrier of gold metal makes it easy for electron transfer. The adsorption of protein A on the Au surface formed an insulating layer that reduces electron transfer leading to a lower peak current of the Au/PrA electrode (Ipeak,Au/PrA=129.2μA). The attachment of glutaraldehyde linkers onto the PrA/Au electrode also resulted in a reduction of Ipeak (Ipeak,Au/PrA/GA=110.0μA). Glutaraldehyde plays a cross-linking role between protein A and IgY antibody through the formation of imine bonds (C=N bonds) between -CHO groups and –NH2 groups as shown in Figure 2. The immobilization of IgY antibodies caused a further decrease in Ipeak (Ipeak,Au/PrA/GA/IgY=99.0μA).The above discussion revealed that the binding of IgY antibodies on the surface of immunosensor leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current. When the WE was deposited with BSA for blocking unbound sites and the IgY antibody was immobilized on the Au electrode, a slight decrease in the peak current was also observed (Ipeak,PrA−GAimmunosensor=87.3μA).


#### 3.1.2. Antibody Immobilization Using SAM/NHS Approach

As shown in Figure3(b), a self-assembled modification (SAM) of TGA is formed on the Au electrode surface through the strong gold-thiolate bonds leading to the formation of a highly insulating surface layer that blocks almost all the faradic currents [24, 25]. That is why no (current) peak on the curve (B) was observed (Figure 4(b)). The reason for this is that the negatively charged terminal carboxyl groups of TGA (formed by the deprotonation in aqueous solution) prevent the transfer of negative Fe(CN)63-/4- redox couple to the electrode surface creating a depleted layer between the sensor surface and solution. If the TGA layer is activated by DCC and NHS, it forms an NHS ester and the negatively charged carboxyl groups of TGA are replaced by the neutrally charged NHS esters as shown in Figure 3(c). Thus, the transfer of the negative Fe(CN)63-/4- redox couple to the electrode surface makes it advantageous for the current to flow. As a result, the current density is increased as illustrated in Figure 4(b) (C) (Ipeak,Au/TGA/DCC+NHS=132.29μA).The immobilization of IgY antibodies on the immunosensor’s surface in which active NHS ester is replaced by the amine groups of anti-NDV IgY (Figure3(d)) leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current (Ipeak,Au/TGA/DCC+NHS/IgY=124.75μA). When the WE was deposited with BSA for blocking unbound sites, a further slight decrease in the peak current was also observed in Figure 4(b) (E) (Ipeak,SAM−NHSimmunosensor=116.7μA). The two immobilization methods of IgY antibodies on the Au electrode exhibit good repeatability that are demonstrated by small deviation values of Ipeak,immunosensor (as shown in Figure S2 of supplemental data).


### 3.2. Newcastle Disease Virus Detection

The current response of immunosensors after reaction with NDV was also characterized by CV measurements in K3Fe(CN)6/K4Fe(CN)6 solution with a scan rate of 25 mV/s and shown as each curve (F) in Figure 4(a) (PrA/GA immunosensor) and Figure 4(b) (SAM/NHS immunosensor).As observed in Figure4(a), in the case of PrA/GA immunosensor, when specific antibody-virus interaction occurred on the surface of the immunosensor, the peak current (Ipeak,NDV/immunosensor=79.1μA, curve (F)) decreased compared to that of the immunosensor without biological interactions (Ipeak,immunosensor=88.2μA, curve (E)). Performing the same experiment using the SAM/NHS immunosensor (Figure 4(b)), one could obtain the similar result. The peak current (Ipeak,NDV/immunosensor=60.3μA, curve (F)), given by the interaction between specific antibody and virus that occurred on the surface of the immunosensor, was smaller than that with no biological interactions (Ipeak,immunosensor=116.7μA, curve (E)). The above results could explain that NDV are bound on the surface of the immunosensor based on specific antibody-virus interaction and therefore inhibiting the charge transfer process of Fe(CN)63-/4- redox couple to the Au electrode surface, resulting in a decrease in current response.The output signal of the immunosensor is calculated by the following equation:(1)ΔIpeak=Ipeak0−IpeakiIpeak0,where Ipeak0 is the Ipeak of the immunosensor treated with 0 EID50/mL NDV (in 0.01 M PBS, pH=7.4), and Ipeaki is the Ipeak of the immunosensor obtained after incubating with a certain NDV sample.


