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Instrumentation Science & Technology
ISSN: 1073-9149 (Print) 1525-6030 (Online) Journal homepage: https://www.tandfonline.com/loi/list20
Electrochemical, continuous-flow determination
of p-benzoquinone on a gold nanoparticles
poly(propylene-co-imidazole) modified gold
electrode
Seyda Korkut, Sinan Uzuncar, Muhammet Samet Kilic & Baki Hazer
To cite this article: Seyda Korkut, Sinan Uzuncar, Muhammet Samet Kilic & Baki Hazer (2016) Electrochemical, continuous-flow determination of p-benzoquinone on a gold nanoparticles
poly(propylene-co-imidazole) modified gold electrode, Instrumentation Science & Technology, 44:6, 614-628, DOI: 10.1080/10739149.2016.1184161
To link to this article: https://doi.org/10.1080/10739149.2016.1184161
Accepted author version posted online: 11 May 2016.
Published online: 07 Jun 2016. Submit your article to this journal
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2016, VOL. 44, NO. 6, 614–628
http://dx.doi.org/10.1080/10739149.2016.1184161
Electrochemical, continuous-flow determination of
p-benzoquinone on a gold nanoparticles poly(propylene-
co-imidazole) modified gold electrode
Seyda Korkuta, Sinan Uzuncara, Muhammet Samet Kilicb, and Baki Hazerb
aDepartment of Environmental Engineering, Bulent Ecevit University, Zonguldak, Turkey; bDepartment of Chemistry, Bulent Ecevit University, Zonguldak, Turkey
ABSTRACT
A novel continuous flow biosensor based on gold nanoparticles and poly(propylene-co-imidazole) was developed for the online determination of p-benzoquinone. The amperometric response was measured as a function of p-benzoquinone concentration at an applied potential of 50 mV. The hydrogen peroxide concentration was optimized and fixed at 1 mM in samples. The mass transfer resistance of the copolymer film was minimized, and the flow cell was regenerated quickly at 1 mL/min. The resulting device provided good analytical performance based on a linear dynamic range from 5–100 µM, a short response time of 3 s, a detection limit of 3.3 µM, excellent repeatability with a relative standard deviation of 0.82%, long-term stability of 95% after four weeks, and an accuracy of 105%. The gold nanoparticles enhanced the electron transfer rate on the electrode. The apparent Michaelis-Menten constant was 4 mM, showing that the enzyme retained catalytic specificity and provided high activity for p-benzoquinone.
KEYWORDS
Continuous flow biosensor; gold nanoparticle; horseradish peroxidase; p-benzoquinone
Introduction
Phenols are common water pollutants and include a wide variety of organic chemicals. They are of particular environmental importance due to their rela-tively high toxicity at low concentrations. Therefore, sensitive and fast assay of phenolics in environmental samples is receiving more attention.[1] Phenols have been determined by spectrophotometry, gas and liquid chromatography, and capillary electrophoresis.[2–5] However, the high cost, pretreatment of sample and slow turnaround times of these methods[6] indicate a need for more sophisticated analytical techniques. Biosensors, which combine a bio-logical component with a physicochemical detector, are devices employed to convert biochemical reactions into electrical signals. These instruments are becoming increasingly important in environmental analysis[7] because they are simple to use, inexpensive, highly sensitive, selective, and respond quickly in comparison to the conventional measurement methods.
CONTACT Seyda Korkut s.korkut@beun.edu.tr Department of Environmental Engineering, Bulent Ecevit University, 67100, Zonguldak, Turkey.
