DOI: 10.1002/chem.201501691
Full Paper
& Graphene
Chemically Modified Graphene: The Influence of Structural Properties on the Assessment of Antioxidant Capacity Kai Hwee Hui, Martin Pumera, and Alessandra Bonanni*[a] Abstract: Graphene materials obtained by different synthetic routes possess dissimilar amount of defects and surface functionalities, which can influence their electrochemical performance towards the detection of electroactive probes. Oxygen-containing groups can be either detrimental to the heterogeneous charge transfer or promote favorable interactions between the graphene surface and the analyte of interest, depending on the structure of the latter. Here, we compared three chemically modified graphenes, obtained
Introduction Since its first characterization in 2004, graphene has been attracting overwhelming interest in material science and technology.[1] Given its great potential in several fields such as energy storage,[2] solar cells,[3] electronics, and electrochemistry,[4, 5] various protocols have been developed for the production of graphene in recent years.[6, 7] To obtain bulk amounts at reduced price, the top-down strategy is the most preferred. Both oxidized and reduced graphenes obtained with this approach have been used for electrochemical purposes and for the study of various analytical probes.[8–12] It was demonstrated that either the different synthetic route for graphite oxidation, which is the first step of the top-down approach, or the protocols used to reduce the graphene and restore the sp2 network can have an immense influence on the final properties of the material.[13, 14] This in turn can tune the electrochemical performance of the graphene material used as platform for the detection of electroactive probes (analytes). In addition, depending on the analyte structure and chemical composition, the favorable interaction with the graphene platform strongly depends on the surface properties and functionalization of the latter.[15, 16] A comprehensive study on the use of different graphene platforms for the detection of specific probes is lacking. Depending on the graphene materials produced by following var[a] K. H. Hui, Prof. M. Pumera, Dr. A. Bonanni Division of Chemistry & Biological Chemistry School of Physical and Mathematical Sciences Nanyang Technological University, Singapore 637371 (Singapore) Fax: (+ 65) 6791-1961 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501691. Chem. Eur. J. 2015, 21, 11793 – 11798
by various procedures and carrying different amounts of oxygen functionalities, for the detection of standard gallic acid, a compound commonly used as an index of the antioxidant capacity of food and beverages. We found that electrochemically reduced graphene provided the best electrochemical performance in terms of calibration sensitivity, selectivity, and linearity of response. Our findings are important in order to understand the suitability of graphene platforms for the assessment of food quality.
ious pathways and possessing diverse features such as amount of defects and oxygen functionalities, the electrochemistry of different probes can be profoundly influenced. For these reasons there is an urgent need to study and compare those materials in order to see which one is more suitable to the specific purpose. In this work, three chemically modified graphene materials, namely graphene oxide, chemically reduced graphene oxide, and electrochemically reduced graphene oxide, each possessing different amount of defects and oxygen functionalities, are compared to study the electrochemical oxidation of gallic acid, a probe which is commonly used to assess the antioxidant capacity of food and beverages. The graphene platform providing the best electroanalytical performance will then be used for real sample analysis.
Experimental Section Materials Gallic acid, N,N-dimethylformamide (DMF), potassium phosphate dibasic, sodium phosphate monobasic, sodium chloride, potassium chloride, hydrochloric acid (37 %), sodium hydroxide, and sulfuric acid (95–98 %) were obtained from Sigma-Aldrich (Singapore). Graphite (natural, 45 mm) was purchased from Asbury Carbons (NJ, USA). Nitric acid (> 90 %) was purchased from J.T. Baker (Singapore). Hydrazine monohydrate and potassium chlorate (98 %) were purchased from Alfa Aesar (Singapore). Red and white wine samples were purchased from a local supermarket. Glassy carbon (GC) electrodes (diameter: 3 mm) were obtained from Autolab (Eco Chemie, The Netherlands). GC electrode surfaces were renewed using a polishing cloth with alumina powder (0.05 mm).
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper Instruments All voltammetric experiments were performed using a mAutolab type III electrochemical analyser (Eco Chemie, The Netherlands). All analytical parameters were controlled by General Purpose Electrochemical Systems Version 4.9 software (Eco Chemie, The Netherlands). The electrochemical measurements were carried out in a voltammetric cell at room temperature employing a three-electrode configuration. A glassy carbon electrode was used as a working electrode, a platinum electrode served as an auxiliary electrode, and an Ag/AgCl electrode as a reference electrode. Modification of glassy carbon electrode with chemically modified graphenes (CMGs): Graphene oxide (GO) was obtained by ultrasonication (37 kHz) of a disper- Scheme 1. Schematic for the synthesis of CMG materials. sion of graphite oxide (GPO; 1 mg mL¢1) for 2 h in DMF. GPO was synthesized using the HofSubsequently, the suspension (1 mL) was transferred onto a remann method.[17, 18] Sulfuric acid (98 %, 87.5 mL) and nitric acid newed GC electrode surface and allowed to dry at room tempera(68 %, 27 mL) were added to a Pyrex beaker containing a thermomture. eter. The mixture was subsequently cooled to 0 8C and graphite (5 g) was added to the mixture under vigorous stirring to obtain Chemically modified graphene characterization: Material characa homogenous dispersion. Potassium chlorate (55 g) was slowly terization was performed by transmission electron microscopy added to the mixture at 0 8C during 30 min. Once the potassium (TEM), X-ray photoelectron spectroscopy (XPS), and Raman specchlorate had completely dissolved, the reaction flask was loosely troscopy[8, 21, 22] (please refer to the Supporting Information for TEM capped to allow chlorine dioxide gas to escape, and the mixture micrographs, XPS, and Raman spectra). was then stirred vigorously for 96 h at room temperature. Upon From TEM micrographs (see Figure S1, Supporting Information) it completion of the reaction, the mixture was poured into deionized was observed that all materials show mono- or few-layer structure. water (3 L) and decanted. GPO was redispersed in HCl solution For Raman spectroscopy, a 514 nm laser excitation was employed. (5 %), continuously centrifuged, and redispersed in deionized water Typically, in a Raman spectrum two prominent peaks are shown: until a negative