### 3.3. Effect of Virus Incubation Time on the Output Signal of the Immunosensors

In both cases of PrA/GA immunosensors and SAM-NHS immunosensors, the current responses increase accordingly with the incubation time andΔIpeak reaches its maximum value (ΔIpeak=0.166 and ΔIpeak=0.370, respectively) at 60 minutes for the immunoreaction (Figures 5(a) and 5(b)). Further increase in incubation time did not cause significant change in ΔIpeak for both types of immunosensors.
(2)IgYAntibody+AntigenNDVirus→ImmunecomplexFigure 5
The output signal of immunosensor depends on incubation time: (a) PrA/GA immunosensor (with 106 EID50/mL NDV) and (b) SAM/NHS immunosensor (with 102 EID50/mL NDV).
(a)
(b)This is due to the nonlinear kinetic of the reversible immunoreaction as shown below that reaches its fastest rate of the association in the first hour [26]. Therefore, for further experiments, 60 min is selected as the virus incubation time for both types of immunosensors.


### 3.4. Specificity of the ND Virus Immunosensors

In order to evaluate the specificity of the PrA/GA and SAM/NHS immunosensors for the detection of NDV,ΔIpeak induced by PBS as the blank and several nonspecific viruses was measured. Nonspecific viruses, including H5N1 and H5N2 avian influenza A virus, and specific NDV were all prepared in 102 EID50/mL. The immunoreactions were carried out with NDV, PBS, AIV/H5N1, and AIV/H5N2 samples for 1 h at 25°C in the microchamber. The specificity of the PrA/GA and SAM/NHS immunosensors for the NDV is exhibited in Figures 6(a) and 6(b). No obvious ΔIpeak response could be found under the blank (PBS). Similarly, at the same concentration, ΔIpeak obtained in the NDV measurement was much higher than that in cases of AIV/H5N1 and of AIV/H5N2. This implies that only the interaction between IgY antibody and NDV can cause significant current changes, another way to say that the developed PrA/GA and SAM/NHS immunosensors offer a good specificity to NDV.Figure 6
Specificity of the PrA/GA (a) and SAM/NHS (b) immunosensors for NDV detection. The output signals,ΔIpeak, of the immunosensors induced by 102 EID50/mL NDV and nonspecific samples such as 0.01 M PBS (pH=7.4), 102 EID50/mL AIV/H5N1, and 102 EID50/mL AIV/H5N2.
(a)
(b)


