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2009, 113, 14071–14075 Published on Web 07/17/2009
Direct Electrochemical Reduction of Single-Layer Graphene Oxide and Subsequent Functionalization with Glucose Oxidase Zhijuan Wang, Xiaozhu Zhou, Juan Zhang, Freddy Boey, and Hua Zhang* School of Materials Science and Engineering, Nanyang Technological UniVersity, 50 Nanyang AVenue, Singapore 639798, Singapore ReceiVed: July 06, 2009
A direct electrochemical method to reduce single-layer graphene oxide (GO) adsorbed on the 3-aminopropyltriethoxysilane (APTES)-modified conductive electrodes is proposed. The reduced GO adsorbed on glassy carbon electrode was modified with glucose oxidase (GOx) by covalent bonding via a polymer generated by electrografting N-succinimidyl acrylate (NSA). The direct electron transfer between the electrode and GOx molecules was realized. The bioactivity of GOx maintains very well on the electrode. The thus-prepared GOx-modified electrode was successfully used to detect glucose. Introduction Graphene, a new class of two-dimensional nanostructures, has attracted a great deal of interest due to its unique properties and potential applications in capacitors, cell images, sensors, devices, drug delivery, and solar cell.1-6 Currently, the major methods, including mechanical exfoliation,7 epitaxial growth,8 and chemical reduction of graphene oxide (GO) suspension,9 have been used to prepare graphene. Among them, the reproducibility of the mechanical exfoliation method is quite low. The epitaxial growth method does not produce graphene with uniform thickness, and thus, the application is limited. The chemical reduction method is reliable, scalable, and low cost.10 This kind of method,11 based on the Hummers’ method,12 produces GO by the strong oxidation of flake graphite with acid followed by subsequent chemical reduction with reducing agent such as hydroquinone,13 NaBH4,14 hydrazine,15 and hydrazine with NH3.16 These agents, particularly hydrazine, are highly toxic, and their use should be with extreme care and minimized.17 The chemical process may also leave some residual epoxide groups on the reduced GO sheets, which leads to some loss in electron mobility. GO contains oxygen functional groups, such as epoxides, -OH, and -COOH groups,18,19 which make it hydrophilic and disperse in water very well. However, GO is incompatible with most organic polymers.9 Chemically reduced GO also tends to aggregate or restack irreversibly to form graphite through van der Waals interactions.16,20 To avoid this, it can be functionalized to improve its dispersion. Functionalization can also enhance the application of the reduced GO, for example, in cellular imaging4,21 and drug delivery.3,22 The materials used to functionalize the reduced GO include aryldiazonium,14 polystyrene-polyacrylamine copolymer,20 alkaline earth metal ions,23 metal nanoparticles,24-28 and metal oxides.29 However, to date, there are very few reports that show the modification of reduced GO with biomolecules. * To whom correspondence should be addressed. Tel: (+65) 67905175. Fax: (+65) 67909081. E-mail:
[email protected]. Website: http://www.ntu.edu.sg/home/hzhang.
10.1021/jp906348x CCC: $40.75
SCHEME 1: Process of Electrochemical Reduction of GO and the Subsequent Modification of rGO with GOx
Herein, different from the existing methods mentioned above, we propose a novel method to directly reduce GO by using an electrochemical method, which is fast, clean, and nondestructive for controlled modification and reduction of materials.30 The electrochemically reduced GO, referred to as rGO herein, is chemically modified with the glucose oxidase (GOx) via a polymer generated by electrografting N-succinimidyl acrylate (NSA) (Scheme 1). The chemically modified biomolecules, for example, GOx, on rGO can be used for biosensing applications. Results and Discussion The experimental design and strategy is shown in Scheme 1. After single-layer GO sheets24 were adsorbed on the surface of 3-aminopropyltriethoxysilane (APTES)-modified glassy carbon electrode (GCE-APTES), referred to as GCE-APTES-GO, they were reduced by the electrochemical method, and rGO sheets were obtained, referred to as GCE-APTES-rGO (Figure 1A, solid line). The reduction was performed by scanning the potential from 0.7 to -1.1 V, and a reduction peak appeared at -0.87 V. In order to find out why and where the reduction peak 2009 American Chemical Society
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J. Phys. Chem. C, Vol. 113, No. 32, 2009
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Figure 1. Cyclic voltammograms of (A) GCE-APTES-GO (first cycle, solid line: curve 1), GCE-APTES-rGO (next 2 cycles, solid lines: curves 2 and 3), and HOPG (dashed line) and (B) ITO-APTES-GO (first cycle, solid line: curve 1), ITO-APTES-rGO (second cycle, solid lines: curves 2), and chemically reduced ITO-APTES-GO (dashed line). Electrolyte: 0.5 M NaCl aqueous solution saturated with N2. Scan rate: 50 mV s-1.