reaction on chloride and sulfate ions was obthe D-band at approximately 1350 cm¢1, which correlates to the served. Lastly, the GPO slurry was dried in a vacuum oven at 50 8C presence of sp3 carbons or defects in the graphene network, and for 48 h. the G-band at approximately 1560 cm¢1, which represents the sp2 Electrochemically reduced graphene oxide (ERGO) was obtained by pristine graphene. The ratio between the intensity of these two applying a potential of ¢1.2 V for 900 s to a GC electrode coated bands, also called D/G ratio, can be taken as an indication of the with 1 mg GO. The reduction was carried out in 8 mL of 0.1 m phosdegree of disorders in the graphene structure. For XPS spectrosco[19] phate buffer solution, pH 7.2. py, the C/O ratio calculated by survey spectra can be used as an inFor the preparation of chemically reduced graphene oxide (CRGO), dication of the relative amount of carbon and oxygen on the gragraphite oxide obtained with Staudenmaier method was emphene material; the materials with the largest amount of oxygenployed.[20] In brief, sulfuric acid (95–98 %, 17.5 mL) and nitric acid containing groups will show the lowest C/O ratio (see Table 1). (9 mL) were added into a flask and stirred at 0 8C for 15 min. Graphite (1 g) was added, followed by potassium chlorate (11 g). Cyclic and differential pulse voltammetry: For gallic acid detecUpon completion of the reaction the obtained GPO was purified. tion, cyclic voltammograms (CV) were obtained in the 0–1.2 V poThe prepared GPO (50 mg) was added to ultrapure water to obtain tential range at a scan rate of 20 mV s-1. Differential pulse voltama 1.0 mg mL¢1 colloidal solution and placed in an ultrasonicator mograms (DPV) were obtained in the 0–1.2 V potential range with (150 W) for 3 h. After hydrazine monohydrate (2 mL, 32.1 mmol) 50 ms modulation time and 25 mV modulation amplitude. A basewas added dropwise, the mixture was stirred under reflux for 24 h line correction with a peak width of 0.01 was applied to all DPV and subsequently allowed to cool to room temperature. The reacscans. tion mixture was filtered using polytetrafluoroethylene (PTFE) membranes (0.2 mm) with methanol and ultrapure water and then Sample preparation and quantification of gallic acid: Diluted red dried at 50 8C for five days. A schematic of the CMG synthetic wine (70 mL, 1:10 from the stock solution) or 100 mL of white wine routes is depicted in Scheme 1. was added into the electrochemical cell containing 8 mL of 0.1 m Prior to measurement, 1 mg mL¢1 suspensions of the chemically modified graphenes (CMGs) in DMF were ultrasonicated for 5 min. Chem. Eur. J. 2015, 21, 11793 – 11798
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phosphate buffer solution, pH 2.5. The differential pulse voltammograms were recorded in the range 0.3–0.9 V with 50 ms modula-
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Full Paper quinone radical form and then into quinone form.[23, 24] For this reason, the whole process is strongly influenced by the pH of the buffer used for the measurement. In general, the shifting Material D/G Ratio C/O Ratio of the peak is consistent for all materials, as well as the peak intensity trend, with the exception of CRGO, which at pH 7.2 GO 1.05 2.8 ERGO 1.08 5.1 showed a larger signal than expected. This may be due to the CRGO 1.42 16.3 favorable formation of H-bonds between gallic acid and the CRGO surface at pH 7.2. Given the obtained results, a phosphate buffer solution at pH 2.5 was used for the detection of tion time and 25 mV modulation amplitude. The concentration of gallic acid on the different CMG platforms. gallic acid was determined by the standard addition method. For a better comparison of the graphene materials, a study of the voltammetric response of CMGs towards the detection of 0.1 mm gallic acid solution was conducted and presented in Results and Discussion Figure 2. The average peak height, potential, and relative standard deviation (RSD) values were recorded in Table 2. The aim of the present work was to study the electrochemical As shown in Figure 2, ERGO provided the highest current reperformance of three chemically modified graphene materials, sponse for gallic acid oxidation, followed by CRGO, GC, and namely graphene oxide (GO), chemically reduced graphene GO. With the exception of GO, the CMGs modified electrodes oxide (CRGO), and electrochemically reduced graphene oxide exhibited higher peak currents than those recorded on bare (ERGO) towards the oxidation of gallic acid. GC electrode. In the case of both electrochemically and chemiIn order to optimize the conditions for gallic acid detection, cally reduced CMGs, this can be explained by the larger electhe effect of pH on the voltammetric response given by all troactive surface area and better electrical conductivity of reCMGs was investigated, as seen in Figure 1. The study was perduced graphenes compared to the bare GC electrode.[8] On the formed in a pH range between 2.5 and 10. As depicted in other end, the lower peak current obtained on GO-modified Figure 1, with decreasing pH a sharper oxidation peak together electrode can be attributed to the larger concentration of with an increased peak current was obtained. In addition, oxygen functionalities present on the GO surface, which makes a shift of peak potential towards more positive values was also the material poorly conductive and has a detrimental effect on observed as pH was decreased from 10 to 2.5. These phenomthe electron-transfer kinetics.[25] In fact, at pH > 4, both gallic ena, as observed by other authors, are due to the fact that the acid and the graphene surface are negatively charged due to oxidation of gallic acid involves protonation reactions for both the deprotonation of carboxylic groups, and this in turn can oxidation steps in which gallic acid is first converted into semicontribute to the weakening of the p–p interaction network between the electrochemical platform and the analyzed probe, thus resulting in a lower electrochemical signal.[26, 27] As for the comparison between the two reduced materials, based on the XPS and Raman characterization, one would expect a higher peak current from CRGO, which is the material possessing the lowest amount of oxygen functionalities (i.e., larger C/O ratio) and the largest density of defects (i.e., larger D/G ratio). Clearly, in the case of gallic acid oxidation, the presence of oxygen functionalities on the ERGO platform that cannot be reduced during the electrochemical treatment plays a major role in favoring the interactions between the graphene surface and the analyte. It Figure 1. Effect of pH variation on differential pulse voltammograms obtained for the detection of 0.1 mm gallic is in fact well-known that among acid on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO. pH values: 2.5, 5.0, 7.2, and 10.0. all oxygen functionalities that Table 1. Raman spectra D/G ratios and XPS spectra C/O ratios of graphene materials.
