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Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode
Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode Srinivasan Kesavan, S. Abraham John PII: DOI: Reference:
S1572-6657(15)30217-4 doi: 10.1016/j.jelechem.2015.11.039 JEAC 2386
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
21 July 2015 12 November 2015 25 November 2015
Please cite this article as: Srinivasan Kesavan, S. Abraham John, Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode, Journal of Electroanalytical Chemistry (2015), doi: 10.1016/j.jelechem.2015.11.039
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ACCEPTED MANUSCRIPT Stable determination of paracetamol in the presence of uric acid in human
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urine sample using melamine grafted graphene modified electrode
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Srinivasan Kesavan and S. Abraham John* Centre for Nanoscience and Nanotechnology Department of Chemistry, Gandhigram Rural Institute Gandhigram–624 302, Dindigul, Tamilnadu, India
Corresponding author:
Tel: +91 451 245 2371 Fax : + 91 451 245 3031 E-mail : [email protected], [email protected]
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ACCEPTED MANUSCRIPT Abstract Electrochemically reduced graphene oxide (ERGO) on aminotriazine (AT) grafted glassy
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carbon (AT/GC) electrode was prepared by electrochemical reduction of graphene oxide (GO)
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attached through 2,4-diamino-1,3,5-triazine on GC electrode and the resulting electrode was
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utilized for the selective determination of paracetamol (PA) in the presence higher concentration uric acid (UA). The GO attached on AT/GC (GO/AT/GC) and the ERGO films on AT/GC
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(ERGO/AT/GC) electrodes were characterized by scanning electron microscopy (SEM), Raman spectroscopy and cyclic voltammetry (CV). The ERGO modified electrode showed excellent
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electrocatalytic activity towards PA and UA. This electrode not only enhanced the oxidation currents of PA and UA but also shifted their oxidation potentials toward less positive potentials
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in contrast to bare GC, AT/GC and GO/AT/GC electrodes. Bare and GO modified electrodes failed to separate the voltammetric signals of UA and PA. However, the ERGO/AT/GC electrode
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successfully separated the voltammetric signals of them in a mixture and hence used for the simultaneous determination. Further, the modified electrode was effectively used for the selective determination of PA in the presence of 50-fold excess of UA. Using amperometry, detection of 40 nM PA was achieved. The current response of PA was increased linearly while increasing its concentration from 4.0 × 10−8-1.0 × 10−4 mol/L and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The practical application of the present modified electrode was demonstrated by simultaneously determining the concentrations of PA and UA in human urine samples. Keywords: Electrografting; melamine; graphene oxide; self-assembly; Raman spectroscopy; paracetamol.
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ACCEPTED MANUSCRIPT 1. Introduction Paracetamol (PA) (N-acetyl-p-aminophenol or Acetaminophen) (Chart 1A) is an effective
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analgesic and antipyretic drug used for the reduction of fever [1]. It acts as a painkiller by
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inhibiting prostaglandin’s synthesis in the central nervous system and reduces fever by sedating
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hypothalamic heat regulating center [2]. It is also a useful drug for the treatment of osteoarthritis therapy, headache, backache, arthritis and postoperative pain [3-7]. At usual therapeutic doses,
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PA is rapidly and completely metabolized by undergoing glucuronidation and sulfation to
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inactive metabolites which are eliminated in the urine [1,6]. However, PA produces toxic metabolite accumulation at higher doses that mainly causes severe fatal hepatoxicity and
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nephrotoxicity [8,9] besides skin rashes and inflammation of the pancreas [10].
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Uric acid (UA) (Chart 1B) is the main end product of purine metabolism in the human
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body [11]. Increased uptake of serum UA acts as a scavenger of radicals and thus preventing from Parkinson's disease [11]. Low concentration of UA is associated with multiple sclerosis [12]. UA is a major interfering agent for the determination of PA in human urine [1]. The electrochemical method has many advantages over the traditional methods due to its high sensitivity, selectivity, low cost and less time consuming. But, it is very difficult to separate UA and PA using conventional electrodes because both of them are oxidized at similar potential. However, accurate and selective determination of PA in human urine sample is essential for the clinical point of view. Therefore, it is a challenging task for the electrochemists to find a suitable electrode system for the determination of PA in the presence of UA. While searching the literature, few reports are available in the literature for the determination of PA and UA using multiwalled carbon nanotube (MWCNTs)/chitosan composite [13], nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet/chitosan [14], ionic liquid/ 3
ACCEPTED MANUSCRIPT MWCNTs/chitosan modified GCE [15], cetylpyridinium bromide/MWCNTs paste electrode [16] and MWCNTs-cobalt nanoparticles modified electrode [17] prepared by drop cast method.
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However, these methods either involve tedious procedures or series of steps involved to fabricate composite films on electrode surface besides the stability of these film is questionable. Further,
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the drop-casting method has intrinsic drawbacks such as lack of control over film thickness and
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less reproducibility. Hence, it is essential for the researchers to develop a simple method for the
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simultaneous determination of PA and UA in clinical point of view.
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Chemical grafting of aryldiazonium salts derivatives is a widely used method for the modification of conducting substrates over the last two decades [18]. The methodology involves
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reduction of an aryldiazonium ion followed by elimination of dinitrogen to give an aryl radical
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which attacks the substrate with a formation of a covalent bond [18,19]. Grafting of diazonium salts is a powerful method to graft aryl groups bearing a wide variety of functionalities on
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different substrates like carbon [20,21], semiconductor [22], gold nanoparticles [23,24], graphene [25,26] and single-walled carbon nanotubes [27]. Graphene, an allotrope of carbon has received much attention because of its excellent electronic conductivity, high mechanical strength, large specific surface area and high stability [28]. It plays a predominant role in the fields of sensors, nanoelectronics, batteries, solar cells and electrochemical double-layer capacitors [29-31]. Graphene modified electrodes show excellent electrocatalytic activity towards biomolecules and toxic chemicals [32-34]. Most of the researchers reported drop casting method for the fabrication of graphene on different electrode substrates [34-40]. But, selfassembly is the best method to fabricate graphene because film thickness can be easily controlled [41].
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ACCEPTED MANUSCRIPT Melamine (1,3,5-triazine-2,4,6-triamine) is mainly used as a precursor for pyrolysis processes and permits the insertion of nitride-like units in the carbon structure [42]. Be´langer et
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al. reported the selective diazotization of one amine group of melamine to graft on carbon
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powder [20]. However, grafting of melamine on electrode surface has not been reported so far. The presence of three amine functionalities in the melamine molecule could be useful for
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chemical grafting. In the present study, the GO is assembled on melamine grafted GC electrode.
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Then, the GO was electrochemically reduced at more negative potential to retain the aromatic backbone of graphene and used for the selective determination of PA in the presence high
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concentration of UA.
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2.1. Chemicals
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2. Experimental
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Graphite powder was purchased from Alfa Aesar. Uric acid (UA) and paracetamol (PA) were purchased from Sigma-Aldrich and were used as received. Potassium permanganate (KMnO4), sodium nitrate (NaNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), melamine, sodium nitrite (NaNO2) and hydrochloric acid (HCl) were purchased from Merck (India). The selective diazotation of one amine group of melamine has been described in the reported patent [43]. 1 equivalent melamine was solubilized in 0.5 mol/L HCl, to which 1 equivalent cold NaNO2 was added drop wise to generate the aryldiazonium cations in situ in the electrochemical cell. Indium tin oxide (ITO) substrates were purchased from Asahi Beer Optical Ltd, Japan. GC plates were purchased from Sigma-Aldrich. 0.2 mol/L phosphate buffer (PB) solution (pH 7.2) was prepared by using Na2HPO4 and NaH2PO4. All other chemicals were of analytical reagent grade and were used as received. Double distilled water was used to prepare the solutions.
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ACCEPTED MANUSCRIPT 2.2. Instrumentation Raman spectra were recorded on a Nanofinder ® HE (Tokyo Instruments, INC), 532 nm
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YAG Laser. SEM measurements were carried out by using VEGA3 TESCAN. Electrochemical measurements were performed in a conventional two compartment three electrode cell with a
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mirror polished 3 mm glassy carbon electrode (GCE) as a working electrode, Pt wire as a counter
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electrode and a NaCl saturated Ag/AgCl as a reference electrode. All the electrochemical
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measurements were carried out with CHI model 634B (Austin, TX, USA) Electrochemical Workstation. For differential pulse voltammetry (DPV) measurements, pulse width of 0.06 s,
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amplitude of 0.05 V, sample period of 0.02 s and pulse period of 0.2 s were used. All the
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electrochemical measurements were carried out under nitrogen atmosphere at room temperature.