comes, a control experiment with a highly ordered pyrolytic graphite (HOPG) as the working electrode was carried out under the same experimental conditions. The result is shown in Figure 1A (dashed line), where no reduction peak is found. Given the high number of functional groups in the GO, such as -OH, -COOH, and epoxides, compared to those in HOPG, the reduction peak shown in Figure 1A (solid line: curve 1) could be attributed to the reduction of the functional groups on GO sheets. Interestingly, the reduction peak was only produced during the first cycle of scanning and disappeared completely in the next cycles. This important observation indicates that the electrochemical reduction of GO is not reversible. Therefore, after the first cycle of scanning (curve 1 in Figure 1A), GO was reduced, that is, rGO was obtained. The next two cycles (curves 2 and 3 in Figure 1A) show the electrochemical behavior of the obtained GCE-APTES-rGO. Another control experiment was carried out to confirm the effect of the electrochemical reduction. After the single-layer GO sheets were adsorbed on the APTES-modified ITO surface (ITO-APTES), referred to as ITO-APTES-GO, the electrochemical behavior, similar to that of GCE-APTES-GO, shows that the GO sheets were reduced by the electrochemical method (Figure 1B, first cycle, solid line: curve 1). However, after the ITO-APTES-GO was chemically reduced with the hydrazine vapor, no reduction peak appeared in the first scanning cycle (dashed line in Figure 1B). The curves are similar to ITOAPTES-GO from the second cycle (i.e., ITO-APTES-rGO in Figure 1B, solid line: curve 2) and hydrazine-reduced GO (Figure 1B, dashed line), indicating that the result of the electrochemical reduction of GO is similar to that of the chemical reduction. AFM images shown in Figure S1 (Supporting Information) indicate that after electrochemical reduction, the rGO sheets are still adsorbed on the ITO-APTES surface (Figure S1B, Supporting Information), similar to GO adsorbed on ITO-APTES surface (Figure S1A, Supporting Information). Although the chemical reduction of GO in aqueous solution leads to the aggregation,16,20 our result shows that no aggregation happened after electrochemical reduction of GO, indicating that the assembly of GO sheets on a solid substrate is a good way to prevent the aggregation of GO after reduction. In addition, the results of Raman spectroscopy (Figure S2, Supporting Information) and X-ray photoelectron spectroscopy (XPS, Figure S3, Supporting Information) indicate that the direct electrochemical reduction of GO has been successful and effective. The [Fe(CN)6]3-/4- couple is widely used as an electrochemical probe to investigate the characteristics of the modified electrode.31 In Figure 2, the peak-to-peak potential difference of the [Fe(CN)6]3-/4- couple changes at different modified
Figure 2. Cyclic voltammograms recorded at GCE (dotted line), GCEAPTES (dash-dot line), GCE-APTES-GO (dashed line), and GCEAPTES-rGO (solid line) for 5 mM [Fe(CN)6]3-/4- in 0.1 M KCl solution at a scan rate of 50 mV s-1.
electrodes. The bare GCE gives the reversible electrochemical response for the [Fe(CN)6]3-/4- couple (dotted line in Figure 2). After GCE was functionlized with APTES, redox peak disappeared due to the weak conductivity of the APTES selfassembled monolayers (SAMs) (dash-dot line in Figure 2). The GCE-APTES-GO electrode (dashed line in Figure 2) gave a cyclic voltammetry curve sismlar to that of GCE-APTES. Two probable reasons should be considered. First, GO is electrical insulator due to the oxidization of graphene sheets.6 Second, GO is negatively charged in solution due to the ionized functional groups such as -COO-, which generate the repulsive force over the negatively charged [Fe(CN)6]3-/4-. After the electrochemical reduction of GCE-APTES-GO, the electrochemical behavior of the [Fe(CN)6]3-/4- couple was significantly improved due to the high conductivity of rGO (solid line in Figure 2). In addition, after the electrochemical reduction, the rGO sheets remain neutral, leading to no repulsion between the [Fe(CN)6]3-/4- couple and GCE-APTES-rGO electrode. The redox peak of [Fe(CN)6]3-/4- is almost the same as that of bare GCE, indicating that the conductivity of the electrochemically reduced GO, that is, rGO, is as good as that of GCE (Figure 2, dotted line). After polymerization of N-succinimidyl acrylate (pNSA) on the GCE-APTES-rGO electrode by electrografting,32 the glucose oxidase (GOx) was immobilized on pNSA through covalent bonding,33 referred to as GCE-APTES-rGO-GOx, Scheme 1. The electrochemical activity of GOx on the GCE-APTES-rGOGOx electrode was investigated using cyclic voltammetry. In Figure 3, a pair of well-defined and symmetric redox peaks showed up after GOx immobilized on the GCE-APTES-rGO electrode. The cathodic and anodic peaks were at -0.46 and -0.43 V, respectively, which is consistent with the value of
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Figure 3. Cyclic voltammetric measurements at bare GCE (dotted line), GCE-APTES-rGO (dashed line), and GCE-APTES-rGO-GOx (solid line) in 0.01 M PBS buffer (pH 7.4) saturated with N2. Scan rate: 50 mV s-1.