Tyrosinase, laccase, and horseradish peroxidase are enzymes for the oxidation of phenols used in amperometric biosensors for the determination of phenolic compounds.[8,9] In horseradish peroxidase-based biosensors, the enyzme is oxidized by peroxides and re-reduced by phenols. Phenoxy radicals formed during the enzymatic oxidation of phenolics are reduced electro-chemically on the electrode surface under a proper applied potential. The reducing current is proportional to the phenol concentration in the bulk medium. However, the sensitivity, repeatability, reproducibility, and response time need to be improved for most biosensors. Increasing attention has been focused on using supporting polymeric materials with nanoparticles to immobilize enzymes on electrode surfaces to overcome these limitations. Nanomaterials are favorable candidates to facilitate efficient signal transduc-tion and molecular recognitransduc-tion because they have unique structural and electronic features. Nanomaterials such as graphene, ZnO, hydroxyapatite, and carbon nanotubes have been used for the determination of phenols, offering promising advantages in terms of sensitivity and analysis time in comparison to most biosensors.[10] Gold nanoparticles are the most stable and have better biocompatibility for bioanalytical devices.[11–13] Poly-propylene is a versatile hydrophobic polymer that offers low cost, good mechanical properties, easy processing, excellent recyclability,[14–21] and wide usage in medical and industrial applications due these properties.[22] Generation of new polypropylene-based nanocomposites with excellent characteristics was achieved by combining gold nanoparticles with the poly-propylene chain.[23] In our previous study, we reported a compartment-less enzymatic fuel cell constructed by using poly(propylene-co-imidazole) as the supporting layer which showed excellent adsorption and provided rapid electron transfer, yielding efficient energy production from municipal wastewater.[24]
Integration of the enzyme electrode in a continuous flow cell is preferred to determine phenols in environmental samples to reduce interferences[25] and electrode surface fouling by the enzymatic reaction products. The flow cell electrode surface contacts the substrate for a short time only. When a buffer contacts the enzyme surface, the substrate concentration is reduced to zero and disappears on the enzyme-electrode in the continuous flow biosensors. The flow system presents the advantage of a reduction in analysis time allowing high sample throughput in comparison to a batch system.
However, few reports are available for flow injection phenol biosensors.[26,27] For example, Dantoni et al.[26] developed a flow injection catechol biosensor based on carbon paste-DNA-tyrosinase electrode. The response of the device decreased due to enzyme inhibition, structural changes, or leaching by the continuous flow. In this study, a poly(propylene-co- imidazole)/horseradish peroxidase film electrode was prepared to combine the advantages of gold nanoparticles with the copolymer and flow injection
for online p-benzoquinone determination. The influence of flow rate on the performance was evaluated and analytical figures of merit were reported. Experimental
Reagents
Chlorinated polypropylene (molecular weight of 150,000 Da, three repeating units have 1 Cl on average), imidazole, toluene (99.9% HPLC grade), tetrahy-drafurane (99.9% GC grade), sodium hydride, gold(III) chloride solution (HAuCl4), horseradish peroxidase (E.C.1.11.1.7) with an activity of
�1000 U/vial, phenol, catechol, and p-benzoquinone were obtained from Sigma. Lithium perchlorate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and 35% hydrogen peroxide were purchased from Merck. Stock solutions of horseradish peroxidase and phenolic compounds were freshly prepared before measurements.
Flow injection system and electrodes
A CH Instruments 1040C electrochemical analyzer was used for ampero-metric measurements. The flow-injection system included an HPLC pump (Waters 515), an injection valve (Shimadzu), a sample loop (7725i), and a flow cell (CH Instruments) with a three-electrode system. Samples were introduced into the flowing carrier solution (buffer) with an injection valve including a sample loop with a volume of 1 mL. Gold working (3 mm diameter), Pt wire counter, and Ag/AgCI (3 M NaCI) reference electrodes were used in the con-tinuous flow cell. The HPLC pump was adjusted to deliver the carrier solution at a flow rate of 1 mL/min. The carrier solution was composed of 100 mM pH 7 potassium phosphate buffer including 0.7 mg/mL LiCIO4 as supporting
electrolyte according to the our previous horseradish peroxidase studies in which the buffer conditions were optimized.[28,29] The experimental setup is presented in Figure 1.