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2.3. Synthesis of GO
GO was synthesized using the Hummer’s method with a slight modification [44].
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Concentrated H2SO4 (12 mL) was added to flake graphite (0.5 g) and NaNO3 (0.25 g) and the mixture was cooled to 0 °C. KMnO4 was added slowly into the mixture and the temperature was maintained at 20 °C. The reaction mixture was warmed to 35 °C and stirred for 30 min. Then, 23 mL of water was added, which produce an exotherm to 98 °C and the temperature was maintained for 15 min by external heating. The reaction was terminated by the large addition of water (72 mL) and 0.5 mL of 30% H2O2. Finally, the reaction mixture was cooled, washed with 0.1 mol/L HCl and water to remove the metal ions and then dried. 2.4. Fabrication of AT grafted ERGO electrode The fabrication of the AT grafted ERGO electrode is schematically shown in Scheme 1. 2,4-diamino-1,3,5-triazine moieties were introduced on the GC electrode by the in situ generation based on the diazotization of melamine. The resulting AT grafted electrode was
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ACCEPTED MANUSCRIPT termed as AT/GC electrode. It was rinsed with water and then immersed into exfoliated GO solution (1 mg/mL) for 12 h. The exfoliation of GO was achieved by the sonication of GO for 45
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min. The GO was self-assembled on AT/GC modified electrode via electrostatic interaction
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between positively charged amine groups and negatively charged carboxyl groups present in the GO solution. In addition to that hydrogen bonding between the amine group in melamine and
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oxygen containing groups in GO and π-π interaction between the GO and triazine ring of
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melamine are also possible. This electrode is termed as GO/AT/GC electrode. Then, the GO modified electrode was electrochemically reduced in PB solution (pH 7) for the fabrication of
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ERGO on GC electrode. After electrochemical reduction of GO, the oxygen functional groups are reduced and strong π-π interaction between ERGO and triazine ring of melamine will anchor
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it on the electrode surface. This electrode is termed as ERGO/AT/GC electrode.
3. Results and discussion
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3.1. Grafting of AT and electrostatic assembly of GO and its electrochemical reduction on GCE Fig. 1A shows the CV obtained for the reduction of 2,4-diamino-1,3,5-triazine diazonium cations generated in situ from melamine. The CV shows a broad reduction wave at -0.47 V which was assigned to the formation of the aminotriazine radical and which attaches aminotriazine group on the GCE [19,21]. A similar behavior has been previously reported for the electrochemical reduction of several diazonium salts on various substrates [18,45]. We have chosen the electrografted GCE formed by 1 cycle for the attachment of GO because more than one cycle leads to the formation of multilayers, which has been explained by a polymerization type reaction between the aminotriazine grafted layer and aminotriazine free radicals in solution [18]. Moreover, while increasing the number of grafting cycles, the cross-linked multilayer formation also increases, which will decrease the attachment of GO on grafted GCE. The GO
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ACCEPTED MANUSCRIPT was assembled on AT grafted GCE by the electrostatic interaction between the negatively charged GO and the positively charged amine terminal of grafted AT layer. The surface attached
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GO was electrochemically reduced to remove the oxygen functional groups of GO present on the
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surface by potential cycling between 0 and −1.4 V for 15 cycles (Fig. 1B). In the first cycle, a cathodic peak at -1.05 V was appeared which might be due to the reduction oxygen functional
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groups present on the surface of GO [46]. From the second cycle onwards, the reduction peak
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was completely vanished. This indicates the complete reduction of oxygen functional groups present on the surface of GO. Due to the removal of oxygen functional groups by
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electrochemical reduction, aromatic backbone of graphene must be regenerated on the surface of
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grafted GCE.
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3.2. Characterization by Raman spectroscopy
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Raman spectroscopy is a very important tool to characterize carbon products due to increase in Raman intensities by the conjugated carbon-carbon double bonds. Fabrication of GO followed by its electrochemical reduction was confirmed by Raman spectroscopy. For Raman spectral measurements, GO and ERGO modified ITO plates prepared by the same procedure were used. Since electrografting is mainly based on radical mechanism the mode of attachment of aryl groups on GC and ITO electrodes is likely the same. Fig. 2 shows the Raman spectra of GO/AT/ITO modified substrate before and after the electrochemical reduction. The GO/AT/ITO modified substrate shows two peaks at 1340 and 1605 cm−1 corresponding to D and G peaks of GO (curve a). After the electrochemical reduction, the D and G peaks were obtained at 1343 and 1602 cm−1, respectively (curve b). A small shift in the G band after electrochemical reduction is due to regeneration of aromatic backbone of graphene [29]. On the other hand, the intensity ratio of D and G bands was increased from 0.90 to 1.10 after the electrochemical reduction indicating 8
ACCEPTED MANUSCRIPT a decrease in the average size of sp2 hybridized lattice of graphene [47]. These results confirm the successful attachment of GO on AT grafted electrode surface by electrostatic assembly and
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its electrochemical reduction removed the oxygen functional groups from the surface of GO.
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3.3. Morphological characterization by SEM
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Fig. 3 shows the SEM images obtained for AT/GC, GO/AT/GC and ERGO/AT/GC substrates. The bare GC plate shows no particles on the surface (Fig. 3A) whereas the AT/GC
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substrate shows a layer like structure [19,21] (Fig. 3B). In contrast to AT/GC substrate, the SEM
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image of the GO/AT/GC substrate shows a layered structure of GO sheets (Fig. 3C). On the other hand, ERGO/AT/GC substrate shows more disordered and aggregated structures which
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appear to be very thin and nothing but a few layers of graphene (Fig. 3D). It also indicates the
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presence of high density edge planes of graphene sheets. This is attributed to an increase in van der Waals attraction between adjacent layers due to the removal of oxygen functional groups
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during electrochemical reduction [29,47]. 3.4. Electrocatalytic activity of ERGO modified electrode towards PA and UA Fig. 4A shows the DPVs obtained for 0.5 mmol/L PA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 M PB solution (pH 5). The bare GCE shows an oxidation peak at 0.51 V (curve a: solid line) whereas AT/GC (curve b: solid line) and GO/AT/GC show oxidation peaks at 0.50 and 0.52 V, respectively (curve c: solid line) for PA. In the subsequent scans, the oxidation peak currents decreased at these electrodes (curve a, b, c: dotted line). This may be due to the surface fouling effect caused by the oxidation products of PA [48]. On the other hand, ERGO/AT/GC modified electrode shows a sharp oxidation peak for PA at 0.49 V (curve d: solid line) with 2-fold, 1.8-fold and 1.7-fold higher current than bare GC, AT/GC and GO/AT/GC electrodes. The oxidation peak was stable even after 5 scans (curve d: 9
ACCEPTED MANUSCRIPT dotted line). The ERGO/AT/GC modified electrode does not show any electrochemical response in the absence of PA in a 0.2 mol/L solution of PB at pH 5 (curve e). It is likely that high
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conducting nature of ERGO/AT/GC electrode effectively prevented the surface fouling effect caused by the oxidation products of PA [29]. The facile electron transfer at ERGO films and the
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π–π interaction between ERGO and PA enhanced the oxidation current of PA with less positive
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potential shift. Compared to bare GC, AT/GC and GO/AT/GC electrodes, the ERGO/AT/GC
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modified electrode shows a stable oxidation with greater enhancement of peak current for PA oxidation. Hence, ERGO modified electrode was highly suitable for the determination of PA.
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Fig. 4B shows the DPVs obtained for 0.5 mmol/L UA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 M PB solution (pH 5). Bare GC, AT/GC and
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GO/AT/GC electrodes show broad oxidation waves for UA at 0.43 V, 0.42 V and 0.42 V, respectively. However, an enhanced oxidation current was observed at 0.41 V for UA at the
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ERGO/AT/GC modified electrode. The oxidation peak of UA was stable even after five scans (curve 4B d: dotted line). Two protons and two electrons are involved in the redox reaction of PA and UA (Scheme 2). The effect of scan rate on the oxidations of PA and UA was studied at ERGO/AT/GC modified electrode. The oxidation peak currents of both PA and UA were increased while increasing the scan rates from 25 to 250 mVs-1 (Fig. S1, Supplementary material). A good linearity was obtained while plotting the anodic peak current against the square root of scan rate with correlation coefficients of 0.9998 for PA (Inset, Fig. S1A) and 0.9998 for UA (Inset, Fig. S1B) indicating that the oxidations of PA and UA at ERGO/AT/GC modified electrode were diffusion controlled process. 3.5. Electrochemical behavior of PA and UA in a mixture at ERGO modified electrode
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ACCEPTED MANUSCRIPT Fig. 5 shows the DPVs obtained for the oxidation of each 0.5 mmol/L PA and UA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 mol/L PB solution (pH
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5). The oxidation signal of PA was observed at 0.52 V whereas the oxidation signal of UA was
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not observed at bare GCE (curve a: solid line). After five scans, the oxidation current was decreased and shifted to more positive potential (curve a: dotted line). Similar to bare GCE, the
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oxidation signal of UA was not resolved at AT/GC electrode (curve b: solid line). Although a
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slight enhancement of oxidation current of PA was observed at AT/GC electrode compared to bare GCE, its oxidation peak was not stable on repetitive potential scans (curve b: dotted line).
