Nitrogen-Doped Graphene as a Robust Scaffold for the Homogeneous

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Nitrogen-doped graphene as a robust scaffold for the homogenous deposition of copper nanostructures: A non-enzymatic disposable glucose sensor N.S.K. Gowthaman, M. Amal Raj, and S. Abraham John ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b02390 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 10, 2017

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ACS Sustainable Chemistry & Engineering

Nitrogen-doped graphene as a robust scaffold for the homogenous deposition of copper nanostructures: A non-enzymatic disposable glucose sensor N.S.K. Gowthamana, M. Amal Rajb and S. Abraham Johna* a

Centre for Nanoscience and Nanotechnology Department of Chemistry, The Gandhigram Rural Institute Gandhigram-624 302, Dindigul, India. b

Present Address: Department of Chemistry, Loyola College Chennai - 600 034, Tamil Nadu, India.

*E-mail: [email protected]; [email protected] *Tel: +91 451 245 2371; Fax: + 91 451 245 3031 KEYWORDS: Nitrogen-doped graphene; copper nanostructures; electrodeposition; scanning electron microscopy; electrocatalytic activity; glucose sensor; screen printed carbon electrode

ABSTRACT: The attachment of nitrogen-doped graphene (NG) on glassy carbon electrode (GCE) followed by electrodeposition of copper nanostructures (CuNS) were described in this paper. Nitrogen-doped graphene oxide (NGO) was prepared by intercalating melamine into graphene oxide (GO) by sonication. The doping of nitrogen was confirmed from the characteristic peaks at 285.3 and 399 eV in the XPS corresponding to C-N bond and nitrogen, respectively. The presence of amine groups on the N-GO was exploited to attach them on GCE via Michael’s reaction. Subsequently, N-GO was electrochemically reduced to form NG by reducing the oxygen functionalities present on the N-GO. Then, the CuNS on NG modified electrode was prepared by electrodeposition at various applied potentials with different deposition times. The homogeneous deposition of cubic, spherical, quasi-dendritic and dendritic NS at the applied potentials of 0, -0.10, -0.30 and -0.40 V, respectively was evidenced from scanning electron microscopy (SEM) studies. The surface energy of the system can be reduced by the intercalated nitrogen in the graphene layer via doping. Hence, the NG layers with large surface area act as a robust scaffold for the deposition of CuNS homogeneously. Further, the electrocatalytic activity of the NG-CuNS modified GCE towards glucose oxidation was studied. Compared to NG and CuNS, the NG-CuNS exhibited 2-fold higher oxidation current. Further, it was found that the electrocatalytic activity of the composite electrode depends on the shape of the CuNS. Among the different CuNS, the NG-dendritic CuNS electrode exhibited higher electrocatalytic activity. Finally, the practical applicability of the present sensor was demonstrated by fabricating NG-dendritic CuNS on screen printed carbon electrode for the determination of glucose in human blood serum and urine samples.

1. INTRODUCTION An incredible scientific and technological attraction on graphene has been witnessed in recent ACS Paragon Plus Environment


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years due to its unique electronic, optical and chemical properties.1 The graphene acts as a receptor to adsorb molecules in terms of π-π interactions and charge transfer which leads to construct high sensitive electrochemical sensors for several biomolecules.1,2 It is a open structured two dimensional plenary sheet, which is distinct from the rolled structured carbon nanotubes and thus it could be effectively exploited for catalysis.3,4 Generally, graphene oxide (GO), prepared by Staudenmaier and Hummer’s methods,5-7 has been used as a precursor for the synthesis of graphene. The GO can be reduced by both chemical and electrochemical methods using reducing agents and applying more negative potential, respectively to retain its aromatic backbone.2, 8-12 It has been shown that the electrochemically reduced graphene sheets have higher conductivity than that of the chemically reduced graphene because of the higher oxygen/carbon ratio of the former than the later.10,13 Graphene was fabricated on different electrodes by various approaches for the construction of sensor devices which include electrodeposition of GO,3,13 drop-casting of graphene, spray- and spincoating, Langmuir-Blodgett, layer-by-layer assembly3 and drop-casting of GO and its subsequent electrochemical reduction.13,14 Although graphene films were successfully fabricated by the above strategies, the thickness and uniformity cannot be controlled by these methods. Self-assembly method can be used to attach nanomaterials successfully on solid substrates.15 Few researchers have reported the attachment of graphene on different solid substrates by self-assembly method.16-19 Recently, self-assembly of GO on GCE through 1,6-hexadiamine SAM was reported from our group.18-20 Copper and copper oxide materials have received enormous attention owing to their excellent electrocatalytic activity towards glucose oxidation besides their low cost and significant catalytic activities.21-23 In contrast to other carbon materials, graphene possesses massive surface area and immense electrical conductivity. Therefore, it is desirable to deposit copper nanostructures (CuNS) on graphene sheets for the construction of an electrochemical glucose sensor.24-26 It is expected that the synergistic catalytic activity of the composite electrode stems from the combination of huge electrochemically active surface area and high conductivity of the carbon materials besides high catalytic activity of CuNS.24-26 Doping of nitrogen in graphene not only leads to significant changes in the semiconducting property27,28 but also leads to the formation of composite materials by interacting with transition metals.29-31 Further, the surface energy of the system can be reduced by the doped nitrogen atoms ingrained in the graphene lattice. Hence, N-doped graphene acts as a strong platform for the uniform dispersion of metal nanoparticles.29-32 Recently, nitrogen-doped graphene-copper composite materials for different applications were reported.2932

