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Novel label-free electrochemical aptasensor for determination of Diazinon using gold nanoparticles-modified screen-printed gold electrode
Author’s Accepted Manuscript Novel Label-Free Electrochemical Aptasensor for Determination of Diazinon Using Gold Nanoparticles-Modified Screen-Printed Gold Electrode Shokoufeh Hassani, Milad Rezaei Akmal, Armin Salek-Maghsoudi, Soheila Rahmani, Mohammad Reza Ganjali, Parviz Norouzi, Mohammad Abdollahi
PII: DOI: Reference:
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S0956-5663(18)30639-0 https://doi.org/10.1016/j.bios.2018.08.041 BIOS10705
To appear in: Biosensors and Bioelectronic Received date: 25 June 2018 Revised date: 27 July 2018 Accepted date: 17 August 2018 Cite this article as: Shokoufeh Hassani, Milad Rezaei Akmal, Armin SalekMaghsoudi, Soheila Rahmani, Mohammad Reza Ganjali, Parviz Norouzi and Mohammad Abdollahi, Novel Label-Free Electrochemical Aptasensor for Determination of Diazinon Using Gold Nanoparticles-Modified Screen-Printed Gold Electrode, Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2018.08.041 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 galley proof before it is published in its final citable 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.
Novel Label-Free Electrochemical Aptasensor for Determination of Diazinon Using Gold NanoparticlesModified Screen-Printed Gold Electrode Shokoufeh Hassani1,2, Milad Rezaei Akmal3, Armin Salek-Maghsoudi2, Soheila Rahmani1, Mohammad Reza Ganjali3, Parviz Norouzi3, Mohammad Abdollahi1,2* 1
Toxicology and Diseases Group, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences (TUMS), Tehran, Iran 2
Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of
Medical Sciences (TUMS), Tehran, Iran 3
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran,
Iran *
Corresponding author: The Institute of Pharmaceutical Sciences (TIPS) and Faculty of
Pharmacy,
Tehran
University
of
Medical
Sciences,
Tehran,
[email protected] or [email protected]
Page 1 of 22
1417614411,
Iran,
Abstract The present study aimed to develop a highly sensitive label-free electrochemical aptasensor for the detection of diazinon (DZN), as one of the most widespread organophosphorus compounds. The aptasensor was assembled using screen-printed gold electrode modified by thiolated aptamers which were immobilized on gold nanoparticles (Au NPs). Optimum deposition time, in which the highest electrochemical response occurred, was found in 150s. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize electrochemical properties of the novel aptasensor. Electrochemical detection was carried out through differential pulse voltammetry in [Fe(CN)6]3-/4- solution. Fluctuation of the current was examined in the DZN concentration range of 0.1 to 1000 nM. According to the results, the designed aptasensor provided an extremely lower limit of detection (0.0169 nM) compared with HPLC and other colorimetric techniques for DZN detection. The present highly specific designed aptasensor doesn’t interact with other analytes in the real sample. Consequently, the present aptasensor is easy to use and relatively inexpensive with a good sensitivity, stability, and reproducibility for this application. We are now evaluating all approaches to make a portable device for fast and sensitive quantification of DZN and related OPs.
Keywords: Electrochemical aptasensor, Diazinon, Gold nanoparticles, Screen-printed gold electrode, Analytical Toxicology, Organophosphorus compounds
1. Introduction Pesticides as a large and heterogeneous group of agricultural chemicals have been widely used to control, kill or repel pests in different fields (Mostafalou and Abdollahi 2017; Balali-Mood and Abdollahi, 2014). Their ubiquitous use leads to inevitable consequences for public health of the countries where they are used (Shadboorestan et al., 2016). Detrimental effects on main organs including endocrine, respiratory, renal, cardiovascular, and immune systems through irreversible inhibition of acetylcholine esterase (AChE) result in development of serious human diseases such as Parkinson’s, Alzheimer’s, Multiple Sclerosis, diabetes, and renal failure (Mostafalou and Abdollahi, 2018; Abdollahi et al. 2004). Contamination of drinking water and food sources, even at very low degree of organophosphorus compounds (OPs) is considered as a serious issue Page 2 of 22
requiring sophisticated resolution (Kazemi et al. 2012). In this regard, fabrication of reliable, rapid, and inexpensive analytical tools in order to detect such small amounts of pesticides in the environment is crucial (Mostafalou and Abdollahi 2013). Conventional analytical methods for determination of pesticides comprise gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC-MS) (Choi et al. 2015; García-Valcárcel and Tadeo 2009; Páleníková et al. 2015; Vuković et al. 2012), capillary electrophoresis (Guler et al. 2010; Regueiro et al. 2015), pressurized liquid extraction (Du et al. 2012), and fluorimetry (Pacioni and Veglia 2003). Despite accuracy and selectivity, these methods have several drawbacks such as exorbitance, time consuming process, complexity, and use of hazardous reagents that make them unsuitable for routine use (Regueiro et al. 2015; Verma and Bhardwaj 2015). Therefore, innovative techniques are needed to minimize the time of analysis and limit the use of hazardous chemicals. Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate) is a common OP with broad agricultural and household use which its remaining trace amounts in the environment can exert detrimental effects in humans and animals (Pakzad et al. 2013; Shah and Iqbal 2010; Zhou et al. 2011). It is evidenced by the health organizations that the accepted maximum residue limits (MRLs) of DZN, in soil (European Union), water (Food and Agriculture Organization of the United Nations/World Health Organization) and vegetables (European Union) is 0.04 μg/kg, 0.1 μg/L, and 0.04 μg/g, respectively (Bhattacharjee et al. 2012; Akan et al. 2013). Thus, it is desirable to set up a sensitive and low cost method for DZN detection. Electrochemical strategies with potential specificity, sensitivity, stability, and low limit of detection seem an excellent option for tracing OPs. Biosensors have renowned recently for their approachability, simplicity, portability, and great selectivity and sensitivity. Accordingly, their application in detection of DZN and other OPs can be proposed as an efficient and promising alternative to conventional analytical methods. Recently, different types of biosensors for OPs detection in the environmental samples have been designed. For example, analysis of triazophos based on acetylcholine esterase inhibition measurement with amperometry transducer (Reybier et al. 2002), detection of parathion and methylparathion with a cell-based biosensor based on the potentiometric method (Bhadekar et al. 2011), and antibody-based biosensor for detection of chlorpyrifos via voltammetric methods (Talan et al. 2018). Nevertheless, since their biological recognition components such as antibody or enzyme are instable, use of biosensors as common Page 3 of 22
detection equipment has been restrained (Hammond et al. 2016; Mehrotra 2016; Mishra et al. 2018). Aptamers are short single-stranded oligonucleotides or peptides which are generated by SELEX (systematic evolution of ligands by exponential enrichment) library that can bind specifically to different elements such as toxicants, drugs, proteins, peptides, vitamins, cells, and small compounds (Saha et al. 2012; Stoltenburg et al. 2007). The main beneficial features of aptamers compared to antibodies include inconsiderable toxicity and immunogenicity, inexpensiveness, feasible production, rapid response, and high thermal and chemical stability. Moreover, in comparison to optical sensors, electrochemical aptasensors require the least amount of analyte for recognition. Possessing these prominent features, aptamers have been widely employed in various analytical techniques (Li et al. 2010; Li et al. 2011; Mokhtarzadeh et al. 2015; Taghdisi et al. 2016). To enhance the efficiency and sensitivity of the device, several techniques such as exploiting nanostructures have been also utilized. Nanostructures with their crucial properties including greater thermal and chemical stability, large surface area, high tensile strength, and elasticity have attracted much attention in designing biosensors (Farghali and Ahmed 2015; Mehlhorn et al. 2018). Immobilization of DNA aptamers and modification of electrode surface with nanoparticles may be the most effective application (Cao et al. 2011; Yang et al. 2008). Metal nanoparticles such as gold nanoparticles can be used to improve electron transfer in electrochemical biosensors (Cao et al. 2011; Zhao et al. 2015). Gold nanoparticles (Au NPs) have surpassing capability to immobilize biomolecules and also can be simply functionalized through thiolation (Yasun et al. 2012). Screen printed electrodes (SPE) which are applicable in modern sensors provide a vast range of advantages over the conventional electrodes (Wan et al. 2015). Capability of large scale manufacturing, technical feasibility, costeffectiveness and disposability are some of these benefits that make SPEs the favorable candidates for practical use in different types of biosensors (Renedo and Martínez 2007; Sanllorente‐Méndez et al. 2009; Wang et al. 1998). Moreover, their versatile features such as composing of various electrode inks (gold, carbon, silver, etc.) allow them to be modified via nanoparticles with the aim of the operation enhancement (Barton et al. 2016; Li et al. 2012; Taleat et al. 2014). Recently, the application of biosensor technologies in the detection of OPs in the environment was reported (Hassani et al. 2017; Salek-Maghsoudi et al. 2018). So far, only one aptasensor for DZN detection has been designed which was based on the colorimetric method (Jokar et al. Page 4 of 22
2017). Herein, we developed a novel label-free electrochemical thiolated aptasensor which was immobilized on gold nanoparticle (Au NP)-modified screen printed gold electrodes (SPGEs) in order to detect minimum levels of DZN in biological samples with high sensitivity and fast performance.
