electrochemically reduced graphene oxide modified electrode

Accepted Manuscript Title: Determination of tetracycline in the presence of major interference in human urine samples using polymelamine/electrochemically reduced graphene oxide modified electrode Author: Srinivasan Kesavan Deivasigamani Ranjith Kumar Jae-Jin Shim PII: DOI: Reference:

S0925-4005(16)31718-X http://dx.doi.org/doi:10.1016/j.snb.2016.10.091 SNB 21149

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

7-7-2016 6-10-2016 19-10-2016

Please cite this article as: Srinivasan Kesavan, Deivasigamani Ranjith Kumar, JaeJin Shim, Determination of tetracycline in the presence of major interference in human urine samples using polymelamine/electrochemically reduced graphene oxide modified electrode, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.10.091 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of tetracycline in the presence of major interference in human urine samples using polymelamine/electrochemically reduced graphene oxide modified electrode

Srinivasan Kesavana, Deivasigamani Ranjith Kumarb, Jae-Jin Shima,b*

a

School of Chemical Engineering, Yeungnam University, Gyeongsan, Gyeongbuk 38541,

Republic of Korea b

Clean Energy Priority Center, Yeungnam University, Gyeongsan, Gyeongbuk 38541,

Republic of Korea

*Corresponding author: Tel: +82 53 810 2587; Fax: +82 53 810 4631; E-mail: [email protected]

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Graphical Abstract

UA

TET

0.5

Potential/V vs (SCE)

Highlights 

Polymelamine film was fabricated on ERGO/GC electrode by potentiodynamic method.

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Simultaneous determination of TET and UA was performed for the first time.

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Selective determination of TET in the presence of 50-fold excess UA was achieved.

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The p-Mel@ERGO/GC electrode was successfully tested using human urine samples.

Abstract A polymelamine (p-Mel) film on electrochemically reduced graphene oxide (ERGO) on a glassy carbon (GC) electrode (p-Mel@ERGO/GC) was designed using a potentiodynamic method for the simultaneous and selective determination of tetracycline (TET) in the presence of a major interference, uric acid (UA). The modified surfaces were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, Raman

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spectroscopy, and cyclic voltammetry. The surface coverage of the modified electrode was found to be 3.98 × 10−11 mol cm−2. The modified p-Mel@ERGO/GC electrode not only enhanced the oxidation currents of the TET and UA, but also shifted their oxidation potential toward a less positive direction compared to the bare GC, GO/GC, ERGO/GC, and p-Mel/GC electrodes. The modified electrode was used effectively for the selective determination of TET in the presence of a 50-fold excess of UA. Differential pulse voltammetry revealed a detection limit of 5 µM TET. The present modified electrode can be applied to the simultaneous determination of TET and UA in human urine samples. To the best of the authors’ knowledge, this is the first report of the simultaneous determination of TET and UA. Keywords: Electrochemically reduced graphene oxide; polymelamine; tetracycline; uric acid; differential pulse voltammetry; human urine samples

1. Introduction Tetracycline (TET) (Fig. S1) is an important antibiotic that is generally used to treat bacterial diseases, such as urinary tract infections, chlamydia, and acne [1]. TET and its derivatives show activity against a wide range of gram-positive and gram-negative aerobic as well as anaerobic bacteria, such as Spirochete, Actinomyces, Rickettsia, and Mycoplasma [2,3]. TET binds to the ribosome and blocks protein building [3]; it can also act as an antiprotozoal, anticancer, and antimalarial agent [3]. TET and its derivatives are also used extensively as animal feed additives for cattle, sheep, poultry, pigs, and fish as well as veterinary medicines due mainly to their broad antibacterial activity, low toxicity, and low cost [3]. The widespread use of TET in veterinary medicines has led to the generation of its residues in food products, including milk, meat, and honey [2]. The risks of TET on human health include the expression of antibiotic-resistant genes, liver damage, allergic reactions, 2

inhibition of intestinal microbes, vision problems, and tooth discoloration [2-4]. TET is also a hepatotoxic agent, particularly for pregnant women [5]. Therefore, it is clinically important to develop reliable analytical methods to determine the TET concentration. Uric acid (UA) (Fig. S1) is the main end product of the purine nucleotide catabolism in the human body. Studies have suggested that UA can have either beneficial or deleterious effects depending on its concentration. The increased consumption of serum UA has been linked to the prevention of Parkinson's disease; UA above normal concentrations acts as a radical scavenger [6]. Serum UA is a deciding factor for oxidative stress [7]; however, elevated levels of serum UA have been related to a risk of cardio-vascular diseases [8]. Low concentrations of UA is associated with multiple sclerosis [9]. TET generally penetrates moderately well into the body fluids and tissues and is excreted in the urine [3]. UA is a major interference in human urine samples. Therefore, a sufficiently accurate determination of TET and UA in human urine samples is very important from a clinical perspective. The electrochemical method has many advantages over traditional methods, including convenience of measurement, reproducibility, high sensitivity, selectivity, low cost, and less time consumption. The electrochemical determination of TET has been reported [10-19] but there is no report on the simultaneous determination of TET and UA. Chatten et al. demonstrated TET determination using Au and Pt electrodes oxidized at a higher oxidation potential of +1.6 V [20]. Quan et al. used gold-modified microelectrode for the determination of TET at +1.5 V [21]. Masawat and Slater showed TET determination at +1.2 V using screen-printed gold electrode [22]. In the above three cases, higher oxidation potentials can lead to the serious problem of Au and Pt electrode poisoning in their oxidized form [20]. Hence, it is better to find an electrode probe for TET determination other than Au

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and Pt. In this regard carbon-based electrode materials might be apt for TET determination. Wang et al. demonstrated the suitability of molecularly-imprinted polymer-modified carbon nanotube-gold nanoparticle electrode for TET determination, and they observed that the polymer has significantly improved the electrode’s sensor ability [23]. The group reported the TET oxidation potential at +1.1 V [23]. Kushikawa et al. developed Pt nanoparticles and carbon powder-modified GC electrode for the determination of TET at +0.95 V [11]. Recently, Calixto et al. demonstrated TET determination in human bovine samples using graphite-polyurethane composite electrode at +0.94 V [15]. Guo et al. developed multiwalled carbon nanotubes and graphene composite based electrocatalytic aptasensor for TET [24]. The above discussion on electrode probes for TET determination signifies that carbon and polymer composite based electrode probes can be reliable for TET determination. These reports, however, focused on the determination of TET only. The present work gives emphasis on the simultaneous determination of TET and UA. In addition, to improve the stability of the electrode and reduce the oxidation potential of TET, we have used graphene and polymelamine composite electrode. Because, graphene has attracted considerable interest over the last decade owing to its excellent properties, such as good electronic conductivity, high mechanical strength, large specific surface area, high stability, high mechanical strength, tunable band gap, and astonishing electron mobility at room temperature [25-32]. The material has potential applications in various fields, such as biosensors [33-35], electrocatalysis [36, 37], nanoelectronics [38], batteries [39], solar cells [40], fuel cells [41], and supercapacitors [42, 43]. The electrochemical reduction of graphene oxide has received much interest in recent years, because it is considered a fast and green approach [33, 34]. Chemical reduction of GO typically uses hydrazine hydrate, requires a high temperature treatment, and requires multiple washing steps, and it is a time consuming process [44, 45]. In contrast electrochemical reduction of graphene oxide can offer thin layer formation [33,