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The, GO/AT/GC electrode shows the oxidation peak for UA and PA at 0.41 and 0.48 V, respectively (curve c: solid line). After five scans, the oxidation peak potentials of them were
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shifted to more positive potentials with significant decrease in oxidation peak currents (curve c: dotted line). This clearly suggested that bare GC, AT/GC and GO/AT/GC electrodes are not
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suitable for the determination of PA and UA in a mixture. On the other hand, ERGO/AT/GC electrode clearly resolved the oxidation peaks of PA and UA with a potential difference of 90 mV between UA and PA. The oxidation peaks of UA and PA were observed at 0.40 V and 0.49 V, respectively with 1.7-fold higher oxidation currents compared to GO/AT/GC electrode (curve d: solid line). Further, the oxidation peaks were not shifted to more positive potentials even after five potential scans (curve d: dotted line). Hence, ERGO/AT/GC electrode was highly suitable for the simultaneous determination of PA and UA in a mixture. The ERGO/AT/GC modified electrode does not show any electrochemical response in the absence of PA and UA in 0.2 mol/L solution of PB at pH 5 (curve e). 3.6. Simultaneous determination of PA and UA at ERGO/AT/GC electrode Since the ERGO/AT/GC modified electrode successfully separated the oxidation signals of PA and UA, it was used for the simultaneous determination of them. Fig. 6 shows the 11
ACCEPTED MANUSCRIPT simultaneous determination of PA and UA in 0.2 mol/L PB solution (pH 5). A well-defined voltammetric signal was observed for 7 µmol/L UA and 8 µmol/L PA at 0.40 V and 0.49 V,
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respectively (curve a). When the concentration of UA was increased from 7 µmol/L to 161
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µmol/L (curves a-w) and PA was increased from 8 µmol/L to 184 µmol/L (curves a-w), the peak currents of the respective analytes were increased linearly with a correlation coefficient of
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0.9982 for UA and 0.9990 for PA (Insets: Fig. 6). The oxidation potentials of UA and PA were
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not affected while increasing their concentrations. The above results suggested that simultaneous
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determination of PA and UA is possible at ERGO/AT/GC electrode. 3.7. Selective determination of PA and UA at ERGO/AT/GC electrode
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The main objective of the present work is to selectively determine PA in the presence of
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high concentration of UA. Generally, high concentration of UA presents in human urine [1,3]. Therefore, it is very much essential to determine PA in the presence of high concentration of UA.
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Fig. 7A shows the DPVs obtained for the increment of 10 µmol/L of PA to a solution of 500 µmol/L UA. A well defined oxidation peak was observed for PA even in the presence of 50-fold excess of UA. While increasing the PA concentration in the range of 10-220 µmol/L, a linear current enhancement was observed with a correlation coefficient of 0.9992 (Inset, Fig. 7A) without affecting UA peak current. These results show that the ERGO modified electrode can be used to selectively determine PA even in the presence of high concentration of UA. Further, we have also attempted to selectively determine UA in the presence of high concentration of PA to examine the performance of the electrode. Fig. 7B shows the DPVs for the increment of 15 µmol/L of UA to a solution of 400 µmol/L PA. A well defined oxidation peak was observed for 15 µmol/L UA in the presence of 27-fold excess of PA. This indicates that the detection of low concentration of UA is possible in the presence of high concentration of 12
ACCEPTED MANUSCRIPT PA. A linear current enhancement was observed while increasing the UA concentrations in the range of 15-345 µmol/L with a correlation coefficient of 0.9990 (Inset, Fig. 7B) while the peak
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current of PA was unchanged. These results show that the ERGO modified electrode was more
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selective towards the oxidation of UA even in the presence of high concentration of PA.
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3.8. Amperometric determination of PA
Fig. 8A shows the amperometric i-t curve for PA at ERGO/AT/GC modified electrode in a
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homogenously stirred 0.2 mol/L PB solution (pH 5) by applying a potential of +0.6 V. The
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ERGO/AT/GC modified electrode shows the current response for each addition of 40 nmol/L PA. The current response increases and the steady state current response was attained within 3 s
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for further addition of 40 nmol/L PA in each step with a sample interval of 50 s. The dependence
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of response current with respect to concentration of PA was linear from 40 nmol/L to 360 nmol/L with a correlation coefficient of 0.9992 (Inset of Fig. 8A). The amperometric i-t curve for
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each addition of 40 nmol/L PA showed linear current increase without noise. Fig. 8B shows that the amperometric current response was increased linearly with increasing PA concentration in the range of 4.0 × 10−8-1.0 × 10−4 mol/L with a correlation coefficient of 0.9990 (Inset of Fig. 8B) and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The observed linear range and the lowest detection limit of PA at the ERGO/AT/GC modified electrode were compared with the reported modified electrodes [49-55] and are given in Table 1. It is evident from Table 1 that the present modified electrode showed the lowest detection limit (6.8 × 10−10 mol/L) and a wide range determination of PA (4.0 × 10−8-1.0 × 10−4 mol/L) compared to reported papers [49-55]. The anti-interference ability of the ERGO/AT/GC modified electrode was tested towards the detection of PA and UA from common interferents such as Na+, Ca2+, Mg2+, K+, NH4+, Cl-,
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ACCEPTED MANUSCRIPT F-, CO32-, SO42-, glucose, urea and oxalate by DPV. No change in the current response was observed for 40 nmol/L each PA and UA in the presence of 30 µmol/L of the interferents,
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demonstrating that the present modified electrode is highly selective towards PA and UA. In
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order to study the stability of the ERGO/AT/GC modified electrode, the DPVs for 0.5 mmol/L PA in 0.2 mol/L PB solution (pH 5) were recorded for every 5 min interval. It was found that
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anodic peak current remained same with a relative standard deviation of 1.3% for 15 times
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repetitive measurements indicating that this electrode has a good reproducibility and does not experience surface fouling during the voltammetric measurements. The current response
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decreased about 2.5% in 1 week and 4.3% in 2 weeks when the electrode was kept in PB solution
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(pH 5).
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3.9. Determination of PA and UA in human urine samples The practical application of the present ERGO/AT/GC modified electrode was
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demonstrated by determining the concentration of PA and UA in human urine samples. The human urine samples were collected from laboratory co-workers and were diluted to 50 times using 0.2 mol/L PB solution (pH 5). The standard addition technique was used for the determination of PA and UA in urine samples. DPV of human urine sample in PB solution at ERGO/AT/GC modified electrode shows an oxidation peak at +0.41 V and the obtained peak is due to the oxidation of UA (curve a in Fig. 9). For the simultaneous determination of PA and UA in human urine, commercial samples of PA and UA were spiked to urine sample. After the addition of 10 µmol/L PA and 10 µmol/L UA, the oxidation peak current at +0.41 V was increased which is due to the oxidation of UA whereas new oxidation peak was obtained at +0.49 V which might be due to the oxidation of PA (curve b in Fig. 9). The modified electrode shows good recovery for the determination of PA and UA in human urine samples (Table 2).
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ACCEPTED MANUSCRIPT Thus, the present modified electrode can be utilized for the determination of PA and UA in real samples.
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4. Conclusions
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We have demonstrated the simultaneous determination of PA and UA using ERGO
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modified AT grafted GCE. Raman and SEM results confirmed the successful attachment of GO and ERGO on AT grafted GCE. The ERGO modified electrode enhanced the oxidation currents
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of PA and UA when compared to bare GC, AT/GC and GO/AT/GC electrodes. The facile
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electron transfer at ERGO film prevented the surface fouling effect and hence the oxidation peaks were stable for several potential scans. Further, the present modified electrode separated
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the oxidation signals of PA and UA whereas bare GC, AT/GC and GO/AT/GC electrodes failed
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to resolve the oxidation signals of them. Hence, ERGO modified electrode was successfully used for the simultaneous determination of PA and UA. Selective determination of PA in the presence
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of 50-fold higher concentration of UA was also demonstrated using the ERGO/AT/GC electrode. Using amperometry, detection of 40 nmol/L of PA was achieved and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The practical application of the present modified electrode was demonstrated by simultaneously determining the concentrations of PA and UA in human urine samples. This is the first report in which melamine grafted ERGO film modified electrode used for the selective determination of PA in the presence of high concentration UA.