2. Experimental 2.1. Materials All reagents were of analytical grade purchased from Sigma-Aldrich (Darmstadt, Germany) unless otherwise stated. Diazinon, chloropyrifos, malathion, deltamethrin (purity: 98%), gold chloride trihydrate (HAuCl4.3H2O), H2SO4, 6-Mercapto-1-Hexanol (MCH), potassium ferricyanide and ferrocyanide, phosphate-buffered saline (PBS), NaCl, KCl, MgCl2 and Tris-HCl were used in this study. Solutions were prepared using ultra-pure water with a resistance of 18MΩ.cm (Millipore, USA). The protocol of the study was approved by the Institute Ethical Committee under code number IR.TUMS.VCR.REC.1395.1129 and all ethical issues on the use of animals were followed. Thiolated aptamer DNA oligonucleotides (DF20) were purchased from MWG-BIOTECH, Germany with following base sequences (Bruno and Chanpong 2012): 5-(SH)-(CH2)6ATCC TCACACCT CTCTAATATA A T TT
TATT CTCTT
ACAA
TACA
AT
CTCCC TAT- .
The stock solution of aptamer (20 µM) was prepared in the binding buffer by Tris-HCl buffer (20 mM Tris-HCl, 0.1M NaCl, 0.2 M KCl, 5 mM MgCl2, pH 7.4) and 10 mM Tris–HCl containing 0.1 M sodium chloride, 5.0 mM magnesium chloride (pH 7.4) was used as the washing buffer and stored at -20 °C.
2.2. Equipment Electrochemical measurements were performed with AUTOLAB PGSTAT 101 (Methrohm Autolab BV, Utrecht, The Netherlands), controlled by NOVA 2.1 Software. Electrochemical characterizations of modified surfaces were followed-up by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) assays that carried out in a redox electrolyte solution of 5 mM [Fe(CN)6] Page 5 of 22
−/4−
in 0.1 M KCl solution. EIS
measurements were conducted using a frequency range from 100 mHz to 100 kHz at a direct current (DC( potential of +0.13 V. The obtained data are represented in Nyquist plots. DPV was recorded within the potential range of 0.45 to -0.05 V under modulation amplitude of 50 mV. CV was employed to establish the fabrication of aptasensor. The measured potential range was between -0.2 and +0.5 V, with a scan rate of 0.1 V/s. SPGE composed of integrated working, reference, and counter electrodes in which the counter electrode and working electrodes (4 mm diameter) were made of gold and the reference electrode and electrical contacts were made of silver (DropSens, S.L., Spain). The morphology of Au NPs after the deposition procedure was assessed using the Field emission scanning electron microscopy (FESEM; A4160 system, Hitachi, Japan).
2.3. Au NP-modified SPGEs assembly The surface of the working electrode was modified by Au NPs through the electrodeposition process using a solution of 5 mM HAuCl4 in 0.5 M H2SO4 (Wan et al. 2015). At first, SPGE surface was activated via H2SO4 solution. Before the activation, the SPGE was characterized by CV in the range of 0.2 - 0.5 V in 5 mM K3Fe(CN)6 (0.1 M KCl) solution with scan rate of 0.1 V/s. Afterwards, the activation process was performed in 0.5 M sulfuric acid with CV scanning in the range of -0.1 - 1.3 V with scan rate of 0.1 V/s in order to obtain a steady voltammogram. We optimized the deposition time for Au NPs modification and compared the performance between Au NPs modified SPGE and solely activated SPGE. Following activation of the electrode, deposition process of Au NPs was carried out under potential of -0.4 V in 5 mM HAuCl4/ 0.5 M H2SO4 solution with different deposition time ranging from 30 - 150 s. After providing each deposition, the SPGE was analyzed by CV in 5 mM K3Fe(CN)6 (0.1 M KCl) with a scan rate of 0.1 V/s. Lastly, the surface structure of Au NPs modified SPGE was characterized through FESEM. The stock solution of Au NPs was stored in a refrigerator at 4 °C for further assessments.
2.4. Immobilization of the aptamer onto Au NP/ SPGEs surface In order to immobilize the aptamer, 10 µL of 2.5 μM aptamer stock solution dropped onto the Au NP-modified SPGE and the self-assembled monolayer (SAM) was spontaneously formed through 16 h incubation in a dark place at 4 °C. The blockade of the non-modified sites was Page 6 of 22
carried out using 5 mM MCH solution with 1 h incubation at room temperature. The electrode was then rinsed with washing buffer and then ultrapure water and blown dry under the nitrogen atmosphere after each incubation step.
2.5 Detection of DZN with the nano-Aptasensor Proceeding for detection, DZN determination in binding buffer solution (pH 7.4) was applied onto the surface of fabricated aptasensor (aptamer/ Au NP/ SPGEs). Following 30 min incubation at 37 °C, the aptasensor was rinsed gently with ultrapure water. Afterwards, the nanoaptasensor was placed into the electrochemical chamber (congaing 5 mM of K3Fe(CN)6 (0.1M KCl) solution), the DPV measurements were conducted for different DZN concentrations at separate SPGEs (experimental condition: modulation time= 0.05 s, interval time= 0.2 s, potential range of 0.45 to -0.05 V). The general protocol of electrochemical aptasensor development has been illustrated in Scheme 1.
Scheme 1. Schematic presentation of the aptasensor fabrication for detection of Diazinon.
3. Results and Discussion 3.1 Characterization of the Au NPs FESEM images of the Au NPs modified SPGEs and bare SPGE are shown in Figure 1A and B. We used Au NPs to immobilize thiolated aptamer which enhanced the signal transduction by means of increasing the surface area of the electrode. Page 7 of 22
Before the activation, the SPGE was characterized by CV (-0.2 to +0.5 V) in 5 mM K3Fe (CN)6 (0.1 M KCl) with scan rates of 0.05 V/s and 0.1 V/s. Next, the activation process was carried out in 0.5 M sulfuric acid solution with CV scanning (-0.1 to +1.3 V) with a scan rate of 0.1 V/s until the steady voltammograms were acquired. It is evidenced by the previous studies that the removal of oxidation layer and the exposure of the most active sites on the electrode surface after the activation could result in a remarkable improvement in the noticeably enhancement of electrochemical performance (Fanjul-Bolado et al. 2008; Su et al. 2011). Deposition time of Au NPs could significantly influence the deposition extent and size of Au NPs on the SPGE (Renedo and Martínez 2007). Regarding the maximum obtained responses, the deposition time of Au NPs was set to 150 s in order to get the most suitable electrochemical behavior of the SPGE, After Au NPs modification, CV was conducted with Au NPs modified and solely activated SPGEs in 5 mM K3Fe(CN)6 (0.1M KCl) (Figure 2).