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34]. Also, ERGO has some unreduced hydroxyl groups which might help in the electrostatic interaction between the ERGO and the monomer, which is useful for electropolymerization. Conducting polymers have attracted considerable interest over the past decade owing to their unique electrical conductivity [46]. Melamine (1,3,5-triazine-2,4,6-triamine) is an organic base and a trimer of cyanamide with a triazine ring. The three amine groups with a triazine ring make the molecule useful for polymerization [47-49], and the nitrogen rich matrix with many π-electrons can interact with the target molecules [33, 34]. Furthermore, the agglomeration of ERGO layers can be stabilized by polymelamine, and the oxygenated functional moieties of ERGO can provide an increased number of active sites for the electropolymerization of polymelamine [50]. Therefore, ERGO and polymelamine can have a synergistic effect that results in enhanced electrochemical activity. Thus, the aim of this work was to determine TET in the presence of UA using a p-Mel@ERGO/GC film modified electrode.

2. Experimental 2.1. Chemicals TET, UA and xanthine were purchased from Sigma-Aldrich and used as received. Graphite, disodium hydrogen phosphate (Na2HPO4), and monosodium hydrogen phosphate (NaH2PO4) were obtained from Alfa Aesar and used as received. 0.2 M phosphate buffer (PB) solution (pH 7.2) was prepared using Na2HPO4 and NaH2PO4. Deionized water (DI H2O) was used for all solution preparation. Type 1 pieces of a 1 cm2 glassy carbon plate (1 mm thick) from Alfa Aesar were used for field emission scanning electron microscopy (FE-SEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) analyses. 2.2. Instrumentation The morphology of the samples were analyzed by FESEM (Hitachi S4800). The Raman spectra were collected using an XploRA PLUS Raman microscope (Horiba) at an 5

excitation wavelength of 532 nm. XPS was carried out on a K-Alfa system (Thermo Scientific). The spectra were calibrated with the C1s peak (284.6 eV) of a pure carbon sample and analyzed using the XPSPEAK41 software. After subtracting the Shirley background, all spectra were fitted with a convolution of Gaussian functions. All electrochemical experiments were carried out on an Autolab PGSTAT302N electrochemical workstation (MetrohmAutolab BV, Netherlands). A conventional three-electrode electrochemical cell was used for all electrochemical measurements. A mirror polished, 3-mm glassy carbon electrode (GCE), Pt wire, and a saturated calomel electrode (SCE) were used as the working electrode, counter electrode, and reference electrode, respectively. All electrochemical measurements were carried out under a nitrogen atmosphere at room temperature. 2.3. Fabrication of p-Mel@ERGO/GCE Graphene oxide (GO) was synthesized using a slight modification of the Hummers’ method [51]. Scheme 1 presents a schematic diagram of the fabrication of polymelamine on the ERGO modified GC electrode. Prior to modification, the GC electrode was polished with emery paper and then with 0.5 and 0.05 μm alumina slurries, followed by rinsing thoroughly with water. GO was mixed with ethanol (1 mg mL-1) containing 5 μL of a 5% Nafion® solution, and the mixture was sonicated to obtain a homogeneous suspension and 10 μL drop casted onto the GCE surface. This electrode is termed GO/GC. The surface-attached GO was reduced electrochemically by potential cycling between 0 and −1.5 V at a scan rate of 50 mV s-1 for 15 cycles. This electrode is termed the ERGO/GC electrode. The electropolymerization of melamine on the ERGO/GC electrode was carried out by 15 successive potential sweeps between 0 and +1.6 V at a scan rate of 50 mV s−1 using 1 mM melamine in a 0.1 M HCl solution [52]. This electrode was termed the p-Mel@ERGO/GC electrode. For SEM, Raman and XPS measurements, GO/GC, ERGO/GC, p-Mel/GC and pMel@ERGO/GC plates prepared by the same procedure were used.

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2.4. Real sample analysis Human urine samples were obtained from laboratory co-workers and the samples were stored in the refrigerator at 4 °C. About 25 mL of urine samples were centrifuged for 15 min at 3,000 rpm. One milliliter of the supernatant was collected and diluted 200 times with 0.2 M PB solution (pH 3). The resulting solution was transferred attentively into an electrochemical cell and analyzed without any further pretreatment.

3. Results and discussion 3.1. Electrochemical reduction of GO and fabrication of p-Mel@ERGO film on the GCE surface The surface-attached GO on the GO/GC electrode was cycled electrochemically between 0 and −1.5 V for 15 cycles to reduce the oxygen functional groups present on the GO surface. In the first cycle (Fig. 1A), a reduction peak of -1.3 V appeared, which confirmed the reduction of oxygen functional groups present on the GO surface [53]. On the second cycle, the reduction peak decreased and completely vanished after the third cycle. This can be explained by the complete reduction of oxygen functional groups present on the GO surface. The aromatic backbone of graphene must be regenerated on the GCE surface due to the removal of oxygen functional groups between the GO layers by electrochemical reduction. To form a p-Mel film on the ERGO modified electrode, a potentiodynamic method was used with 0.1 M HCl as the supporting electrolyte. Fig. 1B shows the potentiodynamic cycles (15 cycles) of 1mM melamine in 0.1 M HCl at a potential window of 0 to +1.60 V [52]. During the first cycle, an oxidation peak at +0.69 V along with a sharp peak at +1.5 V and reduction peak at +0.44 V appeared. In the subsequent cycle, the oxidation peak current at +0.69 V increased. From the fourth cycle onwards, both the oxidation current at +0.69 V and the reduction wave current at approximately +0.44 V decreased. In the 15th cycle, welldefined anodic and cathodic peaks were observed at approximately +0.65 V and +0.52 V,

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respectively. This confirmed that the polymelamine adsorbed onto the ERGO/GC surface enhanced the electron transport on the p-Mel@ERGO/GC film modified electrode surface. The oxidation peak at +0.69 V was assigned to the oxidation of amine to form a radical cation; two cation radicals were then coupled to form a dimer via a hydrazo bond (Scheme 2) [54]. In addition, the oxidation of two -NH- groups leads to the formation of dimer species on the electrode surface and the two -NH- groups were oxidized to form a diradical cation (Scheme 2) [54]. The cationic radicals produced from melamine during oxidation couple rapidly to give dimers via an azo bond. The oxidation wave at +1.5 V was assigned to the activation of carbon present on the ERGO surface [55]. Further, the formed radical cations are unstable; they coupled rapidly to form a dimer at the expense of two protons. The reaction was propagated further to form a p-Mel film on the ERGO/GC surface. 3.2. Electrochemical behavior of the p-Mel@ERGO/GC film modified electrode Fig. 2 displays the CVs obtained for the p-Mel@ERGO/GC film modified electrode in 0.1 M H2SO4. The oxidation and reduction peaks were observed at +0.65 and +0.54 V, respectively. The redox reaction in Fig. 2A was assigned due to a proton and electron addition/elimination reaction at the -NH- sites in the p-Mel film [54]. The surface coverage of the p-Mel film on the ERGO modified electrode was calculated from the surface coverage concentration (τ). τ can be calculated from the charge (Q), which indicates the film formation process according to the following equation.

