Materials Science and Engineering C 39 (2014) 281–287
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Graphene–cyclodextrin–cytochrome c layered assembly with improved electron transfer rate and high supramolecular recognition capability Cheng-Bin Gong 1, Cong-Cong Guo 1, Dan Jiang, Qian Tang ⁎, Chang-Hua Liu, Xue-Bing Ma The Key Laboratory of Applied Chemistry of Chongqing Municipality, College of Chemistry and Chemical Engineering, Southwest University, Chongqing, China
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 28 December 2013 Accepted 1 March 2014 Available online 12 March 2014 Keywords: Graphene Cyclodextrins Cytochrome c Graphene–cyclodextrin–cytochrome c Supramolecular recognition
a b s t r a c t This study aimed to develop a new graphene-based layered assembly, named graphene–cyclodextrin–cytochrome c with improved electron transfer rate. This assembly has combined high conductivity of graphene nanosheets (GNs), selectively binding properties and electronegativity of cyclodextrins (CDs), as well as electropositivity of cytochrome c (Cyt c). This assembly can also mimic the confined environments of the intermembrane space of mitochondria. A β-cyclodextrin (β-CD) functionalized GN (GN–CD) assembly was initially prepared by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide with hydrazine hydrate in the presence of β-CD. Cyt c was then intercalated to the GN–CD assembly to form a layered self-assembled structure, GN–CD–Cyt c, through electrostatic interaction. Compared with GNs and GN–CD, GN–CD–Cyt c assembly displayed improved electron transfer rate and high supramolecular recognition capability toward six probe molecules. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Graphene is a two-dimensional sheet of hexagonally arrayed sp2hybridized carbon atoms packed into a honeycomb lattice structure. Graphene has the advantages of high conductivity, large area surface, excellent chemical stability, and high mechanical flexibility [1–3]. Graphene nanosheets (GNs) are gaining attention as materials with wide-ranging applications, such as in sensors [4–7], nanoelectronics [8,9], nanomaterials [10], catalysis [11], and batteries [12]. However, GNs tend to form irreversible agglomerates due to the π–π interaction between individual GNs and cannot be well dispersed in aqueous solution [13]. This shortcoming can be overcome in two ways: First is to hybridize GNs with surfactants or polymers [14], and second is to chemically modify graphene [15,16]. Cyclodextrins (CDs) [17,18] are oligosaccharides composed of six, seven, or eight glucose units (α-, β-, or γ-CD, respectively). They have a hydrophobic inner cavity and a hydrophilic exterior. The hydrophobic inner cavity makes them suitable for the selective binding of various organic, inorganic, and biological molecules (guest) as the main body (host) to form stable host–guest inclusion assemblies or nanostructured supramolecular assemblies. Meanwhile, the hydrophilic exterior improves the solubility and stability of the host–guest inclusion assembly or the nanostructured supramolecular assemblies. Therefore, CDs ⁎ Corresponding author. Tel.: +86 23 68252360. E-mail address: [email protected] (Q. Tang). 1 These two authors equally contributed to this work.
http://dx.doi.org/10.1016/j.msec.2014.03.010 0928-4931/© 2014 Elsevier B.V. All rights reserved.
have been extensively used to functionalize GNs to improve electrochemical performance and molecular recognition [19–25]. Cytochrome c (Cyt c) is confined in the intermembrane space of mitochondria as an electronic transfer protein in the aerobic respiratory chain [26,27]. Belikova et al. [28] reported that the conformation and peroxidase activity of positively charged Cyt c are significantly influenced by the anionic phospholipid membrane mainly through electrostatic interaction. In recent years, nanoparticles [29–32] have been widely used for the fixation of Cyt c to investigate the structural and surface chemistry effects of these nanoparticles on the properties of Cyt c. However, reports on the intercalation of Cyt c into layered/ laminar structures that mimic the confined environments in the intermembrane space of mitochondria are limited [33,34]. To the best of our knowledge, GN–CD–Cyt c assemblies have not been reported. The resulting electron transfer property of Cyt c after it is intercalated into the layered GN–CD assembly to form a tertiary assembly is unknown. The use of this assembly as an electrochemical sensor has also not yet been studied. In this work, a β-CD functionalized GN assembly (GN–CD) was synthesized by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide (GO) with hydrazine hydrate in the presence of β-CD. Then, Cyt c was intercalated to the GN–CD layered structure to form the layered self-assembly structure (GN–CD–Cyt c). This tertiary assembly had the following four advantages: (1) GNs with a unique two-dimensional structure, extraordinary conductivity, and large surface area that are suitable for constructing an electrochemically layered assembly; (2) the negatively charged CD–GN surface
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2.2. Apparatus
Fig. 1. UV–vis absorption spectra of GO dispersions within the reaction period (0–80 min). The inset (upper right corner) shows the relationship between the wavelength at the maximum absorption and reaction time.