### 3.5. Sensitivity of the ND Virus Immunosensors

To determine the sensitivity of the fabricated immunosensors, various samples of inactivated NDV with concentrations varying from 102 to106 EID50/mL (in 0.01 M PBS, pH=7.4) were tested. For each NDV concentration, five CV measurements were performed similarly using five immunosensors. The standard deviation of the measurements is calculated by the following formula:
(3)S=1N−1∑i=1NΔIi−ΔI¯2,where N is the number of measurements corresponding to N immunosensors; ΔIi is the output signal of the ith immunosensor; ΔI¯ is the average value of NΔI values corresponding to N immunosensors; S is the real standard deviation (the absolute deviation), and S/ΔI¯×100% is the relative standard deviation.After acquiring and processing the data, the calibration curves for both corresponding to the two types of fabricated immunosensors that express the relationship betweenΔIpeak and the logarithm of ND virus concentration are presented in Figure 7. As shown in Figure 7, both types of immunosensors showed the linear relationship between ΔIpeak and log[CNDV] in the range of 102 EID50/mL–106 EID50/mL NDV. The linear regression equation of the PrA/GA immunosensor was ΔIpeak=0.0289×logCNDV−0.0099 (R2=0.983) and that of the SAM/NHS immunosensor was ΔIpeak=0.0588×logCNDV+0.261 (R2=0.991). The calibration curve corresponding to the SAM/NHS immunosensor exhibited a higher slope value and ΔIpeak values at each concentration than those of the PrA/GA immunosensor.Figure 7
ΔIpeak is a function of NDV concentrations.According to Swartz [27], the limit of detection (LOD) and the limit of quantification (LOQ) can be calculated using the following equations:
(4)LOD=3.3×σS,LOQ=10×σS,where σ is the standard deviation of the response y-intercept of the regression line and S is the slope of the calibration curve. Table 1 shows the crucial parameters obtained from the two calibration curves corresponding to the two types of electrochemical label-free immunosensors. The LOD and LOQ were 100.95 EID50/mL and 102.88 EID50/mL in the case of PrA/GA immunosensor and 100.67 EID50/mL and 102.03 EID50/mL in the case of SAM/NHS immunosensor, respectively. It can be explained that, at the same measuring conditions and immune reaction (temperature, pH, incubation time, etc.), due to the formation of stable covalent bonds, the density of specific antibodies bound on the SAM/NHS immunosensor’s surface is higher than that of the PrA/GA immunosensor. Moreover, the insulating property of PrA that influences the formation of the antibody-antigen complex partly contributes to lowering sensitivity of the PrA/GA immunosensor as compared to the SAM/NHS immunosensor.Table 1
The crucial parameters of the two types of electrochemical label-free immunosensors.
Parameter
PrA/GA immunosensor
SAM/NHS immunosensor
Linear range of concentration
102-106 EID50/mL
102-106 EID50/mL
Correlation coefficient (R2)
0.983
0.991
Slope (S)
0.0289
0.0588
Standard deviation of the slope (SDS)
0.00188
0.00286
y-intercept (yi)
-0.0099
0.2606
Standard deviation of they-intercept (σ)
0.0080
0.0119
LOD
0.95 (9 EID50/mL)
0.67 (5 EID50/mL)
LOQ
2.88 (102.88 EID50/mL)
2.03 (102.03 EID50/mL)Table2 summarizes some analytical properties of different methods for the detection of NDV. The limits of detection values of the two types of electrochemical label-free immunosensors were comparable to those of the PCR. Moreover, the fabricated immunosensors also showed advantages over conventional ones such as simple process, do not require clean room facilities, quick response, direct detection, and potential miniaturization by integrating with micro reactors (100 μL–1 mL in volume) which may be suitable for future on-site and in-field measurements. In particular, in this work, the chicken egg yolk antibody (IgY) was used as the biological recognition element of the immunosensor instead of using IgG antibody (monoclonal and polyclonal antibody) [16, 28, 29] which requires modern techniques and expensive biologicals and goes through many steps with complicated procedure [19, 20].Table 2
Comparison of analytical properties of different methods for the detection of NDV.
Technique
Immobilization
Type
Antibody
Virus
Limit of detection
Ref.
Optic
APTES/CDI
Indirect
Goat monoclonal IgY
Purified NDV
2 ng/mL
[30]
RT-PCR
Inactivated NDV
5 × 102 ELD50/mL
[31]
RT-PCR
Inactivated NDV
5 × 102 ELD50/mL
[5]
RT-PCR
Inactivated NDV
105.8 ELD50/mL
[6]
RRT-PCR
NDV RNA
10 EID50/mL
[1]
CV
PrA/GA
Direct
IgY
Inactivated NDV
9 EID50/mL
This work
CV
SAM/NHS
Direct
IgY
Inactivated NDV
5 EID50/mL
NDV: Newcastle disease virus; EID: embryo infectious dose; ELD: embryo lethal dose; RT-PCR: reverse transcriptase-polymerase chain reaction; RRT-PCR: real-time reverse transcription PCR.The study about using chicken egg yolk antibody (IgY) as the biological recognition element of the immunosensor may not only reduce analysis costs but also provide an effective solution to detect immediately virus in outbreaks of infectious diseases.