Synthesis of gold nanoparticle-embedded poly(propylene-co-imidazole)
Gold nanoparticle-embedded poly(propylene-co-imidazole) was synthesized according to a procedure in our previous report as follows:[24] 0.25 g of imi-dazole and 1.43 g of chlorinated polypropylene were separately dissolved in 30 mL of purified tetrahydrafurane. 0.20 g of NaH in oil (60 wt%) was added to the imidazole solution. The reaction mixture was stirred at room temperature under argon for 3 hr. The sodium salt of imidazole solution was added to the chlorinated polypropylene solution and stirred for 30 min. The reaction mixture was stirred for one day and poured into 500 mL of
1 M HCl. The precipitated polymer was filtered, washed with distilled water, and dried under vacuum at 50°C for 24 hr. To increase the purity of the pre-cipitate, it was redissolved in chloroform, reprecipitated in 200 mL of meth-anol, and dried under vacuum overnight at room temperature. 100 mg of synthesized pure poly(propylene-co-imidazole) were dissolved in 10 mL of tet-rahydrafuran. 0.05 mL of 0.2 M aqueous HAuCl4 were added and stirred for
30 minutes. The color of gold nanoparticles was observed within 2–3 min. The final solution was stirred for 1 hr and poured into a 7 cm diameter Petri dish to form the film through solvent casting. After a day, gold nanoparticles embedded poly(propylene-co-imidazole) film was removed from the Petri dish. The polymeric film was washed with methanol and dried under vacuum at room temperature for 24 hr. 1 mg/mL of the gold nanoparticles embedded poly(propylene-co-imidazole) solution in toluene was used for electrode surface coating.
Fabrication of poly(propylene-co-imidazole)/horseradish peroxidase electrode
The working electrode surface was cleaned by gamma alumina powder and rinsed with ultrapure water before the fabrication step. 2 µL of the gold nano-particle embedded poly(propylene-co-imidazole) solution was directly added to the surface of the gold working electrode and allowed to dry at room tem-perature. After washing the electrode with ultrapure water, the electrode was immersed in 0.6 mg/mL of horseradish peroxidase for enzyme immobilization via physical adsorption at þ4°C overnight. The electrodes were washed in 5 mL of 100 mM pH 7 phosphate buffer to remove unbound enzyme from the electrode surface.
Results and discussion
Copolymer characterization
Infrared spectroscopy and scanning electron microscopy characterization of the gold nanoparticle embedded poly(propylene-co-imidazole) were provided in our previous report.[24] The propylene/imidazole based copolymer was shown to be synthesized by trapping the gold nanoparticles in the copolymer pores. Cyclic voltammetry was conducted to evaluate the electrochemical characteristic of the horseradish peroxidase immobilized copolymer film coated electrode surface in continuous flow mode at a flow rate of 1 mL/ min. Cyclic voltammetry was performed at potentials from 0.3 to þ 0.3 V at a scan rate of 100 mV/s. Figure 2 shows that the gold nanoparticle embed-ded poly(propylene-co-imidazole) film did not block electron transport between the electrode and the solution. These results show that the metallic nanoparticles provided the electrical conductivity on the working electrode. We previously reported that the poly(propylene-co-imidazole) film electrodes without nanoparticles did not show an applicable cyclic voltammogram since no passage of current was observed on the electrode surface.[24]
Amperometric response
Horseradish peroxidase contains heme as prosthetic group and is commonly used for the determination of phenolics. In the catalytic cycle of horseradish peroxidase reactions, the native enzyme reacts with hydrogen peroxide to
Figure 2. Cyclic voltammogram of the horseradish peroxidase immobilized gold nanoparticle embedded poly(propylene-co-imidazole) film coated electrode at a flow rate of 1 mL/min. The scan rate was 100 mV/s vs. Ag/AgCl.
activate the intermediate enzymatic form. The oxidized state of horseradish peroxidase accepts an aromatic compound (p-benzoquinone) into its active site, and carries out the oxidation. The resulting free phenoxy radical is reduced by accepting 2 electrons from the electrode surface by applying a suit-able potential. The enzyme oxidizes another aromatic molecule; releasing a second free radical into solution and returning the enzyme to its native state, thus completing the cycle. In this study, the amperometric monitoring of the enzymatic reaction was carried out by the reduction of p-benzoquinone. The reduction current was proportional to p-benzoquinone concentration in the carrier solution if an adequate concentration of hydrogen peroxide was present. In our previous work, we reported that the most effective two electron reduction of p-benzoquinone was achieved at an applied potential of 50 mV.[28] Phenols may be oxidized at potential values higher than þ0.8 V vs. the saturated calomel electrode on conventional electrodes. However, higher electric currents are generated than expected due to the dimerization of phenols and production of electroactive species on the elec-trode by applying overpotentials. In addition, the background current and noise level increase at higher potential values by the adsorption of phenolic polymers on the electrode surface.[30]
The hydrogen peroxide concentration is a very important parameter of horseradish peroxidase catalyzed reactions. The oxidized horseradish peroxi-dase may be further oxidized by excess hydrogen peroxide. This oxidized horseradish peroxidase decomposes spontaneously to its native state. How-ever, this decomposition is sufficiently slow and the excessively oxidized enzyme form is severely hampered in carrying out the catalytic oxidation of phenolics.[31] In brief, excessive hydrogen peroxide is expected degrade the enzyme performance. To investigate the effect of hydrogen peroxide concen-tration on the biosensor response, 1 mM p-benzoquinone was treated with hydrogen peroxide concentrations between 0.25 and 2 mM. The samples were injected into the carrier solution successively at a flow rate of 1 mL/ min.