Acknowledgement S. Kesavan thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, India for the award of Senior Research Fellowship (09/715(0016)/2011-EMR-I).
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ACCEPTED MANUSCRIPT Figures
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A B C
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E vs (Ag/AgCl)/V
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Fig. 1. (A) Cyclic voltammograms obtained for grafting of GC electrode at a scan rate of
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mV s-1 using 2 mmol/L each melamine and NaNO2 in 0.5 mol/L HCl. (B) Electrochemical reduction of electrostatically assembled GO on AT/GC electrode (15 cycles) in 0.2 mol/L PB solution (pH 7.2) at a scan rate of 100 mV s-1.
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a b c 2000
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Fig. 3. SEM images obtained for (A) bare GC, (B) AT/GC, (C) GO/AT/GC and (D) ERGO/AT/GC substrates.
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aa bb cc ddee
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Fig. 4. (A) DPVs obtained for 0.5 mmol/L PA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d)
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ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of PA at GC/AT/ERGO electrode. (B) DPVs obtained for 0.5 mmol/L UA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d) ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of UA at ERGO/AT/GC electrode. (solid line: 1st scan; dotted line: 5th scan).
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Fig. 5. DPVs obtained for each 0.5 mmol/L of UA and PA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d) ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of
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Y = 0.04 X + 0.74 2
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Fig. 7. (A) DPVs obtained for each increment of 10 µmol/L PA in 500 µmol/L of UA (curves a– t) at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5). Inset: plot of the concentration of PA vs. current. (B) DPVs obtained for each increment of 15 µmol/L UA in 400 µmol/L of PA (curves a–w) at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5). Inset: plot of the concentration of UA vs. current.
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Time (sec) 0
Fig. 8. (A)
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Concentration of PA (nM)
400
5 50 55
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a ba bc dc de ef gf gh hi ji kj k ab cd e f g h i j k ab cd e f g h i j k aababbccdcdde eef fgf gghhhi iji jkjkk ab cd e f g h i j k aa bb cc dd ee ff gg hh ii jj kk
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Time (sec) the concentration of 40 electrode in 0.2 mol/L PB solution (pH 5). Each addition Time increases (sec)
nmol/L of PA at regular interval of 50 s. Eapp = +0.6 V. (B) Amperometric i-t curve for the determination of PA at ERGO/AT/GC modified electrode in 0.2 M PB solution (pH 5). Each addition increases the concentrations of (a) 0.04, (b) 0.1, (c) 0.2, (d) 0.5, (e) 1, (f) 2, (g) 5, (h) 10, (i) 20, (j) 50 and (k) 100 µmol/L PA at ERGO/AT/GC modified electrode in 0.2 M PB solution (pH 5) at a regular interval of 50 s. Eapp = +0.6 V. Insets (a) and (b): Plot of concentration of PA vs. current.
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Fig. 9. DPVs obtained for (a) human urine and (b) after spiked with commercial 10 µmol/L of UA and 10 µmol/L of PA at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5).
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Chart 1. Structures of (A) Paracetamol and (B) Uric acid.
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Scheme 1. Schematic representation of the modification of ERGO on melamine grafted GCE.
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Scheme 2. The proposed mechanism for the oxidations of PA and UA.
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Table 1
Electrode
Linear range (mol/L)
References
1
Electrochemically reduced graphene/Ni2O3-NiO/GCE
4.0 × 10-8- 1.0 × 10-4
2.0 × 10-8
[49]
2
SWCNT/graphene nanoshett/GCE
5.0 × 10-8- 6.45 × 10-5
3.8 × 10-8
[50]
3
Graphene/GCE
1.0 × 10-7- 2.0 × 10-5
3.2 × 10-8
[51]
4
Graphene/carbon paste electrode
2.5 × 10-6- 1.43 × 10-4
6.0 × 10-7
[52]
5
Nafion/ TiO2/graphene/GCE
1.0 × 10-6- 1.0 × 10-4
2.1 × 10-7
[53]
6
Fe3O4-poly(diallyldimethyl ammonium chloride)/GCE
1.0 × 10-7- 1.0 × 10-4
3.7 × 10-8
[54]
7
Electrochemically reduced graphene/GCE
5.0 × 10-8- 8.0 × 10-4
2.3 × 10-9
[55]
8
ERGO/AT/GCE
4.0 × 10-8- 1.0 × 10-4
6.8 × 10-10
This work
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Detection limit (mol/L)
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Comparison of the linear range and detection limit of PA with graphene modified electrodes
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Urine 2
PA
UA
PA
27.40 a
---
10
20
23.70 a
24.30 a
---
15
25
---
20
30
Found (µmol/L) and (Recoveries) UA PA
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UA
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Urine 3
Added (µmol/L)
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Urine 1
Original (µmol/L)
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Samples
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a
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Simultaneous determination of PA and UA in human urine samples
37.14
19.8
99.3%
99.0%
38.6
24.9
99.7%
99.6%
44.1 99.5%
29.8 99.3%
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represents the concentration of UA found in 0.2 mL urine solution which was diluted to 10 mL by PBS.
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Graphical abstract
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Electrochemically reduced graphene oxide fabricated on melamine grafted GCE and then used
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for the stable determination of paracetamol in presence of uric acid.
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Electrochemically reduced graphene oxide on aminotriazine grafted GCE was prepared Stable determination of paracetamol in presence of uric acid was demonstrated Selective determination of PA in the presence of 50-fold excess UA was achieved Using amperometry, detection of 40 nmol/L PA was obtained Practical application was demonstrated in human urine samples
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• • • • •
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S1572-6657(15)30217-4 doi: 10.1016/j.jelechem.2015.11.039 JEAC 2386
To appear in:
Journal of Electroanalytical Chemistry
Received date: Revised date: Accepted date:
21 July 2015 12 November 2015 25 November 2015
Please cite this article as: Srinivasan Kesavan, S. Abraham John, Stable determination of paracetamol in the presence of uric acid in human urine sample using melamine grafted graphene modified electrode, Journal of Electroanalytical Chemistry (2015), doi: 10.1016/j.jelechem.2015.11.039
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ACCEPTED MANUSCRIPT Stable determination of paracetamol in the presence of uric acid in human
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urine sample using melamine grafted graphene modified electrode
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Srinivasan Kesavan and S. Abraham John* Centre for Nanoscience and Nanotechnology Department of Chemistry, Gandhigram Rural Institute Gandhigram–624 302, Dindigul, Tamilnadu, India
Corresponding author:
Tel: +91 451 245 2371 Fax : + 91 451 245 3031 E-mail : [email protected], [email protected]
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ACCEPTED MANUSCRIPT Abstract Electrochemically reduced graphene oxide (ERGO) on aminotriazine (AT) grafted glassy
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carbon (AT/GC) electrode was prepared by electrochemical reduction of graphene oxide (GO)
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attached through 2,4-diamino-1,3,5-triazine on GC electrode and the resulting electrode was
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utilized for the selective determination of paracetamol (PA) in the presence higher concentration uric acid (UA). The GO attached on AT/GC (GO/AT/GC) and the ERGO films on AT/GC
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(ERGO/AT/GC) electrodes were characterized by scanning electron microscopy (SEM), Raman spectroscopy and cyclic voltammetry (CV). The ERGO modified electrode showed excellent
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electrocatalytic activity towards PA and UA. This electrode not only enhanced the oxidation currents of PA and UA but also shifted their oxidation potentials toward less positive potentials
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in contrast to bare GC, AT/GC and GO/AT/GC electrodes. Bare and GO modified electrodes failed to separate the voltammetric signals of UA and PA. However, the ERGO/AT/GC electrode
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successfully separated the voltammetric signals of them in a mixture and hence used for the simultaneous determination. Further, the modified electrode was effectively used for the selective determination of PA in the presence of 50-fold excess of UA. Using amperometry, detection of 40 nM PA was achieved. The current response of PA was increased linearly while increasing its concentration from 4.0 × 10−8-1.0 × 10−4 mol/L and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The practical application of the present modified electrode was demonstrated by simultaneously determining the concentrations of PA and UA in human urine samples. Keywords: Electrografting; melamine; graphene oxide; self-assembly; Raman spectroscopy; paracetamol.