## 3.1. Immobilization of the IgY Antibody from Chicken Egg Yolk on the Au Electrode

Figure4 shows CV characteristics of the WE measured in Fe(CN)63−/4– solution with different IgY antibody immobilization step for PrA/GA immunosensor (Figure 4(a)) and SAM/NHS immunosensor (Figure 4(b)). The difference in the current peak (Ipeak=Ianodic−Icathodic) was used to demonstrate the formation of the layers of the immunosensor during stepwise modification procedures (see Table S1 in supplemental information).Figure 4
CV characterization of modified electrodes measured in the microchamber. (a) PrA/GA immunosensor: CV curve of (A) Au electrode, (B) PrA-Au electrode, (C) GA-PrA-Au electrode, (D) IgY-GA-PrA-Au electrode, and (E) BSA-IgY-GA-PrA-Au electrode and (F) after immunoreaction of 104 EID50/mL NDV solution with PrA/GA immunosensor. (b) SAM/NHS immunosensor: (A) Au electrode, (B) TGA-Au electrode, (C) NHS-TGA-Au electrode, (D) IgY-NHS-TGA-Au electrode, and (E) BSA-IgY-NHS-TGA-Au electrode and (F) after immunoreaction of 104 EID50/mL NDV solution with SAM/NHS immunosensor.
(a)
(b)


### 3.1.1. Antibody Immobilization Using PrA/GA Approach

As observed in Figure4(a), the highest Ipeak (202.8 μA) on the CV curve obtained with the bare Au electrode due to high conductivity and low barrier of gold metal makes it easy for electron transfer. The adsorption of protein A on the Au surface formed an insulating layer that reduces electron transfer leading to a lower peak current of the Au/PrA electrode (Ipeak,Au/PrA=129.2μA). The attachment of glutaraldehyde linkers onto the PrA/Au electrode also resulted in a reduction of Ipeak (Ipeak,Au/PrA/GA=110.0μA). Glutaraldehyde plays a cross-linking role between protein A and IgY antibody through the formation of imine bonds (C=N bonds) between -CHO groups and –NH2 groups as shown in Figure 2. The immobilization of IgY antibodies caused a further decrease in Ipeak (Ipeak,Au/PrA/GA/IgY=99.0μA).The above discussion revealed that the binding of IgY antibodies on the surface of immunosensor leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current. When the WE was deposited with BSA for blocking unbound sites and the IgY antibody was immobilized on the Au electrode, a slight decrease in the peak current was also observed (Ipeak,PrA−GAimmunosensor=87.3μA).


### 3.1.2. Antibody Immobilization Using SAM/NHS Approach

As shown in Figure3(b), a self-assembled modification (SAM) of TGA is formed on the Au electrode surface through the strong gold-thiolate bonds leading to the formation of a highly insulating surface layer that blocks almost all the faradic currents [24, 25]. That is why no (current) peak on the curve (B) was observed (Figure 4(b)). The reason for this is that the negatively charged terminal carboxyl groups of TGA (formed by the deprotonation in aqueous solution) prevent the transfer of negative Fe(CN)63-/4- redox couple to the electrode surface creating a depleted layer between the sensor surface and solution. If the TGA layer is activated by DCC and NHS, it forms an NHS ester and the negatively charged carboxyl groups of TGA are replaced by the neutrally charged NHS esters as shown in Figure 3(c). Thus, the transfer of the negative Fe(CN)63-/4- redox couple to the electrode surface makes it advantageous for the current to flow. As a result, the current density is increased as illustrated in Figure 4(b) (C) (Ipeak,Au/TGA/DCC+NHS=132.29μA).The immobilization of IgY antibodies on the immunosensor’s surface in which active NHS ester is replaced by the amine groups of anti-NDV IgY (Figure3(d)) leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current (Ipeak,Au/TGA/DCC+NHS/IgY=124.75μA). When the WE was deposited with BSA for blocking unbound sites, a further slight decrease in the peak current was also observed in Figure 4(b) (E) (Ipeak,SAM−NHSimmunosensor=116.7μA). The two immobilization methods of IgY antibodies on the Au electrode exhibit good repeatability that are demonstrated by small deviation values of Ipeak,immunosensor (as shown in Figure S2 of supplemental data).


## 3.1.1. Antibody Immobilization Using PrA/GA Approach

As observed in Figure4(a), the highest Ipeak (202.8 μA) on the CV curve obtained with the bare Au electrode due to high conductivity and low barrier of gold metal makes it easy for electron transfer. The adsorption of protein A on the Au surface formed an insulating layer that reduces electron transfer leading to a lower peak current of the Au/PrA electrode (Ipeak,Au/PrA=129.2μA). The attachment of glutaraldehyde linkers onto the PrA/Au electrode also resulted in a reduction of Ipeak (Ipeak,Au/PrA/GA=110.0μA). Glutaraldehyde plays a cross-linking role between protein A and IgY antibody through the formation of imine bonds (C=N bonds) between -CHO groups and –NH2 groups as shown in Figure 2. The immobilization of IgY antibodies caused a further decrease in Ipeak (Ipeak,Au/PrA/GA/IgY=99.0μA).The above discussion revealed that the binding of IgY antibodies on the surface of immunosensor leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current. When the WE was deposited with BSA for blocking unbound sites and the IgY antibody was immobilized on the Au electrode, a slight decrease in the peak current was also observed (Ipeak,PrA−GAimmunosensor=87.3μA).