Figure 1. FESEM images of gold electrodes; A) Bare gold electrode, B) Au NPs modified SPGEs with 5 mM HAuCl4/0.5 M H2SO4 at optimum deposition time of 150 s. 3.2 Characterization of the Aptasensor (DZN/ thiolated aptamer/ Au NP/ SPGEs) Characterization of the DZN/thiolated aptamer/ Au NP/ SPGE was carried out through CV and EIS by monitoring the processes of the assembly of the aptasensor in each step. All electrochemical measurements were performed in 20 mM Tris-HCl buffer solution (pH 7.4), containing 5 mM K3Fe(CN)6 (0.1 M KCl). Ferri-ferrocyanide redox electrolyte was used for electrochemical transduction (Montrose et al. 2013; Sharma et al. 2010). CV measurements were achieved in the potential range from -0.2 to 0.5 V, with a scan rate of 0.1 V/s. Figure 2 shows the ferri-ferrocyanide CVs of the bare gold SPE (curve a), Au NPs /SPGE (curve b), aptamer/ Au Page 8 of 22
NPs /SPGE (curve c), MCH/ aptamer/ Au NPs /SPGE (curve d) and 10 nM Diazinon / aptamer/ Au NPs /SPGE (curve e). The bare gold SPE showed an appropriate reversible peak, which was attributed to the t occurrence of faster heterogeneous electron transfer kinetics. Definite characteristics of [Fe(CN)6]3-/4- were assessed through this measurement by means of redox peak separation and calculating ΔEp of the cathodic and anodic waves and current response. The CV results of redox elements Fe(CN)6
3-
/Fe(CN)6
4-
at the SPGE indicated a pair
of reversible redox peaks with ΔEp of 0.073 V (Figure 2, curve a). After deposition of Au NPs on the SPGE surface, the signal was considerably intensified ΔEp reduced to 0.070 V (Figure 2, curve b). It confirmed the previous experiments claiming that the modification of the SPE surface with nanoparticles could provide larger exposed area which led to enhance the current response. Self-assembly of the thiolated aptamer onto the Au NP modified SPGE caused the peak currents of electrolyte [Fe(CN)6]3-/4- dropped significantly and a modest ΔEp increase (0.074 V) was observed (Figure 2, curve c). The most probable reason for this current reduction could be the repulsion force induced by the phosphate groups of the aptamer and also its hindrance which interfere with the electrolyte transfer. According to the curve d in Figure 2, MCH addition onto the electrode surface blocked the active sites necessary for electron transfer; this resulted in a decrease in the redox peak current and an increase in the peak separation (ΔEp=0.109 V) (Baghayeri et al.2018). At last we incubated the novel aptasensor with 10 nM DZN for 30 min; the peak currents decreased dramatically in parallel with the striking increase of the peak potential ΔEp
to 0.168 V (Figure 2, curve e). The obtained results are broadly consistent with previous
studies in which the binding the immobilized aptamer to its target led to the folding of aptamer and formation of stem-loop structure which resulted in the current change. According to published reports, the target small molecule-aptasensor complex could modify the electrochemical features of the biosensor through specific changes in the aptamer conformation (Figure S1); however, the precise mechanism of this interaction is yet to be determined (Kim et al. 2010; Kim et al. 2007).
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Figure 2. Cyclic voltammograms of the gold electrode in different steps of the fabrication process (measurement in 20 mM Tris-HCl buffer solution (pH 7.4), containing 0.1 M KCl and 5 mM [Fe (CN)6]3-/4- solution): (a) bare gold SPE, (b) Au NPs /SPGE, (c) aptamer/ Au NPs /SPGE, (d) MCH/ aptamer/ Au NPs /SPGE, (e) 10 nM Diazinon / aptamer/ Au NPs /SPGE. 3.3 Optimization of Experimental Conditions In order to improve the performance of the novel aptasensor in every detailed aspect, various parameters encompass the incubation time, aptamer self-assembled time, deposition time for Au NPs, pH, and aptamer concentration were optimized. Different concentrations of aptamer solution were assessed in order to achieve a high coverage of the electrode surface with aptamers for sensitive target detection. The optimal concentration of aptamer was determined with 10.0 µL drops of aptamer with different concentrations (0.5–10 µM) on the surface of Au NP/ SPGEs. Following an increase in aptamer concentration, the maximum current was reached at 2.5 µM of aptamer solution (Figure S2 A). It is conceivable that a saturation of the Au NP-modified electrode surface with aptamers occurred at 2.5 µM of aptamer and this concentration could be regarded as the optimum aptamer concentration. In order to achieve the optimum time of self-assembly reaction, different time points in the range of 4-24 h were assessed. The maximum enhancement in the DPV current response occurred Page 10 of 22
when the time of self-assembly extended to 16 h (Figure S2 B). Although the prolonged incubation time led to more interactions of the thiolated aptamer with the surface of the gold electrode, the current response reached to a steady state after 16 h. It may be attributed to the saturation of the reactive sites on the electrode surface after certain time. Therefore, we chose 16 h as the optimum time for self-assembly reaction. The impact of DZN incubation time with the novel aptasensor also examined. The DPV response of thiolated aptamer/ Au NP/ SPGEs in the presence of specific concentration of DZN (10 nM) at different time points (from 10 to 45 min) was evaluated ( Figure S2C). The total current response increased considerably with prolongation in DZN incubation and similar to aptamer incubation, it reached a plateau at specific time point (at 30 min). So, the incubation time of 30 min for DZN was considered as optimal time in further experiments. The pH degree of the Tris-HCL buffer solution (20 mM) had a crucial impact on DZN and aptamer reaction and electrochemical behavior of the EC aptasensor. The sensor response by recording DPV of the DZN-aptamer complex at different pH values (from 5 to 8) indicated that the current increased in an acidic pH range with a sharp peak at pH 7.4 and then decreased as pH moved to 8 (basic solution) (Figure. S2D), The proposed mechanism of this phenomenon could be the adsorption of different ionic charge in DZN-aptamer complex in distinct pH. In basic pH, the complex adsorbed the negatively charged OH- ions which resulted in repulsion of redox elements [Fe(CN)6]3-/4- and subsequent current response reduction. On the other hand, H+ ions in the acidic solution led to inverse effects and as a consequence the current response increased. Regarding the explained mechanism, the optimal pH would be 7.4 in which the least interactions occurred and the highest current response was achieved. The cyclic voltammograms of the novel aptasensor at different deposition time of Au NPs treatment was investigated (Figure S3). Parallel to increase of deposition time, the oxidation and reduction currents increased distinctly. Overall, longer deposition time (from 30 to 150 s) resulted in the higher reduction current (Figure S2E). Hence, in order to achieve the optimal state of the nano-aptasensor, 150 s was selected as the best deposition time of the Au NP exposure.
3.4 Electrochemical Impedance Spectroscopy of the Modified Electrode Electrochemical impedance spectroscopy (EIS) experiments were performed to evaluate the exact state of aptasensor in each step of assembly. The impedance spectrum is known as Nyquist Page 11 of 22
plot, including two different parts, a semicircle part at high frequencies and a linear one at lower frequencies. The semicircle diameter represents the charge-transfer resistance (Rct) which shows the blocking behavior of the electrode surface towards the redox couple whereas the linear part is representative of the diffusion process. EIS measurements were conducted at a DC potential of 0.13 V with AC amplitude of 0.01 V, in which the Nyquist plots were recorded over a frequency range from 100 mHz to 100 kHz. Figure 3 illustrates the Nyquist plots of faradic impedance spectra in the presence of 20 mM Tris-HCl buffer solution (pH 7.40) containing 5 mM K3Fe(CN)6 (0.1M KCl). According to the figure 3 and the curve a, a roughly straight line corresponded to the impedance of the bare SPGE indicating an electron-transfer resistance (Rct) of about 1.
KΩ at the electrode surface.
Subsequent Au NP deposition of SPGE resulted in the reduced Rct level to 0.4 KΩ (Figure , curve b) and enhancement of the conductivity which confirmed the CV measurements. As mentioned earlier, this improvement in the current response could be contributed to the expansion of the available surface area of the electrode. The successful immobilization of the thiolated aptamer onto the Au NP-modified SPGE was asserted through the calculation of the Rct, which changed to a semi-circle curve; its significant increase (28.75 KΩ) and the following decreases in the current response could be assigned to the blockade of electrolyte transfer occurred by the immobilized aptamer (Figure 3, curve c). Finally, when the immobilized aptamer attached to its target DZN (10 nM DZN for 30 min incubation), the highly significant hindrance effect of the aptamer-DZN complex could disrupt the electrolyte [Fe(CN)6]3-/4- transduction which resulted in the remarkable increase in Rct ( 1.7 KΩ) and decrease in the current response. Further, the semicircle domain increased (Figure 3, curve d).