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ACCEPTED MANUSCRIPT 1. Introduction Paracetamol (PA) (N-acetyl-p-aminophenol or Acetaminophen) (Chart 1A) is an effective
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analgesic and antipyretic drug used for the reduction of fever [1]. It acts as a painkiller by
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inhibiting prostaglandin’s synthesis in the central nervous system and reduces fever by sedating
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hypothalamic heat regulating center [2]. It is also a useful drug for the treatment of osteoarthritis therapy, headache, backache, arthritis and postoperative pain [3-7]. At usual therapeutic doses,
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PA is rapidly and completely metabolized by undergoing glucuronidation and sulfation to
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inactive metabolites which are eliminated in the urine [1,6]. However, PA produces toxic metabolite accumulation at higher doses that mainly causes severe fatal hepatoxicity and
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nephrotoxicity [8,9] besides skin rashes and inflammation of the pancreas [10].
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Uric acid (UA) (Chart 1B) is the main end product of purine metabolism in the human
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body [11]. Increased uptake of serum UA acts as a scavenger of radicals and thus preventing from Parkinson's disease [11]. Low concentration of UA is associated with multiple sclerosis [12]. UA is a major interfering agent for the determination of PA in human urine [1]. The electrochemical method has many advantages over the traditional methods due to its high sensitivity, selectivity, low cost and less time consuming. But, it is very difficult to separate UA and PA using conventional electrodes because both of them are oxidized at similar potential. However, accurate and selective determination of PA in human urine sample is essential for the clinical point of view. Therefore, it is a challenging task for the electrochemists to find a suitable electrode system for the determination of PA in the presence of UA. While searching the literature, few reports are available in the literature for the determination of PA and UA using multiwalled carbon nanotube (MWCNTs)/chitosan composite [13], nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet/chitosan [14], ionic liquid/ 3
ACCEPTED MANUSCRIPT MWCNTs/chitosan modified GCE [15], cetylpyridinium bromide/MWCNTs paste electrode [16] and MWCNTs-cobalt nanoparticles modified electrode [17] prepared by drop cast method.
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However, these methods either involve tedious procedures or series of steps involved to fabricate composite films on electrode surface besides the stability of these film is questionable. Further,
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the drop-casting method has intrinsic drawbacks such as lack of control over film thickness and
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less reproducibility. Hence, it is essential for the researchers to develop a simple method for the
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simultaneous determination of PA and UA in clinical point of view.
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Chemical grafting of aryldiazonium salts derivatives is a widely used method for the modification of conducting substrates over the last two decades [18]. The methodology involves
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reduction of an aryldiazonium ion followed by elimination of dinitrogen to give an aryl radical
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which attacks the substrate with a formation of a covalent bond [18,19]. Grafting of diazonium salts is a powerful method to graft aryl groups bearing a wide variety of functionalities on
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different substrates like carbon [20,21], semiconductor [22], gold nanoparticles [23,24], graphene [25,26] and single-walled carbon nanotubes [27]. Graphene, an allotrope of carbon has received much attention because of its excellent electronic conductivity, high mechanical strength, large specific surface area and high stability [28]. It plays a predominant role in the fields of sensors, nanoelectronics, batteries, solar cells and electrochemical double-layer capacitors [29-31]. Graphene modified electrodes show excellent electrocatalytic activity towards biomolecules and toxic chemicals [32-34]. Most of the researchers reported drop casting method for the fabrication of graphene on different electrode substrates [34-40]. But, selfassembly is the best method to fabricate graphene because film thickness can be easily controlled [41].
4
ACCEPTED MANUSCRIPT Melamine (1,3,5-triazine-2,4,6-triamine) is mainly used as a precursor for pyrolysis processes and permits the insertion of nitride-like units in the carbon structure [42]. Be´langer et
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al. reported the selective diazotization of one amine group of melamine to graft on carbon
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powder [20]. However, grafting of melamine on electrode surface has not been reported so far. The presence of three amine functionalities in the melamine molecule could be useful for
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chemical grafting. In the present study, the GO is assembled on melamine grafted GC electrode.
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Then, the GO was electrochemically reduced at more negative potential to retain the aromatic backbone of graphene and used for the selective determination of PA in the presence high
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concentration of UA.
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2.1. Chemicals
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2. Experimental
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Graphite powder was purchased from Alfa Aesar. Uric acid (UA) and paracetamol (PA) were purchased from Sigma-Aldrich and were used as received. Potassium permanganate (KMnO4), sodium nitrate (NaNO3), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), melamine, sodium nitrite (NaNO2) and hydrochloric acid (HCl) were purchased from Merck (India). The selective diazotation of one amine group of melamine has been described in the reported patent [43]. 1 equivalent melamine was solubilized in 0.5 mol/L HCl, to which 1 equivalent cold NaNO2 was added drop wise to generate the aryldiazonium cations in situ in the electrochemical cell. Indium tin oxide (ITO) substrates were purchased from Asahi Beer Optical Ltd, Japan. GC plates were purchased from Sigma-Aldrich. 0.2 mol/L phosphate buffer (PB) solution (pH 7.2) was prepared by using Na2HPO4 and NaH2PO4. All other chemicals were of analytical reagent grade and were used as received. Double distilled water was used to prepare the solutions.
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ACCEPTED MANUSCRIPT 2.2. Instrumentation Raman spectra were recorded on a Nanofinder ® HE (Tokyo Instruments, INC), 532 nm
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YAG Laser. SEM measurements were carried out by using VEGA3 TESCAN. Electrochemical measurements were performed in a conventional two compartment three electrode cell with a
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mirror polished 3 mm glassy carbon electrode (GCE) as a working electrode, Pt wire as a counter
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electrode and a NaCl saturated Ag/AgCl as a reference electrode. All the electrochemical
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measurements were carried out with CHI model 634B (Austin, TX, USA) Electrochemical Workstation. For differential pulse voltammetry (DPV) measurements, pulse width of 0.06 s,
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amplitude of 0.05 V, sample period of 0.02 s and pulse period of 0.2 s were used. All the
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electrochemical measurements were carried out under nitrogen atmosphere at room temperature.
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2.3. Synthesis of GO
GO was synthesized using the Hummer’s method with a slight modification [44].
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Concentrated H2SO4 (12 mL) was added to flake graphite (0.5 g) and NaNO3 (0.25 g) and the mixture was cooled to 0 °C. KMnO4 was added slowly into the mixture and the temperature was maintained at 20 °C. The reaction mixture was warmed to 35 °C and stirred for 30 min. Then, 23 mL of water was added, which produce an exotherm to 98 °C and the temperature was maintained for 15 min by external heating. The reaction was terminated by the large addition of water (72 mL) and 0.5 mL of 30% H2O2. Finally, the reaction mixture was cooled, washed with 0.1 mol/L HCl and water to remove the metal ions and then dried. 2.4. Fabrication of AT grafted ERGO electrode The fabrication of the AT grafted ERGO electrode is schematically shown in Scheme 1. 2,4-diamino-1,3,5-triazine moieties were introduced on the GC electrode by the in situ generation based on the diazotization of melamine. The resulting AT grafted electrode was
6
ACCEPTED MANUSCRIPT termed as AT/GC electrode. It was rinsed with water and then immersed into exfoliated GO solution (1 mg/mL) for 12 h. The exfoliation of GO was achieved by the sonication of GO for 45
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min. The GO was self-assembled on AT/GC modified electrode via electrostatic interaction
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between positively charged amine groups and negatively charged carboxyl groups present in the GO solution. In addition to that hydrogen bonding between the amine group in melamine and
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oxygen containing groups in GO and π-π interaction between the GO and triazine ring of
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melamine are also possible. This electrode is termed as GO/AT/GC electrode. Then, the GO modified electrode was electrochemically reduced in PB solution (pH 7) for the fabrication of
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ERGO on GC electrode. After electrochemical reduction of GO, the oxygen functional groups are reduced and strong π-π interaction between ERGO and triazine ring of melamine will anchor
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it on the electrode surface. This electrode is termed as ERGO/AT/GC electrode.