Electrochemical measurements were carried out using CHI 660A Electrochemical Workstation (Shanghai Chenhua Instruments Co., Ltd., Shanghai, China): A three-compartment electrochemical cell containing a modified glassy carbon electrode (GCE; φ = 4 mm) as a working electrode, a platinum wire as an auxiliary electrode and a saturated calomel electrode as a reference electrode. Electrochemical impedance spectroscopy (EIS) data were collected for the 100 kHz to 0.1 Hz frequency range with an AC amplitude of 10 mV peak to peak. The surface morphologies of GNs, GN–CD, and GN–CD–Cyt c were identified by scanning electron microscopy (SEM; S-4800, Hitachi, Tokyo, Japan) and atomic force microscopy (AFM; Veeco Instruments Inc., USA). Ultraviolet–visible (UV–vis) absorption spectra were recorded on a UV-4802 spectrophotometer (Shanghai UNICO Instruments Co., Ltd., Shanghai, China). Fourier-transform infrared (FT-IR) spectra were recorded on a PerkinElmer Model GX Spectrometer using a KBr pellet method with polystyrene as a standard. Zeta potential was measured by dynamic light scattering (DLS) (VEBA/LMU, UK). All experiments were carried out at room temperature. 2.3. Preparation of GNs and GN–CD
promotes the self-assembly of positively charged Cyt c through electrostatic interaction; (3) β-CD has a hydrophobic inner cavity that can enable to accomplish host–guest recognition and enrichment; and (4) the intercalated structure similar to the phospholipid membrane confers onto Cyt c excellent electrical properties. These made the GN– CD–Cyt c assembly particularly suitable for ultrasensitive determination as electrochemical sensors.
2. Experimental 2.1. Reagents and materials Graphite oxide (GO) was purchased from Xian Feng Nano Co., Ltd. (Nanjing, China). Cyt c was purchased from Shanghai Ruiqi Biological Co., Ltd. (Shanghai, China). β-CD and hydrazine hydrate solution (50 wt.%) were obtained from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) with various pH values was prepared using stock solution of 0.1 M Na2HPO4, and 0.1 M NaH2PO4 , and the supporting electrolyte was 0.1 M KCl. All chemicals used were of analytical grade and used as received without further purification. Double-distilled water was used throughout the experiment.
GN–CD hybrid nanosheets were prepared according to a previous method with minor modification [15]. In a typical procedure, 30.0 mL of homogeneous GO dispersion (0.5 mg mL−1) and 30.0 mL (3 wt.%) of β-CD aqueous solution were mixed in a 100 mL three-necked flask. The resultant mixture was vigorously stirred overnight at 50 °C and then cooled to room temperature. About 175.0 μL of ammonia solution (25–28 wt.%) and 20.0 μL of hydrazine hydrate solution were introduced, followed by stirring for 5 min. The resultant mixture was heated to 95 °C for 1.5 h, and a stable black dispersion was obtained. The dispersion was filtered through a microporous membrane (0.22 μm), and the solid was throughly washed with double-distilled water, finally the organic–inorganic hybrid nanosheets, GN–CD, were collected. These nanosheets can be readily dispersed in water (0.25 mg mL−1) by ultrasonication. GNs were prepared in an identical fashion except that no β-CD was added. 2.4. Preparation of GN–CD–Cyt c About 20 μ L (0.5 mg mL−1) of Cyt c PBS (pH = 7.0) was introduced to 100 μ L (0.25 mg mL−1) of GN–CD solution. The mixture was placed in a refrigerated chamber for 24 h. A black GN–CD–Cyt c suspension was obtained after decanting the supernatant. 2.5. Preparation of GCE modified GO, GNs, GN–CD, and GN–CD–Cyt c The GCE was polished with 0.3 and 0.05 μm alumina slurry to mirror-like quality, sonicated in ethanol and double-distilled water for several minutes, and dried in N2. About 10 μL (0.25 mg mL−1) of Table 1 Zeta potential of GO, GNs, GN–CD and GN–CD–Cytc c. No
Zeta(mV)
Average value (mV)
GO
−50.4 −52.0 −51.7 −33.9 −34.1 −33.0 −45.6 −46.0 −45.0 −29.5 −28.1 −30.0
−51.4
GNs
GN–CD
GN–CD–Cytc c Fig. 2. FT-IR spectra of GO, GNs, and GN–CD.