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Figure 3. Nyquist plots of electrochemical impedance spectra of modified SPGE under different conditions (measurement in 20 mM Tris-HCl buffer solution (pH 7.4), containing 0.1 M KCl and 5 mM [Fe (CN)6]3-/4- solution): (a) bare gold SPE, (b) Au NPs /SPGE, (c) aptamer/Au NPs/SPGE, (d) 10 nM Diazinon captured on the aptamer/Au NPs/SPGE. 3.5 Electrochemical response to DZN Due to more sensitivity of the DPV technique in comparison to CV, it is recommended to perform the quantitative analyses by DPV (Yang et al. 2016). Hence, we measured the DPV signals for DZN detection in the same solution used for CV assay under the optimal experimental conditions (modulation time= 0.05 s, interval time= 0.2 s, potential range of 0.45 to -0.05 V, modulation amplitude= 0.05 V). The aptasensor was incubated for 30 min in a series concentration of DZN in 20 mM Tris-HCl buffer solution (pH 7.4) and subsequently detected in 5 mM of K3Fe(CN)6 (0.1M KCl) solution. As shown in Figure 4, there is a significant decrease in the corresponding peak current with increasing DZN concentrations. This occurred because the hindrance effect of aptamer–DZN complex disrupted the normal flow rate of redox ([Fe(CN)6]3-/4-) toward the electrode surface.
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Figure 4. Differential pulse voltammograms of the sensor incubated with different concentrations of Diazinon [measurement in 20 mM Tris-HCl buffer solutions (pH 7.4)], containing 0.1M KCl and 5 mM [Fe (CN)6]3-/4- solution, (a–f): 0, 0.1, 1, 10, 100 and 1000 nM. The linear relationship between the peak cathodic current of the aptasensor and the DZN concentration was observable in the range of 0.1 - 1000 nM. The aptasensor differential responses versus DZN concentrations are illustrated in Figure 5. A logarithmic correlation between the changes in the DPV peak currents (ΔIs) and DZN concentration (CDZN) is seen. The inset of Figure 5 implicates the linear relationship between ΔI (µA) and log (CDZN) (nM) in the range of 0.1 nM to 1000 nM with the linear regression equation of ΔI (µA)= 2.1447 log(CDZN) (nM) + 3.9762 and correlation coefficient of 0.9952. The calculated limit of detection was 0.0169 nM (defined as S/N=3) (Swartz and Krull 2012).
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Figure 5. Variation of the aptasensor response against the DZN concentrations in the range of 0.1 nM to 1000 nM. The inset shows the linear relationship between the peak currents and DZN. The illustrated error bars represent the standard deviation of three repetitive measurements. Reproducibility of the aptasensor was tested using three assembled SPGEs at the same conditions for the detection of DZN at 0.1 nM. The acceptable relative standard deviation (RSD) value (2.7%) revealed that the assay was reproducible under the tested conditions. To investigate the storage stability of fabricated aptasensors, three SPGEs were kept at 4 °C for two weeks without any preservatives. No significant change in the initial signal (less than 10%) was found in the frequent assays (every 3 days) indicating that no significant decomposition occurred in long term storage. Table 1 provided a brief comparison of the available methods for DZN detection and our novel aptasensor. Compared to the published studies, the main favorable characteristics of our aptasensor would be very low LOD and linear regression in the range of small concentrations.
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Table 1: Comparison of presenting aptasensor with other reported analytical methods for the
determination of Diazinon. Determination method
Commercial sample
Linear range
LOD
HPLC
Plasma of Male Wistar rat
200-6400 ng/mL
137.42 ng/mL
0.9992
(Hassani et al. 2018)
Colorimetric
Water
0.042-0.197 ng/mL
5.44 ng/mL
0.9928
(Jokar et al. 2017)
PIM-GC-MS
Water
0.005-1 ng/mL
0.003 ng/mL
0.9988
(Vera et al. 2018)
DI–SPME–GCFPD
Vegetables
0.3-100 ng/mL
0.07 ng/mL
0.998
(Sapahin et al. 2014)
MIP-SPE-HPLCUV
Soil media and cucumber tissue
20-5000 ng/mL
5 ng/mL
0.997
(Zare et al. 2015)
MEPS-CD-IMS
Water
0.2-20 ng/mL
0.07 ng/mL
0.999
(Saraji et al. 2018)
GC-FPD
Milk
2.5-25 ng/mL
0.66 ng/mL
0.998
(Naksen et al. 2016)
Water
0.3-3 ng/mL
MIP and MWCNTs
R2
0.998 0.124 ng/mL
Urine samples
15.2-304 ng/mL
Nano-MIP-CP electrode
Well water Apple fruit
0.76-30.4 ng/mL
Electrochemical aptasensor
Plasma of Male Wistar rat
0.0304-304 ng/mL
Ref
(Khadem et al. 2017) 0.999 0.997 0.983
(Motaharian et al. 2016)
0.9952
This project
0.79 ng/mL 30.4-608 ng/mL
0.005 ng/mL
3.6. Selectivity of the aptasensor The specificity of the biosensor plays an important role in analyzing of real samples. The selectivity of the novel aptasensor was examined using different pesticides other than DZN including Malathion, Chloropyrifos and Deltamethrin, with the same analysis condition. After
Page 16 of 22
binding to 1000 nM of the target, the aptasensor was incubated for 30 min at 37 °C and subsequently DPV measurements were performed. According to the obtained results, insignificant responses of the tested samples could be attributed to negligible affinity of the novel aptasensor to such samples and the high specificity for DZN (Figure 6). 12
10
ΔI (µA)
8
6
4
2
0
Figure 6. The relative magnitudes of the signals obtained from the aptasensor after incubation in 1000 nM of Diazinon, Malathion, Chloropyrifos and Deltamethrin. Error bars were achieved based on three independent measurements. 3.7. Real sample analysis To evaluate the capability of the novel aptasensor in detecting DZN in the real samples, three different spiked concentrations of DZN in control plasma of Male Wistar rat samples were analyzed. According to the routine procedure, the plasma was diluted with the buffer solution (pH 7.4) firstly and then the standard solutions of DZN were added to each sample. As shown in Table 2, the recovery value was in the range of 96.0 – 99.3%, the RSD varied from 2.60% to 3.72%, which indicates that the developed assay might be applied for the determination of DZN in rat plasma samples. The acquired data revealed that the determined levels of DZN in real Page 17 of 22
samples were highly consistent with the HPLC technique (Hassani et al. 2018). The novel aptasensor can detect precisely the ultra-trace levels of DZN in the complex matrices such as plasma.
Table 2. Recoveries of Diazinon in plasma of Male Wistar rats with detection of the EC aptasensor. Sample
Added (nM)
Found (nM)
Recovery (%)
RSD (%) (N=3)
1
100
99.3
99.30
2.60
2
10
9.6
96.0
3.15
3
1
0.98
98.0
3.72
4. Conclusion The present study provided a simple and straightforward method for the assembly of novel label free electrochemical nano-aptasensor for the highly sensitive and rapid detection of DZN. Novel Au NP modified SPGE aptasensor will be able to determine the least level of DZN compared to the other reported methods. Interestingly, it has exhibited the minimum limit of detection for DZN (0.0169 nM). Moreover, superb selectivity and excellent performance in real samples with no considerable interaction with other components were achieved. This innovaitve fabrication strategy for the development of label-free electrochemical aptasensors as portable devices would be a promising approach in fast and precise detection of various small molecules such as DZN in the near future. However, some issues are still remaining to be addressed in the field of aptasensor application as an analythical approach. Simultaneous detection of different chemicals in one sample such as plasma or water would be one of the most challenging concerns. Development of such aptasensors capable of rapid and concurrent detection of the small amounts of different contaminants in food or biological samples will be the most predominant perspective in this area.
Acknowledgment: This study was partly supported by a grant from TUMS with code number 96-0145-34625. The study was also self-supported by the correspondin author directed general grant
Page 18 of 22
from Iran National Science Foundation. Authors are grateful to Dr Raheleh Torabi from University of Tehran for her assistance in docking part of the study. Authors appreciate Dr John G. Bruno from Operational Technologies Corporation, San Antonio, United States) for his advice to select the best aptamer sequence. The present electrochemical aptasensor determination of diazinonhas been domestically patented in Iranian Intellectual Proprty Center by the code number of 96108 on July 4, 2018.
Authors’ contribution: Professor MA and PN supervised whole study. MRG was an advisor. SH performed the study and drafted the article. MRA helped in data analysis and their interpretation. ASM helped in performing the experimental part of the study. SR helped in experimental part and edited the article. All authors have read and approved the final version.
Conflict of interest: The authors declare no conflict of interest.