Zhou et al. reported an efficient oxygen reduction catalyst based on copper oxide/N-doped reduced

graphene oxide (N-rGO) nanocomposite at low temperature (90º) aqueous process (mixture of aqueous GO and NH3).29 Zhang et al. constructed a N-doped graphene (NG) supported few-layer graphene shells incorporated copper nanoparticles via in-situ chemical vapor deposition for SERS application.30 Kumar et

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al. studied the interaction of [Cu(bpy)2(H2O)2] complex with NG for photocatalytic reduction of CO2.31 Jiang et al. demonstrated the copper nanoparticles decorated NG by annealing of GO, copper nitrate and glycine at 500º C as a non-enzymatic glucose sensor.32 Since the reported methods are based on thermal annealing and hydrothermal process, they require more time and also high cost. Herein, we have reported an economically viable, less time consuming synthesis of N-GO via intercalation of melamine with GO by sonication for the first time. Moreover, the electrocatalytic activity with respect to copper morphology in the nanocomposite is not yet reported. Thus, the present study aims to deposit different copper nanostructures on NG modified glassy carbon electrode (GCE) and study the effect of CuNS morphology on glucose oxidation. In the present study, NG films were fabricated on GCE by attaching the N-GO through electrostatic interaction. Subsequently, the N-GO was electroctrochemically reduced to form NG. The different shapes of CuNS were electrodeposited on the NG modified GCE at +0.20, +0.10, 0.0, -0.10, -0.20, -0.30, -0.40, -0.50 and -0.60 V. The resulting electrodes were characterized by different spectroscopic, microscopic and electrochemical techniques. Further, the oxidation of glucose was studied at different shapes of CuNS modified NG electrodes. Interestingly, dendritic CuNS modified NG electrode shows superior electrocatalytic activity towards the oxidation of glucose in contrast to other shapes. 2. EXPERIMENTAL SECTION 2.1. Preparation of N-GO. The GO suspension (1mg/1mL water) was exfoliated in basic medium (pH 9) by adding sodium carbonate and sonicated for 30 min for the complete exfoliation of GO. Then, a mixture of 15 mM of melamine and the GO suspension was sonicated for 1 h to intercalate melamine with GO. It is expected that the amine functionality of melamine was attached to GO layers via nucleophilic addition to the epoxy functionality of GO. The melamine intercalated GO is termed as N-GO (Scheme 1A). For the SEM analysis, the as-synthesized N-GO suspension was centrifuged and the resulting powder was dried in a stream of N2 gas before measurement. 2.2. Fabrication of GCE with N-GO and NG. The GCE was cleaned according to the reported procedure.18 For the fabrication of N-GO on GCE, the GCE was immersed into a solution of exfoliated NGO for 12 h. The N-GO was attached on GCE through Michael’s reaction. This electrode is designated as GCE/N-GO. Then, the resultant electrode was electrochemically cycled between 0 to -1.3 V in 0.2 M PB solution (pH 7) for the fabrication of NG.20,34 This electrode is designated as GCE/NG (Scheme 1B). 2.3. Electrodeposition of copper nanostructures on GC/NG electrode. The copper nanostructures (CuNS) were electrodeposited on NG modified GC electrode using 0.01 M CuSO4 in 0.1 M H2SO4 at the applied potentials of +0.20, +0.10, 0, -0.10, -0.20, -0.30, -0.40, -0.50 and -0.60 V (vs. Ag/AgCl) for 400 s. This electrode is represented as GCE/NG-CuNS (Scheme 1B). To compare the performance of the NG/CuNS electrode with electrochemically reduced graphene oxide (ERGO), ERGO was fabricated on



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GCE using the same procedure. But, instead of taking GO with melamine, the GO was initially assembled on the GCE using 1,6-hexanediamine as a linker. The GC/GO electrode was then electrochemically reduced to form ERGO on GCE. For Raman, XPS, XRD, SEM and EDAX measurements, indium-tin-oxide (ITO) was used whereas GC plate was used for ATR-FT-IR measurement. For the determination of glucose in real samples, NG-dendritic CuNS were fabricated on screen printed carbon electrode. 3. RESULTS AND DISCUSSION 3.1. Fabrication of N-GO on GCE and its electrochemical reduction. The N-GO was attached on GCE through the Michael’s nucleophilic addition in which the attachment of free amine groups of NGO on olefinic double bonds of GCE.35,36 The presence of oxygen functionalities including epoxide, carboxyl and hydroxyl on the surface of N-GO were electrochemically reduced by cycling the GC/N-GO electrode between 0 and -1.30 V for 15 cycles and thereby the aromatic lattice of graphene was retained (Figure 1). The observed reduction peak at -1.15 V in the first cycle is corresponding to the reduction of oxygen functionalities and it was drastically decreased in the next cycle and completely vanished after six cycles. This indicates the regeneration of sp2 backbone of graphene after the electrochemical reduction of N-GO. 3.2. Characterization by ATR-FT-IR spectroscopy. The attachment of N-GO followed by its electrochemical reduction were confirmed by ATR-FT-IR spectroscopy. Figure S1 exhibits the ATR-FTIR spectra obtained for GC/N-GO and GC/NG substrates. The N-GO modified substrate shows the peaks at 1080 (ν(C-O)), 1180 (ν(C-N)), 1420 (C=O stretching of carboxylate groups), 1540 (N-H bending), 1618 (ν(C=C)), 1680 (C=O stretching of amide), 3130 (ν(C-H stretching)), 3320 (ν(N-H stretching)) and 3590 cm-1 (ν(O-H)). After electrochemical reduction the peaks at 1080 (ν(C-O)), 1420 (C=O stretching of carboxylate groups) and 3590 cm-1 (ν(O-H)) were disappeared indicating the removal of oxygen functionalities present in the N-GO substrate. The peak assignments are shown in Table S1. The above results confirm the successful fabrication of NG. 3.3. Characterization by Raman spectroscopy. Raman spectroscopy yields information about the defects, ordered and disordered structures.3 Figure 2 shows the Raman spectra of N-GO and NG modified substrates. The N-GO fabricated substrate displays D and G bands at 1349 and 1590 cm-1, respectively (curve a). A noticeable increase in intensity of D band when compared to G band was observed after the electrochemical reduction of N-GO (curve b). The D and G bands were appeared at 1340 and 1581 cm-1, respectively.27 The extent of defects due to vacancies, distortion and edges in carbon materials can be usually identified from the ratio of the intensities of D and G bands (ID/IG). From Figure 2, the ID/IG value was found to be 0.97 and 1.25 for N-GO and NG, respectively. The larger ID/IG (1.25) value clearly reveals the greater sp2 character as well as the decline in the average size of the sp2 domain due to electro-