−33.7
−45.5
−29.2
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Fig. 3. AFM image and section analysis of GNs (A) on mica. AFM image (2-D (left) and 3-D (right)) of Cyt c on mica (B). SEM image of GN–CD (C) and GN–CD–Cyt c (D).
GN–CD–Cyt c aqueous suspension was dropped onto the clean and bright surface of the GCE and dried in air at room temperature to fabricate an electrochemical sensor (GN–CD–Cyt c/GCE). For comparison, GO/GCE, GNs/GCE, and GN–CD/GCE were also prepared in the same way. 3. Results and discussions 3.1. Structural and morphologic characterization The reaction process for the reduction of GO was monitored by UV–vis absorption spectroscopy. The reaction mixture was sampled at an interval of 10 min. Fig. 1 shows that at the beginning (at 0 min), the maximum absorption at 231 nm and the shoulder absorption at 300 nm can be attributed to the π–π* transition of aromatic C_C and the n–π* transition of the C_O bond of GO, respectively [20,35]. With increased reaction time, the color of the dispersion changed from light yellow to dark brown and finally to black. The absorption peak of GO at 231 nm gradually shifted to 267 nm, and the shoulder absorption at 300 nm gradually decreased until it disappeared. Moreover, the absorbance throughout the entire spectral region increased with an increased reaction time,
demonstrating that the oxygen-containing groups (e.g., \COOH, \OH) were reduced by hydrate hydrazine and the electronic conjugation within GNs was restored. After 70 min, the system reached a photostationary state, indicating the completion of reduction reaction. FT-IR spectra of GO, GNs and GN–CD are shown in Fig. 2. After reduction of GO to GNs, the adsorption peaks of O\H (ν O\H at 2900–3700 cm− 1) and C_O in carboxylic acid and carbonyl moieties (νC_O at 1720 cm−1) significantly decreased [13]. After functionalization with β-CD, the FT-IR spectrum of GN–CD displayed the typical CD absorption peaks of ring vibrations at 585, 875 and 950 cm−1, the coupled C\O\C stretching/O\H bending vibrations at 1120 cm−1, the CH2 stretching vibrations at 2990 cm−1, C\H/O\H bending vibrations at 1400 cm−1, and O\H stretching vibrations at 3477 cm−1 [20]. The redshift of O\H stretching vibrations from 3700 cm−1 (free OH) to 3477 cm−1 demonstrated the formation of hydrogen bond between CD molecules and some oxygen-containing groups of GNs [36]. The electronegativity of GO, GNs, GN–CD and GN–CD–Cyt c was detected by DLS, and the results are shown in Table 1. GO's zeta potential (−51.4 mV) was markedly lower than −30 mV, so it stably existed in aqueous solution [37]. The zeta potential of GNs (−33.7 mV) increased
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because of the hydrophilic exterior of β-CDs. This negative zeta potential (more negative than −30 mV) stabilized the GN–CD nanosheets from aggregation in solution. The negative charges on the surface of GN–CD facilitated the assembly process with Cyt c. The zeta potential increased to − 29.2 mV after the combination of GN–CD with the positively charged Cyt c through electrostatic interaction. Fig. 3A shows a typical AFM image of GNs. The average thickness of GNs measured from the height profile of the AFM image was about 0.79 nm, which was close to the theoretical values of two graphene layers [3]. The AFM image of Cyt c is shown in Fig. 3B, which showed the typical morphology of globular proteins. After functionalization with β-CD (Fig. 3C), small CDs adsorbed on both sides of GNs were clearly observed. After self-assembly with Cyt c through electrostatic interaction (Fig. 3D), graphene became thicker because of the agglomeration of GN–CD–Cyt c, which was due to the increase in zeta potential, and the surface was covered by Cyt c. 3.2. Electrochemical characterization
Fig. 4. (A) Cyclic voltammograms of GNs/GCE (curve a), GN–CD/GCE (curve b), and GNs– aqueous solution. (B) EIS plot of GNs (curve a), CD–Cyt c/GCE (curve c) in Fe(CN)3−/4− 6 aqueous solution: inset is GN–CD (curve b), and GN–CD–Cyt c (curve c) in Fe(CN)3−/4− 6 fitting of impedance spectra for Fe(CN)3−/4− at GN–CD/GCE and GN–CD–Cyt c/GCE. 6 (C) The equivalent circuit.