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Highlights: Electrochemical Aptasensor for Determination of Diazinon.
Gold Nanoparticles-Modified Screen-Printed Gold Electrode. To make a portable and fast sensitive device.
Page 22 of 22
PII: DOI: Reference:
www.elsevier.com/locate/bios
S0956-5663(18)30639-0 https://doi.org/10.1016/j.bios.2018.08.041 BIOS10705
To appear in: Biosensors and Bioelectronic Received date: 25 June 2018 Revised date: 27 July 2018 Accepted date: 17 August 2018 Cite this article as: Shokoufeh Hassani, Milad Rezaei Akmal, Armin SalekMaghsoudi, Soheila Rahmani, Mohammad Reza Ganjali, Parviz Norouzi and Mohammad Abdollahi, Novel Label-Free Electrochemical Aptasensor for Determination of Diazinon Using Gold Nanoparticles-Modified Screen-Printed Gold Electrode, Biosensors and Bioelectronic, https://doi.org/10.1016/j.bios.2018.08.041 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 galley proof before it is published in its final citable 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.
Novel Label-Free Electrochemical Aptasensor for Determination of Diazinon Using Gold NanoparticlesModified Screen-Printed Gold Electrode Shokoufeh Hassani1,2, Milad Rezaei Akmal3, Armin Salek-Maghsoudi2, Soheila Rahmani1, Mohammad Reza Ganjali3, Parviz Norouzi3, Mohammad Abdollahi1,2* 1
Toxicology and Diseases Group, The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences (TUMS), Tehran, Iran 2
Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of
Medical Sciences (TUMS), Tehran, Iran 3
Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran,
Iran *
Corresponding author: The Institute of Pharmaceutical Sciences (TIPS) and Faculty of
Pharmacy,
Tehran
University
of
Medical
Sciences,
Tehran,
[email protected] or [email protected]
Page 1 of 22
1417614411,
Iran,
Abstract The present study aimed to develop a highly sensitive label-free electrochemical aptasensor for the detection of diazinon (DZN), as one of the most widespread organophosphorus compounds. The aptasensor was assembled using screen-printed gold electrode modified by thiolated aptamers which were immobilized on gold nanoparticles (Au NPs). Optimum deposition time, in which the highest electrochemical response occurred, was found in 150s. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize electrochemical properties of the novel aptasensor. Electrochemical detection was carried out through differential pulse voltammetry in [Fe(CN)6]3-/4- solution. Fluctuation of the current was examined in the DZN concentration range of 0.1 to 1000 nM. According to the results, the designed aptasensor provided an extremely lower limit of detection (0.0169 nM) compared with HPLC and other colorimetric techniques for DZN detection. The present highly specific designed aptasensor doesn’t interact with other analytes in the real sample. Consequently, the present aptasensor is easy to use and relatively inexpensive with a good sensitivity, stability, and reproducibility for this application. We are now evaluating all approaches to make a portable device for fast and sensitive quantification of DZN and related OPs.
Keywords: Electrochemical aptasensor, Diazinon, Gold nanoparticles, Screen-printed gold electrode, Analytical Toxicology, Organophosphorus compounds
1. Introduction Pesticides as a large and heterogeneous group of agricultural chemicals have been widely used to control, kill or repel pests in different fields (Mostafalou and Abdollahi 2017; Balali-Mood and Abdollahi, 2014). Their ubiquitous use leads to inevitable consequences for public health of the countries where they are used (Shadboorestan et al., 2016). Detrimental effects on main organs including endocrine, respiratory, renal, cardiovascular, and immune systems through irreversible inhibition of acetylcholine esterase (AChE) result in development of serious human diseases such as Parkinson’s, Alzheimer’s, Multiple Sclerosis, diabetes, and renal failure (Mostafalou and Abdollahi, 2018; Abdollahi et al. 2004). Contamination of drinking water and food sources, even at very low degree of organophosphorus compounds (OPs) is considered as a serious issue Page 2 of 22
requiring sophisticated resolution (Kazemi et al. 2012). In this regard, fabrication of reliable, rapid, and inexpensive analytical tools in order to detect such small amounts of pesticides in the environment is crucial (Mostafalou and Abdollahi 2013). Conventional analytical methods for determination of pesticides comprise gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC-MS) (Choi et al. 2015; García-Valcárcel and Tadeo 2009; Páleníková et al. 2015; Vuković et al. 2012), capillary electrophoresis (Guler et al. 2010; Regueiro et al. 2015), pressurized liquid extraction (Du et al. 2012), and fluorimetry (Pacioni and Veglia 2003). Despite accuracy and selectivity, these methods have several drawbacks such as exorbitance, time consuming process, complexity, and use of hazardous reagents that make them unsuitable for routine use (Regueiro et al. 2015; Verma and Bhardwaj 2015). Therefore, innovative techniques are needed to minimize the time of analysis and limit the use of hazardous chemicals. Diazinon (O,O-Diethyl O-[4-methyl-6-(propan-2-yl)pyrimidin-2-yl] phosphorothioate) is a common OP with broad agricultural and household use which its remaining trace amounts in the environment can exert detrimental effects in humans and animals (Pakzad et al. 2013; Shah and Iqbal 2010; Zhou et al. 2011). It is evidenced by the health organizations that the accepted maximum residue limits (MRLs) of DZN, in soil (European Union), water (Food and Agriculture Organization of the United Nations/World Health Organization) and vegetables (European Union) is 0.04 μg/kg, 0.1 μg/L, and 0.04 μg/g, respectively (Bhattacharjee et al. 2012; Akan et al. 2013). Thus, it is desirable to set up a sensitive and low cost method for DZN detection. Electrochemical strategies with potential specificity, sensitivity, stability, and low limit of detection seem an excellent option for tracing OPs. Biosensors have renowned recently for their approachability, simplicity, portability, and great selectivity and sensitivity. Accordingly, their application in detection of DZN and other OPs can be proposed as an efficient and promising alternative to conventional analytical methods. Recently, different types of biosensors for OPs detection in the environmental samples have been designed. For example, analysis of triazophos based on acetylcholine esterase inhibition measurement with amperometry transducer (Reybier et al. 2002), detection of parathion and methylparathion with a cell-based biosensor based on the potentiometric method (Bhadekar et al. 2011), and antibody-based biosensor for detection of chlorpyrifos via voltammetric methods (Talan et al. 2018). Nevertheless, since their biological recognition components such as antibody or enzyme are instable, use of biosensors as common Page 3 of 22
detection equipment has been restrained (Hammond et al. 2016; Mehrotra 2016; Mishra et al. 2018). Aptamers are short single-stranded oligonucleotides or peptides which are generated by SELEX (systematic evolution of ligands by exponential enrichment) library that can bind specifically to different elements such as toxicants, drugs, proteins, peptides, vitamins, cells, and small compounds (Saha et al. 2012; Stoltenburg et al. 2007). The main beneficial features of aptamers compared to antibodies include inconsiderable toxicity and immunogenicity, inexpensiveness, feasible production, rapid response, and high thermal and chemical stability. Moreover, in comparison to optical sensors, electrochemical aptasensors require the least amount of analyte for recognition. Possessing these prominent features, aptamers have been widely employed in various analytical techniques (Li et al. 2010; Li et al. 2011; Mokhtarzadeh et al. 2015; Taghdisi et al. 2016). To enhance the efficiency and sensitivity of the device, several techniques such as exploiting nanostructures have been also utilized. Nanostructures with their crucial properties including greater thermal and chemical stability, large surface area, high tensile strength, and elasticity have attracted much attention in designing biosensors (Farghali and Ahmed 2015; Mehlhorn et al. 2018). Immobilization of DNA aptamers and modification of electrode surface with nanoparticles may be the most effective application (Cao et al. 2011; Yang et al. 2008). Metal nanoparticles such as gold nanoparticles can be used to improve electron transfer in electrochemical biosensors (Cao et al. 2011; Zhao et al. 2015). Gold nanoparticles (Au NPs) have surpassing capability to immobilize biomolecules and also can be simply functionalized through thiolation (Yasun et al. 2012). Screen printed electrodes (SPE) which are applicable in modern sensors provide a vast range of advantages over the conventional electrodes (Wan et al. 2015). Capability of large scale manufacturing, technical feasibility, costeffectiveness and disposability are some of these benefits that make SPEs the favorable candidates for practical use in different types of biosensors (Renedo and Martínez 2007; Sanllorente‐Méndez et al. 2009; Wang et al. 1998). Moreover, their versatile features such as composing of various electrode inks (gold, carbon, silver, etc.) allow them to be modified via nanoparticles with the aim of the operation enhancement (Barton et al. 2016; Li et al. 2012; Taleat et al. 2014). Recently, the application of biosensor technologies in the detection of OPs in the environment was reported (Hassani et al. 2017; Salek-Maghsoudi et al. 2018). So far, only one aptasensor for DZN detection has been designed which was based on the colorimetric method (Jokar et al. Page 4 of 22
2017). Herein, we developed a novel label-free electrochemical thiolated aptasensor which was immobilized on gold nanoparticle (Au NP)-modified screen printed gold electrodes (SPGEs) in order to detect minimum levels of DZN in biological samples with high sensitivity and fast performance.
