- Email: [email protected]
Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode
Accepted Manuscript Title: Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode Author: Hedieh Haji-Hashemi Parviz Norouzi Mohammad Reza Safarnejad Mohammad Reza Ganjali PII: DOI: Reference:
S0925-4005(16)32106-2 http://dx.doi.org/doi:10.1016/j.snb.2016.12.135 SNB 21509
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
24-9-2016 14-12-2016 28-12-2016
Please cite this article as: Hedieh Haji-Hashemi, Parviz Norouzi, Mohammad Reza Safarnejad, Mohammad Reza Ganjali, Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.12.135 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode
Hedieh Haji-Hashemi1, Parviz Norouzi*1,2, Mohammad Reza Safarnejad3, Mohammad Reza Ganjali1,2 1
Center of Excellence in Electrochemistry, University of Tehran, Tehran, Iran
2
Endocrinology & Metabolism Research Center, Tehran University of Medical Sciences, Tehran,
Iran 3
Agricultural research, education and extension organization, Iranian Research Institute of
Plant Protection, Tehran, Iran
*
Corresponding author. Tel.: +98-21-61112788; Fax: +98-21-66495291 E-mail address: [email protected]
Highlights
The first electrochemical immunosensor for citrus tristeza virus (CTV) detection.
Simplification of the operation as a result of labeling steps elimination.
Excellent specificity of proposed sensor against interferences from real samples.
Outstanding LOD that is noticeably lower than previous reports for CTV detection.
Abstract This paper presents the first label-free and sensitive electrochemical immunosensor for efficient and rapid detection of citrus tristeza virus (CTV). The specific antibody against coat protein (CP) of CTV was successfully immobilized on 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) modified gold electrode via carbodiimide coupling reaction using
N-(3-dimethylaminopropyl)-N′-ethyl
carbodiimide
hydrochloride
(EDC)
and
N-
HydroxySuccinamide (NHS). Electrochemical characterizations of the immunosensor (antiCTV/MUA-MPA/Au electrode) were carried out by cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical detection was performed by differential pulse voltammetry in [Fe(CN)6]3−/4− solution. The decrease in the [Fe(CN)6]3−/4− current upon increasing antigen concentrations on the immunosensor surface was determined in the range of 1 nM to 5 µM. The developed immunosensor provided a much lower limit of detection (LOD=0.27 nM) than ELISA and other reported immunoassay techniques for CTV detection. The specificity of the immunosensor was also examined and no significant interference from other analytes in the real sample was observed. The proposed immunosensor is simple, relatively inexpensive, and shows good sensitivity for this application. Keywords: Label-free immunosensor; Citrus tristeza virus; Self assembled monolayer; Electrochemical detection.
1. Introduction Citrus tristeza virus (CTV) is one of the most economically important and damaging diseases in citrus trees [1]. It can be spread quickly and do damage by killing trees with sour orange rootstock [2]. CTV epidemics have caused significant losses in fruit yield and death of millions of citrus trees in many regions all over the world [3]. Therefore, rapid detection of this virus in early stages of infection is significantly critical for producing of virus free plants in nurseries and managing of disease in gardens. To date, several methods have been developed for the detection of CTV such as biological indexing [4], serological and immunoassay [5, 6] and polymerase chain reaction based techniques (PCR) [7, 8]. Enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) are the most frequent techniques in CTV detection but these techniques are costly, time consuming and multistep process [7]. Recently, a number of immunosensors have been developed for CTV detection [9, 10]. Despite the advantage of high sensitivity, these sensors require labeling steps that make the analytical procedure more complicated, time consuming and laborious. Therefore, developments of label free immunosensors have attracted a great deal of attention. In fabrication of label-free immunosensors, various transduction methods such as electrochemical, piezoelectric, surface plasmon resonance and cantilevers have been used [1114]. Among these methods, electrochemical immunosensors offer good possibilities for sensitive, low cost, fast and simple detection of unlabeled proteins [15-19]. In electrochemical label-free immunosensors, the formation of antibody–antigen complex at the electrode surface changes the mass and thickness of the electrode surface, which in turn blocks the electron transfer of the redox probe to the electrode surface [20, 21]. Therefore, the key factor here is the immobilization of the antibody or antigen on the electrode surface with no non-specific binding [22]. Attachment of proteins to the carboxylic acid end-groups of self-assembled monolayers (SAM) of alkyl thiols on the surface of gold electrode has been widely used for antibodies immobilization [23-25]. Functionalization of gold surfaces with highly ordered monolayer of alkyl thiols can be efficiently achieve via strong S-Au bond [22, 26]. SAMs reduce random orientation of the antibodies binding to the surface and enhance the sensitivity and selectivity of electrochemical immunosensor [27].