after reduction of GO, which was probably due to the decrease in oxygen containing groups, making GO less hydrophilic. The zeta potential of GN–CD decreased to −45.5 mV after combination of β-CD, demonstrating that GN–CD possessed numerous negative charges on the surface
The cyclic voltammetric response of GCE modified with GNs, GN–CD redox couple in and GN–CD–Cyt c was examined using an Fe(CN)3−/4− 6 containing 0.1 M KCl (Fig. 4A). neutral solution of standard Fe(CN)3−/4− 6 Compared with the GNs, GN–CD showed slightly better electrochemical response. This result indicated that the rate of electron transfer on the electrode surface was enhanced after the functionalization of GNs with CD. This finding illustrated that the enrichment of β-CD molecules between the electrochemical probe Fe(CN)36 −/4 − and the surface of GCE aided to the conductivity [20]. After the combination of Cyt c with GN–CD through electrostatic interaction, the tertiary assembly, GN–CD–Cyt c showed the highest current, demonstrating that GN–CD–Cyt c possessed a higher electron transfer rate than GNs and GN–CD. EIS was used to evaluate the surface layer kinetics for solution containing 0.1 M KCl as a redox probe. Fig. 4B repFe(CN)3−/4− 6 resents a Nyquist plot for a GNs/GCE (curve a), GN–CD/GCE (curve b) and GN–CD–Cyt c/GCE (curve c). It was found that GNs/GCE displayed a semicircle at high frequency region and a straight line at low frequency region. However, GN–CD/GCE and GN–CD–Cyt c/GCE showed a low frequency straight line with a very small semi-circle at high frequency on region, indicating a diffusion controlled process for the Fe(CN)3−/4− 6 GN–CD/GCE and GN–CD–Cyt c/GCE. The Randles equivalent circuit was used for GNs/GCE analysis (Fig. 4C), where Rs, Rct, Rw, and CPE represent the solution resistance, the charge-transfer resistance, the Warburg resistance, and the constant phase element, respectively. The charge transfer resistance at GN–CD/GCE is higher than that at GN–CD–Cyt c. The much lower Rct value on GN–CD–Cyt c/GCE illustrates that the electron transfer is quasi-reversible. Namely, GN–CD–Cyt c acreaction for Fe(CN)3−/4− 6 celerated the electron transfer between the electrochemical probe and the GCE. This phenomenon can be attributed to the Fe(CN)3−/4− 6 confined environment of Cyt c that was similar to the phospholipid membrane and conferred Cyt c with excellent electrical property. Fig. 5A depicts the cyclic voltammetric responses of GN–CD–Cyt c/GCE in PBS (pH = 7.0) at various scan rates. With increased scanning speed, the redox peak current (Fig. 5B) linearly increased and the redox peak potential slightly moved, indicating a typical adsorptioncontrolled process. 3.3. Optimization of the determination conditions
Fig. 5. Cyclic voltammograms (A) and Imax (B) (inset) of the GN–CD–Cyt c/GCE at different scan rates in 0.1 M PBS (pH = 7.0).
To obtain the best properties, experimental conditions such as the assembly time, pH, detection temperature, and dosage of GN–CD–Cyt c were optimized in PBS solution. Assembly time plays an important role in the assembly layer of GN–CD–Cyt c. Fig. 6A shows that the current value of electrode modified with GNs–CD–Cyt c initially increased and then decreased with increased assembly time, and the maximum value was obtained at 36 h. This phenomenon can be explained as follows. At the first stage (0–36 h),
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Fig. 6. Effect of assembly time (A), pH (B), temperature (C) and amount of GN–CD–Cyt c (D) on the GN–CD–Cyt c/GCE response in 0.1 M PBS. The insets in (B), (C) and (D) show the current versus pH, temperature and volume of GNs–CD–Cyt c solution, respectively.
Fig. 7. Procedure for preparing GNs, GN–CD, GN–CD–Cyt c nanosheets, and sensing guest molecules by an electrochemical method.
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2.0 to 7.0, reaching a maximum at pH 7.0. The possible reason is that Cyt c possesses better redox potential at neutral pH [38]. The influence of experimental temperature (0–55 °C) was investigated in buffer solution at pH 7. Fig. 6C shows that the current peak reached the maximum value between 45 and 50 °C. However, 37 °C (close to the human body temperature) was selected as the optimum temperature to maintain the bioactivity of Cyt c as long as possible. Finally, the optimum dosage of GN–CD–Cyt c was investigated. Fig. 6D shows that the maximum current was obtained when the volume of GNs–CD–Cyt c solution is ≥7 mL. In summary, the optimum experimental conditions for obtaining the best GN–CD–Cyt c properties were assembly duration of 36 h, pH = 7.0, 37 °C, and 7 μL of GNs–CD–Cyt c solution.