2. Experimental 2.1. Materials All reagents were of analytical grade purchased from Sigma-Aldrich (Darmstadt, Germany) unless otherwise stated. Diazinon, chloropyrifos, malathion, deltamethrin (purity: 98%), gold chloride trihydrate (HAuCl4.3H2O), H2SO4, 6-Mercapto-1-Hexanol (MCH), potassium ferricyanide and ferrocyanide, phosphate-buffered saline (PBS), NaCl, KCl, MgCl2 and Tris-HCl were used in this study. Solutions were prepared using ultra-pure water with a resistance of 18MΩ.cm (Millipore, USA). The protocol of the study was approved by the Institute Ethical Committee under code number IR.TUMS.VCR.REC.1395.1129 and all ethical issues on the use of animals were followed. Thiolated aptamer DNA oligonucleotides (DF20) were purchased from MWG-BIOTECH, Germany with following base sequences (Bruno and Chanpong 2012): 5-(SH)-(CH2)6ATCC TCACACCT CTCTAATATA A T TT
TATT CTCTT
ACAA
TACA
AT
CTCCC TAT- .
The stock solution of aptamer (20 µM) was prepared in the binding buffer by Tris-HCl buffer (20 mM Tris-HCl, 0.1M NaCl, 0.2 M KCl, 5 mM MgCl2, pH 7.4) and 10 mM Tris–HCl containing 0.1 M sodium chloride, 5.0 mM magnesium chloride (pH 7.4) was used as the washing buffer and stored at -20 °C.
2.2. Equipment Electrochemical measurements were performed with AUTOLAB PGSTAT 101 (Methrohm Autolab BV, Utrecht, The Netherlands), controlled by NOVA 2.1 Software. Electrochemical characterizations of modified surfaces were followed-up by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) assays that carried out in a redox electrolyte solution of 5 mM [Fe(CN)6] Page 5 of 22
−/4−
in 0.1 M KCl solution. EIS
measurements were conducted using a frequency range from 100 mHz to 100 kHz at a direct current (DC( potential of +0.13 V. The obtained data are represented in Nyquist plots. DPV was recorded within the potential range of 0.45 to -0.05 V under modulation amplitude of 50 mV. CV was employed to establish the fabrication of aptasensor. The measured potential range was between -0.2 and +0.5 V, with a scan rate of 0.1 V/s. SPGE composed of integrated working, reference, and counter electrodes in which the counter electrode and working electrodes (4 mm diameter) were made of gold and the reference electrode and electrical contacts were made of silver (DropSens, S.L., Spain). The morphology of Au NPs after the deposition procedure was assessed using the Field emission scanning electron microscopy (FESEM; A4160 system, Hitachi, Japan).
2.3. Au NP-modified SPGEs assembly The surface of the working electrode was modified by Au NPs through the electrodeposition process using a solution of 5 mM HAuCl4 in 0.5 M H2SO4 (Wan et al. 2015). At first, SPGE surface was activated via H2SO4 solution. Before the activation, the SPGE was characterized by CV in the range of 0.2 - 0.5 V in 5 mM K3Fe(CN)6 (0.1 M KCl) solution with scan rate of 0.1 V/s. Afterwards, the activation process was performed in 0.5 M sulfuric acid with CV scanning in the range of -0.1 - 1.3 V with scan rate of 0.1 V/s in order to obtain a steady voltammogram. We optimized the deposition time for Au NPs modification and compared the performance between Au NPs modified SPGE and solely activated SPGE. Following activation of the electrode, deposition process of Au NPs was carried out under potential of -0.4 V in 5 mM HAuCl4/ 0.5 M H2SO4 solution with different deposition time ranging from 30 - 150 s. After providing each deposition, the SPGE was analyzed by CV in 5 mM K3Fe(CN)6 (0.1 M KCl) with a scan rate of 0.1 V/s. Lastly, the surface structure of Au NPs modified SPGE was characterized through FESEM. The stock solution of Au NPs was stored in a refrigerator at 4 °C for further assessments.
2.4. Immobilization of the aptamer onto Au NP/ SPGEs surface In order to immobilize the aptamer, 10 µL of 2.5 μM aptamer stock solution dropped onto the Au NP-modified SPGE and the self-assembled monolayer (SAM) was spontaneously formed through 16 h incubation in a dark place at 4 °C. The blockade of the non-modified sites was Page 6 of 22
carried out using 5 mM MCH solution with 1 h incubation at room temperature. The electrode was then rinsed with washing buffer and then ultrapure water and blown dry under the nitrogen atmosphere after each incubation step.
2.5 Detection of DZN with the nano-Aptasensor Proceeding for detection, DZN determination in binding buffer solution (pH 7.4) was applied onto the surface of fabricated aptasensor (aptamer/ Au NP/ SPGEs). Following 30 min incubation at 37 °C, the aptasensor was rinsed gently with ultrapure water. Afterwards, the nanoaptasensor was placed into the electrochemical chamber (congaing 5 mM of K3Fe(CN)6 (0.1M KCl) solution), the DPV measurements were conducted for different DZN concentrations at separate SPGEs (experimental condition: modulation time= 0.05 s, interval time= 0.2 s, potential range of 0.45 to -0.05 V). The general protocol of electrochemical aptasensor development has been illustrated in Scheme 1.
Scheme 1. Schematic presentation of the aptasensor fabrication for detection of Diazinon.
3. Results and Discussion 3.1 Characterization of the Au NPs FESEM images of the Au NPs modified SPGEs and bare SPGE are shown in Figure 1A and B. We used Au NPs to immobilize thiolated aptamer which enhanced the signal transduction by means of increasing the surface area of the electrode. Page 7 of 22
Before the activation, the SPGE was characterized by CV (-0.2 to +0.5 V) in 5 mM K3Fe (CN)6 (0.1 M KCl) with scan rates of 0.05 V/s and 0.1 V/s. Next, the activation process was carried out in 0.5 M sulfuric acid solution with CV scanning (-0.1 to +1.3 V) with a scan rate of 0.1 V/s until the steady voltammograms were acquired. It is evidenced by the previous studies that the removal of oxidation layer and the exposure of the most active sites on the electrode surface after the activation could result in a remarkable improvement in the noticeably enhancement of electrochemical performance (Fanjul-Bolado et al. 2008; Su et al. 2011). Deposition time of Au NPs could significantly influence the deposition extent and size of Au NPs on the SPGE (Renedo and Martínez 2007). Regarding the maximum obtained responses, the deposition time of Au NPs was set to 150 s in order to get the most suitable electrochemical behavior of the SPGE, After Au NPs modification, CV was conducted with Au NPs modified and solely activated SPGEs in 5 mM K3Fe(CN)6 (0.1M KCl) (Figure 2).