Chem. Eur. J. 2015, 21, 11793 – 11798
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Full Paper sponding differential pulse voltammograms of gallic acid oxidation at bare GC electrode and GC electrode modified with GO, ERGO, and CRGO are also shown in Figure 3. For ease of comparison, Table 3 tabulated the slope, R2, and peak width at half-height values for all the materials. In terms of calibration sensitivity, ERGO-modified electrode provided the most sensitive response to gallic acid oxidation, with the highest slope of
Table 3. Comparison of analytical parameters for the detection of gallic acid at bare GC electrode and GC electrodes modified with GO, ERGO, and CRGO.
Figure 2. Differential pulse voltammograms for the detection of 0.1 mm gallic acid on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO. All measurements were performed in 0.1 m phosphate buffer solution, pH 2.5.
Table 2. Analytical parameters for the detection of 0.1 mm gallic acid at bare GC electrode and GC electrodes modified with GO, ERGO, and CRGO; values are represented as average of three replicates. Material
Peak height [mA]
Peak potential [V]
RSD [%]
GC GO ERGO CRGO
1.41 1.14 7.23 4.62
0.453 0.453 0.433 0.433
14 6 9 13
are expected to be present on the graphene oxide surface (e.g., epoxide, carbonyl, hydroxyl, aldehyde, carboxyl, hydroperoxy, peroxy, ether, and ester groups), only the reductions of aldehydes, epoxides, and peroxides can occur at the potential range of ¢0.9 to ¢1.5 V (vs. Ag/AgCl) in neutral pH buffers.[25, 28–30] Based on these considerations it is evident that the presence of nonelectrochemically reducible oxygen-containing groups can promote the stacking of the electrochemical probe (analyte) on the graphene surface.[16, 31, 32] Scheme 2 depicts the possible interactions between the oxygen-containing groups on the graphene surface and gallic acid. For a detailed assessment on the performance of the different CMGs, calibration curves of voltammetric peak height versus concentration of gallic acid were obtained. The corre-
Material
Slope [mAmm¢1]
R2
W1/2 [mV]
GC GO ERGO CRGO
0.0119 0.0067 0.0571 0.0218
0.9523 0.9144 0.9670 0.9524
70 98 60 63
0.0571 mA mM¢1, followed by CRGO, GC, and GO. The linearity of response was then evaluated considering the value of R2 obtained from the calibration curves of gallic acid using different CMG platforms. GO presented the poorest linear relationship between oxidation peak and concentration (R2 = 0.9144), whereas the other three materials displayed relatively good linear response (R2 > 0.9523). On the basis of selective response towards gallic acid evaluated by measuring the peak width at half height, ERGO showed the highest selectivity with the smallest value of 60 mV. Moreover, a detection limit of 0.436 mg L¢1, calculated by considering the residual standard deviation of the regression, was obtained when using the ERGO platform. Given the favorable analytical performances shown by ERGO in terms of calibration sensitivity, linearity, and selectivity of response, the material was selected for the quantification of gallic acid in wine samples. The obtained results from the quantitative analysis of gallic acid in red and white wine samples using an ERGO-modified electrode are displayed in Table 4. For every sample, gallic acid concentration was obtained using the standard addition method. As depicted in Table 4, red wine exhibited significantly higher antioxidant activity than white wine, based on gallic acid equivalents. Generally, a good linear relationship (R2 > 0.9432) and reproducibility were obtained for all samples. These results are in agreement with those obtained by using standard methods on the same samples.[23, 33]
Table 4. Gallic acid equivalents (mg L¢1) in wine samples using an ERGOmodified electrode. All measurements were performed in 0.1 m phosphate buffer solution, pH 2.5.
Scheme 2. Schematic of interactions occurring between oxygen-containing groups on graphene surface and gallic acid. Chem. Eur. J. 2015, 21, 11793 – 11798
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Wine sample
GAE [mg L¢1]
RSD [%]
R2
Cabernet Sauvignon red wine Shiraz Cabernet red wine Chardonnay white wine Sauvignon Blanc white wine
6266 5893 282 285
19 25 17 20
0.9432 0.9802 0.9897 0.9835
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Full Paper way for the use of graphene materials in the quality assessment of food and beverages.