Figure 3 showed the dependence of the enzymatic reaction on hydrogen peroxide concentration at an applied potential of 50 mV. The results show that the p-benzoquinone reduction current increased up to a hydrogen per-oxide concentration of 1 mM and decreased at higher concentrations due to its suppression on the horseradish peroxidase reactions. An optimized hydrogen peroxide concentration of 1 mM was added in subsequent measurements.
Optimization of flow rate studies and phenols on the response
The flow rate directly affects the enzymatic reaction parameters and hence the biosensor sensitivity, detection limit, and sample throughput. The choice of optimum flow rate is necessary to obtain accurate and precise results. The
effect of flow rate on the amperometric response of the biosensor was inves-tigated at flow rates from 0.1–10 mL/min for 1 mM of p-benzoquinone at an applied potential of 50 mV. The response of the biosensor as a function of flow rate is shown in Figure 4. The reduction currents decreased with increas-ing flow rate as a result of the shorter substrate retention time for the enzy-matic reaction in the flow cell. However, the reduction current did not change with flow rates higher than 1 mL/min. Low flow rates (0.1–1 mL/ min) provided low sample throughput, increased sample dispersion, and high response time due to the long retention time of p-benzoquinone. Imidazole chains provided a larger surface area for the polypropylene-based copolymer film for the gold nanoparticles and immobilizing the enzyme deeply along with the long thrums of the copolymer. The enzyme reacted with substrate to use the entire working electrode area. The enhanced mass transfer with increasing flow rate provides an extra driving force for substrate in a continu-ous flow system. The mass transfer resistance of the polymeric layer was mini-mized at 1 mL/min on the electrode surface since the same reduction current was observed at higher flow rates. However, many samples may be analyzed because sharp peaks were formed within short response times at flow rates higher than 1 mL/min. The system was regenerated as rapidly as possible for real-time analysis. Regeneration involves passing the carrier solution without substrate through the system. The speed of cleaning depends on flow rate.[32] The flow cell was regenerated rapidly at 1 mL/min.
Figure 3. Optimization of hydrogen peroxide concentration for the biosensor response using 1 mM p-benzoquinone.
The selectivity of a horseradish peroxidase-based biosensor depends on the stability of the phenoxy radicals generated by enzymatic reaction, electrode material, polymeric matrix, enzyme immobilization method, and the magni-tude of applied potential.[29] The substrate selectivity of the enzyme for differ-ent phenols was consistdiffer-ent with the ability of the substitudiffer-ents for forming electron-donor conjugation. Usually, stronger electron conjugation results in a higher response. Figure 5 shows the amperometric response of the bio-sensor for 1 mM p-benzoquinone, phenol, and catechol at 1 mL/min and an applied potential of 50 mV. The results show that the response was less sensitive to phenol and catechol due to the lower currents in comparison to
p-benzoquinone. The results show that the enzyme exhibited higher affinity
for p-benzoquinone. This result is consistent with our previous report where we calculated a lower apparent Michaelis-Menten constant for
p-benzoquinone in comparison to catechol.[33] In another report, lactoperox-idase and chloroperoxlactoperox-idase electrodes provided higher responses for phenol and catechol, and lower responses for other phenolics in comparison to a horseradish peroxidase electrode.[9]
Analytical figures of merit
p-Benzoquinone detection was performed at the flow rate of 1 mL/min
with an applied potential of 50 mV. The continuous flow system was
Figure 4. Optimization of the flow rate for the amperometric response of the poly(propylene-co- imidazole)/horseradish peroxidase film electrode. The flows from left to right were 0.1, 0.2, 0.3, 0.5, 1, 2, 4, 6, 8, and 10 mL/min.
allowed to reach steady-state conditions before the introduction of sample. Concentrations of p-benzoquinone between 5 µM and 16 mM were introduced successively. The amperometric current was recorded for each
p-benzoquinone injection and presented in Figure 6. Proportionally increas-ing p-benzoquinone reduction currents were generated with the
Figure 5. Response of the biosensor to various phenolics at a flow rate of 1 mL/min.