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chemical reduction. Moreover, it confirms that the electrochemical reduction of N-GO not only leads to alteration in its structure but also introduces some structural defects.37 The obtained results reveal the successful formation of N-GO and NG. From the D and G band intensities, the average crystalline size of the N-GO and NG modified substrates was calculated using Eqn. 1.38

(1) where, La and λ represent the average crystalline size and the laser wavelength (532 nm), respectively. The average crystalline size was found to be 19.80 and 15.38 nm for N-GO and NG modified substrates, respectively. The obtained results suggest that the average crystalline size of N-GO was decreased after electrochemical reduction. 3.4. Characterization of NG modified substrate by SEM and XPS. SEM images of N-GO and NG modified substrates are shown in Figure 3. The N-GO film modified substrate shows homogeneously ordered structure (Figure 3a) whereas NG shows very thin isolated layers which are nothing but few layers of graphene (Figure 3b). Further, the NG layers appeared as folded into a typical crumpled structure after the electrochemical reduction of N-GO. It is attributed that aggregation of GO takes place during electrochemical reduction due to the increased π-π interaction between the graphene layers.1 The grainy and heterogeneous nature of NG films might be due to high density of edge planes of graphene layers. The fabrication of NG on GCE was further characterized by XPS. Figure 4A shows XPS survey spectrum of NG modified substrate. It shows an asymmetric peak at 284 eV along with peaks at higher binding energy side. The observed peaks at 284, 531 and 399 eV correspond to the C 1s peak of sp2 carbon, O 1s spectrum of various oxygen functionalities and the amine functionality of the melamine intercalated with the GO, respectively. Figure 4B shows the C 1s spectra of NG fabricated substrate. The C 1s spectra show three peaks at 284.5, 285.3 and 288.1 eV corresponding to sp2 carbon, C–N bond and C=O bond, respectively. The peak at 285.3 eV due to C-N bond, which confirms the intercalation of melamine into GO. Further, the intensity of sp2 carbon was higher than the oxygen-containing functional groups (Figure 4C). This suggests that the electrochemical reduction of N-GO results the removal of oxygen functionalities.10 The bonding configurations of nitrogen atoms in NG substrate were characterized from N1s spectra. The N 1s spectrum of NG substrate can be deconvoluted into four peaks at 398.2, 399.2, 401.1 and 402.6 eV (Figure 4D). In general, the pyridine-like and pyrrole-like nitrogens provide the πconjugated system with a pair of π-electrons in the GO layers, which appeared at 398.2 and 399.2 eV, respectively. Carbon atoms present on the GO layers were replaced with nitrogen atoms in the form of ‘graphitic’ nitrogen.27 The corresponding peak for graphitic nitrogen is expected to appear in the region of 400.8-401.3 eV. The peak at the region of 402.3-402.9 eV is due to oxidized nitrogen.27,28 The deconvo-



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luted N1s spectra of NG substrate shows the peaks at 401.1 and 402.6 eV in the higher binding energy region corresponding to graphitic nitrogen and the oxidized nitrogen, respectively. This result is well supported with the earlier reports and confirms the doping of nitrogen via intercalation of melamine to the graphene layers.27-29 3.5. Characterization of NG-CuNS modified substrate by SEM and XPS. Figure 5a shows the SEM image obtained for electrodeposition of copper on ITO surface at an applied potential of -0.10 V for 400 s. When copper was deposited directly on ITO surface, agglomerated spherical particles with the size of 400 nm were formed. On the other hand, deposition of copper at NG modified ITO surface leads to the formation of uniform and isotropic CuNPs under identical conditions (Figure 5b). Interestingly, the growth and size of CuNPs was controlled by NG layers. It has been already reported that the homogeneous deposition of metal nanoparticles on NG film was achieved in the presence of doped nitrogen atoms because it reduces the surface energy of the graphene lattice.29-32 Hence, the N-doped graphene acts as a strong platform for the homogeneous deposition of CuNPs. The size of the deposited CuNPs was in the range of 100 nm. It seems that the CuNPs were not only deposited on the surface of NG layers but also intercalated into the graphene layers. Thus, the CuNPs deposited on NG modified electrode expected to act as a better catalyst for electrocatalytic applications. Further, the applied potential of copper deposition on NG film will affect the morphology of the CuNS which in turn will also affect the electrocatalytic application. Thus, the effect of applied potential of CuNS deposition on the NG film was studied in detail. The electrodeposition of Cu on NG film was also carried out by varying the applied potentials. When the potential was varied from 0.0 to +0.20 V, deposition rate is very low and only few spherical particles were observed (Figures 6a and b). On the other hand, cubic CuNS were formed on the NG surface at 0 V (Figure 6c) and spherical CuNS were deposited at -0.10 V (Figure 6d). The spherical CuNS were aggregated when the potential was switched to -0.10 V (Figure 6e). The density of the spherical CuNS is low and few of them were grown like quasi-dendrites at an applied potential of -0.30 V (Figure 6f). The size of each branch of the dendrites is about 200 nm. Further, when the potential is switched to -0.40 V, well defined dendrites were grown on the surface of the NG with the size of 50 nm of each branch (Figure 6g). Here, homogeneous as well as heterogeneous nucleation may take place concomitantly. For heterogeneous nucleation, the initially deposited particles act as seeds to direct the formation of dendritic morphology whereas the formation of dendrites is a consequence of attachment of primary particles in the homogeneous nucleation.39 Recently, Xie et al. reported one step electrodeposition of rGO/Cu composite film and studied the morphology of the composite film by varying the applied potential. They observed the formation of nanograins and pine tree leaf nanostructures at -1.20 and -0.40 V, respectively.40 While switching the applied potentials to -0.50 and -0.60 V, the well defined dendrites were aggregated (Figures 6h and i). This may be due to overgrowth of dendritic CuNS.