Acknowledgements A.B. acknowledges Nanyang Technological University for the financial support. M.P. acknowledges the Singapore Ministry of Education Academic Research Fund AcRF Tier 1 (2013-T1-001014, RGT1/13) for the funding. Keywords: antioxidant capacity · graphene · gallic acid · oxygen-containing groups [1] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, A. A. Firsov, Science 2004, 306, 666. Figure 3. Differential pulse voltammograms for the determination of gallic acid (concentration range: from 1 mm [2] M. I. Hoffert, K. Caldeira, G. Bento 10 mm) on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO; all measford, D. R. Criswell, C. Green, H. urements were performed in 0.1 m phosphate buffer solution, pH 2.5. Herzog, A. K. Jain, H. S. Kheshgi, K. S. Lackner, J. S. Lewis, H. D. Lightfoot, W. Manheimer, J. C. Mankins, M. E. Mauel, L. J. Perkins, M. E. Conclusion Schlesinger, T. Volk, T. M. L. Wigley, Science 2002, 298, 981. [3] F. Bonaccorso, Z. Sun, T. Hasan, A. C. Ferrari, Nat. Photonics 2010, 4, 611. To summarize, we have studied the effect of the amount of de[4] M. Pumera, Chem. Rec. 2009, 9, 211. fects and surface oxygen functionalities of three different [5] A. Ambrosi, C. K. Chua, A. Bonanni, M. Pumera, Chem. Rev. 2014, 114, chemically modified graphene platforms on the electrochemi7150. [6] W. Choi, I. Lahiri, R. Seelaboyina, Y. S. Kang, Crit. Rev. Solid State Mater. cal detection of gallic acid oxidation. We have demonstrated Sci. 2010, 35, 52. that the presence of defects and oxygen functionalities on gra[7] P. Avouris, C. Dimitrakopoulos, Mater. Today 2012, 15, 86. phene can strongly influence the electrochemical response of [8] A. Ambrosi, A. Bonanni, Z. Sofer, J. S. Cross, M. Pumera, Chem. Eur. J. the material towards the redox activity of the analyte of inter2011, 17, 10763. [9] W. Y. H. Khoo, M. Pumera, A. Bonanni, Anal. Chim. Acta 2013, 804, 92. est. [10] J. Y. Sun, K. J. Huang, S. Y. Wei, Z. W. Wu, F. P. Ren, Colloids Surf. B 2011, The electrochemically reduced graphene oxide provided the 84, 421. best analytical performance in terms of calibration sensitivity, [11] S. Y. Chee, M. Pumera, Electrochem. Commun. 2012, 20, 141. selectivity, and linearity of response, together with good repro[12] S. M. Tan, A. Ambrosi, C. K. Chua, M. Pumera, J. Mater. Chem. A 2014, 2, 10668. ducibility of results. This could be mainly due to the favorable [13] C. E. Chng, M. Pumera, A. Bonanni, Electrochem. Commun. 2014, 46, interaction between the analyte and the oxygen functionalities 137. present on ERGO, which promote the stacking of the analyte [14] A. Y. S. Eng, A. Ambrosi, C. K. Chua, F. Sanek, Z. Sofer, M. Pumera, Chem. on the graphene surface. Eur. J. 2013, 19, 12673. [15] C. S. Lim, A. Ambrosi, M. Pumera, Phys. Chem. Chem. Phys. 2014, 16, ERGO has also revealed to be a suitable platform for real 12178. sample analysis. Four wine samples were analyzed and their [16] S. M. Tan, H. L. Poh, Z. Sofer, M. Pumera, Analyst 2013, 138, 4885. antioxidant capacity, expressed as gallic acid equivalents (GAE), [17] U. Hofmann, R. Holst, Ber. Dtsch. Chem. Ges. 1939, 72, 754. confirmed the trend previously obtained with traditional meth[18] U. Hofmann, E. Konig, Z. Anorg. Allg. Chem. 1937, 234, 311. [19] M. Zhou, Y. L. Wang, Y. M. Zhai, J. F. Zhai, W. Ren, F. A. Wang, S. J. Dong, ods. In addition, the detection limit obtained with the develChem. Eur. J. 2009, 15, 6116. oped platform was two orders of magnitude lower than that [20] L. Staudenmaier, Ber. Dtsch. Chem. Ges. 1898, 31, 1481. achieved with traditional spectrophotometric methods em[21] A. Bonanni, C. K. Chua, M. Pumera, Chem. Eur. J. 2014, 20, 217. ploying Folin–Chocalteu protocol on similar wine samples.[33] [22] C. K. Chua, M. Pumera, J. Mater. Chem. 2012, 22, 23227. [23] L. P. Souza, F. Calegari, A. J. G. Zarbin, L. H. Marcolino, M. F. Bergamini, J. Overall, we have demonstrated the suitability of an electroAgric. Food Chem. 2011, 59, 7620. chemically reduced graphene platform for the determination [24] S. Gunckel, P. Santander, G. Cordano, J. Ferreira, S. Munoz, L. J. Nunezof antioxidant capacity of real food samples. This paves the Vergara, J. A. Squella, Chem.-Biol. Interact. 1998, 114, 45. [25] A. Bonanni, A. Ambrosi, M. Pumera, Chem. Eur. J. 2012, 18, 4541. Chem. Eur. J. 2015, 21, 11793 – 11798
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Full Paper [26] K. K. R. Datta, O. Kozak, V. Ranc, M. Havrdova, A. B. Bourlinos, K. Safarova, K. Hola, K. Tomankova, G. Zoppellaro, M. Otyepka, R. Zboril, Chem. Commun. 2014, 50, 10782. [27] B. Konkena, S. Vasudevan, J. Phys. Chem. Lett. 2012, 3, 867. [28] J. Grimshaw, Electrochemical Reactions and Mechanisms in Organic Chemistry, Elsevier, Amsterdam, 2000, p. 330. [29] L. Xiao, G. G. Wildgoose, A. Crossley, R. G. Compton, Sens. Actuators B 2009, 138, 397. [30] J. H. Wagenknecht, L. Eberson and J. H. P. Utley in Carboxylic Acids and Derivatives, (Eds.: H. Lund and O. Hammerich), Marcel Dekker, Inc., New York, 2001, pp. 454.