In the present study, we have developed a “label-free” electrochemical immunosensor for the direct detection of low concentrations of CTV antigen, using anti-CTV antibody (anti-CTV) immobilized on a SAM modified gold electrode. The interaction of antibody and CTV antigen at the surface of the modified electrode was investigated by differential pulse voltammetry technique in the presence of [Fe(CN)6]3−/4− as a redox probe. The linear dynamic range and limit of detection of the presented immunosensor were compared with ELISA and other recently reported immunoassays for CTV detection.
2. Experimental 2.1.
Materials and reagents
Purified rabbit polyclonal anti-CTV IgG antibody (1 mg/ml) and CTV antigen (coat protein of CTV, CP-CTV, 1 mg/ml) were obtained from department of Plant Viruses, Iranian Institute of Plant Protection, Tehran, Iran. 11-Mercapto Undecanoic acid 95% (MUA), 3-Mercapto Propionic acid 99% (MPA), N-(3-Dimethyla-minopropyl)-N′-ethyl carbodiimide hydrochloride (EDC), N-Hydroxy Succinimide 98% (NHS), bovine serum albumin (BSA), potassium chloride (KCl), potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6) and Tween-20 were obtained from Sigma-Aldrich chemicals. Other chemicals were of analytical grade and used without further purification. Phosphate buffer saline (PBS: 10mM Na2HPO4, 10mM KH2PO4, 150mM NaCl, pH 7.4) and PBST buffer (10mM Na2HPO4, 10mM KH2PO4, 0.05% W/V Tween 20, pH7.4) were prepared in Milli-Q water.
2.2.
Apparatus
All electrochemical experiments involving cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed at room temperature using a PalmSens potentiostat (Palm Instrument BV, the Netherlands). Electrochemical impedance spectroscopy (EIS) measurements were carried out in a faraday cage with a low noise AutoLab Potentiostat PGSTAT302N (Metrohm AutoLab BV, Utrecht, The Netherlands). All measurements were performed in a conventional three-electrode cell configuration consisting of a gold disk electrode (3mm diameter) as working electrode, Ag/AgCl reference electrode and a platinum wire counter electrode.
2.3.
Gold electrode preparation
It was observed that strong cleaning of the gold electrode is required for the reproducible formation of SAMs, so the electrode was cleaned by immersing in piranha’s solution (1:3, 30% H2O2 and concentrated H2SO4, respectively) at 60 °C for 5 min then rinsed with deionized water and repeated the procedure twice (caution: Piranha’s solution is a powerful oxidizing agent and reacts violently with organic compounds. It is necessary to prepare this solution under fume hood conditions and protect hands with gloves). Prior to immobilization, the electrode was also subjected to the electrochemical pretreatment by cycling electrode potential between 0.0 and 1.5 V versus Ag/AgCl reference electrode with a scan rate of 0.1 Vs−1 for 25 scans in 0.5 M sulfuric acid solution. Then the electrode was rinsed with deionized water and dried using pure nitrogen gas.
2.4.
Fabrication of immunosensor
First, the cleaned gold electrode was immersed in 5 mM MUA/MPA (7:3; v/v) solution in ethanol overnight and a SAM of MUA/MPA obtained on the gold electrode surface (MUAMPA/Au electrode) [28]. Then the modified electrode was rinsed with ethanol to remove any unbound molecules. In the next step the MUA-MPA/Au electrode was immersed in a solution of EDC (400 mM) and NHS (100 mM) for 1 h to activate the terminal carboxylic groups to NHS esters, and then it was rinsed with deionized water. For anti-CTV immobilization, a 10 µL of 0.5 mgml-1 of anti-CTV antibody solution in PBS was dropped on the modified gold electrode and incubated for 1.5 h at 37 °C. The excess antibodies were removed by rinsing with PBS. After antibody immobilization, the electrode was incubated with a solution of 1% BSA in PBST for 1 h at room temperature in order to block the unreacted COOH groups and non-specific sites, followed by washing with PBST. Anti-CTV/MUA-MPA/Au electrode was stored in the refrigerator until use for CP-CTV antigen detection. Scheme 1 represents the electrode modification and the immunosensor fabrication steps.