Figure 6. Amperometric response at a flow rate of 1 mL/min of the poly(propylene-co- imidazole)/horseradish peroxidase film electrode to the concentration of p-benzoquinone: (a) 0.005 mM, (b) 0.01 mM, (c) 0.03 mM, (d) 0.05 mM, (e) 0.1 mM, (f) 0.3 mM, (g) 0.5 mM, (h) 0.75 mM, (i) 1 mM, (j) 1.5 mM, (k) 2 mM, (l) 2.5 mM, (m) 3 mM, (n) 5 mM, (o) 7 mM, (p) 10 mM, (q) 12 mM, (r) 14 mM, and (s) 16 mM.
concentration of p-benzoquinone. Stable and well-shaped peak formation may be attributed to the continuous flow conditions since the microchannels of the flow system provided effective injection pulse on the working electrode surface. Rapid system regeneration was observed using the flow conditions. Therefore, a number of samples may be analyzed in a short time period, and electrode surface fouling by the enzymatic reaction products was not observed.
A calibration curve for p-benzoquinone was constructed by using the maximum peak current as a function of the p-benzoquinone concentrations as shown in Figure 6. A linear response for p-benzoquinone was obtained from 5 to 100 µM (r2 ¼0.985). The nonlinearity at higher concentrations was due to decreased peroxidase activity from rapid phenoxy radical polymer-ization on the electrode surface. Quinone-type phenoxy radicals have been shown to reduce the catalytic activities of phenol oxidases.[34] There have been no previous reports on flow-injected biosensors for p-benzoquinone specifi-cally except our previously published paper.[28] Therefore, it is difficult to compare the analytical parameters with previous studies.
An amperometric biosensor based on horseradish peroxidase/carbon nano-tube/polypyrrole nanobiocomposite film was developed for various phenols, and we reported a narrower linear dynamic range for p-benzoquinone than in this study. Sadeghi et al.[35] used a Fe3O4/polyaniline/laccase/chitosan
biocomposite-modified carbon paste electrode for catechol, and reported a short linear range between 0.5 µM and 80 µM. Qiu et al.[36] reported an amperometric biosensor for 4-chlorophenol based on a horseradish peroxi-dase/gold thin film with two linear ranges (2.5–40 µM and 62.5–117.5 µM) at an applied potential of 0.55 V. A linear range between 1 and 30 µM was reported using MnO2 based flow injection for the determination of
p-aminophenol.[37] Yao and Kotegawa[38] used a glassy carbon disc electrode and immobilized laccase with glutaraldehyde. Flow-injection measurements provided a linear range of 2 nM to 2 µM for 2, 4, 6-thrichlorephenol. Ruan and Li[39] constructed a flow-injection biosensor based on chemically immobilized horseradish peroxidase and reported a linear range of 1–100 µM for phenol. Wang and Hasebe[40] immobilized tyrosinase covalently on amino-functionalized carbon felt by ultrasonic treatment with a flow- injection system. These authors reported a linear dynamic range from 0.1– 10 µM for catechol, 0.1–3 µM for p-cresol, 0.1–10 µM for p-chlorophenol, and 0.1–30 µM for phenol.
In another report, a tyrosinase including packed bed flow reactor was employed for phenol for a concentration range from 0.1–2 µM.[25] The detec-tion limit was 3.3 µM. Fe3O4 nanoparticles in a poly(glycidylmethacrylate)/
horseradish peroxidase based gold electrode were reported for phenol by Çevik et al.[41] They reported a detection limit of 500 µM even though they used metallic nanoparticles to accelerate the electron transfer rate. The
biosensor sensitivity was 3 nA/µM across the linear concentration range obtained in our work. The literature has shown that the sensitivity values were between 0.81 and 2.22 nA/µM for phenol even when redox mediators such as osmium and ruthenium were used to accelerate the electron transfer rate.[42–44] The mediated biosensors are expected to provide higher sensitivity based on the direct electron transferring mechanism.