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[31] Z.-H. Sheng, X.-Q. Zheng, J.-Y. Xu, W.-J. Bao, F.-B. Wang, X.-H. Xia, Biosens. Bioelectron. 2012, 34, 125. [32] P. Lazar, F. Karlicky, P. Jurecka, M. Kocman, E. Otyepkova, K. Safarova, M. Otyepka, J. Am. Chem. Soc. 2013, 135, 6372. [33] S. Dobrinas, A. Soceanu, V. D. Artem, Environ. Eng. Manage. J. 2015, 14, 863.
Received: April 30, 2015 Published online on July 1, 2015
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Full Paper
& Graphene
Chemically Modified Graphene: The Influence of Structural Properties on the Assessment of Antioxidant Capacity Kai Hwee Hui, Martin Pumera, and Alessandra Bonanni*[a] Abstract: Graphene materials obtained by different synthetic routes possess dissimilar amount of defects and surface functionalities, which can influence their electrochemical performance towards the detection of electroactive probes. Oxygen-containing groups can be either detrimental to the heterogeneous charge transfer or promote favorable interactions between the graphene surface and the analyte of interest, depending on the structure of the latter. Here, we compared three chemically modified graphenes, obtained
Introduction Since its first characterization in 2004, graphene has been attracting overwhelming interest in material science and technology.[1] Given its great potential in several fields such as energy storage,[2] solar cells,[3] electronics, and electrochemistry,[4, 5] various protocols have been developed for the production of graphene in recent years.[6, 7] To obtain bulk amounts at reduced price, the top-down strategy is the most preferred. Both oxidized and reduced graphenes obtained with this approach have been used for electrochemical purposes and for the study of various analytical probes.[8–12] It was demonstrated that either the different synthetic route for graphite oxidation, which is the first step of the top-down approach, or the protocols used to reduce the graphene and restore the sp2 network can have an immense influence on the final properties of the material.[13, 14] This in turn can tune the electrochemical performance of the graphene material used as platform for the detection of electroactive probes (analytes). In addition, depending on the analyte structure and chemical composition, the favorable interaction with the graphene platform strongly depends on the surface properties and functionalization of the latter.[15, 16] A comprehensive study on the use of different graphene platforms for the detection of specific probes is lacking. Depending on the graphene materials produced by following var[a] K. H. Hui, Prof. M. Pumera, Dr. A. Bonanni Division of Chemistry & Biological Chemistry School of Physical and Mathematical Sciences Nanyang Technological University, Singapore 637371 (Singapore) Fax: (+ 65) 6791-1961 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201501691. Chem. Eur. J. 2015, 21, 11793 – 11798
by various procedures and carrying different amounts of oxygen functionalities, for the detection of standard gallic acid, a compound commonly used as an index of the antioxidant capacity of food and beverages. We found that electrochemically reduced graphene provided the best electrochemical performance in terms of calibration sensitivity, selectivity, and linearity of response. Our findings are important in order to understand the suitability of graphene platforms for the assessment of food quality.
ious pathways and possessing diverse features such as amount of defects and oxygen functionalities, the electrochemistry of different probes can be profoundly influenced. For these reasons there is an urgent need to study and compare those materials in order to see which one is more suitable to the specific purpose. In this work, three chemically modified graphene materials, namely graphene oxide, chemically reduced graphene oxide, and electrochemically reduced graphene oxide, each possessing different amount of defects and oxygen functionalities, are compared to study the electrochemical oxidation of gallic acid, a probe which is commonly used to assess the antioxidant capacity of food and beverages. The graphene platform providing the best electroanalytical performance will then be used for real sample analysis.
Experimental Section Materials Gallic acid, N,N-dimethylformamide (DMF), potassium phosphate dibasic, sodium phosphate monobasic, sodium chloride, potassium chloride, hydrochloric acid (37 %), sodium hydroxide, and sulfuric acid (95–98 %) were obtained from Sigma-Aldrich (Singapore). Graphite (natural, 45 mm) was purchased from Asbury Carbons (NJ, USA). Nitric acid (> 90 %) was purchased from J.T. Baker (Singapore). Hydrazine monohydrate and potassium chlorate (98 %) were purchased from Alfa Aesar (Singapore). Red and white wine samples were purchased from a local supermarket. Glassy carbon (GC) electrodes (diameter: 3 mm) were obtained from Autolab (Eco Chemie, The Netherlands). GC electrode surfaces were renewed using a polishing cloth with alumina powder (0.05 mm).