where Q is the charge under the anodic curve, n is the number of electrons involved in the film formation process, A is the geometric area of the GC (0.0707 cm2), and F is the faraday constant (96485 C mol-1). The surface coverage concentration of p-Mel@ERGO/GC was 3.98×10-11 mol cm-2. The electrochemical behavior of the p-Mel@ERGO/GC film modified

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electrode at different scan rates was studied in 0.1 M H2SO4. When the scan rate was increased from 25 to 250 mV s−1, the oxidation and reduction peak currents were also increased while the potentials were not shifted (Fig. 2B), indicating the reversible redox reaction of the p-Mel film modified electrode. The oxidation current of the pMel@ERGO/GC film modified electrode increased linearly with increasing scan rate with a correlation coefficient of 0.999 (Inset in Fig. 2B), suggesting that the observed redox reaction was due to a surface confined process. 3.3. Morphological characterization by SEM In order to study the surface morphology of the electrode, GO was drop casted onto the GC plates. Electrochemical reduction of the GO was done followed by the electropolymerization of melamine on the ERGO-modified GC plates. The same procedure was used for the Raman and XPS studies. The morphology of the fabricated GO, ERGO, and p-Mel@ERGO on the GC surface was examined by SEM. Fig. 3 displays SEM images of the GO/GC, ERGO/GC, p-Mel/GC and p-Mel@ERGO/GC plates. The GO/GC showed a layered structure (Fig. 3A). On the other hand, the ERGO/GC plate showed reduced GO with a sheetlike structure (Fig. 3B) [33,53]. During electrochemical reduction, the aggregation of GO occurs due to the increased π-π interactions between the layers of graphene. p-Mel/GC exhibits crystalline spherical like clusters, which are inter-linked with one another (Fig. 3C and D) [48]. Furthermore, the p-Mel@ERGO/GC plate shows the disordered aggregated structure of polymelamine on the ERGO surface (Fig. 3E and F). This may be due to the unreduced oxygen functional groups interacting with the amine groups of the melamine and the defects present on the ERGO surface. The SEM images confirm the formation of a p-Mel film on the ERGO electrode surface. 3.4. XPS studies of p-Mel@ERGO/GC film modified electrode

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The fabrication of GO, ERGO, and p-Mel@ERGO film on the GC electrodes were also investigated by XPS. Fig. 4A presents the XPS survey spectrum of GO/GC, ERGO/GC, and p-Mel@ERGO/GC plates. All three modified electrode substrates showed peaks at 285 and 531 eV. The peak at 285 eV corresponds to the C 1s peak of sp2 carbon. The peak at 531 eV was assigned to the O 1s spectrum of different oxygen functional groups. The C/O peak intensity for GO/GC and ERGO/GC were 1.4 and 2.1, respectively. This confirms the successful attachment of GO on the GC electrode surface and its electrochemical reduction of oxygen functionality in GO. The appearance of a sharp peak at 400 eV for the pMel@ERGO/GC plate (Fig. 4A (c)) confirmed the increased nitrogen functional groups due to melamine film formation [33, 56]. Furthermore, electrochemical reduction was also confirmed by fitting the C 1s spectra of GO and ERGO modified plates using the Gaussian functions after a background correction. Fig. 4B and C shows the C 1s spectrum of the GO/GC and ERGO/GC plates. The C 1s spectrum of the GO modified electrode showed peaks at 284.6 eV (-C=C-), 286.5 eV (C-OH), 287.6 eV (C=O), and 289.1 eV (O=C-OH) [33, 34, 53, 56]. On the other hand, after electrochemical reduction of the GO modified plate, the intensities of the oxygen functional groups peaks were decreased significantly (Fig. 4C). This confirms that a large number of oxygen-containing groups were removed, indicating the regeneration of an aromatic lattice after the electrochemical reduction of GO. Table S1 lists the C/O ratio and the peak assignments. The p-Mel@ERGO/GC film modified electrode was next examined. Fig. S2 shows the XPS results obtained for p-Mel@ERGO/GC plate. Table S2 lists the peak-fitting results and their assignments. Melamine film formation was confirmed by fitting the N 1s spectra of the p-Mel@ERGO/GC plate (Fig. S2A) using the Gaussian functions after a background correction. The N 1s spectrum of the pMel@ERGO/GC plate shows four peaks at 398.2, 399.4, 400.2, and 402.2 eV. The peak at 398.2 was attributed to sp2 nitrogen atoms (-N=) linked to two carbon atoms in the triazine

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ring of melamine. The other two peaks at 399.4 and 402.2 eV were assigned to the free amine (-NH2) and positively charged nitrogen (-N+H), respectively [47, 54]. The peak at 400.2 eV confirmed the presence of azo bridges (-N=N-) on the p-Mel@ERGO/GC film modified substrate [54, 56]. This existence of azo bridges supports the p-Mel film formation mechanism (Scheme 2). In addition, the N 1s components confirmed that film formation takes place via the amine groups present on melamine. The C 1s core level spectrum displayed six peaks at 284.6, 286, 286.5, 287.3, 287.6, and 289.1 eV (Fig. S2B). The C 1s component at 287.3 eV was attributed to the -C=N- bond of the triazine ring. The 286.0 eV peak corresponds to the carbon attached to the nitrogen group, which confirms the presence of an amine group [56]. The other peaks were assigned to the aromatic ring and oxygen functional groups. XPS confirmed the successful fabrication of GO on the GC electrode surface, and its electrochemical reduction to form an ERGO and p-Mel formation on the ERGO electrode surface. 3.5. Characterization by Raman spectroscopy Raman spectroscopy is an important tool for characterizing carbon materials. Fig. 5 displays the Raman spectra of the GO/GC and ERGO/GC plate. In general, two main bands exist in the spectra of graphene-based materials, i.e. the G and D bands. The G and D bands of carbon materials were assigned to the E2g phonon mode of sp2 carbon atoms and the breathing mode of the k-point phonons with A1g symmetry. GO/GC showed two peaks at 1351 cm-1 and 1595 cm-1, corresponding to the D and G peaks of GO (curve a). After electrochemical reduction, the ERGO/GC plate showed the D and G peaks at 1344 and 1584 cm-1, respectively (curve b). This confirmed the successful attachment of GO and its electrochemical reduction. In addition, the ERGO/GC plate showed an increase in the intensity of the D band compared to that of the G band, and a shift in the G band confirmed the increasing defects in the carbon basal plane and the regeneration of the aromatic