2.5.
Antigen detection
For antigen detection, 10 µL of CP-CTV standard solutions at different concentrations (1
nM to 10 µM) or 10 µL of spiked samples was applied to the anti-CTV/MUA-MPA/Au electrode surface and incubated for 1h at 37 °C prior to rinsing electrode with PBST. The specificity of the anti-CTV/MUA-MPA/Au electrode was determined by the antigen concentrations that have been spiked in healthy plant sap samples.
2.5.1. Sap extraction from plant materials The infected and healthy samples were collected from citrus fields located in Sari region in Mazandaran province, Iran. Young leaves from four different locations around the canopy were collected and stored at −20 °C until use. The presence and confirmation of CTV infection was performed at the plant virus diseases department of Iranian research institute of plant protection by using ELISA. Plant sap was extracted from healthy citrus trees by crushing 0.1 g leaves in liquid nitrogen followed by suspension in 2000μl Tris-HCL buffer (pH 7.5). The sap samples were then stored at −20 °C until use.
3. Results and discussions 3.1.
Characterization of Anti-CTV/MUA-MPA/Au electrode
Characterization of the anti-CTV/MUA-MPA/Au electrode was carried out by cyclic voltammetry and electrochemical impedance spectroscopy. All electrochemical measurements were performed in PBS solution, pH 7.4, containing 250 mM KCl and 5mM K3[Fe(CN)6] and 5mM K4[Fe(CN)6]. Ferri/ferrocyanide electron transfer kinetics changes at different electrode surfaces and therefore, this redox couple used to investigate the changes in electrode behavior after each surface modification step [29, 30]. Cyclic voltammetry measurements were performed in the potential range from −0.5 to 0.7 V with a scan rate of 0.05 Vs-1. Fig. 1A shows the ferri/ferrocyanide CVs of the bare Au (curve a), MUA-MPA/Au (curve b), EDC/NHS activated MUA-MPA/Au (curve c) and anti-CTV/MUAMPA/Au (curve d) electrodes. As expected, the bare Au electrode showed a well-defined characteristic reversible peak that signifies the fast heterogeneous charge transport kinetics (Fig. 1A, curve a). The homogeneous self-assembled monolayer of long-chain alkane thiols, such as MUA may leads to steric hindrance and disorder of the bulky terminal groups due to its high densities of the surface terminal groups. Therefore, combination of MPA and MUA has been
used to avoid these drawbacks and allowing access of chemical species in solution, such as redox mediator, to the electrode surface [22, 30]. The cyclic voltammogram of
MUA-MPA/Au
electrode presents less faradic currents and more peak separation (ΔEp) between the cathodic and anodic waves, due to repulsive interaction of COO− polyanion and negative redox ions [Fe(CN)6]3−/4− at the surface interface which confirms SAM formation (Fig. 1A, curve b) [31]. The activation with EDC/NHS converts the majority of terminal carboxylic groups to NHS esters, so the repulsive interaction with anionic probe [Fe(CN)6]3−/4− at the electrode interface declines and the transfer of the redox ions to the electrode surface promotes. Consequently, as it can be observed from curve c in Fig. 1A, the formed monolayers became less insulating, and faradic current response increased. As a final point, the covalent immobilization of the anti-CTV antibody onto the activated electrode surface acts as an inert electron transfer blocking layer, and so hinders the diffusion of redox couple towards the electrode surface. For that reason, as shown in curve d, the anti-CTV/MUA-MPA/Au electrode showed less faradic currents than EDC/NHS activated MUA-MPA/Au electrode. EIS experiments were further performed to probe the features of different steps of electrode modification [32-34]. The impedance spectrum known as Nyquist plot includes a semicircle part at high frequencies and a linear part at lower frequencies. The semicircle diameter corresponds to the electron-transfer resistance (Rct) which indicates the blocking behavior of the electrode surface towards the redox couple while the linear part corresponds to the diffusion process. EIS measurements were performed at a DC potential of 0.2 V with AC amplitude of 0.01 V, in which the Nyquist plots were recorded over a frequency range from 0.1Hz to 1 MHz. Fig. 1B illustrates the EIS curves for the electrode modification steps including bare Au (curve a), MUA-MPA/Au (curve b), EDC/NHS activated MUA-MPA/Au (curve c) and antiCTV/MUA-MPA/Au (curve d) electrodes. The Nyquist plot of the bare Au electrode displayed almost straight line which points to existence of diffusion controlled electrochemical process at the electrode surface (Fig. 1B, curve a).