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Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper Instruments All voltammetric experiments were performed using a mAutolab type III electrochemical analyser (Eco Chemie, The Netherlands). All analytical parameters were controlled by General Purpose Electrochemical Systems Version 4.9 software (Eco Chemie, The Netherlands). The electrochemical measurements were carried out in a voltammetric cell at room temperature employing a three-electrode configuration. A glassy carbon electrode was used as a working electrode, a platinum electrode served as an auxiliary electrode, and an Ag/AgCl electrode as a reference electrode. Modification of glassy carbon electrode with chemically modified graphenes (CMGs): Graphene oxide (GO) was obtained by ultrasonication (37 kHz) of a disper- Scheme 1. Schematic for the synthesis of CMG materials. sion of graphite oxide (GPO; 1 mg mL¢1) for 2 h in DMF. GPO was synthesized using the HofSubsequently, the suspension (1 mL) was transferred onto a remann method.[17, 18] Sulfuric acid (98 %, 87.5 mL) and nitric acid newed GC electrode surface and allowed to dry at room tempera(68 %, 27 mL) were added to a Pyrex beaker containing a thermomture. eter. The mixture was subsequently cooled to 0 8C and graphite (5 g) was added to the mixture under vigorous stirring to obtain Chemically modified graphene characterization: Material characa homogenous dispersion. Potassium chlorate (55 g) was slowly terization was performed by transmission electron microscopy added to the mixture at 0 8C during 30 min. Once the potassium (TEM), X-ray photoelectron spectroscopy (XPS), and Raman specchlorate had completely dissolved, the reaction flask was loosely troscopy[8, 21, 22] (please refer to the Supporting Information for TEM capped to allow chlorine dioxide gas to escape, and the mixture micrographs, XPS, and Raman spectra). was then stirred vigorously for 96 h at room temperature. Upon From TEM micrographs (see Figure S1, Supporting Information) it completion of the reaction, the mixture was poured into deionized was observed that all materials show mono- or few-layer structure. water (3 L) and decanted. GPO was redispersed in HCl solution For Raman spectroscopy, a 514 nm laser excitation was employed. (5 %), continuously centrifuged, and redispersed in deionized water Typically, in a Raman spectrum two prominent peaks are shown: until a negative reaction on chloride and sulfate ions was obthe D-band at approximately 1350 cm¢1, which correlates to the served. Lastly, the GPO slurry was dried in a vacuum oven at 50 8C presence of sp3 carbons or defects in the graphene network, and for 48 h. the G-band at approximately 1560 cm¢1, which represents the sp2 Electrochemically reduced graphene oxide (ERGO) was obtained by pristine graphene. The ratio between the intensity of these two applying a potential of ¢1.2 V for 900 s to a GC electrode coated bands, also called D/G ratio, can be taken as an indication of the with 1 mg GO. The reduction was carried out in 8 mL of 0.1 m phosdegree of disorders in the graphene structure. For XPS spectrosco[19] phate buffer solution, pH 7.2. py, the C/O ratio calculated by survey spectra can be used as an inFor the preparation of chemically reduced graphene oxide (CRGO), dication of the relative amount of carbon and oxygen on the gragraphite oxide obtained with Staudenmaier method was emphene material; the materials with the largest amount of oxygenployed.[20] In brief, sulfuric acid (95–98 %, 17.5 mL) and nitric acid containing groups will show the lowest C/O ratio (see Table 1). (9 mL) were added into a flask and stirred at 0 8C for 15 min. Graphite (1 g) was added, followed by potassium chlorate (11 g). Cyclic and differential pulse voltammetry: For gallic acid detecUpon completion of the reaction the obtained GPO was purified. tion, cyclic voltammograms (CV) were obtained in the 0–1.2 V poThe prepared GPO (50 mg) was added to ultrapure water to obtain tential range at a scan rate of 20 mV s-1. Differential pulse voltama 1.0 mg mL¢1 colloidal solution and placed in an ultrasonicator mograms (DPV) were obtained in the 0–1.2 V potential range with (150 W) for 3 h. After hydrazine monohydrate (2 mL, 32.1 mmol) 50 ms modulation time and 25 mV modulation amplitude. A basewas added dropwise, the mixture was stirred under reflux for 24 h line correction with a peak width of 0.01 was applied to all DPV and subsequently allowed to cool to room temperature. The reacscans. tion mixture was filtered using polytetrafluoroethylene (PTFE) membranes (0.2 mm) with methanol and ultrapure water and then Sample preparation and quantification of gallic acid: Diluted red dried at 50 8C for five days. A schematic of the CMG synthetic wine (70 mL, 1:10 from the stock solution) or 100 mL of white wine routes is depicted in Scheme 1. was added into the electrochemical cell containing 8 mL of 0.1 m Prior to measurement, 1 mg mL¢1 suspensions of the chemically modified graphenes (CMGs) in DMF were ultrasonicated for 5 min. Chem. Eur. J. 2015, 21, 11793 – 11798
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phosphate buffer solution, pH 2.5. The differential pulse voltammograms were recorded in the range 0.3–0.9 V with 50 ms modula-
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Full Paper quinone radical form and then into quinone form.[23, 24] For this reason, the whole process is strongly influenced by the pH of the buffer used for the measurement. In general, the shifting Material D/G Ratio C/O Ratio of the peak is consistent for all materials, as well as the peak intensity trend, with the exception of CRGO, which at pH 7.2 GO 1.05 2.8 ERGO 1.08 5.1 showed a larger signal than expected. This may be due to the CRGO 1.42 16.3 favorable formation of H-bonds between gallic acid and the CRGO surface at pH 7.2. Given the obtained results, a phosphate buffer solution at pH 2.5 was used for the detection of tion time and 25 mV modulation amplitude. The concentration of gallic acid on the different CMG platforms. gallic acid was determined by the standard addition method. For a better comparison of the graphene materials, a study of the voltammetric response of CMGs towards the detection of 0.1 mm gallic acid solution was conducted and presented in Results and Discussion Figure 2. The average peak height, potential, and relative standard deviation (RSD) values were recorded in Table 2. The aim of the present work was to study the electrochemical As shown in Figure 2, ERGO provided the highest current reperformance of three chemically modified graphene materials, sponse for gallic acid oxidation, followed by CRGO, GC, and namely graphene oxide (GO), chemically reduced graphene GO. With the exception of GO, the CMGs modified electrodes oxide (CRGO), and electrochemically reduced graphene oxide exhibited higher peak currents than those recorded on bare (ERGO) towards the oxidation of gallic acid. GC electrode. In the case of both electrochemically and chemiIn order to optimize the conditions for gallic acid detection, cally reduced CMGs, this can be explained by the larger electhe effect of pH on the voltammetric response given by all troactive surface area and better electrical conductivity of reCMGs was investigated, as seen in Figure 1. The study was perduced graphenes compared to the bare GC electrode.