## 3.1.2. Antibody Immobilization Using SAM/NHS Approach

As shown in Figure3(b), a self-assembled modification (SAM) of TGA is formed on the Au electrode surface through the strong gold-thiolate bonds leading to the formation of a highly insulating surface layer that blocks almost all the faradic currents [24, 25]. That is why no (current) peak on the curve (B) was observed (Figure 4(b)). The reason for this is that the negatively charged terminal carboxyl groups of TGA (formed by the deprotonation in aqueous solution) prevent the transfer of negative Fe(CN)63-/4- redox couple to the electrode surface creating a depleted layer between the sensor surface and solution. If the TGA layer is activated by DCC and NHS, it forms an NHS ester and the negatively charged carboxyl groups of TGA are replaced by the neutrally charged NHS esters as shown in Figure 3(c). Thus, the transfer of the negative Fe(CN)63-/4- redox couple to the electrode surface makes it advantageous for the current to flow. As a result, the current density is increased as illustrated in Figure 4(b) (C) (Ipeak,Au/TGA/DCC+NHS=132.29μA).The immobilization of IgY antibodies on the immunosensor’s surface in which active NHS ester is replaced by the amine groups of anti-NDV IgY (Figure3(d)) leads to an increase in the charge transfer resistance of Fe(CN)63-/4- redox couple to the Au electrode, thereby reducing the peak current (Ipeak,Au/TGA/DCC+NHS/IgY=124.75μA). When the WE was deposited with BSA for blocking unbound sites, a further slight decrease in the peak current was also observed in Figure 4(b) (E) (Ipeak,SAM−NHSimmunosensor=116.7μA). The two immobilization methods of IgY antibodies on the Au electrode exhibit good repeatability that are demonstrated by small deviation values of Ipeak,immunosensor (as shown in Figure S2 of supplemental data).


## 3.2. Newcastle Disease Virus Detection

The current response of immunosensors after reaction with NDV was also characterized by CV measurements in K3Fe(CN)6/K4Fe(CN)6 solution with a scan rate of 25 mV/s and shown as each curve (F) in Figure 4(a) (PrA/GA immunosensor) and Figure 4(b) (SAM/NHS immunosensor).As observed in Figure4(a), in the case of PrA/GA immunosensor, when specific antibody-virus interaction occurred on the surface of the immunosensor, the peak current (Ipeak,NDV/immunosensor=79.1μA, curve (F)) decreased compared to that of the immunosensor without biological interactions (Ipeak,immunosensor=88.2μA, curve (E)). Performing the same experiment using the SAM/NHS immunosensor (Figure 4(b)), one could obtain the similar result. The peak current (Ipeak,NDV/immunosensor=60.3μA, curve (F)), given by the interaction between specific antibody and virus that occurred on the surface of the immunosensor, was smaller than that with no biological interactions (Ipeak,immunosensor=116.7μA, curve (E)). The above results could explain that NDV are bound on the surface of the immunosensor based on specific antibody-virus interaction and therefore inhibiting the charge transfer process of Fe(CN)63-/4- redox couple to the Au electrode surface, resulting in a decrease in current response.The output signal of the immunosensor is calculated by the following equation:(1)ΔIpeak=Ipeak0−IpeakiIpeak0,where Ipeak0 is the Ipeak of the immunosensor treated with 0 EID50/mL NDV (in 0.01 M PBS, pH=7.4), and Ipeaki is the Ipeak of the immunosensor obtained after incubating with a certain NDV sample.