3. Results and discussion
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3.1. Grafting of AT and electrostatic assembly of GO and its electrochemical reduction on GCE Fig. 1A shows the CV obtained for the reduction of 2,4-diamino-1,3,5-triazine diazonium cations generated in situ from melamine. The CV shows a broad reduction wave at -0.47 V which was assigned to the formation of the aminotriazine radical and which attaches aminotriazine group on the GCE [19,21]. A similar behavior has been previously reported for the electrochemical reduction of several diazonium salts on various substrates [18,45]. We have chosen the electrografted GCE formed by 1 cycle for the attachment of GO because more than one cycle leads to the formation of multilayers, which has been explained by a polymerization type reaction between the aminotriazine grafted layer and aminotriazine free radicals in solution [18]. Moreover, while increasing the number of grafting cycles, the cross-linked multilayer formation also increases, which will decrease the attachment of GO on grafted GCE. The GO
7
ACCEPTED MANUSCRIPT was assembled on AT grafted GCE by the electrostatic interaction between the negatively charged GO and the positively charged amine terminal of grafted AT layer. The surface attached
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GO was electrochemically reduced to remove the oxygen functional groups of GO present on the
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surface by potential cycling between 0 and −1.4 V for 15 cycles (Fig. 1B). In the first cycle, a cathodic peak at -1.05 V was appeared which might be due to the reduction oxygen functional
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groups present on the surface of GO [46]. From the second cycle onwards, the reduction peak
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was completely vanished. This indicates the complete reduction of oxygen functional groups present on the surface of GO. Due to the removal of oxygen functional groups by
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electrochemical reduction, aromatic backbone of graphene must be regenerated on the surface of
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grafted GCE.
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3.2. Characterization by Raman spectroscopy
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Raman spectroscopy is a very important tool to characterize carbon products due to increase in Raman intensities by the conjugated carbon-carbon double bonds. Fabrication of GO followed by its electrochemical reduction was confirmed by Raman spectroscopy. For Raman spectral measurements, GO and ERGO modified ITO plates prepared by the same procedure were used. Since electrografting is mainly based on radical mechanism the mode of attachment of aryl groups on GC and ITO electrodes is likely the same. Fig. 2 shows the Raman spectra of GO/AT/ITO modified substrate before and after the electrochemical reduction. The GO/AT/ITO modified substrate shows two peaks at 1340 and 1605 cm−1 corresponding to D and G peaks of GO (curve a). After the electrochemical reduction, the D and G peaks were obtained at 1343 and 1602 cm−1, respectively (curve b). A small shift in the G band after electrochemical reduction is due to regeneration of aromatic backbone of graphene [29]. On the other hand, the intensity ratio of D and G bands was increased from 0.90 to 1.10 after the electrochemical reduction indicating 8
ACCEPTED MANUSCRIPT a decrease in the average size of sp2 hybridized lattice of graphene [47]. These results confirm the successful attachment of GO on AT grafted electrode surface by electrostatic assembly and
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its electrochemical reduction removed the oxygen functional groups from the surface of GO.
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3.3. Morphological characterization by SEM
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Fig. 3 shows the SEM images obtained for AT/GC, GO/AT/GC and ERGO/AT/GC substrates. The bare GC plate shows no particles on the surface (Fig. 3A) whereas the AT/GC
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substrate shows a layer like structure [19,21] (Fig. 3B). In contrast to AT/GC substrate, the SEM
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image of the GO/AT/GC substrate shows a layered structure of GO sheets (Fig. 3C). On the other hand, ERGO/AT/GC substrate shows more disordered and aggregated structures which
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appear to be very thin and nothing but a few layers of graphene (Fig. 3D). It also indicates the
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presence of high density edge planes of graphene sheets. This is attributed to an increase in van der Waals attraction between adjacent layers due to the removal of oxygen functional groups
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during electrochemical reduction [29,47]. 3.4. Electrocatalytic activity of ERGO modified electrode towards PA and UA Fig. 4A shows the DPVs obtained for 0.5 mmol/L PA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 M PB solution (pH 5). The bare GCE shows an oxidation peak at 0.51 V (curve a: solid line) whereas AT/GC (curve b: solid line) and GO/AT/GC show oxidation peaks at 0.50 and 0.52 V, respectively (curve c: solid line) for PA. In the subsequent scans, the oxidation peak currents decreased at these electrodes (curve a, b, c: dotted line). This may be due to the surface fouling effect caused by the oxidation products of PA [48]. On the other hand, ERGO/AT/GC modified electrode shows a sharp oxidation peak for PA at 0.49 V (curve d: solid line) with 2-fold, 1.8-fold and 1.7-fold higher current than bare GC, AT/GC and GO/AT/GC electrodes. The oxidation peak was stable even after 5 scans (curve d: 9
ACCEPTED MANUSCRIPT dotted line). The ERGO/AT/GC modified electrode does not show any electrochemical response in the absence of PA in a 0.2 mol/L solution of PB at pH 5 (curve e). It is likely that high
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conducting nature of ERGO/AT/GC electrode effectively prevented the surface fouling effect caused by the oxidation products of PA [29]. The facile electron transfer at ERGO films and the
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π–π interaction between ERGO and PA enhanced the oxidation current of PA with less positive
SC
potential shift. Compared to bare GC, AT/GC and GO/AT/GC electrodes, the ERGO/AT/GC
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modified electrode shows a stable oxidation with greater enhancement of peak current for PA oxidation. Hence, ERGO modified electrode was highly suitable for the determination of PA.
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Fig. 4B shows the DPVs obtained for 0.5 mmol/L UA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 M PB solution (pH 5). Bare GC, AT/GC and
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GO/AT/GC electrodes show broad oxidation waves for UA at 0.43 V, 0.42 V and 0.42 V, respectively. However, an enhanced oxidation current was observed at 0.41 V for UA at the
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ERGO/AT/GC modified electrode. The oxidation peak of UA was stable even after five scans (curve 4B d: dotted line). Two protons and two electrons are involved in the redox reaction of PA and UA (Scheme 2). The effect of scan rate on the oxidations of PA and UA was studied at ERGO/AT/GC modified electrode. The oxidation peak currents of both PA and UA were increased while increasing the scan rates from 25 to 250 mVs-1 (Fig. S1, Supplementary material). A good linearity was obtained while plotting the anodic peak current against the square root of scan rate with correlation coefficients of 0.9998 for PA (Inset, Fig. S1A) and 0.9998 for UA (Inset, Fig. S1B) indicating that the oxidations of PA and UA at ERGO/AT/GC modified electrode were diffusion controlled process. 3.5. Electrochemical behavior of PA and UA in a mixture at ERGO modified electrode
10
ACCEPTED MANUSCRIPT Fig. 5 shows the DPVs obtained for the oxidation of each 0.5 mmol/L PA and UA at bare GC, AT/GC, GO/AT/GC and ERGO/AT/GC modified electrodes in 0.2 mol/L PB solution (pH
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5). The oxidation signal of PA was observed at 0.52 V whereas the oxidation signal of UA was
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not observed at bare GCE (curve a: solid line). After five scans, the oxidation current was decreased and shifted to more positive potential (curve a: dotted line). Similar to bare GCE, the
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oxidation signal of UA was not resolved at AT/GC electrode (curve b: solid line). Although a
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slight enhancement of oxidation current of PA was observed at AT/GC electrode compared to bare GCE, its oxidation peak was not stable on repetitive potential scans (curve b: dotted line).
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The, GO/AT/GC electrode shows the oxidation peak for UA and PA at 0.41 and 0.48 V, respectively (curve c: solid line). After five scans, the oxidation peak potentials of them were
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shifted to more positive potentials with significant decrease in oxidation peak currents (curve c: dotted line). This clearly suggested that bare GC, AT/GC and GO/AT/GC electrodes are not
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suitable for the determination of PA and UA in a mixture. On the other hand, ERGO/AT/GC electrode clearly resolved the oxidation peaks of PA and UA with a potential difference of 90 mV between UA and PA. The oxidation peaks of UA and PA were observed at 0.40 V and 0.49 V, respectively with 1.7-fold higher oxidation currents compared to GO/AT/GC electrode (curve d: solid line). Further, the oxidation peaks were not shifted to more positive potentials even after five potential scans (curve d: dotted line). Hence, ERGO/AT/GC electrode was highly suitable for the simultaneous determination of PA and UA in a mixture. The ERGO/AT/GC modified electrode does not show any electrochemical response in the absence of PA and UA in 0.2 mol/L solution of PB at pH 5 (curve e). 3.6. Simultaneous determination of PA and UA at ERGO/AT/GC electrode Since the ERGO/AT/GC modified electrode successfully separated the oxidation signals of PA and UA, it was used for the simultaneous determination of them. Fig. 6 shows the 11
ACCEPTED MANUSCRIPT simultaneous determination of PA and UA in 0.2 mol/L PB solution (pH 5). A well-defined voltammetric signal was observed for 7 µmol/L UA and 8 µmol/L PA at 0.40 V and 0.49 V,
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respectively (curve a). When the concentration of UA was increased from 7 µmol/L to 161
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µmol/L (curves a-w) and PA was increased from 8 µmol/L to 184 µmol/L (curves a-w), the peak currents of the respective analytes were increased linearly with a correlation coefficient of
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0.9982 for UA and 0.9990 for PA (Insets: Fig. 6). The oxidation potentials of UA and PA were
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not affected while increasing their concentrations. The above results suggested that simultaneous
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determination of PA and UA is possible at ERGO/AT/GC electrode. 3.7. Selective determination of PA and UA at ERGO/AT/GC electrode
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The main objective of the present work is to selectively determine PA in the presence of
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high concentration of UA. Generally, high concentration of UA presents in human urine [1,3]. Therefore, it is very much essential to determine PA in the presence of high concentration of UA.