For MUA-MPA/Au electrode, the value of Rct
significantly increased to 6.92 K, due to the high blocking effect of the SAM for [Fe(CN)6]3−/4 ions (Fig. 1B, curve b). However, in case of curve c, the Rct value was dramatically decreased to 1.21 K, after the activation of MUA-MPA/Au electrode surface with EDC/NHS. This may due
to enhancement of the [Fe(CN)6]3/4− redox reaction rate at anti-CTV/MUA-MPA/Au electrode surface, as shown in the CV results. Since the covalent immobilization of the anti-CTV antibody onto the activated electrode surface acts as an inert electron transfer blocking layer, the antiCTV/MUA-MPA/Au electrode showed higher Rct value than EDC/NHS activated MUAMPA/Au electrode (Fig. 1B, curve d).
3.2.
Electrochemical response to antigen
Fig. 2 shows the cyclic voltammograms of the anti-CTV/MUA-MPA/Au electrode in the presence of [Fe(CN)6]3−/4− redox probe before (curve b) and after incubation with 1 µM of CPCTV antigen (curve c). The formation of immunocomplex onto the electrode surface acts as an inert electron transfer blocking layer and so hinders the diffusion of redox couple towards the electrode surface. Therefore, the CP-CTV/anti-CTV/MUA-MPA/Au electrode (Fig. 2, curve c) showed less faradic peak currents and more ΔEp than anti-CTV/MUA-MPA/Au electrode (Fig. 2, curve b). Since DPV is a more sensitive electrochemical technique compare to CV [35, 36], immunodetection of CP-CTV antigen was performed with this method and based on the measurement of faradaic peak current of [Fe(CN)6]3−/4− redox couple. For this purpose, the antiCTV/MUA-MPA/Au electrode was exposed to various concentrations of CP-CTV in the range of 1 nM to 10 µM. DPV measurements were carried out in the same solution used for CV measurements under following conditions; (modulation time= 0.1 s, interval time= 0.2 s, initial
potential= −0.1 V,
end
potential= 0.5 V,
step potential= 0.01 V,
modulation
amplitude= 0.05 V). The height of the resulting peak waveform was recorded and plotted against CP-CTV concentration to give a calibration curve. The DPV signals recorded for increasing concentrations of CP-CTV on anti-CTV/MUAMPA/Au electrode is shown in Fig. 3. The value of the DPV peak current of the redox probe reflects the amount of immunocomplex formation at the electrode surface. Since the antigenantibody coupling at the electrode surface acts as a kinetic barrier for the ionic transfer, a
significant decrease in the DPV peak currents can be observed upon increasing antigen concentrations. Similar behavior of ferro/ferricyanide was reported for other target molecules in the literature [15, 16]. Fig. 4 shows the immunosensor differential responses (DPV response for incubation with CP-CTV antigen subtracted from the response for incubation with PBS) versus CP-CTV concentrations. As shown, there was a logarithmic relationship between the changes in the DPV peak currents (ΔIs) and CP-CTV antigen concentration (CCP_CTV). The inset of Fig. 4 shows the linear relationship between ΔI(µA) and log(CCP_CTV) (nM) in the range of 1 nM to 5 μM with the linear regression equation of ΔI(µA)=11.014 log(CCP_CTV) (nM) + 22.82 and correlation coefficient of 0.996. The limits of detection (LOD) and quantification (LOQ) were 0.27 and 0.97 nM respectively, calculated using the equations: LOD=3s/S and LOQ=10s/S (where s is the standard deviation of the response and S is the slope of the calibration curve)[37]. The reproducibility of the immunosensors was evaluated at 1 nM CP-CTV with five electrodes fabricated at the same conditions. The RSD% value was 3.9%, suggesting that the assay is reproducible under the tested conditions. The storage stability of the immunosensor was investigated over a 30-day period when three immunosensors were stored at 4оC without chemical preservatives and characterized at regular interval times (every 3 days). No obvious change of initial signal (less than 10%) was found over this period, indicating the effective retention of the activity of the immunosensor. Table 1 summarized the performance of the presented immunosensor and other recent immunoassay methods for CP-CTV detection. As it can be seen, the developed electrochemical immunosensor displayed better detection limit, linear range and linear regression coefficient in comparison to ELISA and other immunoassay methods for the CP-CTV detection.