## 3.3. Effect of Virus Incubation Time on the Output Signal of the Immunosensors

In both cases of PrA/GA immunosensors and SAM-NHS immunosensors, the current responses increase accordingly with the incubation time andΔIpeak reaches its maximum value (ΔIpeak=0.166 and ΔIpeak=0.370, respectively) at 60 minutes for the immunoreaction (Figures 5(a) and 5(b)). Further increase in incubation time did not cause significant change in ΔIpeak for both types of immunosensors.
(2)IgYAntibody+AntigenNDVirus→ImmunecomplexFigure 5
The output signal of immunosensor depends on incubation time: (a) PrA/GA immunosensor (with 106 EID50/mL NDV) and (b) SAM/NHS immunosensor (with 102 EID50/mL NDV).
(a)
(b)This is due to the nonlinear kinetic of the reversible immunoreaction as shown below that reaches its fastest rate of the association in the first hour [26]. Therefore, for further experiments, 60 min is selected as the virus incubation time for both types of immunosensors.


## 3.4. Specificity of the ND Virus Immunosensors

In order to evaluate the specificity of the PrA/GA and SAM/NHS immunosensors for the detection of NDV,ΔIpeak induced by PBS as the blank and several nonspecific viruses was measured. Nonspecific viruses, including H5N1 and H5N2 avian influenza A virus, and specific NDV were all prepared in 102 EID50/mL. The immunoreactions were carried out with NDV, PBS, AIV/H5N1, and AIV/H5N2 samples for 1 h at 25°C in the microchamber. The specificity of the PrA/GA and SAM/NHS immunosensors for the NDV is exhibited in Figures 6(a) and 6(b). No obvious ΔIpeak response could be found under the blank (PBS). Similarly, at the same concentration, ΔIpeak obtained in the NDV measurement was much higher than that in cases of AIV/H5N1 and of AIV/H5N2. This implies that only the interaction between IgY antibody and NDV can cause significant current changes, another way to say that the developed PrA/GA and SAM/NHS immunosensors offer a good specificity to NDV.Figure 6
Specificity of the PrA/GA (a) and SAM/NHS (b) immunosensors for NDV detection. The output signals,ΔIpeak, of the immunosensors induced by 102 EID50/mL NDV and nonspecific samples such as 0.01 M PBS (pH=7.4), 102 EID50/mL AIV/H5N1, and 102 EID50/mL AIV/H5N2.
(a)
(b)