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It can be seen from Figure 6, the primary morphology of the deposited CuNS was found to be cubic. While switching the applied potential to more negative, the particle size was decreased monotonically which is escorted by a corresponding increase in the particle number density. Obviously, a more negative applied potential on the working electrode generates more nucleation sites, which is attributed to the increase in the particle number density. In other words, the applied potential via the Nernst equation directs the relative surface electroactive species concentration. Further, the deposition of Cu on NG film follows the instantaneous nucleation mechanism followed by diffusion-limited growth.41,42 The formation of different CuNS over NG film is based on nucleation mechanism as follows: (i) Cu2+ is reduced to form Cu at an appropriate applied potential and formed nuclei after long duration, leading to the regular spherical particles. (ii) The spherical particles tend to join together to form a one-dimensional root and subsequently the nucleation initiated on the active sites from the root, resulting in the branched growth of dendritic NS. (iii) Further increasing the applied potential, the existing nuclei will grow onto the aggregated CuNPs and the free nanoparticles diffused frequently toward the aggregate and further immobilized, forming a large aggregate. It has been already demonstrated that dendritic CuNS act as a better electrocatalyst than other structures.42 This is due to the porous and hierarchical nature of the dendritic CuNS. Hence, the condition from which dendrites were formed is optimized for further studies. The time dependent growth of dendritic CuNS on NG film was monitored by SEM, keeping the applied potential of -0.40 V as constant. At an electrolysis time of 100 s, the density of the deposited NS is very low and most of the particles obtained are spherical crystals with an average size of 100 nm without any dendritic morphology (Figure S2a). When the deposition time was increased to 200 s, the non-uniform quasi-dendritic structures with the average branch size of 200 nm were formed (Figure S2b). Further increasing the electrolysis time to 400 s, more uniform dendritic structures were grown on the NG film and the dendritic coverage on the NG film was very high (Figure S2c). The dendrites were grown as irregular shape and were highly dense and the size of the dendritic branch was increased to 600 nm while increasing the deposition time to 600 s (Figure S2d). This is due to the aggregation of dendrites. The obtained results reveal that 400 s is ideal for the deposition of well defined dendritic CuNS. The dendritic CuNS modified NG film was further characterized by XPS. Figure 7A shows the XPS survey spectrum of NG-dendritic CuNS modified substrate. The peak at 932 eV corresponding to the Cu2p region along with the C1s, O1s and N1s peaks confirmed the deposition of CuNS at NG film. Moreover, XPS is used to find out the nature of copper on the NG modified substrate. The samples were kept under inert atmosphere to avoid copper oxidation. The Cu2p spectra of NG-dendritic CuNS substrate show peaks at 932.5 and 952.8 eV corresponding to 2p3/2 and 2p1/2 signals of elemental Cu, respectively (Figure 7B). The absence of the strong satellite peaks at 935 and 955 eV confirmed the absence of Cu2+. It