S0925-4005(16)32106-2 http://dx.doi.org/doi:10.1016/j.snb.2016.12.135 SNB 21509
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
24-9-2016 14-12-2016 28-12-2016
Please cite this article as: Hedieh Haji-Hashemi, Parviz Norouzi, Mohammad Reza Safarnejad, Mohammad Reza Ganjali, Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.12.135 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Label-free electrochemical immunosensor for direct detection of Citrus tristeza virus using modified gold electrode
Hedieh Haji-Hashemi1, Parviz Norouzi*1,2, Mohammad Reza Safarnejad3, Mohammad Reza Ganjali1,2 1
Center of Excellence in Electrochemistry, University of Tehran, Tehran, Iran
2
Endocrinology & Metabolism Research Center, Tehran University of Medical Sciences, Tehran,
Iran 3
Agricultural research, education and extension organization, Iranian Research Institute of
Plant Protection, Tehran, Iran
*
Corresponding author. Tel.: +98-21-61112788; Fax: +98-21-66495291 E-mail address: [email protected]
Highlights
The first electrochemical immunosensor for citrus tristeza virus (CTV) detection.
Simplification of the operation as a result of labeling steps elimination.
Excellent specificity of proposed sensor against interferences from real samples.
Outstanding LOD that is noticeably lower than previous reports for CTV detection.
Abstract This paper presents the first label-free and sensitive electrochemical immunosensor for efficient and rapid detection of citrus tristeza virus (CTV). The specific antibody against coat protein (CP) of CTV was successfully immobilized on 11-mercaptoundecanoic acid (MUA) and 3-mercapto propionic acid (MPA) modified gold electrode via carbodiimide coupling reaction using
N-(3-dimethylaminopropyl)-N′-ethyl
carbodiimide
hydrochloride
(EDC)
and
N-
HydroxySuccinamide (NHS). Electrochemical characterizations of the immunosensor (antiCTV/MUA-MPA/Au electrode) were carried out by cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical detection was performed by differential pulse voltammetry in [Fe(CN)6]3−/4− solution. The decrease in the [Fe(CN)6]3−/4− current upon increasing antigen concentrations on the immunosensor surface was determined in the range of 1 nM to 5 µM. The developed immunosensor provided a much lower limit of detection (LOD=0.27 nM) than ELISA and other reported immunoassay techniques for CTV detection. The specificity of the immunosensor was also examined and no significant interference from other analytes in the real sample was observed. The proposed immunosensor is simple, relatively inexpensive, and shows good sensitivity for this application. Keywords: Label-free immunosensor; Citrus tristeza virus; Self assembled monolayer; Electrochemical detection.