## 3.5. Sensitivity of the ND Virus Immunosensors

To determine the sensitivity of the fabricated immunosensors, various samples of inactivated NDV with concentrations varying from 102 to106 EID50/mL (in 0.01 M PBS, pH=7.4) were tested. For each NDV concentration, five CV measurements were performed similarly using five immunosensors. The standard deviation of the measurements is calculated by the following formula:
(3)S=1N−1∑i=1NΔIi−ΔI¯2,where N is the number of measurements corresponding to N immunosensors; ΔIi is the output signal of the ith immunosensor; ΔI¯ is the average value of NΔI values corresponding to N immunosensors; S is the real standard deviation (the absolute deviation), and S/ΔI¯×100% is the relative standard deviation.After acquiring and processing the data, the calibration curves for both corresponding to the two types of fabricated immunosensors that express the relationship betweenΔIpeak and the logarithm of ND virus concentration are presented in Figure 7. As shown in Figure 7, both types of immunosensors showed the linear relationship between ΔIpeak and log[CNDV] in the range of 102 EID50/mL–106 EID50/mL NDV. The linear regression equation of the PrA/GA immunosensor was ΔIpeak=0.0289×logCNDV−0.0099 (R2=0.983) and that of the SAM/NHS immunosensor was ΔIpeak=0.0588×logCNDV+0.261 (R2=0.991). The calibration curve corresponding to the SAM/NHS immunosensor exhibited a higher slope value and ΔIpeak values at each concentration than those of the PrA/GA immunosensor.Figure 7
ΔIpeak is a function of NDV concentrations.According to Swartz [27], the limit of detection (LOD) and the limit of quantification (LOQ) can be calculated using the following equations:
(4)LOD=3.3×σS,LOQ=10×σS,where σ is the standard deviation of the response y-intercept of the regression line and S is the slope of the calibration curve. Table 1 shows the crucial parameters obtained from the two calibration curves corresponding to the two types of electrochemical label-free immunosensors. The LOD and LOQ were 100.95 EID50/mL and 102.88 EID50/mL in the case of PrA/GA immunosensor and 100.67 EID50/mL and 102.03 EID50/mL in the case of SAM/NHS immunosensor, respectively. It can be explained that, at the same measuring conditions and immune reaction (temperature, pH, incubation time, etc.), due to the formation of stable covalent bonds, the density of specific antibodies bound on the SAM/NHS immunosensor’s surface is higher than that of the PrA/GA immunosensor. Moreover, the insulating property of PrA that influences the formation of the antibody-antigen complex partly contributes to lowering sensitivity of the PrA/GA immunosensor as compared to the SAM/NHS immunosensor.Table 1
The crucial parameters of the two types of electrochemical label-free immunosensors.
Parameter
PrA/GA immunosensor
SAM/NHS immunosensor
Linear range of concentration
102-106 EID50/mL
102-106 EID50/mL
Correlation coefficient (R2)
0.983
0.991
Slope (S)
0.0289
0.0588
Standard deviation of the slope (SDS)
0.00188
0.00286
y-intercept (yi)
-0.0099
0.2606
Standard deviation of they-intercept (σ)
0.0080
0.0119
LOD
0.95 (9 EID50/mL)
0.67 (5 EID50/mL)
LOQ
2.88 (102.88 EID50/mL)
2.03 (102.03 EID50/mL)Table2 summarizes some analytical properties of different methods for the detection of NDV. The limits of detection values of the two types of electrochemical label-free immunosensors were comparable to those of the PCR. Moreover, the fabricated immunosensors also showed advantages over conventional ones such as simple process, do not require clean room facilities, quick response, direct detection, and potential miniaturization by integrating with micro reactors (100 μL–1 mL in volume) which may be suitable for future on-site and in-field measurements. In particular, in this work, the chicken egg yolk antibody (IgY) was used as the biological recognition element of the immunosensor instead of using IgG antibody (monoclonal and polyclonal antibody) [16, 28, 29] which requires modern techniques and expensive biologicals and goes through many steps with complicated procedure [19, 20].Table 2
Comparison of analytical properties of different methods for the detection of NDV.
Technique
Immobilization
Type
Antibody
Virus
Limit of detection
Ref.
Optic
APTES/CDI
Indirect
Goat monoclonal IgY
Purified NDV
2 ng/mL
[30]
RT-PCR
Inactivated NDV
5 × 102 ELD50/mL
[31]
RT-PCR
Inactivated NDV
5 × 102 ELD50/mL
[5]
RT-PCR
Inactivated NDV
105.8 ELD50/mL
[6]
RRT-PCR
NDV RNA
10 EID50/mL
[1]
CV
PrA/GA
Direct
IgY
Inactivated NDV
9 EID50/mL
This work
CV
SAM/NHS
Direct
IgY
Inactivated NDV
5 EID50/mL
NDV: Newcastle disease virus; EID: embryo infectious dose; ELD: embryo lethal dose; RT-PCR: reverse transcriptase-polymerase chain reaction; RRT-PCR: real-time reverse transcription PCR.The study about using chicken egg yolk antibody (IgY) as the biological recognition element of the immunosensor may not only reduce analysis costs but also provide an effective solution to detect immediately virus in outbreaks of infectious diseases.


## 4. Conclusions

The integrated three-electrode system using fabricated Ag/AgCl electrode as qRE and the microchamber not only simplifies the microelectrode fabrication process in a clean room and reduces the analytical volume but also increases accuracy by enhancing the signal-to-noise ratio and thereby improving the sensitivity of electrochemical immunosensors. The IgY was used as the biological recognition element to replace purified IgG antibody that requires a complex extraction process and is time-consuming. The IgY against NDV was immobilized on the Au electrodes (WE) using PrA/GA and SAM/NHS approaches. Both immunosensors offer linear relationship with log[CNDV] in the range of 102 EID50/mL-106 EID50/mL NDV. The fabricated immunosensors showed advantages over conventional ones such as simple process, do not require clean room facilities, quick response, direct detection, and potential miniaturization by integrating with micro reactors (100 μL–1 mL in volume) which may be suitable for future on-site and in-field measurements.



---

*Source: 3835609-2019-10-10.xml*