Figure 1. FESEM images of gold electrodes; A) Bare gold electrode, B) Au NPs modified SPGEs with 5 mM HAuCl4/0.5 M H2SO4 at optimum deposition time of 150 s. 3.2 Characterization of the Aptasensor (DZN/ thiolated aptamer/ Au NP/ SPGEs) Characterization of the DZN/thiolated aptamer/ Au NP/ SPGE was carried out through CV and EIS by monitoring the processes of the assembly of the aptasensor in each step. All electrochemical measurements were performed in 20 mM Tris-HCl buffer solution (pH 7.4), containing 5 mM K3Fe(CN)6 (0.1 M KCl). Ferri-ferrocyanide redox electrolyte was used for electrochemical transduction (Montrose et al. 2013; Sharma et al. 2010). CV measurements were achieved in the potential range from -0.2 to 0.5 V, with a scan rate of 0.1 V/s. Figure 2 shows the ferri-ferrocyanide CVs of the bare gold SPE (curve a), Au NPs /SPGE (curve b), aptamer/ Au Page 8 of 22
NPs /SPGE (curve c), MCH/ aptamer/ Au NPs /SPGE (curve d) and 10 nM Diazinon / aptamer/ Au NPs /SPGE (curve e). The bare gold SPE showed an appropriate reversible peak, which was attributed to the t occurrence of faster heterogeneous electron transfer kinetics. Definite characteristics of [Fe(CN)6]3-/4- were assessed through this measurement by means of redox peak separation and calculating ΔEp of the cathodic and anodic waves and current response. The CV results of redox elements Fe(CN)6
3-
/Fe(CN)6
4-
at the SPGE indicated a pair
of reversible redox peaks with ΔEp of 0.073 V (Figure 2, curve a). After deposition of Au NPs on the SPGE surface, the signal was considerably intensified ΔEp reduced to 0.070 V (Figure 2, curve b). It confirmed the previous experiments claiming that the modification of the SPE surface with nanoparticles could provide larger exposed area which led to enhance the current response. Self-assembly of the thiolated aptamer onto the Au NP modified SPGE caused the peak currents of electrolyte [Fe(CN)6]3-/4- dropped significantly and a modest ΔEp increase (0.074 V) was observed (Figure 2, curve c). The most probable reason for this current reduction could be the repulsion force induced by the phosphate groups of the aptamer and also its hindrance which interfere with the electrolyte transfer. According to the curve d in Figure 2, MCH addition onto the electrode surface blocked the active sites necessary for electron transfer; this resulted in a decrease in the redox peak current and an increase in the peak separation (ΔEp=0.109 V) (Baghayeri et al.2018). At last we incubated the novel aptasensor with 10 nM DZN for 30 min; the peak currents decreased dramatically in parallel with the striking increase of the peak potential ΔEp
to 0.168 V (Figure 2, curve e). The obtained results are broadly consistent with previous
studies in which the binding the immobilized aptamer to its target led to the folding of aptamer and formation of stem-loop structure which resulted in the current change. According to published reports, the target small molecule-aptasensor complex could modify the electrochemical features of the biosensor through specific changes in the aptamer conformation (Figure S1); however, the precise mechanism of this interaction is yet to be determined (Kim et al. 2010; Kim et al. 2007).
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Figure 2. Cyclic voltammograms of the gold electrode in different steps of the fabrication process (measurement in 20 mM Tris-HCl buffer solution (pH 7.4), containing 0.1 M KCl and 5 mM [Fe (CN)6]3-/4- solution): (a) bare gold SPE, (b) Au NPs /SPGE, (c) aptamer/ Au NPs /SPGE, (d) MCH/ aptamer/ Au NPs /SPGE, (e) 10 nM Diazinon / aptamer/ Au NPs /SPGE. 3.3 Optimization of Experimental Conditions In order to improve the performance of the novel aptasensor in every detailed aspect, various parameters encompass the incubation time, aptamer self-assembled time, deposition time for Au NPs, pH, and aptamer concentration were optimized. Different concentrations of aptamer solution were assessed in order to achieve a high coverage of the electrode surface with aptamers for sensitive target detection. The optimal concentration of aptamer was determined with 10.0 µL drops of aptamer with different concentrations (0.5–10 µM) on the surface of Au NP/ SPGEs. Following an increase in aptamer concentration, the maximum current was reached at 2.5 µM of aptamer solution (Figure S2 A). It is conceivable that a saturation of the Au NP-modified electrode surface with aptamers occurred at 2.5 µM of aptamer and this concentration could be regarded as the optimum aptamer concentration. In order to achieve the optimum time of self-assembly reaction, different time points in the range of 4-24 h were assessed. The maximum enhancement in the DPV current response occurred Page 10 of 22
when the time of self-assembly extended to 16 h (Figure S2 B). Although the prolonged incubation time led to more interactions of the thiolated aptamer with the surface of the gold electrode, the current response reached to a steady state after 16 h. It may be attributed to the saturation of the reactive sites on the electrode surface after certain time. Therefore, we chose 16 h as the optimum time for self-assembly reaction. The impact of DZN incubation time with the novel aptasensor also examined. The DPV response of thiolated aptamer/ Au NP/ SPGEs in the presence of specific concentration of DZN (10 nM) at different time points (from 10 to 45 min) was evaluated ( Figure S2C). The total current response increased considerably with prolongation in DZN incubation and similar to aptamer incubation, it reached a plateau at specific time point (at 30 min). So, the incubation time of 30 min for DZN was considered as optimal time in further experiments. The pH degree of the Tris-HCL buffer solution (20 mM) had a crucial impact on DZN and aptamer reaction and electrochemical behavior of the EC aptasensor. The sensor response by recording DPV of the DZN-aptamer complex at different pH values (from 5 to 8) indicated that the current increased in an acidic pH range with a sharp peak at pH 7.4 and then decreased as pH moved to 8 (basic solution) (Figure. S2D), The proposed mechanism of this phenomenon could be the adsorption of different ionic charge in DZN-aptamer complex in distinct pH. In basic pH, the complex adsorbed the negatively charged OH- ions which resulted in repulsion of redox elements [Fe(CN)6]3-/4- and subsequent current response reduction. On the other hand, H+ ions in the acidic solution led to inverse effects and as a consequence the current response increased. Regarding the explained mechanism, the optimal pH would be 7.4 in which the least interactions occurred and the highest current response was achieved. The cyclic voltammograms of the novel aptasensor at different deposition time of Au NPs treatment was investigated (Figure S3). Parallel to increase of deposition time, the oxidation and reduction currents increased distinctly. Overall, longer deposition time (from 30 to 150 s) resulted in the higher reduction current (Figure S2E). Hence, in order to achieve the optimal state of the nano-aptasensor, 150 s was selected as the best deposition time of the Au NP exposure.
3.4 Electrochemical Impedance Spectroscopy of the Modified Electrode Electrochemical impedance spectroscopy (EIS) experiments were performed to evaluate the exact state of aptasensor in each step of assembly. The impedance spectrum is known as Nyquist Page 11 of 22
plot, including two different parts, a semicircle part at high frequencies and a linear one at lower frequencies. The semicircle diameter represents the charge-transfer resistance (Rct) which shows the blocking behavior of the electrode surface towards the redox couple whereas the linear part is representative of the diffusion process. EIS measurements were conducted at a DC potential of 0.13 V with AC amplitude of 0.01 V, in which the Nyquist plots were recorded over a frequency range from 100 mHz to 100 kHz. Figure 3 illustrates the Nyquist plots of faradic impedance spectra in the presence of 20 mM Tris-HCl buffer solution (pH 7.40) containing 5 mM K3Fe(CN)6 (0.1M KCl). According to the figure 3 and the curve a, a roughly straight line corresponded to the impedance of the bare SPGE indicating an electron-transfer resistance (Rct) of about 1.
KΩ at the electrode surface.
Subsequent Au NP deposition of SPGE resulted in the reduced Rct level to 0.4 KΩ (Figure , curve b) and enhancement of the conductivity which confirmed the CV measurements. As mentioned earlier, this improvement in the current response could be contributed to the expansion of the available surface area of the electrode. The successful immobilization of the thiolated aptamer onto the Au NP-modified SPGE was asserted through the calculation of the Rct, which changed to a semi-circle curve; its significant increase (28.75 KΩ) and the following decreases in the current response could be assigned to the blockade of electrolyte transfer occurred by the immobilized aptamer (Figure 3, curve c). Finally, when the immobilized aptamer attached to its target DZN (10 nM DZN for 30 min incubation), the highly significant hindrance effect of the aptamer-DZN complex could disrupt the electrolyte [Fe(CN)6]3-/4- transduction which resulted in the remarkable increase in Rct ( 1.7 KΩ) and decrease in the current response. Further, the semicircle domain increased (Figure 3, curve d).