the native molecule.34 Meanwhile, the peak-to-peak separation was 0.03 V, and the ratio of the redox peaks was close to 1, indicating that both peaks arise from the redox reaction of the active site of GOx.34,35 In addition, the current of electrocatalysis of GCE-APTES-rGO-GOx toward glucose increased with the glucose concentration (Figure S4, Supporting Information). Therefore, the GOx immobilized on the GCE-APTES-rGO electrode underwent the direct electron transfer between the active site of GOx and the electrode. Also, the effect of the scan rate on anodic and cathodic peak currents was measured, suggesting that the thin-layer electrochemical behavior is a surface-confined process (Figure S5, Supporting Information). In order to confirm the enzymatic activity of the immobilized GOx on an electrode, the electrocatalysis of GOx toward glucose was measured (Figure 4). In the air-saturated PBS solution, the reduction peak of glucose was at -0.29 V, and the reduction peak current decreased upon adding glucose to the air-saturated PBS solution (Figure 4A). The amperometric response was linear to the concentration of glucose, ranging from 0 to 24 mM (Figure 4B). All of these results confirm that the bioactivity of GOx is retained well after immobilization on the surface of GCE-APTES-rGO, which is consistent with the previous report.34 Conclusion In summary, we proposed a direct electrochemical method to reduce single-layer GO adsorbed on APTES-modified conductive electrodes. Thus-reduced GO was modified with pNSA by electrografting, followed by the covalent bonding with the GOx molecules. Importantly, the direct electron transfer between GOx molecules and the electrode was realized. The bioactivity of GOx maintained very well on the electrode. In addition, the obtained GCE-APTES-rGO-GOx was successfully used to detect glucose, which opens up a potential application in biosensing. Our method provided a simple and nontoxic way to reduce and then functionalize GO. Methods Materials. Nature graphite was purchased from Bay Carbon (Bay City, Michigan) and used for synthesizing graphene oxide (GO). 3-Aminopropyltriethoxysilane (APTES), H2O2 (30%), H2SO4 (98%), phosphate buffered saline, K4[Fe(CN)6] (99.9%), K3[Fe(CN)6] (99%), and alpha-D-glucose (96%) were purchased from Sigma-Aldrich (Milwaukee, WI) and used as received. 2-(N-Morpholino)ethanesulfonic acid (MES monohydrate, g99.5%, Fluka, St. Gallen, Switzerland), NH3 · H2O (28%, J. T. Baker, Phillipsburg, NJ), HCl (37%, Merck, Darmstadt, Germany), NaCl (99.5%, Merck, Darmstadt, Germany), glucose
J. Phys. Chem. C, Vol. 113, No. 32, 2009 14073 oxidase from Aspergillus niger (EC 1.1.3.4, ∼25 units/mg, Fluka, St. Gallen, Switzerland) were used as received. 1-Butyl3-methylimidazolium hexafluorophosphate (BMIMPF6, 97%, Sigma-Aldrich, Milwaukee, WI) was purified before use.36 Toluene was taken from a solvent purification system (PS-4005, Innovative Technology Inc.). ITO (10 ohm/sq, thickness: 0.7 mm) was purchased from Kintec Company (Hong Kong, China). N-Succinimidyl acrylate (NSA) was provided by Mr. Yifeng Wang (School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore). The 10 × 10 × 1 mm3 highly ordered pyrolytic graphite (HOPG) samples (Grade SPI2) were purchased from SPI Supplies (West Chester, PA). Ultrapure Milli-Q water (Milli-Q System, Millipore, Billerica, MA) was used in all experiments. Synthesis of Graphene Oxide (GO). GO was synthesized based on our previous report.24 In brief, 0.3 g of graphite was added into a mixture of 2.4 mL of 98% H2SO4, 0.5 of g K2S2O8, and 0.5 g of P2O5, and the solution was kept at 80 °C for 4.5 h. The resulting preoxidized product was cleaned by water and dried. After the preoxidized product was added into 12 mL of 98% H2SO4, followed by slow addition of 1.5 g of KMnO4 with the temperature kept at