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backbone of graphene [25, 33, 56]. The ID/IG ratio of the carbon products provides useful information, such as the extent of chemical modification [27, 33, 56]. The calculated ID/IG values for the GO/GC and ERGO/GC plates were 0.79 and 1.26, respectively. The increase in the ID/IG ratio after electrochemical reduction suggests the retained sp2 aromatic backbone of graphene. Table S1 summarizes the peak assignments and the ID/IG values. Using the ID/IG values, the average crystalline size of the carbon material was calculated using the following equation [57].

where, La is the average crystalline size and λ is the laser wavelength in nm. The calculated average crystalline sizes of the GO/GC and ERGO/GC plates were 24.33 and 15.26 nm, respectively. The decrease in the average crystalline size of the ERGO/GC plate can be explained by the formation of numerous new graphitic domains that are smaller than the ones present in the GO/GC plate. 3.6. Electrochemical activity of the p-Mel@ERGO/GC film modified electrode towards TET and UA The aim of this work was to determine TET in the presence of its major interference, UA, using a p-Mel@ERGO/GC film-modified electrode. Fig. 6A and Fig. S3A show the CVs obtained for 0.5 mM TET at the bare GC, GO/GC, ERGO/GC, p-Mel/GC, and pMel@ERGO/GC film modified electrodes in a 0.2 M PB solution (pH 3). The bare GCE showed an oxidation peak for TET at +0.97 V (curve a: solid line). In the subsequent cycles, oxidation peak of TET was shifted to a more positive potential with a decrease in the oxidation peak current (curve a: dotted line). In addition, the GO/GC, ERGO/GC, and pMel/GC electrodes showed oxidation peaks at +0.94, +0.94, and +0.97 V, respectively, for TET (curves e, f, and b: solid line). After 5 cycles, the oxidation peak current of TET 12

decreased at these electrodes (curves e, f, and b: dotted line). These results suggest that the bare GC, GO/GC, ERGO/GC, p-Mel/GC electrodes are unsuitable for the determination of TET. The decrease in oxidation current may be explained by surface fouling caused by the oxidation products of TET. On the other hand, the p-Mel@ERGO/GC film modified electrode showed an oxidation peak for TET at +0.92 V (curve c: solid line), which was a 50, 20, 20, and 50 mV less positive potential and a 1.6-, 1.6-, 1.6-, and 5-fold higher current than the bare GC, GO/GC, ERGO/GC, and p-Mel/GC electrodes, respectively. The pMel@ERGO/GC film modified electrode shows a stable oxidation peak even after 5 cycles (curve c: dotted line). The p-Mel@ERGO/GC film modified electrode efficiently prevented the surface fouling effect caused by the oxidation product of TET. The enhance oxidation current and less positive potential shift can be explained by the synergic effects of the p-Mel film and ERGO modified electrode. Moreover, the π- π interaction between TET and the pMel@ERGO film and facile electron transfer at the ERGO film and TET can also cause an enhanced less positive potential shift of TET [33, 34]. The redox peak with a lower peak current was observed at the potential region of +0.43 to +0.26 V in the absence of TET in the 0.2 M PB solution at the p-Mel@ERGO film modified electrode (curve d). The observed redox behavior was attributed to a proton and electron elimination/addition reaction at the –NH– sites presents on the p-Mel film. One proton and one electron was involved in the redox reaction of TET (Scheme 3). Therefore, p-Mel@ERGO film modified electrode was highly suitable for the stable determination of TET. In previous studies, carbon based electrode materials such as MWCNTs and graphene modified electrode were used for TET determination. Vega et al. used MWCNT/GC modified electrode and obtained good linear range but the TET operating oxidation potential was at +1.2 V [18]. Calixto et al. utilized graphite-polyurethane composite electrode which resulted in a lower operating potential of +0.96 V as compared to MWCNT [15]. Wang et al. demonstrated the use of molecularly

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imprinted polymer modified MWCNT-gold nanoparticles electrode for TET determination at +1.1 V [23]. In all these attempts, the MWCNTs and graphene modified electrode were utilized to reduce the TET oxidation peak potential and improve the electronic conductivity of the composites. In contrast, p-Mel@ERGO/GC modified electrode showed an operating potential at +0.92 V for TET, and it was also used for the simultaneous determination of TET and UA. Fig. 6B and Fig. S3B shows the CV obtained for 0.5 mM UA at bare GC, GO/GC, ERGO/GC, p-Mel/GC, and p-Mel@ERGO/GC film modified electrodes in a 0.2 M PB solution (pH 3). The bare GC, GO/GC, ERGO/GC, and p-Mel/GC electrodes show oxidation peaks for UA at +0.41, +0.41, +0.39, and +0.41 V, respectively (curves a, e, f, and b: solid line). The oxidation current obtained for UA decreased after 5 cycles (curves a, e, f, and b: dotted line). On the other hand, the p-Mel@ERGO/GC film modified electrode showed an oxidation peak for UA at +0.38 V (curve c: solid line), which is a 30, 30,10, and 30 mV lower positive potential and a 1.6-, 1.2-, 1.3-, and 1.28-fold higher current than the bare GC, GO/GC, ERGO/GC, and p-Mel/GC electrodes, respectively. The oxidation peak UA was stable, even after 5 cycles (curve c: dotted line). The absence of UA in the pMel@ERGO/GC film modified electrode showed the redox reaction of the –NH– sites present on the p-Mel film (curve d). These results suggest that the p-Mel@ERGO/GC film modified electrode not only increased the oxidation currents of TET and UA, but also shifted their oxidation potentials toward less positive values. Therefore, the p-Mel@ERGO/GC film modified electrode is suitable for the stable determination of TET and UA. The effects of the scan rate on the oxidation of TET and UA at the p-Mel@ERGO/GC film modified electrode were studied. The oxidation peak currents of TET and UA increased with increasing scan rates from 50 mV s−1 to 250 mV s−1 (Fig. S4). Good linearity was observed while plotting the anodic peak current as a function of the square root of the scan rate with a correlation coefficient of 0.999 for TET (inset, Fig. S4A) and 0.999 for UA (inset,

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Fig. S4B), indicating that the oxidation of TET and UA at p-Mel@ERGO/GC film modified electrode were diffusion controlled processes. 3.7. Electrochemical behavior of TET and UA in a mixture at the p-Mel@ERGO/GC film modified electrode Fig. S5A and S5B shows the CVs obtained for a mixture of 0.5 mM each TET and UA at the bare GC, GO/GC, ERGO/GC, p-Mel/GC, and p-Mel@ERGO/GC film modified electrodes in 0.2 M PB solution (pH 3). The bare GC electrode showed an oxidation peak at +0.45 V for UA and +0.9 V for TET in the first cycle (curve a: solid line). After five cycles, the oxidation peak currents of TET and UA decreased (curve a: dotted line). Therefore, the bare GC electrode is unsuitable for the stable determination of TET and UA in a mixture. In the first cycle, the GO/GC, ERGO/GC, and p-Mel/GC electrodes showed oxidation peaks at +0.41, +0.41, and +0.42 V, corresponding to the oxidation of UA, and +0.85, +0.85 and +0.88 V corresponding to the oxidation of TET, respectively (curves e, f, and b: solid line). After 5 cycles, the oxidation peak potentials were shifted with a significant decrease in the peak current (curves e, f, and b: dotted line). On the other hand, the p-Mel@ERGO/GC film modified electrode showed oxidation peaks at +0.40 V and +0.81 V for UA and TET, respectively, with enhanced oxidation currents and a peak separation of 410 mV between them (curve c: solid line). The p-Mel@ERGO/GC film modified electrode showed higher oxidation peak currents for UA and TET, compared to the bare GC, GO/GC, ERGO/GC, and p-Mel/GC electrodes. Furthermore, compared to the bare GC, GO/GC, ERGO/GC and pMel/GC modified electrodes, the p-Mel@ERGO/GC film modified electrode shifted the oxidation potential toward a less positive potential by 50, 10, 10, and 20 mV for UA and 90, 40, 40, and 70 mV for TET, respectively. The oxidation peaks of TET and UA were stable even after 5 cycles (curve c: dotted line), showing that the simultaneous determination of TET and UA was possible at the p-Mel@ERGO/GC film-modified electrode. The absence of 15