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is known that Cu+ has the same binding energy with the Cu(0). It is distinguished from Cu(0) by the presence of two weak satellite peaks at 939-945 eV. As shown in Figure 7B, the weak satellite peak at 939945 eV was absent. This confirms the absence of Cu+. The above XPS results suggest that the copper present on the NG substrate is in the form of Cu(0). 3.6. Characterization by XRD and EDAX. Further, the dendritic CuNS modified NG film was characterized by XRD and EDAX analysis. Figure S3 shows the XRD patterns of NG and NG-dendritic CuNS. Figure S3a shows a broad peak around 26.0° corresponding to crystalline nature of graphene. The peaks at 42.8°, 49° and 73.7° are the characteristics of (111), (200) and (220) planes of Cu (Figure S3b) (JCPDS File No. 85-1326). The peaks obtained at 30.57°, 35.32°, 49.58° and 50.85° were corresponding to ITO substrate (JCPDS File No. 89–4599). The obtained XRD results confirm the successful formation of NG and dendritic CuNS. The doping of nitrogen via intercalation of melamine to the GO and deposition of dendritic CuNS on NG film was further confirmed by EDAX analysis. Figure S4 shows the EDAX spectra obtained for N-GO, NG and NG-dendritic CuNS modified substrates. N-GO shows the peaks at 0.27 and 0.53 keV correspond to carbon and oxygen, respectively. The peak corresponding to nitrogen at 0.39 keV confirms the doping of nitrogen to GO (Figure S4A). It was found that the oxygen intensity was decreased after the electrochemical reduction (Figure S4B) and the presence of nitrogen confirms the fabrication of NG on the electrode surface. The characteristics peaks at 0.92 and 8.0 keV show the electrochemically deposited Cu (Figure S4C). These results suggest that nitrogen was doped to the GO and the CuNS were successfully deposited on NG film. 3.7. Characterization of GC/NG-CuNS electrode by EIS and chronocoulometry. The conducting nature of NG-CuNS modified electrode was examined by electrical impedance spectroscopy (EIS) study. Figure S5 shows the Nyquist plots obtained for bare GCE, ERGO and NG fabricated GCE and CuNS deposited graphene and NG films in 1 mM [Ru(NH3)6]3+ containing 0.2 M PB solution (pH 3) from 0.01 to 100,000 Hz. A Randles circuit model [RS(C-RCT)] (inset: Figure S5) was employed to fit the impedance data, where RS is the solution resistance and C is the capacitance. The semicircle obtained from the Nyquist plot can be used to calculate the charge transfer resistance (RCT). The bare GCE shows a large semicircle of about 27.06 kΩ (curve a). When it is fabricated with ERGO film, RCT value of 24.12 kΩ was obtained (curve b), whereas the GCE/NG exhibits 22.64 kΩ (curve c). The decrease in the RCT value is attributed to the high surface area provided by NG film. Interestingly, when CuNS were deposited on the ERGO and NG film, the RCT value was drastically decreased to 1.84 and 1.44 kΩ, respectively (curve d and e). The higher conductivity of the NG-CuNS is attributed to the doping of N on the graphene lattice, which in turn reduce the surface energy to favor the deposition of CuNS on its lattice. These results reveal that the NG acts as a strong platform for the uniform dispersion of CuNS. The obtained RS, C,


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and RCT values were given in Table S2. The heterogeneous electron-transfer rate constant (ket) was calculated using the equation (2).43

ket =

RT n2F 2ARCT C 0

(2)

where RCT is charge transfer resistance, A and C0 represent the electrode area (0.07 cm2) and the concentration of the redox couple in the bulk solution (1 mM), respectively and n refers to number of electrons transferred per molecule of the redox probe (n = 1 for [Ru(NH3)6]3+) and rest of the symbols have their usual representations. The calculated ket values are 1.40 × 10−4, 1.57 × 10−4, 1.68 × 10−4, 2.06 × 10−3 and 2.63 × 10−3 cm s−1 for bare GCE, ERGO, NG, ERGO/CuNS and NG/CuNS modified electrodes, respectively. The obtained higher ket value for GCE/NG/CuNS reveals that the electron transfer reaction was faster at this electrode compared to graphene/CuNS electrode. Further, the electrochemically active surface area (A) of the ERGO, NG and CuNS fabricated ERGO and NG electrodes was calculated from the Anson equation (3)44,45 as follows:

a A =

2 n F CD1/2 π1/2

(3)

where, a is the slope obtained from Anson plot (Q vs. t1/2), which is directly obtained from chronocoulometric experiment, n represents the number of electrons transferred, F is the Faraday’s constant, C and D represents the concentration of the redox couple in the bulk solution (1 mM) and diffusion coefficient (8.4 × 10-6 cm2 s-1), respectively. The electrochemically active surface area was found to be 0.018, 0.022, 0.561 and 0.781 cm2 for ERGO, NG, ERGO/CuNS and NG/CuNS fabricated GCEs, respectively. The lone pair electron present on nitrogen interacts with the deposited CuNS to form the composite material. Moreover, the surface energy of the system is expected to be reduced by the doped nitrogen atoms ingrained in the graphene lattice and hence it provides higher electrochemically active surface area.27-31 3.8. Electrocatalytic oxidation of glucose. It has been well established that graphene and CuNPs exhibit good electrical conductivity24-26,30-32 and therefore the electrodes modified with them can be effectively utilized for electrocatalytic applications. In this study, the electrocatalytic activity of CuNS deposited NG film was investigated using glucose as a probe. Figure 8 shows the CVs obtained for 1.0 mM glucose at bare GC, GC/NG, GC/CuNPs, GC/ERGO-CuNPs and GC/NG-CuNPs electrodes in 0.1 M NaOH at 50 mV s-1. As shown in Figure 8, neither the bare GC (curve a) nor the NG modified GC (curve b) electrode shows response for glucose oxidation. In the anodic scan, the ramp in current starting about +0.30 V with a shoulder oxidation hump at +0.55 V due to glucose oxidation was observed at GC/CuNPs elec-