The response of the biosensor rapidly reached a steady-state current in approximately 3 s at a flow rate of 1 mL/min, showing that a number of
p-benzoquinone determinations may be performed in 1 hr. The rapid response
indicated fast electron exchange between horseradish peroxidase and the elec-trode due to the gold nanoparticles embedded into the copolymer. Sadeghi et al.[35] reported a response time of 8 s at 40°C using a laccase-based biosensor. A long response time of 80 s was reported for a tyrosinase/Fe2þ/polypyrrole/ indium tin oxide electrode in another study.[45] The accuracy was 105% for p-benzoquinone calibration.[29] The relationship between the current response and the substrate concentration followed the Michaelis-Menten kinetic mech-anism. The apparent Michaelis-Menten constant (Kmapp) was estimated from
the electrochemical analog of the Lineweaver-Burk equation:[46]
1 I ¼ I1max þ Kmapp Imax � 1 S; ð1Þ
where I is the steady-state current of the substrate addition, S is the bulk con-centration of the substrate, and Imax is the maximum current measured under
saturated substrate condition. The slope and intercept in the plot of 1/I vs. 1/S provide values of Imax and Kmapp for the horseradish peroxidase immobilized
working electrode (graph not shown). Here, the Imax and Kmapp values were
calculated to be 4.95 × 10 6 A and 4 mM at the flow rate of 1 mL/min, respectively. The Lineweaver-Burk equation was employed to obtain
1
I ¼ 2:02 � 10
5þ 8 � 108� 1
S: ð2Þ
In previous papers, Km values of 5 mM for 2, 3-dimethoxyphenol,[47] 24 mM
for 3, 4-dihydroxyphenylacetic acid and guaiacol,[48] 9.1 mM for
o-cresol,[49] 3.59 mM for phenol,[50] and 55.4 mM for pyrogallol[51] were reported for free peroxidases. The Kmapp value obtained in this work was small
in comparison to these reports. The smaller Kmapp value showed that the
horseradish peroxidase immobilized on the gold nanoparticle embedded poly(propylene-co-imidazole) film-coated electrode had high catalytic activity and exhibited a higher affinity for p-benzoquinone.
Repeatability and stability
Continuous repeatability of the designed working electrode for the same measurements in one operation was investigated by six repetitive injections
of 1 mM p-benzoquinone at 1 mL/min. The relative standard deviation of these measurements was 0.82%. After measurements, electrode was rinsed with pH 7 phosphate buffer and stored at 4°C. Higher relative standard devia-tions were reported between 1.2 and 3.6% in similar biosensor studies.[37–39]
p-Benzoquinone calibrations were performed three times in one day. The
slopes of the calibration curves (biosensor sensitivity) did not change up to the end of third day but decreased during the fourth day.
The long-term stability was studied over four weeks by monitoring the gen-erated current from the injection of 1 mM p-benzoquinone with use every two to three days and storage in 100 mM pH 7 phosphate buffer. The electrode response was 95% of its initial value at the end of the fourth week. The results demonstrated that gold nanoparticle embedded poly(propylene-co-imidazole) film was efficient at retaining the activity of horseradish peroxidase. The improved stability is attributed to the immobilization by trapping the enzyme deeply on the surface of the copolymeric film under mild conditions. Conclusions
This article described the fabrication of a novel poly(propylene-co-imidazole)- based continuous flow device for online monitoring of p-benzoquinone. Gold nanoparticles were successfully embedded into the copolymer. Horseradish peroxidase was adsorbed on the long imidazole thrums of the copolymer. The influence of the flow conditions on the determination of p-benzoquinone was investigated. The flow cell was rapidly regenerated at a flow rate of 1 mL/ min with reduced electrode fouling by the enzymatic reaction products to determine p-benzoquinone in a short time. The fabricated device presented favorable analytical figures of merit for p-benzoquinone including the linear dynamic range, detection limit, response time, repeatability, and stability. The gold nanoparticles embedded into the copolymeric film promoted elec-tron transfer between horseradish peroxidase and the working electrode. Funding
This work was supported by the Bulent Ecevit University Research Fund under Grant [number BEU-2012-10-03-13], under Grant [number BEU-2013-77047330-01], and under Grant [number BEU-2014-72118496-01].
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