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Figure 3. Nyquist plots of electrochemical impedance spectra of modified SPGE under different conditions (measurement in 20 mM Tris-HCl buffer solution (pH 7.4), containing 0.1 M KCl and 5 mM [Fe (CN)6]3-/4- solution): (a) bare gold SPE, (b) Au NPs /SPGE, (c) aptamer/Au NPs/SPGE, (d) 10 nM Diazinon captured on the aptamer/Au NPs/SPGE. 3.5 Electrochemical response to DZN Due to more sensitivity of the DPV technique in comparison to CV, it is recommended to perform the quantitative analyses by DPV (Yang et al. 2016). Hence, we measured the DPV signals for DZN detection in the same solution used for CV assay under the optimal experimental conditions (modulation time= 0.05 s, interval time= 0.2 s, potential range of 0.45 to -0.05 V, modulation amplitude= 0.05 V). The aptasensor was incubated for 30 min in a series concentration of DZN in 20 mM Tris-HCl buffer solution (pH 7.4) and subsequently detected in 5 mM of K3Fe(CN)6 (0.1M KCl) solution. As shown in Figure 4, there is a significant decrease in the corresponding peak current with increasing DZN concentrations. This occurred because the hindrance effect of aptamer–DZN complex disrupted the normal flow rate of redox ([Fe(CN)6]3-/4-) toward the electrode surface.
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Figure 4. Differential pulse voltammograms of the sensor incubated with different concentrations of Diazinon [measurement in 20 mM Tris-HCl buffer solutions (pH 7.4)], containing 0.1M KCl and 5 mM [Fe (CN)6]3-/4- solution, (a–f): 0, 0.1, 1, 10, 100 and 1000 nM. The linear relationship between the peak cathodic current of the aptasensor and the DZN concentration was observable in the range of 0.1 - 1000 nM. The aptasensor differential responses versus DZN concentrations are illustrated in Figure 5. A logarithmic correlation between the changes in the DPV peak currents (ΔIs) and DZN concentration (CDZN) is seen. The inset of Figure 5 implicates the linear relationship between ΔI (µA) and log (CDZN) (nM) in the range of 0.1 nM to 1000 nM with the linear regression equation of ΔI (µA)= 2.1447 log(CDZN) (nM) + 3.9762 and correlation coefficient of 0.9952. The calculated limit of detection was 0.0169 nM (defined as S/N=3) (Swartz and Krull 2012).
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Figure 5. Variation of the aptasensor response against the DZN concentrations in the range of 0.1 nM to 1000 nM. The inset shows the linear relationship between the peak currents and DZN. The illustrated error bars represent the standard deviation of three repetitive measurements. Reproducibility of the aptasensor was tested using three assembled SPGEs at the same conditions for the detection of DZN at 0.1 nM. The acceptable relative standard deviation (RSD) value (2.7%) revealed that the assay was reproducible under the tested conditions. To investigate the storage stability of fabricated aptasensors, three SPGEs were kept at 4 °C for two weeks without any preservatives. No significant change in the initial signal (less than 10%) was found in the frequent assays (every 3 days) indicating that no significant decomposition occurred in long term storage. Table 1 provided a brief comparison of the available methods for DZN detection and our novel aptasensor. Compared to the published studies, the main favorable characteristics of our aptasensor would be very low LOD and linear regression in the range of small concentrations.
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Table 1: Comparison of presenting aptasensor with other reported analytical methods for the
determination of Diazinon. Determination method
Commercial sample
Linear range
LOD
HPLC
Plasma of Male Wistar rat
200-6400 ng/mL
137.42 ng/mL
0.9992
(Hassani et al. 2018)
Colorimetric
Water
0.042-0.197 ng/mL
5.44 ng/mL
0.9928
(Jokar et al. 2017)
PIM-GC-MS
Water
0.005-1 ng/mL
0.003 ng/mL
0.9988
(Vera et al. 2018)
DI–SPME–GCFPD
Vegetables
0.3-100 ng/mL
0.07 ng/mL
0.998
(Sapahin et al. 2014)
MIP-SPE-HPLCUV
Soil media and cucumber tissue
20-5000 ng/mL
5 ng/mL
0.997
(Zare et al. 2015)
MEPS-CD-IMS
Water
0.2-20 ng/mL
0.07 ng/mL
0.999
(Saraji et al. 2018)
GC-FPD
Milk
2.5-25 ng/mL
0.66 ng/mL
0.998
(Naksen et al. 2016)
Water
0.3-3 ng/mL
MIP and MWCNTs
R2
0.998 0.124 ng/mL
Urine samples
15.2-304 ng/mL
Nano-MIP-CP electrode
Well water Apple fruit
0.76-30.4 ng/mL
Electrochemical aptasensor
Plasma of Male Wistar rat
0.0304-304 ng/mL
Ref
(Khadem et al. 2017) 0.999 0.997 0.983
(Motaharian et al. 2016)
0.9952
This project
0.79 ng/mL 30.4-608 ng/mL
0.005 ng/mL
3.6. Selectivity of the aptasensor The specificity of the biosensor plays an important role in analyzing of real samples. The selectivity of the novel aptasensor was examined using different pesticides other than DZN including Malathion, Chloropyrifos and Deltamethrin, with the same analysis condition. After
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binding to 1000 nM of the target, the aptasensor was incubated for 30 min at 37 °C and subsequently DPV measurements were performed. According to the obtained results, insignificant responses of the tested samples could be attributed to negligible affinity of the novel aptasensor to such samples and the high specificity for DZN (Figure 6). 12
10
ΔI (µA)
8
6
4
2
0
Figure 6. The relative magnitudes of the signals obtained from the aptasensor after incubation in 1000 nM of Diazinon, Malathion, Chloropyrifos and Deltamethrin. Error bars were achieved based on three independent measurements. 3.7. Real sample analysis To evaluate the capability of the novel aptasensor in detecting DZN in the real samples, three different spiked concentrations of DZN in control plasma of Male Wistar rat samples were analyzed. According to the routine procedure, the plasma was diluted with the buffer solution (pH 7.4) firstly and then the standard solutions of DZN were added to each sample. As shown in Table 2, the recovery value was in the range of 96.0 – 99.3%, the RSD varied from 2.60% to 3.72%, which indicates that the developed assay might be applied for the determination of DZN in rat plasma samples. The acquired data revealed that the determined levels of DZN in real Page 17 of 22
samples were highly consistent with the HPLC technique (Hassani et al. 2018). The novel aptasensor can detect precisely the ultra-trace levels of DZN in the complex matrices such as plasma.
Table 2. Recoveries of Diazinon in plasma of Male Wistar rats with detection of the EC aptasensor. Sample
Added (nM)
Found (nM)
Recovery (%)
RSD (%) (N=3)
1
100
99.3
99.30
2.60
2
10
9.6
96.0
3.15
3
1
0.98
98.0
3.72
4. Conclusion The present study provided a simple and straightforward method for the assembly of novel label free electrochemical nano-aptasensor for the highly sensitive and rapid detection of DZN. Novel Au NP modified SPGE aptasensor will be able to determine the least level of DZN compared to the other reported methods. Interestingly, it has exhibited the minimum limit of detection for DZN (0.0169 nM). Moreover, superb selectivity and excellent performance in real samples with no considerable interaction with other components were achieved. This innovaitve fabrication strategy for the development of label-free electrochemical aptasensors as portable devices would be a promising approach in fast and precise detection of various small molecules such as DZN in the near future. However, some issues are still remaining to be addressed in the field of aptasensor application as an analythical approach. Simultaneous detection of different chemicals in one sample such as plasma or water would be one of the most challenging concerns. Development of such aptasensors capable of rapid and concurrent detection of the small amounts of different contaminants in food or biological samples will be the most predominant perspective in this area.
Acknowledgment: This study was partly supported by a grant from TUMS with code number 96-0145-34625. The study was also self-supported by the correspondin author directed general grant
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from Iran National Science Foundation. Authors are grateful to Dr Raheleh Torabi from University of Tehran for her assistance in docking part of the study. Authors appreciate Dr John G. Bruno from Operational Technologies Corporation, San Antonio, United States) for his advice to select the best aptamer sequence. The present electrochemical aptasensor determination of diazinonhas been domestically patented in Iranian Intellectual Proprty Center by the code number of 96108 on July 4, 2018.
Authors’ contribution: Professor MA and PN supervised whole study. MRG was an advisor. SH performed the study and drafted the article. MRA helped in data analysis and their interpretation. ASM helped in performing the experimental part of the study. SR helped in experimental part and edited the article. All authors have read and approved the final version.
Conflict of interest: The authors declare no conflict of interest.
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Highlights: Electrochemical Aptasensor for Determination of Diazinon.
Gold Nanoparticles-Modified Screen-Printed Gold Electrode. To make a portable and fast sensitive device.
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