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trode (curve c). This suggests the essential role of CuNPs in the electrochemical oxidation of glucose. On the other hand, the GC/ERGO-CuNPs electrode exhibited noticeable increase in current with 80 mV less positive potential shift (curve d). The observed higher electrocatalytic activity is due to huge surface area possessed by ERGO, confirming the important role of the graphene sheets towards glucose oxidation, besides CuNPs. Interestingly, when the CuNPs were deposited on NG film, the glucose oxidation current was substantially increased by 2-fold with 60 mV less positive potential (curve e). The above results clearly depicts that the GC/NG-CuNPs electrode is a superior electrocatalyst for glucose oxidation when compared to GC/CuNPs and GC/ERGO/CuNPs electrodes. This is mainly due to not only the huge surface area provided by NG but also the higher conductivity and fast electron transfer rate offered by NG film, as evidenced from EIS and chronocoulometry. The effect of morphology of the different CuNS on the oxidation of glucose was examined at NG film fabricated electrode. Figure S6 shows the CVs of 1.0 mM glucose at cubic, spherical, quasi-dendritic and dendritic CuNS modified GC/NG electrodes in 0.1 M NaOH. The cubic CuNS (Eapp = 0 V) shows an anodic peak at +0.52 V (curve a) whereas the spherical CuNS (Eapp = -0.10 V) show oxidation at +0.54 V with enhanced current (curve b). When the electrode was modified with quasi-dendritic CuNS (Eapp = -0.30 V), the oxidation potential was shifted towards less positive potential but the oxidation current was decreased compared to spherical and cubic NS, which is due to less population of dendritic CuNS over the NG film (curve c). The dendritic CuNS formed on the NG electrode (Eapp = -0.40 V) exhibited the glucose oxidation at less positive potential with enhanced oxidation current in contrast to cubic, spherical and quasidendritic CuNS (curve d). It shows glucose oxidation at +0.42 V with 2-fold increase in current when compared to cubic nanostructures. The enhanced electrocatalytic activity is attributed to the porous nature of dendritic CuNS which serves large specific surface area along with abundant active sites and sharp edges. The obtained results clearly indicated that the dendritic CuNS showed excellent electrocatalytic activity in contrast to other CuNS. The effect of scan rate on the glucose oxidation at GC/NG-dendritic CuNS electrode in 0.1 M NaOH was also investigated. The anodic peak current (Ipa) due to glucose oxidation was increased linearly with scan rates from 10 to 150 mVs-1 (Figure S7). The plot of anodic peak current against the square root of scan rate shows a good linearity (R2 = 0.9977; Figure S7, inset), indicating glucose oxidation at GC/NG-dendritic CuNS electrode is a diffusion controlled process. Figure 9 shows the DPVs for each addition of 20 µM glucose at GC/NG-dendritic CuNS electrode in 0.1 M NaOH. While adding 20 µM glucose, the GC/NG-dendritic CuNS electrode exhibits the oxidation peak at +0.39 V. Further, addition of glucose in the range of 20-400 µM increases the oxidation peak current of glucose linearly with a correlation coefficient of 0.9930 (Figure 9, inset). The performance of GC/NG-dendritic CuNS electrode was further evaluated by amperometry. Figure 10A exhibits i-t curve


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for glucose at GC/NG-dendritic CuNS electrode in a stirred 0.1 M NaOH solution with an applied potential of +0.50 V. The GC/NG-dendritic CuNS electrode shows current response for the initial addition of 2 µM glucose. Further addition of 2 µM glucose in each step increases the current response with an interval of 50 s and reaches saturation in 3 s. The dependence of the response current with respect to the concentration of glucose was linear from 2 to 22 µM with a correlation coefficient of 0.9989 (Figure 10A, inset). Further, glucose was determined in a wide range of concentration. While the concentration increases from 500 nM to 5.0 mM, the amperometric current response was increased linearly (Figure 10B) at an applied potential of +0.50 V. The limit of detection for glucose is 14.4 ×10-9 mol/L (S/N = 3) and the sensitivity is 1848 µA mM-1 cm-2. The performance of the NG-CuNS modified electrode towards the oxidation of glucose is compared with the earlier reports32,39,47-53 and are given in Table 1. When compared with other glucose sensors (Table 1), the GC/NG-dendritic CuNS electrode displays a good linearity in a wide range glucose concentration and lowest limit of detection. Further, we have studied the effect of interferents on the determination of glucose by adding the urea, oxalic acid and common metal ions such as Na+, Mg2+ and Ca2+. The i–t curve for glucose at GC/NG-dendritic CuNS electrode in the presence of interferences in a stirred 0.1 M NaOH is shown in Figure S8. The initial current response was increased for the addition of 2 µM glucose (a) and a linear current increment was observed for further additions. The addition of 0.5 mM of (c) Ca2+, (d) Mg2+, (e) K+, (f) Na+, (g) SO42- and (h) Cl- and 0.1 mM of (i) urea, (j) oxalic acid, (k) uric acid and (l) ascorbic acid to the same solution does not provoke the current response and it was again responded while adding 5 µM glucose (b). These results suggest that it is possible to determine 2 µM glucose even in the presence of 100-fold excess of common interferents. In order to examine the real applicability of the present sensor, the NG-dendritic CuNS was fabricated on screen printed carbon electrode (SPCE) with the same procedure adopted for GC electrode. The SPC/NG-dendritic CuNS electrode was utilized to determine glucose in human blood serum and urine samples. The SPCE/NG-dendritic CuNS does not show any response in the absence of blood serum in 0.1 M NaOH (Figure S9A, curve a) and in the presence of serum it shows the oxidation peak at +0.45 V. The obtained peak may be due to the oxidation of glucose present in the serum sample (Figure S9A, curve b). Further, the commercial glucose was spiked to the serum solution. The addition of 100 µM of glucose to the serum sample shows the enhanced oxidation peak current at +0.45 V which confirms that it is due to glucose oxidation (Figure S9A, curve c and d). The modified electrode was also used to determine glucose in human urine samples (Figure S9B). The proposed method exhibits good recoveries for the determination of glucose in human blood serum and urine samples (Table S3). Thus, the SPC/NG-dendritic CuNS electrode can be effectively utilized to determine glucose in real samples. The present method of electrode