1. Introduction Citrus tristeza virus (CTV) is one of the most economically important and damaging diseases in citrus trees [1]. It can be spread quickly and do damage by killing trees with sour orange rootstock [2]. CTV epidemics have caused significant losses in fruit yield and death of millions of citrus trees in many regions all over the world [3]. Therefore, rapid detection of this virus in early stages of infection is significantly critical for producing of virus free plants in nurseries and managing of disease in gardens. To date, several methods have been developed for the detection of CTV such as biological indexing [4], serological and immunoassay [5, 6] and polymerase chain reaction based techniques (PCR) [7, 8]. Enzyme-linked immunosorbent assay (ELISA) and direct tissue blot immunoassay (DTBIA) are the most frequent techniques in CTV detection but these techniques are costly, time consuming and multistep process [7]. Recently, a number of immunosensors have been developed for CTV detection [9, 10]. Despite the advantage of high sensitivity, these sensors require labeling steps that make the analytical procedure more complicated, time consuming and laborious. Therefore, developments of label free immunosensors have attracted a great deal of attention. In fabrication of label-free immunosensors, various transduction methods such as electrochemical, piezoelectric, surface plasmon resonance and cantilevers have been used [1114]. Among these methods, electrochemical immunosensors offer good possibilities for sensitive, low cost, fast and simple detection of unlabeled proteins [15-19]. In electrochemical label-free immunosensors, the formation of antibody–antigen complex at the electrode surface changes the mass and thickness of the electrode surface, which in turn blocks the electron transfer of the redox probe to the electrode surface [20, 21]. Therefore, the key factor here is the immobilization of the antibody or antigen on the electrode surface with no non-specific binding [22]. Attachment of proteins to the carboxylic acid end-groups of self-assembled monolayers (SAM) of alkyl thiols on the surface of gold electrode has been widely used for antibodies immobilization [23-25]. Functionalization of gold surfaces with highly ordered monolayer of alkyl thiols can be efficiently achieve via strong S-Au bond [22, 26]. SAMs reduce random orientation of the antibodies binding to the surface and enhance the sensitivity and selectivity of electrochemical immunosensor [27].
In the present study, we have developed a “label-free” electrochemical immunosensor for the direct detection of low concentrations of CTV antigen, using anti-CTV antibody (anti-CTV) immobilized on a SAM modified gold electrode. The interaction of antibody and CTV antigen at the surface of the modified electrode was investigated by differential pulse voltammetry technique in the presence of [Fe(CN)6]3−/4− as a redox probe. The linear dynamic range and limit of detection of the presented immunosensor were compared with ELISA and other recently reported immunoassays for CTV detection.
2. Experimental 2.1.
Materials and reagents
Purified rabbit polyclonal anti-CTV IgG antibody (1 mg/ml) and CTV antigen (coat protein of CTV, CP-CTV, 1 mg/ml) were obtained from department of Plant Viruses, Iranian Institute of Plant Protection, Tehran, Iran. 11-Mercapto Undecanoic acid 95% (MUA), 3-Mercapto Propionic acid 99% (MPA), N-(3-Dimethyla-minopropyl)-N′-ethyl carbodiimide hydrochloride (EDC), N-Hydroxy Succinimide 98% (NHS), bovine serum albumin (BSA), potassium chloride (KCl), potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6) and Tween-20 were obtained from Sigma-Aldrich chemicals. Other chemicals were of analytical grade and used without further purification. Phosphate buffer saline (PBS: 10mM Na2HPO4, 10mM KH2PO4, 150mM NaCl, pH 7.4) and PBST buffer (10mM Na2HPO4, 10mM KH2PO4, 0.05% W/V Tween 20, pH7.4) were prepared in Milli-Q water.
2.2.
Apparatus
All electrochemical experiments involving cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed at room temperature using a PalmSens potentiostat (Palm Instrument BV, the Netherlands). Electrochemical impedance spectroscopy (EIS) measurements were carried out in a faraday cage with a low noise AutoLab Potentiostat PGSTAT302N (Metrohm AutoLab BV, Utrecht, The Netherlands). All measurements were performed in a conventional three-electrode cell configuration consisting of a gold disk electrode (3mm diameter) as working electrode, Ag/AgCl reference electrode and a platinum wire counter electrode.
2.3.