TET and UA in the p-Mel@ERGO/GC film modified electrode showed a redox reaction of –NH– sites present on the p-Mel film (curve d). 3.8. Simultaneous and selective determination of TET and UA at p-Mel@ERGO/GC film modified electrode Because the p-Mel@ERGO/GC film modified electrode effectively separated the oxidation signals of TET and UA, it was used for the simultaneous determination of them. Fig. 7 shows the DPVs obtained for the simultaneous determination of TET and UA in a 0.2 M PB solution (pH 3) at the p-Mel@ERGO/GC film modified electrode. Well-defined voltammetry signals were observed for 10 µM UA and 10 µM TET at +0.42 and +0.85 V, respectively (curve a). When the concentration of UA was increased from 10 µM to 80 µM (curves a-h) and TET was increased from 10 µM to 80 µM (curves a-h), the peak current of the respective analytes increased linearly with a correlation coefficient of 0.995 for UA and 0.994 for TET (Inset, Fig. 7). The oxidation potentials of TET and UA were unaffected while increasing their concentrations. The above results suggest that the simultaneous determination of TET and UA is possible at the p-Mel@ERGO/GC film modified electrode. One of the objectives of the present work was to selectively determine TET in the presence of a high concentration of UA. Generally, a high concentration of UA is present in human urine [7]. Therefore, it is essential to determine TET in the presence of high concentrations of UA. Fig. 8 shows the DPVs obtained for the increment of 10 µM TET to a solution of 500 µM UA. A well-defined oxidation peak was observed for TET even in the presence of a 50-fold excess of UA. Linear current enhancement was observed while increasing the TET concentration in the range of 10-70 µM with a correlation coefficient of 0.996 without affecting the UA peak current (Inset, Fig. 8). These results suggest that that the p-Mel@ERGO/GC film modified electrode was selective towards the oxidation of TET, even in the presence of high concentrations of UA. 16

3.9. Sensitive determination of TET Fig. S6 shows the sensitive determination of TET in a 0.2 M PB solution (pH 3) at the p-Mel@ERGO/GC film modified electrode. A clear voltammetry signal was observed at 0.88 V for the addition of 5 µM TET. For each addition of TET, the current increased linearly in the range of 5.0 × 10−6 to 2.25× 10−4 M with a correlation coefficient of 0.994 (Inset of Fig. S6) and the detection limit was found to be 2.2 × 10−6 M (S/N = 3). It is important to compare the TET oxidation potential, detection limit and wide linear range concentration of TET obtained in the present study with the reported modified electrodes (Table 1). It can be seen from Table 1 that the p-Mel@ERGO/GC film modified electrode showed better oxidation peak potential and comparable detection limit when compared to previous studies, and it exhibited a wide concentration range. The anti-interference ability of the p-Mel@ERGO/GC film modified electrode was tested towards the detection of TET and UA in the presence of common interferents, such as Na+, Ca2+, Mg2+, K+, NH4+, Cl-, F-, CO32-, SO42-, glucose, urea, and oxalate by amperometry. No change in the current response was observed for 30 nM UA and 500 nM TET in the presence of 30 µM of the interferents, showing that the present modified electrode is highly selective towards TET and UA. To study the stability of the p-Mel@ERGO/GC film modified electrode, the DPVs for 0.5 mM TET in a 0.2 M PB solution (pH 3) were recorded at 5 min intervals. The anodic peak current remained the same with a relative standard deviation of 1.2% for 15 repetitive measurements, indicating that this electrode has good reproducibility and does not experience surface fouling during the voltammetry measurements. The current response decreased by approximately 1.9% in 1 week and 2.8% in 2 weeks when the electrode was kept in PB (pH 3). 3.10. Real sample analysis

17

To demonstrate the practical applications of the p-Mel@ERGO/GC film modified electrode, the concentrations of TET and UA were determined simultaneously in human urine samples. The human urine samples were collected from laboratory co-workers. The prepared human urine sample was placed into the electrochemical cell without any chemical treatment. Aliquot amounts of 0.01 M TET and UA stock solutions were added to the urine samples according to the amounts shown in Table S3. The standard addition technique was used to determine TET and UA in human urine samples. The DPV of the urine sample in the PB solution on the p-Mel@ERGO/GC film modified electrode showed two oxidation peaks at +0.43 V and +0.77 V, which correspond to the oxidation of UA and xanthine (XN), respectively (curve a in Fig. S7). In order to demonstrate that these peaks are due to UA and XN, we have added commercially available UA and XN to the above solution. After adding 10 µM UA and 10 µM XN, the oxidation peaks at +0.43 and +0.77 V increased (curve b in Fig. S7), which confirms the presence of UA and XN in urine sample. For the simultaneous determination of TET and UA in human urine, commercial samples of TET and UA were spiked in the urine sample. After adding 10 µM UA and 20 µM TET, the oxidation peaks were obtained at +0.43 and +0.87 V, which can be attributed to the oxidation of UA and TET, respectively (curve b in Fig. S8). The p-Mel@ERGO/GC film modified electrode showed good recovery for the determination of TET and UA in human urine samples (Table S3). The proposed method showed good recovery for the spiked TET and UA in human urine samples. These results suggest that the present modified electrode can be used effectively for the simultaneous determination of TET and UA in real samples.

4. Conclusions A simple approach was established for the fabrication of polymelamine onto an electrochemically reduced graphene oxide surface using a potentiodynamic method. The p-

18

Mel@ERGO/GC film modified electrode showed better electrochemical activity towards TET and UA. The modified electrode was used successfully for the simultaneous determination of TET and UA. Furthermore, the modified electrode was utilized successfully for the selective determination of TET in the presence of a 50-fold excess of UA. Using differential pulse voltammetry, the current response of TET increased linearly with increasing concentration from 5.0 × 10−6 to 2.25 × 10−4 M. The detection limit was found to be 2.2 × 10-6 M (S/N = 3). The practical applications of the present modified electrode was demonstrated by measuring the concentrations of TET and UA in human urine samples.