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Fig. 7A shows the DPVs obtained for the increment of 10 µmol/L of PA to a solution of 500 µmol/L UA. A well defined oxidation peak was observed for PA even in the presence of 50-fold excess of UA. While increasing the PA concentration in the range of 10-220 µmol/L, a linear current enhancement was observed with a correlation coefficient of 0.9992 (Inset, Fig. 7A) without affecting UA peak current. These results show that the ERGO modified electrode can be used to selectively determine PA even in the presence of high concentration of UA. Further, we have also attempted to selectively determine UA in the presence of high concentration of PA to examine the performance of the electrode. Fig. 7B shows the DPVs for the increment of 15 µmol/L of UA to a solution of 400 µmol/L PA. A well defined oxidation peak was observed for 15 µmol/L UA in the presence of 27-fold excess of PA. This indicates that the detection of low concentration of UA is possible in the presence of high concentration of 12
ACCEPTED MANUSCRIPT PA. A linear current enhancement was observed while increasing the UA concentrations in the range of 15-345 µmol/L with a correlation coefficient of 0.9990 (Inset, Fig. 7B) while the peak
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current of PA was unchanged. These results show that the ERGO modified electrode was more
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selective towards the oxidation of UA even in the presence of high concentration of PA.
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3.8. Amperometric determination of PA
Fig. 8A shows the amperometric i-t curve for PA at ERGO/AT/GC modified electrode in a
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homogenously stirred 0.2 mol/L PB solution (pH 5) by applying a potential of +0.6 V. The
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ERGO/AT/GC modified electrode shows the current response for each addition of 40 nmol/L PA. The current response increases and the steady state current response was attained within 3 s
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for further addition of 40 nmol/L PA in each step with a sample interval of 50 s. The dependence
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of response current with respect to concentration of PA was linear from 40 nmol/L to 360 nmol/L with a correlation coefficient of 0.9992 (Inset of Fig. 8A). The amperometric i-t curve for
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each addition of 40 nmol/L PA showed linear current increase without noise. Fig. 8B shows that the amperometric current response was increased linearly with increasing PA concentration in the range of 4.0 × 10−8-1.0 × 10−4 mol/L with a correlation coefficient of 0.9990 (Inset of Fig. 8B) and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The observed linear range and the lowest detection limit of PA at the ERGO/AT/GC modified electrode were compared with the reported modified electrodes [49-55] and are given in Table 1. It is evident from Table 1 that the present modified electrode showed the lowest detection limit (6.8 × 10−10 mol/L) and a wide range determination of PA (4.0 × 10−8-1.0 × 10−4 mol/L) compared to reported papers [49-55]. The anti-interference ability of the ERGO/AT/GC modified electrode was tested towards the detection of PA and UA from common interferents such as Na+, Ca2+, Mg2+, K+, NH4+, Cl-,
13
ACCEPTED MANUSCRIPT F-, CO32-, SO42-, glucose, urea and oxalate by DPV. No change in the current response was observed for 40 nmol/L each PA and UA in the presence of 30 µmol/L of the interferents,
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demonstrating that the present modified electrode is highly selective towards PA and UA. In
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order to study the stability of the ERGO/AT/GC modified electrode, the DPVs for 0.5 mmol/L PA in 0.2 mol/L PB solution (pH 5) were recorded for every 5 min interval. It was found that
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anodic peak current remained same with a relative standard deviation of 1.3% for 15 times
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repetitive measurements indicating that this electrode has a good reproducibility and does not experience surface fouling during the voltammetric measurements. The current response
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decreased about 2.5% in 1 week and 4.3% in 2 weeks when the electrode was kept in PB solution
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(pH 5).
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3.9. Determination of PA and UA in human urine samples The practical application of the present ERGO/AT/GC modified electrode was
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demonstrated by determining the concentration of PA and UA in human urine samples. The human urine samples were collected from laboratory co-workers and were diluted to 50 times using 0.2 mol/L PB solution (pH 5). The standard addition technique was used for the determination of PA and UA in urine samples. DPV of human urine sample in PB solution at ERGO/AT/GC modified electrode shows an oxidation peak at +0.41 V and the obtained peak is due to the oxidation of UA (curve a in Fig. 9). For the simultaneous determination of PA and UA in human urine, commercial samples of PA and UA were spiked to urine sample. After the addition of 10 µmol/L PA and 10 µmol/L UA, the oxidation peak current at +0.41 V was increased which is due to the oxidation of UA whereas new oxidation peak was obtained at +0.49 V which might be due to the oxidation of PA (curve b in Fig. 9). The modified electrode shows good recovery for the determination of PA and UA in human urine samples (Table 2).
14
ACCEPTED MANUSCRIPT Thus, the present modified electrode can be utilized for the determination of PA and UA in real samples.
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4. Conclusions
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We have demonstrated the simultaneous determination of PA and UA using ERGO
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modified AT grafted GCE. Raman and SEM results confirmed the successful attachment of GO and ERGO on AT grafted GCE. The ERGO modified electrode enhanced the oxidation currents
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of PA and UA when compared to bare GC, AT/GC and GO/AT/GC electrodes. The facile
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electron transfer at ERGO film prevented the surface fouling effect and hence the oxidation peaks were stable for several potential scans. Further, the present modified electrode separated
D
the oxidation signals of PA and UA whereas bare GC, AT/GC and GO/AT/GC electrodes failed
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to resolve the oxidation signals of them. Hence, ERGO modified electrode was successfully used for the simultaneous determination of PA and UA. Selective determination of PA in the presence
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of 50-fold higher concentration of UA was also demonstrated using the ERGO/AT/GC electrode. Using amperometry, detection of 40 nmol/L of PA was achieved and the detection limit was found to be 6.8 × 10−10 mol/L (S/N = 3). The practical application of the present modified electrode was demonstrated by simultaneously determining the concentrations of PA and UA in human urine samples. This is the first report in which melamine grafted ERGO film modified electrode used for the selective determination of PA in the presence of high concentration UA.
Acknowledgement S. Kesavan thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, India for the award of Senior Research Fellowship (09/715(0016)/2011-EMR-I).
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[29] M.J. Allen, V.C. Tung, R.B. Kaner, Honeycomb carbon: a review of graphene, Chem. Rev.
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321-325.
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[36] Z. Wang, J. Xia, L. Zhu, F. Zhang, X. Guo, Y. Li, Y. Xia, The fabrication of poly (acridine
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[37] J. Gong, T. Zhou, D. Song, L. Zhang, Monodispersed Au nanoparticles decorated graphene as an enhanced sensing platform for ultrasensitive stripping voltammetric detection of
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ACCEPTED MANUSCRIPT [42] H. Liu, Y. Zhang, R. Li, X. Sun, S. Désilets, H. Abou-Rachid, M. Jaidann, L.-S. Lussier, Structural and morphological control of aligned nitrogen-doped carbon nanotubes, Carbon
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intermediate for ammeline (cyanurodiamide) preparation, USSR patent SU565033, 1977.
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aryldiazonium salt seed layers, J. Mater. Chem. 18 (2008) 5913-5920. [46] 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. [47] S. Stankovich, D.A. Dikin, R.D. Piner, K.A. Kohlhaas, A. Kleinhammes, Y. Jia, Y. Wu, S.T. Nguyen, R.S. Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558-1565. [48] S. Kesavan, S.B. Revin, S.A. John, Potentiodynamic formation of gold nanoparticles film on glassy carbon electrode using aminophenyl diazonium cations grafted gold nanoparticles: Determination of histamine H2 receptor antagonist, Electrochim. Acta. 119 (2014) 214-224.
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ACCEPTED MANUSCRIPT [49] G.T. Liu, H.F. Chen, G.M. Lin, P.P. Ye, X.P. Wang, Y.Z. Jiao, X.Y. Guo, Y. Wen, H.F. Yang, One-step electrodeposition of graphene loaded nickel oxides nanoparticles for
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acetaminophen detection, Biosens. Bioelectron. 56 (2014) 26-32.
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Sens. Actuators, B 161 (2012) 648-654.
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[51] X. Kang, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, A graphene-based electrochemical sensor for sensitive detection of paracetamol, Talanta 81 (2010) 754-759.