Gold electrode preparation
It was observed that strong cleaning of the gold electrode is required for the reproducible formation of SAMs, so the electrode was cleaned by immersing in piranha’s solution (1:3, 30% H2O2 and concentrated H2SO4, respectively) at 60 °C for 5 min then rinsed with deionized water and repeated the procedure twice (caution: Piranha’s solution is a powerful oxidizing agent and reacts violently with organic compounds. It is necessary to prepare this solution under fume hood conditions and protect hands with gloves). Prior to immobilization, the electrode was also subjected to the electrochemical pretreatment by cycling electrode potential between 0.0 and 1.5 V versus Ag/AgCl reference electrode with a scan rate of 0.1 Vs−1 for 25 scans in 0.5 M sulfuric acid solution. Then the electrode was rinsed with deionized water and dried using pure nitrogen gas.
2.4.
Fabrication of immunosensor
First, the cleaned gold electrode was immersed in 5 mM MUA/MPA (7:3; v/v) solution in ethanol overnight and a SAM of MUA/MPA obtained on the gold electrode surface (MUAMPA/Au electrode) [28]. Then the modified electrode was rinsed with ethanol to remove any unbound molecules. In the next step the MUA-MPA/Au electrode was immersed in a solution of EDC (400 mM) and NHS (100 mM) for 1 h to activate the terminal carboxylic groups to NHS esters, and then it was rinsed with deionized water. For anti-CTV immobilization, a 10 µL of 0.5 mgml-1 of anti-CTV antibody solution in PBS was dropped on the modified gold electrode and incubated for 1.5 h at 37 °C. The excess antibodies were removed by rinsing with PBS. After antibody immobilization, the electrode was incubated with a solution of 1% BSA in PBST for 1 h at room temperature in order to block the unreacted COOH groups and non-specific sites, followed by washing with PBST. Anti-CTV/MUA-MPA/Au electrode was stored in the refrigerator until use for CP-CTV antigen detection. Scheme 1 represents the electrode modification and the immunosensor fabrication steps.
2.5.
Antigen detection
For antigen detection, 10 µL of CP-CTV standard solutions at different concentrations (1
nM to 10 µM) or 10 µL of spiked samples was applied to the anti-CTV/MUA-MPA/Au electrode surface and incubated for 1h at 37 °C prior to rinsing electrode with PBST. The specificity of the anti-CTV/MUA-MPA/Au electrode was determined by the antigen concentrations that have been spiked in healthy plant sap samples.
2.5.1. Sap extraction from plant materials The infected and healthy samples were collected from citrus fields located in Sari region in Mazandaran province, Iran. Young leaves from four different locations around the canopy were collected and stored at −20 °C until use. The presence and confirmation of CTV infection was performed at the plant virus diseases department of Iranian research institute of plant protection by using ELISA. Plant sap was extracted from healthy citrus trees by crushing 0.1 g leaves in liquid nitrogen followed by suspension in 2000μl Tris-HCL buffer (pH 7.5). The sap samples were then stored at −20 °C until use.
3. Results and discussions 3.1.
Characterization of Anti-CTV/MUA-MPA/Au electrode
Characterization of the anti-CTV/MUA-MPA/Au electrode was carried out by cyclic voltammetry and electrochemical impedance spectroscopy. All electrochemical measurements were performed in PBS solution, pH 7.4, containing 250 mM KCl and 5mM K3[Fe(CN)6] and 5mM K4[Fe(CN)6]. Ferri/ferrocyanide electron transfer kinetics changes at different electrode surfaces and therefore, this redox couple used to investigate the changes in electrode behavior after each surface modification step [29, 30]. Cyclic voltammetry measurements were performed in the potential range from −0.5 to 0.7 V with a scan rate of 0.05 Vs-1. Fig. 1A shows the ferri/ferrocyanide CVs of the bare Au (curve a), MUA-MPA/Au (curve b), EDC/NHS activated MUA-MPA/Au (curve c) and anti-CTV/MUAMPA/Au (curve d) electrodes. As expected, the bare Au electrode showed a well-defined characteristic reversible peak that signifies the fast heterogeneous charge transport kinetics (Fig. 1A, curve a). The homogeneous self-assembled monolayer of long-chain alkane thiols, such as MUA may leads to steric hindrance and disorder of the bulky terminal groups due to its high densities of the surface terminal groups. Therefore, combination of MPA and MUA has been