[8] On the formed in a pH range between 2.5 and 10. As depicted in other end, the lower peak current obtained on GO-modified Figure 1, with decreasing pH a sharper oxidation peak together electrode can be attributed to the larger concentration of with an increased peak current was obtained. In addition, oxygen functionalities present on the GO surface, which makes a shift of peak potential towards more positive values was also the material poorly conductive and has a detrimental effect on observed as pH was decreased from 10 to 2.5. These phenomthe electron-transfer kinetics.[25] In fact, at pH > 4, both gallic ena, as observed by other authors, are due to the fact that the acid and the graphene surface are negatively charged due to oxidation of gallic acid involves protonation reactions for both the deprotonation of carboxylic groups, and this in turn can oxidation steps in which gallic acid is first converted into semicontribute to the weakening of the p–p interaction network between the electrochemical platform and the analyzed probe, thus resulting in a lower electrochemical signal.[26, 27] As for the comparison between the two reduced materials, based on the XPS and Raman characterization, one would expect a higher peak current from CRGO, which is the material possessing the lowest amount of oxygen functionalities (i.e., larger C/O ratio) and the largest density of defects (i.e., larger D/G ratio). Clearly, in the case of gallic acid oxidation, the presence of oxygen functionalities on the ERGO platform that cannot be reduced during the electrochemical treatment plays a major role in favoring the interactions between the graphene surface and the analyte. It Figure 1. Effect of pH variation on differential pulse voltammograms obtained for the detection of 0.1 mm gallic is in fact well-known that among acid on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO. pH values: 2.5, 5.0, 7.2, and 10.0. all oxygen functionalities that Table 1. Raman spectra D/G ratios and XPS spectra C/O ratios of graphene materials.
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Full Paper sponding differential pulse voltammograms of gallic acid oxidation at bare GC electrode and GC electrode modified with GO, ERGO, and CRGO are also shown in Figure 3. For ease of comparison, Table 3 tabulated the slope, R2, and peak width at half-height values for all the materials. In terms of calibration sensitivity, ERGO-modified electrode provided the most sensitive response to gallic acid oxidation, with the highest slope of
Table 3. Comparison of analytical parameters for the detection of gallic acid at bare GC electrode and GC electrodes modified with GO, ERGO, and CRGO.
Figure 2. Differential pulse voltammograms for the detection of 0.1 mm gallic acid on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO. All measurements were performed in 0.1 m phosphate buffer solution, pH 2.5.
Table 2. Analytical parameters for the detection of 0.1 mm gallic acid at bare GC electrode and GC electrodes modified with GO, ERGO, and CRGO; values are represented as average of three replicates. Material
Peak height [mA]
Peak potential [V]
RSD [%]
GC GO ERGO CRGO
1.41 1.14 7.23 4.62
0.453 0.453 0.433 0.433
14 6 9 13
are expected to be present on the graphene oxide surface (e.g., epoxide, carbonyl, hydroxyl, aldehyde, carboxyl, hydroperoxy, peroxy, ether, and ester groups), only the reductions of aldehydes, epoxides, and peroxides can occur at the potential range of ¢0.9 to ¢1.5 V (vs. Ag/AgCl) in neutral pH buffers.[25, 28–30] Based on these considerations it is evident that the presence of nonelectrochemically reducible oxygen-containing groups can promote the stacking of the electrochemical probe (analyte) on the graphene surface.[16, 31, 32] Scheme 2 depicts the possible interactions between the oxygen-containing groups on the graphene surface and gallic acid. For a detailed assessment on the performance of the different CMGs, calibration curves of voltammetric peak height versus concentration of gallic acid were obtained. The corre-
Material
Slope [mAmm¢1]
R2
W1/2 [mV]
GC GO ERGO CRGO
0.0119 0.0067 0.0571 0.0218
0.9523 0.9144 0.9670 0.9524
70 98 60 63
0.0571 mA mM¢1, followed by CRGO, GC, and GO. The linearity of response was then evaluated considering the value of R2 obtained from the calibration curves of gallic acid using different CMG platforms. GO presented the poorest linear relationship between oxidation peak and concentration (R2 = 0.9144), whereas the other three materials displayed relatively good linear response (R2 > 0.9523). On the basis of selective response towards gallic acid evaluated by measuring the peak width at half height, ERGO showed the highest selectivity with the smallest value of 60 mV. Moreover, a detection limit of 0.436 mg L¢1, calculated by considering the residual standard deviation of the regression, was obtained when using the ERGO platform. Given the favorable analytical performances shown by ERGO in terms of calibration sensitivity, linearity, and selectivity of response, the material was selected for the quantification of gallic acid in wine samples. The obtained results from the quantitative analysis of gallic acid in red and white wine samples using an ERGO-modified electrode are displayed in Table 4. For every sample, gallic acid concentration was obtained using the standard addition method. As depicted in Table 4, red wine exhibited significantly higher antioxidant activity than white wine, based on gallic acid equivalents. Generally, a good linear relationship (R2 > 0.9432) and reproducibility were obtained for all samples. These results are in agreement with those obtained by using standard methods on the same samples.[23, 33]
Table 4. Gallic acid equivalents (mg L¢1) in wine samples using an ERGOmodified electrode. All measurements were performed in 0.1 m phosphate buffer solution, pH 2.5.
Scheme 2. Schematic of interactions occurring between oxygen-containing groups on graphene surface and gallic acid. Chem. Eur. J. 2015, 21, 11793 – 11798
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Wine sample
GAE [mg L¢1]
RSD [%]
R2
Cabernet Sauvignon red wine Shiraz Cabernet red wine Chardonnay white wine Sauvignon Blanc white wine
6266 5893 282 285
19 25 17 20
0.9432 0.9802 0.9897 0.9835
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Full Paper way for the use of graphene materials in the quality assessment of food and beverages.