Acknowledgement This work was supported by the 2015 Yeungnam University Research Grant.

References [1]

J.W. Fritz, Y. Zuo, Simultaneous determination of tetracycline, oxytetracycline, and 4-epitetracycline in milk by high-performance liquid chromatography, Food Chem. 105 (2007) 1297-1301.

[2]

B.G. Katzung, A.J. Trevor, S.B. Masters, Basic and Clinical Pharmacology, New York: McGraw-Hill, 2006.

[3]

I. Chopra, M. Roberts, Tetracycline antibiotics: modeofaction, applications, molecular biology, and epidemiology of bacterial resistance, Microbiol. Mol. Biol. Rev. 65 (2001) 232-260.

[4]

M. Jeon, J. Kim, K.J. Paeng, S.W. Park, I.R. Paeng, Biotin-avidin mediated competitive enzyme-linked immunosorbent assay to detect residues of tetracyclines in milk, Microchem. J. 88 (2007) 26-31. 19

[5]

M.C. Gwee, Can tetracycline-induced fatty liver in pregnancy be attributed to choline deficiency? Med Hypotheses. 9 (1982)157-162.

[6]

E. Andreadou, C. Nikolaou, F. Gournaras, M. Rentzos, F. Boufidou, A. Tsoutsou, C. Zournas, V. Zissimopoulos, D. Vassilopoulos, Serum uric acid levels in patients with Parkinson's disease: their relationship to treatment and disease duration, Clin. Neurol. Neurosurg. 111 (2009) 724-728.

[7]

P. Strazzullo, J.G. Puig, Uric acid and oxidative stress: relative impact on cardiovascular risk? Nutr. Metab. Cardiovasc. Dis. 17 (2007) 409-414.

[8]

J. Vekic, Z.J. Ivanovic, V.S. Kalimanovska, L. Memon, A. Zeljkovic, N.B. Stanojevic, S. Spasic, High serum uric acid and low-grade inflammation are associated with smaller LDL and HDL particles, Atherosclerosis 203 (2009) 236-242.

[9]

M. Rentzos, C. Nikolaou, M. Anagnostouli, A. Rombos, K. Tsakanikas, M. Economou, A. Dimitrakopoulos, M. Karouli, D. Vassilopoulos, Serum uric acid and multiple sclerosis, Clin. Neurol. Neurosurg. 108 (2006) 527-531.

[10]

Y. Xu, M. Gao, G. Zhang, X. Wang, J. Li, S. Wang, Electrochemically reduced graphene oxide with enhanced electrocatalytic activity toward tetracycline detection, Chin. J. Catal. 36 (2015) 1936-1942.

[11]

R.T. Kushikawa, M.R. Silva, A.C.D. Angelo, M.F.S. Teixeira, Construction of an electrochemical sensing platform based on platinum nanoparticles supported on carbon for tetracycline determination, Sens. Actuators, B 228 (2016) 207-213.

[12]

A. Wong, M. Scontri, E.M. Materon, M.R.V. Lanza, M.D.P.T. Sotomayor, Development and application of an electrochemical sensor modified with multiwalled carbon nanotubes and graphene oxide for the sensitive and selective detection of tetracycline, J. Electroanal. Chem. 757 (2015) 250-257.

20

[13]

X. Liu, S. Zheng, Y. Hu, Z. Li, F. Luo, Z. He, Electrochemical immunosensor based on the chitosan-magnetic nanoparticles for detection of tetracycline, Food Anal. Methods (2016) 1-7.

[14]

X. Zhan, G. Hu, T. Wagberg, S. Zhan, H. Xu, P. Zhou, Electrochemical aptasensor for tetracycline using a screen-printed carbon electrode modified with an alginate film containing reduced graphene oxide and magnetite (Fe3O4) nanoparticles, Microchim. Acta 183 (2016) 723-729.

[15]

C.M.F. Calixto, É.T.G. Cavalheiro, Determination of tetracyclines in bovine and human urine using a graphite-polyurethane composite electrode, Anal. Lett. 48 (2015) 1454-1464.

[16]

A.A. Yakout, D.A. El-Hady, A combination of β-cyclodextrin functionalized magnetic graphene oxide nanoparticles with β-cyclodextrin-based sensor for highly sensitive and selective voltammetric determination of tetracycline and doxycycline in milk samples, RSC Adv. 6 (2016) 41675-41686.

[17]

S. Jahanbani, A. Benvidi, Comparison of two fabricated aptasensors based on modified carbon paste/oleic acid and magnetic bar carbon paste/Fe3O4@oleic acid nanoparticle electrodes for tetracycline detection, Biosens. Bioelectron. 85 (2016) 553-562.

[18]

D. Vega, L. Agui, A. G. Cortes, P. Y. Sedeno, J. M. Pingarron, Voltammetry and amperometric detection of tetracyclines at multi-wall carbon nanotube modified electrodes, Anal. Bioanal. Chem. 389 (2007) 951–958.

[19]

T. Gan, Z. Shi, J. Sun, Y. Liu, Simple and novel electrochemical sensor for the determination of tetracycline based on iron/zinc cations-exchanged montmorillonite catalyst, Talanta 121 (2014) 187–193.

21

[20]

L.G. Chatten, M. Fleischmann, D. Pletcher, The anodic oxidation of some tetracyclines, J. Electroanal. Chem. 102 (1979) 407-413.

[21]

H. Wang, H. Zhao, X. Quan, Gold modified microelectrode for direct tetracycline detection, Front. Environ. Sci. Eng. 6 (2012) 313–319.

[22]

P. Masawat, J.M. Slater, The determination of tetracycline residues in food using a disposable screen-printed gold electrode (SPGE), Sens. Actuators, B 124 (2007) 127132.

[23]

H. Wang, H. Zhao, X. Quan, S. Chen, Electrochemical determination of tetracycline using molecularly imprinted polymer modified carbon nanotube-gold nanoparticles electrode, Electroanalysis 23 (2011) 1863-1869.

[24]

Y. Guo, G. Shen, X. Sun, X. Wang, Electrochemical aptasensor based on multiwalled carbon nanotubes and graphene for tetracycline detection, IEEE Sens. J. 15 (2015) 1951-1958.

[25]

V. Georgakilas, J.N. Tiwari, K.C. Kemp, J.A. Perman, A.B. Bourlinos, K.S. Kim, R. Zboril, Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials, Biosensing, Catalytic, and Biomedical Applications, Chem. Rev. 116 (2016) 5464-5519.

[26]

C. Saldano, A. Mahmood, E. Dujardin, Production, properties and potential of graphene, Carbon 48 (2010) 2127-2150.

[27]

M.J. Allen, V.C. Tung, R.B. Kaner, Honeycomb carbon: a review of graphene, Chem. Rev. 110 (2010) 132-145.

[28]

C. Lee, X. Wei, J.W. Kysar, J. Hone, Measurement of the elastic properties and intrinsic strength of monolayer graphene, Science 321 (2008) 385-388.