used to avoid these drawbacks and allowing access of chemical species in solution, such as redox mediator, to the electrode surface [22, 30]. The cyclic voltammogram of
MUA-MPA/Au
electrode presents less faradic currents and more peak separation (ΔEp) between the cathodic and anodic waves, due to repulsive interaction of COO− polyanion and negative redox ions [Fe(CN)6]3−/4− at the surface interface which confirms SAM formation (Fig. 1A, curve b) [31]. The activation with EDC/NHS converts the majority of terminal carboxylic groups to NHS esters, so the repulsive interaction with anionic probe [Fe(CN)6]3−/4− at the electrode interface declines and the transfer of the redox ions to the electrode surface promotes. Consequently, as it can be observed from curve c in Fig. 1A, the formed monolayers became less insulating, and faradic current response increased. As a final point, the covalent immobilization of the anti-CTV antibody onto the activated electrode surface acts as an inert electron transfer blocking layer, and so hinders the diffusion of redox couple towards the electrode surface. For that reason, as shown in curve d, the anti-CTV/MUA-MPA/Au electrode showed less faradic currents than EDC/NHS activated MUA-MPA/Au electrode. EIS experiments were further performed to probe the features of different steps of electrode modification [32-34]. The impedance spectrum known as Nyquist plot includes a semicircle part at high frequencies and a linear part at lower frequencies. The semicircle diameter corresponds to the electron-transfer resistance (Rct) which indicates the blocking behavior of the electrode surface towards the redox couple while the linear part corresponds to the diffusion process. EIS measurements were performed at a DC potential of 0.2 V with AC amplitude of 0.01 V, in which the Nyquist plots were recorded over a frequency range from 0.1Hz to 1 MHz. Fig. 1B illustrates the EIS curves for the electrode modification steps including bare Au (curve a), MUA-MPA/Au (curve b), EDC/NHS activated MUA-MPA/Au (curve c) and antiCTV/MUA-MPA/Au (curve d) electrodes. The Nyquist plot of the bare Au electrode displayed almost straight line which points to existence of diffusion controlled electrochemical process at the electrode surface (Fig. 1B, curve a).
For MUA-MPA/Au electrode, the value of Rct
significantly increased to 6.92 K, due to the high blocking effect of the SAM for [Fe(CN)6]3−/4 ions (Fig. 1B, curve b). However, in case of curve c, the Rct value was dramatically decreased to 1.21 K, after the activation of MUA-MPA/Au electrode surface with EDC/NHS. This may due
to enhancement of the [Fe(CN)6]3/4− redox reaction rate at anti-CTV/MUA-MPA/Au electrode surface, as shown in the CV results. Since the covalent immobilization of the anti-CTV antibody onto the activated electrode surface acts as an inert electron transfer blocking layer, the antiCTV/MUA-MPA/Au electrode showed higher Rct value than EDC/NHS activated MUAMPA/Au electrode (Fig. 1B, curve d).
3.2.
Electrochemical response to antigen
Fig. 2 shows the cyclic voltammograms of the anti-CTV/MUA-MPA/Au electrode in the presence of [Fe(CN)6]3−/4− redox probe before (curve b) and after incubation with 1 µM of CPCTV antigen (curve c). The formation of immunocomplex onto the electrode surface acts as an inert electron transfer blocking layer and so hinders the diffusion of redox couple towards the electrode surface. Therefore, the CP-CTV/anti-CTV/MUA-MPA/Au electrode (Fig. 2, curve c) showed less faradic peak currents and more ΔEp than anti-CTV/MUA-MPA/Au electrode (Fig. 2, curve b).
potential= −0.1 V,
end
potential= 0.5 V,
step potential= 0.01 V,
modulation
amplitude= 0.05 V). The height of the resulting peak waveform was recorded and plotted against CP-CTV concentration to give a calibration curve. The DPV signals recorded for increasing concentrations of CP-CTV on anti-CTV/MUAMPA/Au electrode is shown in Fig. 3. The value of the DPV peak current of the redox probe reflects the amount of immunocomplex formation at the electrode surface. Since the antigenantibody coupling at the electrode surface acts as a kinetic barrier for the ionic transfer, a
significant decrease in the DPV peak currents can be observed upon increasing antigen concentrations. Similar behavior of ferro/ferricyanide was reported for other target molecules in the literature [15, 16].