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fabrication is simple and any one can easily determine glucose level in blood serum and urine using SPCE/NG-dendritic CuNS. 4. CONCLUSIONS In the present work, fabrication of nitrogen-doped graphene-CuNS composite film (NG-CuNS) on GCE was achieved by electrochemical reduction of pre-assembled N-GO followed by electrodeposition of copper. The N-GO was prepared by intercalating melamine into GO and attach them on GCE via Michael’s reaction. The aromatic backbone of graphene was retained by the electrochemical reduction of oxygen functionalities of N-GO to form NG. Further, nitrogen doping via the intercalation of melamine with GO was confirmed by XPS. Then, the CuNS on NG modified electrode was prepared by electrodepositing Cu at various applied potentials with different deposition times and were characterized by XRD, XPS and SEM. The SEM images showed that the deposited cubic, spherical, quasi-dendritic and dendritic NS at the applied potentials of 0, -0.10, -0.30 and -0.40 V, respectively on NG film were uniform. The electrocatalytic activity of the GC/NG-CuNS electrode towards glucose oxidation depends on the shape of the CuNS. Among the different CuNS, the NG-dendritic CuNS electrode showed higher oxidation current for glucose when compared to other CuNS. Finally, the NG-dendritic CuNS were fabricated on screen printed carbon electrode and was successfully utilized to determine glucose in human blood serum and urine samples. The facile and economically viable fabrication of the present composite material makes it as an ideal candidate for the glucose analysis. ASSOCIATED CONTENT Supporting Information. Experimental details and ATR-FT-IR spectra obtained for N-GO and NG modified substrates, SEM images obtained for the time dependent growth of dendritic CuNS on NG film, XRD Pattern and EDAX spectra obtained for N-GO, NG and NG-dendritic CuNS modified substrate, Characterization of NG and NG/CuNS by EIS study, CVs obtained for 1.0 mM glucose at cubic, spherical, quasi-dendritic and dendritic CuNS modified GC/NG electrodes and at different scan rates, amperometric determination of glucose in the presence of its major interferents and determination of glucose in human blood serum and urine samples and impedance spectral data. “This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] *Tel: +91 451 245 2371; Fax: + 91 451 245 3031 ACKNOWLEDGMENT


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N.S.K. Gowthaman thanks the University Grants Commission (UGC), New Delhi, India for the award of a Meritorious Student Fellowship (F. 7-225/2009(BSR) dated 15.01.2013). Financial support from Department of Biotechnology (BT/PR10372/PFN/20/904/2013), New Delhi, India is gratefully acknowledged. The authors thank Dr. T.G. Satheesh Babu, Amrita Vishwa Vidyapeetham, Coimbatore for providing screen printed carbon electrodes. REFERENCES (1) Georgakilas, V.; Tiwari, J.N.; Kemp, K.C.; Perman, J.A.; Bourlinos, A.B.; Kim, K.S.; Zboril, R. Noncovalent Functionalization of Graphene and Graphene Oxide for Energy Materials, Biosensing, Catalytic and Biomedical Applications. Chem. Rev. 2016, 116, 5464-5519. (2) Ambrosi, A.; Chua, C.K.; Bonanni, A.; Pumera, M. Electrochemistry of Graphene and Related Materials. Chem. Rev. 2014, 114, 7150-7188. (3) Li, Z.; Liu, Z.; Sun, H.; Gao, C. Superstructured Assembly of Nanocarbons: Fullerenes, Nanotubes, and Graphene. Chem. Rev. 2015, 115, 7046-7117. (4) Zhou, Y.G.; Chen,; Wang, F.B.; Sheng, Z.H.; Xia, X.H. A Facile Approach to the Synthesis of Highly Electroactive Pt Nanoparticles on Graphene as an Anode Catalyst for Direct Methanol Fuel Cells. Chem. Commun. 2010, 46, 5951-5953. (5) Chen, D.; Feng, H.; Li, J.; Graphene Oxide: Preparation, Functionalization, and Electrochemical Applications. Chem. Rev. 2012, 112, 6027-6053. (6) Staudenmaier, L. Verfahren zur Darstellung der Graphitsaure. Ber. Dtsch. Chem. Ges. 1898, 31, 1481-1487. (7) Hummers, W.S.; Oﬀeman, R.E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339-1339. (8) Zhou, X.; Zhang, J.; Wu, H.; Yang, H.; Zhang, J.; Guo. S. Reducing Graphene Oxide via Hydroxylamine: A Simple and Efficient Route to Graphene. J. Phys. Chem. C 2011, 115, 1195711961. (9) Merino, F.M.; Guardia, L.; Paredes, J.I.; Rodil, S.V.; Fernandez, S.P.; Alonso, M.A.; Tascon, JD. Vitamin C Is an Ideal Substitute for Hydrazine in the Reduction of Graphene Oxide Suspensions. J. Phys. Chem. C 2010, 114, 6426-6432. (10) Raj, M.A.; John, S.A. Fabrication of Electrochemically Reduced Graphene Oxide Films on Glassy Carbon Electrode by Self-Assembly Method and Their Electrocatalytic Application. J. Phys. Chem. C 2013, 117, 4326-4335.