Acknowledgements A.B. acknowledges Nanyang Technological University for the financial support. M.P. acknowledges the Singapore Ministry of Education Academic Research Fund AcRF Tier 1 (2013-T1-001014, RGT1/13) for the funding. Keywords: antioxidant capacity · graphene · gallic acid · oxygen-containing groups [1] K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, A. A. Firsov, Science 2004, 306, 666. Figure 3. Differential pulse voltammograms for the determination of gallic acid (concentration range: from 1 mm [2] M. I. Hoffert, K. Caldeira, G. Bento 10 mm) on bare GC electrode and GC electrodes modified with 1 mg of CMGs: GO, ERGO, and CRGO; all measford, D. R. Criswell, C. Green, H. urements were performed in 0.1 m phosphate buffer solution, pH 2.5. Herzog, A. K. Jain, H. S. Kheshgi, K. S. Lackner, J. S. Lewis, H. D. Lightfoot, W. Manheimer, J. C. Mankins, M. E. Mauel, L. J. Perkins, M. E. Conclusion Schlesinger, T. Volk, T. M. L. Wigley, Science 2002, 298, 981. [3] F. Bonaccorso, Z. Sun, T. Hasan, A. C. Ferrari, Nat. Photonics 2010, 4, 611. To summarize, we have studied the effect of the amount of de[4] M. Pumera, Chem. Rec. 2009, 9, 211. fects and surface oxygen functionalities of three different [5] A. Ambrosi, C. K. Chua, A. Bonanni, M. Pumera, Chem. Rev. 2014, 114, chemically modified graphene platforms on the electrochemi7150. [6] W. Choi, I. Lahiri, R. Seelaboyina, Y. S. Kang, Crit. Rev. Solid State Mater. cal detection of gallic acid oxidation. We have demonstrated Sci. 2010, 35, 52. that the presence of defects and oxygen functionalities on gra[7] P. Avouris, C. Dimitrakopoulos, Mater. Today 2012, 15, 86. phene can strongly influence the electrochemical response of [8] A. Ambrosi, A. Bonanni, Z. Sofer, J. S. Cross, M. Pumera, Chem. Eur. J. the material towards the redox activity of the analyte of inter2011, 17, 10763. [9] W. Y. H. Khoo, M. Pumera, A. Bonanni, Anal. Chim. Acta 2013, 804, 92. est. [10] J. Y. Sun, K. J. Huang, S. Y. Wei, Z. W. Wu, F. P. Ren, Colloids Surf. B 2011, The electrochemically reduced graphene oxide provided the 84, 421. best analytical performance in terms of calibration sensitivity, [11] S. Y. Chee, M. Pumera, Electrochem. Commun. 2012, 20, 141. selectivity, and linearity of response, together with good repro[12] S. M. Tan, A. Ambrosi, C. K. Chua, M. Pumera, J. Mater. Chem. A 2014, 2, 10668. ducibility of results. This could be mainly due to the favorable [13] C. E. Chng, M. Pumera, A. Bonanni, Electrochem. Commun. 2014, 46, interaction between the analyte and the oxygen functionalities 137. present on ERGO, which promote the stacking of the analyte [14] A. Y. S. Eng, A. Ambrosi, C. K. Chua, F. Sanek, Z. Sofer, M. Pumera, Chem. on the graphene surface. Eur. J. 2013, 19, 12673. [15] C. S. Lim, A. Ambrosi, M. Pumera, Phys. Chem. Chem. Phys. 2014, 16, ERGO has also revealed to be a suitable platform for real 12178. sample analysis. Four wine samples were analyzed and their [16] S. M. Tan, H. L. Poh, Z. Sofer, M. Pumera, Analyst 2013, 138, 4885. antioxidant capacity, expressed as gallic acid equivalents (GAE), [17] U. Hofmann, R. Holst, Ber. Dtsch. Chem. Ges. 1939, 72, 754. confirmed the trend previously obtained with traditional meth[18] U. Hofmann, E. Konig, Z. Anorg. Allg. Chem. 1937, 234, 311. [19] M. Zhou, Y. L. Wang, Y. M. Zhai, J. F. Zhai, W. Ren, F. A. Wang, S. J. Dong, ods. In addition, the detection limit obtained with the develChem. Eur. J. 2009, 15, 6116. oped platform was two orders of magnitude lower than that [20] L. Staudenmaier, Ber. Dtsch. Chem. Ges. 1898, 31, 1481. achieved with traditional spectrophotometric methods em[21] A. Bonanni, C. K. Chua, M. Pumera, Chem. Eur. J. 2014, 20, 217. ploying Folin–Chocalteu protocol on similar wine samples.[33] [22] C. K. Chua, M. Pumera, J. Mater. Chem. 2012, 22, 23227. [23] L. P. Souza, F. Calegari, A. J. G. Zarbin, L. H. Marcolino, M. F. Bergamini, J. Overall, we have demonstrated the suitability of an electroAgric. Food Chem. 2011, 59, 7620. chemically reduced graphene platform for the determination [24] S. Gunckel, P. Santander, G. Cordano, J. Ferreira, S. Munoz, L. J. Nunezof antioxidant capacity of real food samples. This paves the Vergara, J. A. Squella, Chem.-Biol. Interact. 1998, 114, 45. [25] A. Bonanni, A. Ambrosi, M. Pumera, Chem. Eur. J. 2012, 18, 4541. Chem. Eur. J. 2015, 21, 11793 – 11798
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Received: April 30, 2015 Published online on July 1, 2015
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