22

[29]

Y. Zhang, T.T. Tang, C. Girit, Z. Hao, M.C. Martin, A. Zettl, M.F. Crommie, Y.R. Shen, F. Wang, Direct observation of a widely tunable bandgap in bilayer graphene, Nature 459 (2009) 820-823.

[30]

M.Y. Han, B. Oezyilmaz, Y. Zhang, P. Kim, Energy band-gap engineering of graphene nanoribbons, Phys. Rev. Lett. 98 (2007) 206805.

[31]

A.K. Geim, K.S. Novoselov, The rise of graphene, Nat. Mater. 6 (2007) 183-191.

[32]

S.V. Morozov, K.S. Novoselov, M.I. Katsnelson, F. Schedin, D.C. Elias, J.A. Jaszczak, A.K. Geim, Giant intrinsic carrier mobilities in graphene and its bilayer, Phys. Rev. Lett. 100 (2008) 016602.

[33]

S. Kesavan, S.A. John, Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode, J. Electroanal. Chem. 760 (2016) 6-14.

[34]

S. Kesavan, M.A. Raj, S.A John, Formation of electrochemically reduced graphene oxide on melamine electrografted layers and its application toward the determination of methylxanthines, Anal. Biochem. 496 (2016) 14-24.

[35]

P. Rafighi, M. Tavahodi, B. Haghighi, Fabrication of a third-generation glucose biosensor using graphene-polyethyleneimine-gold nanoparticles hybrid, Sens. Actuators, B, 232 (2016) 454-461.

[36]

D. Higgins, P. Zamani, A. Yu, Z. Chen, The application of graphene and its composites in oxygen reduction electrocatalysis: a perspective and review of recent progress, Energy Environ. Sci., 9 (2016) 357-390.

[37]

T.T. Nguyena, V.H. Nguyena, R.K. Deivasigamania, D. Kharismadewia, Y. Iwaic, J.J. Shim, Facile synthesis of cobalt oxide/reduced graphene oxide composites for electrochemical capacitor and sensor applications, Solid State Sci. 53 (2016) 71-77.

23

[38]

C.H. Liu, Q. Chen, C.H. Liu, Z. Zhong, Graphene ambipolar nanoelectronics for high noise rejection amplification, Nano Lett. 16 (2016) 1064-1068.

[39]

I. Roy, G. Sarkar, S. Mondal, D. Rana, A. Bhattacharyya, N.R. Saha, A. Adhikari, D. Khastgir, S. Chattopadhyay, D. Chattopadhyay, Synthesis and characterization of graphene from waste dry cell battery for electronic applications, RSC Adv. 6 (2016) 10557-10564.

[40]

N.T. Khoa, D.V. Thuan, S.W. Kim, S. Park, T.V. Tam, W.M. Choi, S. Cho, E. J. Kim, S.H. Hahn, Facile fabrication of thermally reduced graphene oxide-platinum nanohybrids and their application in catalytic reduction and dye-sensitized solar cells, RSC Adv. 6 (2016) 1535-1541.

[41]

M. Vinothkannan, R. Kannan, A.R. Kim, G.G. Kumar, K.S. Nahm, D.J. Yoo, Facile enhancement in proton conductivity of sulfonated poly (ether ether ketone) using functionalized graphene oxide-synthesis, characterization, and application towards proton exchange membrane fuel cells, Colloid. Polym. Sci. (2016) 1-11.

[42]

D.R. Kumar, T.T. Nguyen, C. Lamiel, J.-J. Shim, Layered 2-D Bi2Se3 nanosheets intercalated by Ni(OH)2 and their supercapacitor performance, Mater. Lett. 165 (2016) 257-262.

[43]

V.H. Nguyen, C. Lamiel, J.-J. Shim, 3D hierarchical mesoporous NiCo2S4@Ni(OH)2 core-shell nanosheet arrays for high performance supercapacitors, New J. Chem. 40 (2016) 4810-4817.

[44]

L. Chen, Y. Tang, K. Wang, C. Liu, S. Luo, Direct electrodeposition of reduced graphene oxide on glassy carbon electrode and its electrochemical application, Electrochem. Commun. 13 (2011) 133-137.

[45]

J. Yang, S. Gunasekaran, Electrochemically reduced graphene oxide sheets for use in high performance supercapacitors, Carbon 51 (2013) 36-44.

24

[46]

S. Shrivastava, N. Jadon, R. Jain, Next-generation polymer nanocomposite-based electrochemical sensors and biosensors: A review, TrAC, Trends Anal. Chem. 82 (2016) 55-67.

[47]

S. Baskar, C.-W. Liao, J.-L Chang, J.-M. Zen, Electrochemical synthesis of electro active poly(melamine) with mechanistic explanation and its applicability to functionalize carbon surface to prepare nanotube–nanoparticles hybrid, Electrochim. Acta 88 (2013) 1-5.

[48]

P. Gupta, R.N. Goyal, Polymelamine modified edge plane pyrolytic graphite sensor for the electrochemical assay of serotonin, Talanta 120 (2014) 17-22.

[49]

X. Liu, L. Luo, Y. Ding, Q. Wu, Y. Wei, D. Ye, A highly sensitive method for determination of guanine, adenine and epinephrine using poly-melamine film modified glassy carbon electrode, J. Electroanal. Chem. 675 (2012) 47-53.

[50]

H.J. Salavagione, G. Martínez, G. Ellis, Recent advances in the covalent modification of graphene with polymers, Macromol. Rapid Commun. 32 (2011) 1771-1789.

[51]

S. William, J.R. Hummers, E.O. Richard, Preparation of Graphitic Oxide, J. Am. Chem. Soc. 80 (1958) 1339.

[52]

S. Baskar, J.L. Chang, J.M. Zen, Extremely stable copper–polymelamine composite material for amperometric hydrogen peroxide sensing, J. Polym. Sci., Part B: Polym. Phys. 51(2013) 1639-1646.

[53]

M.A. Raj, S.A. John, Fabrication of electrochemically reduced graphene oxide films on glassy carbon electrode by self-assembly method and their electrocatalytic application, J. Phys. Chem. C 117 (2013) 4326-4335.

[54]

S. Kesavan, S.B. Revin, S.A. John, Potentiodynamic formation of gold nanoparticles film on glassy carbon electrode using aminophenyl diazonium cations grafted gold

25

nanoparticles: Determination of histamine H2 receptor antagonist, Electrochim. Acta 119 (2014) 214-224. [55]

M. Nose, T. Kinumoto, H.-S.Choo, K. Miyazaki, T. Abe, Z. Ogumi, Electrochemical oxidation of highly oriented pyrolytic graphite in sulphuric acid solution under potential pulse condition, Fuel Cells 9 (2009) 284-290.

[56]

J.F. Moulder, W.F. Stickle, P.E. Sobol, K.D. Bomben, Handbook of X-Ray Photoelectron Spectroscopy, Eden Prarie, MN, 1992.

[57]

M.A. Pimenta, G. Dresselhaus, M.S. Dresselhaus, L.G. Cancado, A. Jorio, R. Saito, Studying disorder in graphite-based systems by Raman spectroscopy, Phys. Chem. Chem. Phys. 9 (2007) 1276-1290.