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Figure Captions Scheme 1. Schematic representations of (A) synthesis of N-GO and (B) fabrication of NG-CuNS on GCE. Figure1. Electrochemical reduction of electrostatically assembled N-GO at GCE (15 cycles) in 0.2 M PB solution (pH 7.2) at a scan rate of 100 mV s-1. Figure 2. Raman spectra obtained for (a) N-GO and (b) NG modified substrates. Figure 3. SEM images obtained for (a) N-GO and (b) NG modified substrates. Figure 4. (A) XPS survey spectrum, (B) C 1s, (C) O 1s and (D) N 1s spectra obtained for NG modified substrate. Figure 5. SEM images obtained for (a) CuNPs and (b) NG-CuNPs modified substrates at an applied potential of -0.1 V. Figure 6. SEM images obtained for CuNS deposited NG modified substrate at different applied potentials of (a) +0.20, (b) +0.10, (c) 0, (d) -0.10, (e) -0.20, (f) -0.30, (g) -0.40, (h) -0.50 and (i) -0.60 V. Figure 7. (A) XPS survey spectrum and (B) deconvoluted Cu 2p spectrum obtained for NG-CuNS modified substrate. Figure 8. CVs obtained for 1 mM glucose at (a) bare, (b) NG, (c) CuNPs, (d) ERGO-CuNPs and (e) NGCuNPs modified GCE in 0.1 M NaOH at a scan rate of 50 mV s-1. Figure 9. DPVs obtained for each 20 µM addition of glucose at GC/NG-dendritic CuNS electrode in 0.1 M NaOH. Inset: Calibration plot for oxidation current vs. concentration of glucose. Figure 10. Amperometric i-t curves obtained for (A) each 2 µM addition of glucose and (B) addition of (a) 0.0005, (b) 0.001, (c) 0.005, (d) 0.01, (e) 0.05, (f) 0.1, (g) 0.2, (h) 0.5, (i) 1.0, (j) 2.0 and (k) 5.0 mM of glucose at the time interval of 50 s at GC/NG-dendritic CuNS electrode in 0.1 M NaOH (Eapp = +0.50 V). Insets A and B: calibration plots obtained for current vs. concentration of glucose. Table 1. Comparison of sensitivity, linear range and limit of detection of glucose obtained from the present modified electrode with the reported modified electrodes.


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A

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Scheme 1 N.S.K. Gowthaman, M. Amal Raj and S. Abraham John



0

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–10

–1

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E/V vs. Ag/AgCl (NaCl sat)

Figure 1 N.S.K. Gowthaman, M. Amal Raj and S. Abraham John


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D G

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sp2 C

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399.2 eV 402.6 eV 401.1 eV

a 525

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a

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0.6

0.8

E/V vs. Ag/AgCl (NaCl sat)




Y = 13.9192 X + 8.2785 2

AAAA / I

30

µ

30 R = 0.9930

u

20

10 0

Ι/µΑ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 32

100

200

[Glucose]/µM

300

b

400

20

10

a

0 0.2

0.4

0.6

E/V vs Ag/AgCl (NaCl sat)



Page 29 of 32

300

1.6

I/µ A

2

300

y = 0.1131x + 0.7547 2 R = 0.9989

y = 61.62x + 9.23 2 R = 0.9887

200

I/µ A

2.0

100

1.2

0

5

10

15

1

200

20

[Glucose]/µM

2

3

[Glucose]/mM

d

600

i

c 2

a

b e

1 1100

1200

(a–e) 400

5

3

100

A

4

4

Ι/µΑ 1

200

j

0

0.8

Ι/µΑ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1300

f

g

h

k

B

0 800

Time (Sec)

1200

1400

1600

Time (Sec)




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Page 30 of 32

Table 1. Comparison of sensitivity, linear range and limit of detection of glucose obtained from the present modified electrode with the reported modified electrodes. S.No

Electrodes

Medium

Sensitivity -1

Linear range -2

(µA mM cm )

(mM)

Detection lim-

Reference

it (µM)

1

Nafion/Cu-NGa/GCE

NaOH

48.13

0.004-4.5

1.3

[32]

2

Cu-GENTFsd/GCE

KOH

11.3

0.9-11

1.0

[39]

3

graphene/pectinCuNPs/GCE

NaOH

35.2

0.001-1

0.35

[47]

4

NAe/NiONFf-rGO/GCE

NaOH

36

0.002-0.6

0.77

[48]

5

Graphene/CuNPs

NaOH

-

up to 4.5

0.5

[49]

6

Graphene/CuNPs

NaOH

607

0.005-1.4

0.2

[50]

NaOH

285

0.002-0.87

0.4

[51]

Cu2O/NiOx/graphene ox-

7

ide/GCE 8

CuO/graphene/GCE

NaOH

1065

0.001-8.0

1.0

[52]

9

Pencil graphite/CuNPs

NaOH

1467.5

1.0-100

0.44

[53]

10

NG-CuNS/GCE

NaOH

1848

0.0005-5.0

0.014

This work

a e

nitrogen-doped graphene/copper nanoparticles, bcopper oxide, cpolypyrrole, dreduced graphene oxide, nafion, fnickel oxide nanofibres


Page 31 of 32

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(For Table of Contents Use Only)

Nitrogen-doped graphene as a robust scaffold for the homogenous deposition of copper nanostructures: A non-enzymatic disposable glucose sensor

N.S.K. Gowthamana, M. Amal Rajb and S. Abraham Johna*

Nitrogen-doped graphene-CuNS were fabricated on screen printed carbon electrode for the determination of glucose in human blood serum and urine samples.



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Page 32 of 32

Graphical Abstract Nitrogen doped graphene (NG) was prepared by intercalating melamine into graphene oxide (GO) via sonication followed by electrochemical reduction. Different copper nanostructures (CuNS) were electrodeposited on NG by varying the applied potential. Dendritic CuNS showed greater electrocatalytic activity towards the oxidation of glucose compared to other CuNS. The NGdendritic CuNS were fabricated on screen printed carbon electrode for the determination of glucose in human blood serum and urine samples.


Nitrogen-Doped Graphene as a Robust Scaffold for the Homogeneous

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