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[52] H. Bahramipur, F. Jalali, Sensitive determination of paracetamol using a graphenemodified carbon-paste electrode, Afr. J. Pharm. Pharmacol. 6 (2012) 1298-1305.
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[53] Y. Fan, J.H. Liu, H.T. Lu, Q. Zhang, Electrochemical behavior and voltammetric
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ACCEPTED MANUSCRIPT Figures
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A B C
A B C 0
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–0.5
0
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E vs (Ag/AgCl)/V
–1
0
E vs (Ag/AgCl)/V
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Fig. 1. (A) Cyclic voltammograms obtained for grafting of GC electrode at a scan rate of
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mV s-1 using 2 mmol/L each melamine and NaNO2 in 0.5 mol/L HCl. (B) Electrochemical reduction of electrostatically assembled GO on AT/GC electrode (15 cycles) in 0.2 mol/L PB solution (pH 7.2) at a scan rate of 100 mV s-1.
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a b c 2000
a b c
D G
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D G
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a0 b c
ID/IG = 1.10
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1000
Raman Shift (cm–1 )
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ID/IG = 0.90
2000
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0
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a b c
2000
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1000
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Intensity (a.u)
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0
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Fig. 2. Raman spectra obtained for (a) GO/AT/ITO and (b) ERGO/AT/ITO substrates.
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AB C D
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Fig. 3. SEM images obtained for (A) bare GC, (B) AT/GC, (C) GO/AT/GC and (D) ERGO/AT/GC substrates.
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10
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aa bb cc ddee
10 10
abcde abcde
0 0
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0.6 0.6
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PA abcde
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b
a e
0
0.2
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E vs (Ag/AgCl)/V
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E vs (Ag/AgCl)/V
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Fig. 4. (A) DPVs obtained for 0.5 mmol/L PA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d)
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ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of PA at GC/AT/ERGO electrode. (B) DPVs obtained for 0.5 mmol/L UA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d) ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of UA at ERGO/AT/GC electrode. (solid line: 1st scan; dotted line: 5th scan).
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d
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c
10
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PA
a
b
e
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0 0.2
0.4
0.6
E vs (Ag/AgCl)/V
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Fig. 5. DPVs obtained for each 0.5 mmol/L of UA and PA at (a) bare GC, (b) AT/GC (c) GO/AT/GC and (d) ERGO/AT/GC electrodes in 0.2 mol/L PB solution (pH 5). (e) Absence of
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UA and PA at ERGO/AT/GC electrode. (solid line: 1st scan; dotted line: 5th scan).
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Y = 0.04 X + 0.74 2
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PA
R = 0.9982
6
UA
w
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3
0
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5
a
0.4
40
80
120
a 160
Concentration of UA()
Y = 0.05 X + 0.52 2
R = 0.9990 6
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50
100
150
b
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Concentration of PA()
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0.5
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E vs (Ag/AgCl)/V Fig. 6. DPVs obtained for the simultaneous increment of 7 µmol/L of UA and 8 µmol/L of PA (curves a–w) at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5). Insets: Plot of concentration of (a) UA and (b) PA vs. current.
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0 100
4
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Concentration of PA()
PA
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t
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10
PA
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A B C
Y = 0.08 X + 1.68
2
R = 0.9992
A B UA C
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a
0 0.3
0.6
E vs (Ag/AgCl)/V
0.4
0.5
0.6
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Fig. 7. (A) DPVs obtained for each increment of 10 µmol/L PA in 500 µmol/L of UA (curves a– t) at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5). Inset: plot of the concentration of PA vs. current. (B) DPVs obtained for each increment of 15 µmol/L UA in 400 µmol/L of PA (curves a–w) at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5). Inset: plot of the concentration of UA vs. current.
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A B C
Y = 0.086 X + 0.01
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ab cd e f g h i j k
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b
60
90
0.5
5R
5
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0
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a
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0
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200200
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Time (sec)
Time (sec)
0 0 0
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modified
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200 400 600 200 400400 600600 400 200 600 Time (sec) Time (sec) 400 Time (sec)600 0 Time200 0 (sec) 0 200 600 Time (sec) 0 0 400 0 200 600 400 600 200 400 200 400 Time (sec) 600 200 400 600 200 400 600 200 400 600 0 (sec) Time (sec) Time Amperometric i-t curve forTime the determination of PA at ERGO/AT/GC 0 (sec) Time (sec) Time (sec) 0 Time 400 (sec) 200 600 200 400 600 200 400 Time (sec) 600
Time (sec) 0
Fig. 8. (A)
0.05
5 5 5
Concentration of PA (nM)
400
5 50 55
= 0.9992
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5
5
Y = 0.024 X + 0.30 2
0.1
5 5 5
5
a ba bc dc de ef gf gh hi ji kj k ab cd e f g h i j k ab cd e f g h i j k aababbccdcdde eef fgf gghhhi iji jkjkk ab cd e f g h i j k aa bb cc dd ee ff gg hh ii jj kk
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I/nA
I/nA
Concentration of PA (M)
Time (sec) the concentration of 40 electrode in 0.2 mol/L PB solution (pH 5). Each addition Time increases (sec)
nmol/L of PA at regular interval of 50 s. Eapp = +0.6 V. (B) Amperometric i-t curve for the determination of PA at ERGO/AT/GC modified electrode in 0.2 M PB solution (pH 5). Each addition increases the concentrations of (a) 0.04, (b) 0.1, (c) 0.2, (d) 0.5, (e) 1, (f) 2, (g) 5, (h) 10, (i) 20, (j) 50 and (k) 100 µmol/L PA at ERGO/AT/GC modified electrode in 0.2 M PB solution (pH 5) at a regular interval of 50 s. Eapp = +0.6 V. Insets (a) and (b): Plot of concentration of PA vs. current.
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1.5
0.5
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1
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b
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0.2
PA
a 0.4
0.6
E vs (Ag/AgCl)/V
Fig. 9. DPVs obtained for (a) human urine and (b) after spiked with commercial 10 µmol/L of UA and 10 µmol/L of PA at ERGO/AT/GC electrode in 0.2 mol/L PB solution (pH 5).
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AC CE P
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A B
Chart 1. Structures of (A) Paracetamol and (B) Uric acid.
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A B
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Scheme 1. Schematic representation of the modification of ERGO on melamine grafted GCE.
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Scheme 2. The proposed mechanism for the oxidations of PA and UA.
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Table 1
Electrode
Linear range (mol/L)
References
1
Electrochemically reduced graphene/Ni2O3-NiO/GCE
4.0 × 10-8- 1.0 × 10-4
2.0 × 10-8
[49]
2
SWCNT/graphene nanoshett/GCE
5.0 × 10-8- 6.45 × 10-5
3.8 × 10-8
[50]
3
Graphene/GCE
1.0 × 10-7- 2.0 × 10-5
3.2 × 10-8
[51]
4
Graphene/carbon paste electrode
2.5 × 10-6- 1.43 × 10-4
6.0 × 10-7
[52]
5
Nafion/ TiO2/graphene/GCE
1.0 × 10-6- 1.0 × 10-4
2.1 × 10-7
[53]
6
Fe3O4-poly(diallyldimethyl ammonium chloride)/GCE
1.0 × 10-7- 1.0 × 10-4
3.7 × 10-8
[54]
7
Electrochemically reduced graphene/GCE
5.0 × 10-8- 8.0 × 10-4
2.3 × 10-9
[55]
8
ERGO/AT/GCE
4.0 × 10-8- 1.0 × 10-4
6.8 × 10-10
This work
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Detection limit (mol/L)
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Comparison of the linear range and detection limit of PA with graphene modified electrodes
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Urine 2
PA
UA
PA
27.40 a
---
10
20
23.70 a
24.30 a
---
15
25
---
20
30
Found (µmol/L) and (Recoveries) UA PA
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UA
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Urine 3
Added (µmol/L)
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Urine 1
Original (µmol/L)
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Samples
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a
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Simultaneous determination of PA and UA in human urine samples
37.14
19.8
99.3%
99.0%
38.6
24.9
99.7%
99.6%
44.1 99.5%
29.8 99.3%
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represents the concentration of UA found in 0.2 mL urine solution which was diluted to 10 mL by PBS.
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Graphical abstract
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Electrochemically reduced graphene oxide fabricated on melamine grafted GCE and then used
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for the stable determination of paracetamol in presence of uric acid.
2000
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Electrochemically reduced graphene oxide on aminotriazine grafted GCE was prepared Stable determination of paracetamol in presence of uric acid was demonstrated Selective determination of PA in the presence of 50-fold excess UA was achieved Using amperometry, detection of 40 nmol/L PA was obtained Practical application was demonstrated in human urine samples
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• • • • •
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