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Author Biographies

Srinivasan Kesavan is a Research professor in the School of Chemical Engineering, Yeungnam University, South Korea. He received his B.Sc.Ed. (PCM) degree from University of Mysore in 2008 and M.Sc. degree from Madurai Kamaraj University in 2010. He received his Ph.D. degree in 2015 from Gandhigram Rural University, Tamil Nadu, India. His current research interests are the fabrication and development of electrode substrate by grafting, self-assembly, metal oxides, and polymer composites materials for the electrochemical sensing of biomolecules and energy applications.

Deivasigamani Ranjith Kumar is a Research professor in the Clean Energy Priority Research Center at Yeungnam University, Republic of Korea. He received his M.Sc. and Ph.D. degrees in Physical Chemistry from the University of Madras, Chennai, India in 2009 and 2014, respectively. His research interests include the electroanalytical chemistry, energy storage, study of new electrode materials for development of electrochemical sensors and biosensors.

Jae-Jin Shim received his B.S. in Chemical Engineering from Seoul National University in 1980, his M.S. in Chemical Engineering from Korea Advanced Institute of Science and Technology in 1982, and his Ph.D. in Chemical Engineering from the University of Texas at Austin in 1990. He is a professor in the School of Chemical Engineering at Yeungnam University, Republic of Korea. His current research focuses on energy storage materials and materials for gas sensor and electrochemical sensor.

27

Figures:

0

A

B

A

B 5

−100

0

15





200

th

−5

100

cycle

−200

−10 0.4

1 st

0.6

0.8

0

−300 −1.5

−1

−0.5

0

0

0.5

1

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Potential (V vs. SCE)

Potential (V vs. SCE)

Figure 1. Cyclic voltammograms (CVs) obtained for (A) the electrochemical reduction of GO on glassy carbon (GC) electrode (15 cycles) in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s-1 and (B) polymelamine film formation on ERGO modified electrode (15 cycles) in 0.1 M HCl at a scan rate of 50 mV s-1. (Red line: 1-3 cycles, green line: 4-14 cycles, black line: 15th cycle).

28

2

A

B

C

D

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C

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j

D

5

a 

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0

0 Y=0.026X+0.45 6 R2=0.999



−1

−5

4 2 0

−2 0.4

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1

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0.4

0.6

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100

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Figure 2. (A) CV of p-Mel@ERGO/GC film modified electrode in 0.1 M H2SO4 at a scan rate of 50 mV s -1. (B) CVs of p-Mel@ERGO/GC film modified electrode in 0.1 M H2SO4 at scan rates of a-j = 25-250 mV s−1. Inset: plot of the anodic peak current vs. scan rate.

29

1

A B C D E F A B C D E F

A B C D E F

C D E F

A B C D E F

A B C D E F

Figure 3. SEM images obtained for (A) GO/GC, (B) ERGO/GC, (C) p-Mel/GC, (D) high magnification of p-Mel/GC, (E) p-Mel@ERGO/GC, and (F) high magnification of pMel@ERGO/GC plates.

30

a b c

a b c

N 1s

Intensity (a.u)

C 1s

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−C=C− −C−OH O=C−OH −C=O

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ERGO

−C=C− −C=O HO−C=O −C−OH

290

280

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285

280

Binding Energy (eV)

Figure 4. (A) XPS survey spectra of (a) GO/GC, (b) ERGO/GC and (c) p-Mel@ERGO/GC plates. XPS of C 1s spectra obtained for (B) GO/GC and (C) ERGO/GC plates.

31

D

Intensity (a.u)

400

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−1

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G D

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GO

1000 1000

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a b shift (cm−1) Raman Figure 5. Raman spectra of (a) GO/GC and (b) ERGO/GC plates.

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2000 2000

20

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cD

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d

d

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Potential (V vs. SCE)

0.2

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Potential (V vs. SCE)

Figure 6. CVs of 0.5 mM (A) TET and (B) UA at (a) bare GC, (b) p-Mel/GC and (c) pMel@ERGO/GC film modified electrodes in 0.2 M PB solution (pH 3) at a scan rate of 50 mV s-1. (d) p-Mel@ERGO/GC film modified electrode in the absence of TET and UA (solid line: 1st cycle; dotted line: 5th cycle).

33

UA

Y=0.018X+1.12 2 R =0.995

2.5

2

I ( A)

h TET

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60

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0.3

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Potential/V vs (SCE)

I ( A)

0.36

0.30

b 0 0

0

0.5

1

30

60

Concentration of TET (M)

Potential (V vs. SCE) Figure 7. DPVs obtained for each increment of 10 µM TET and 10 µM UA (curves a-h) at the p-Mel@ERGO/GC film modified electrode in a 0.2 M PB solution (pH 3). Insets: (a) Plot of concentration of UA vs. current. (b) Plot of concentration of TET vs. current.

34

90

10

UA

g 0.6

0.4

0.3 0.75



5

0.8

0.36

0.30 20

40

60

Concentration of TET ()





I ( A)

0.42

Y=0.002X+0.29 2 R =0.996

a 0.85

Potential/V vs (SCE)

TET

0.4

0 0

0.5

1

Potential (V vs. SCE) 0.7 0.8 Figure 8. DPVs obtained for each increment of 10 µM TET to 500 µM UA (curves a-g) at p-

Potential/V vs (SCE)

Mel@ERGO/GC film modified electrode in 0.2 M PB solution (pH 3). Inset: Plot of the concentration of TET vs. current.

35

0.9

Scheme 1. Schematic representation of polymelamine film formation on the ERGO modified GC electrode surface.

36

Scheme 2. Mechanism for the potentiodynamic film formation of melamine on an ERGO surface.

37

Scheme 3. Proposed mechanism for the oxidation of TET.

38

Table 1 Comparison of the proposed sensor with other methods described in the literature.

Electrode

Linear range (mol L−1)

Potential (V)

Detection (mol L−1)

PtNPS/C/GCEa

9.99 × 10−6–4.4 × 10−5

0.95

Graphite/polyurethaneb

3.8 × 10−6–3.8 × 10−5

GCE (Fe/Zn-MMT)c GMEd Screen-printed electrode

gold

p-Mel@ERGO/GCE

pH

Reference

4.28 × 10−6

3

[11]

0.94

2.6 × 10−6

2.5

[15]

3.0 × 10−7–5.2 × 10−5

1.17

1.0 × 10−7

1

[19]

2.0 × 10−5–2.0 × 10−4

1.5

1.8 × 10−7

1

1.0 × 10−6–5.0 × 10−4

1.2

9.6 × 10−7

2

[22]

5.0 × 10−6–2.25 × 10−4

0.92

2.2 × 10−6

3

This work

a

platinum nanoparticles supported on carbon (PtNPs/C) coated on GCE

b

Graphite-polyurethane composite electrode

c

Iron/zinc cation-exchanged electrode(GCE) d

montmorillonite

Gold modified microelectrode.

39

(Fe/Zn-MMT)

catalyst

limit

on

[21]

glassy

carbon