3.4. Supramolecular recognition of GN–CD–Cyt c assembly
Fig. 8. (A) Cyclic voltammograms of 50 μM glycine at GCE (a), GNs/GCE (b), GN–CD– Cyt c/GCE (c), and GN–CD–Cyt c/GCE (d) in 0.1 M phosphate buffer (pH 7.0) at a scan rate of 50 mV/s. (B) Cyclic voltammograms of GN–CD–Cyt c/GCE at different concentrations of glycine. (C) Imax versus glycine concentration. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
more Cyt c molecules were adsorbed and the number of assembled layers increased. The electrical properties of GN–CD–Cyt c gradually improved and finally reached the maximum value. At the second stage (N36 h), the thickness of GN–CD–Cyt c was too large, which impeded the electron transmission and markedly reduced conductive performance. This finding was similar to that of a cell membrane, whose maximum permeability is obtained only at the optimal thickness [26,27]. The pH markedly influences the bioactivity of protein and enzyme. The influence of pH values on the cyclic voltammetric response of GN–CD–Cyt c was investigated from pH 2.0 to 12.0 in 0.1 M PBS. As shown in Fig. 6B, the peak current increased with increased pH from
As described above, the tertiary assembly GN–CD–Cyt c showed improved electron transfer rate. Furthermore, the good biocompatibility of Cyt c, and non-cytotoxicity of carbon [39] made the layered GN–CD–Cyt c assembly particularly suitable for ultrasensitive determination because of host–guest recognition and enrichment of CD. To verify our assumption (Fig. 7), the electrochemical behaviors of GN–CD–Cyt c toward six kinds of small biomolecules (glycine, alanine, valine, proline, phenylalanine, and tryptophane) including the simplest, aromatic, and heterocyclic amino acids were investigated. Glycine was chosen as a representative analyte, because it is generally used as a food additive to relieve the sour and bitter taste (caused by saccharin), thereby increasing the sweet taste of food. However, excessive intake of glycine breaks the absorption equilibrium of amino acids and affects the absorption of other amino acids for humans, which can result in nutrient imbalance and affect human health. Therefore, the detection or sensing of glycine has important scientific significance and practical value. Fig. 8A shows the cyclic voltammograms of glycine at bare GCE (curve a, black), GNs/GCE (curve b, red), GN–CD/GCE (curve c, green), and GN–CD–Cyt c/GCE (curve d, blue) in 0.1 M PBS (pH = 7.0) at a scan rate of 50 mV/s. The electrochemical response decreased in the order: GN–CD–Cyt c/GCE N GN–CD/GCE N GNs/GCE N blank (GCE), illustrating favorable catalytic activity of GNs, GN–CD, and GN–CD–Cyt c toward the oxidation of glycine. This finding also demonstrated that β-CD molecules on the surface of GNs with high supramolecular recognition capability can enrich the analyte molecules to the detection surface. GN–CD–Cyt c/GCE electrode showed the best electrochemical properties among the four, demonstrating that the layered GN–CD– Cyt c assembly had all the merits of three constituents like the high conductivity and high surface area of GNs, the host–guest recognition and enrichment of β-CD molecules, as well as the high conductivity of Cyt c. Fig. 8B shows that the cyclic voltammograms of GN–CD–Cyt c modified GCE toward different glycine concentrations. The current increased with an increased glycine concentration. Fig. 8C displays the relationship between Imax and glycine concentration. GNs–CD–Cyt c/GCE had two concentration ranges that linearly responded to glycine. From 0.1 μM to 7.0 μM, the regression equation was ip (mA) = 1.5263 C (μM) + 24.588, and R2 = 0.9941; from 7.0 μM to 20.0 μM, the regression equation was ip (mA) = 0.5418 C (μM) + 31.256, and R2 = 0.9983. The limit of detection for glycine was estimated to be 3.0 × 10− 8 mol L− 1 (S/N = 3). These data further indicated that GCE modified with GN–CD–Cyt c exhibited very high electrochemical performance toward the target molecules. A similar electrochemical behavior of GN– CD–Cyt c toward the other five amino acids (alanine, valine, proline, phenylalanine, and tryptophane) was observed (see Supporting information). These data indicated that using GN–CD–Cyt c/GCE was a simple and reliable electrochemical technique that can be applied to the development of sensitive electrochemical sensors. These sensors can be used to determine a wide variety of electro-active compounds.
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4. Conclusion A new tertiary (graphene, β-CD, and Cyt c) layered assembly named GN–CD–Cyt c was prepared and characterized. GN–CD–Cyt c had combined advantages of all three components and thus showed improved electron transfer rate and high sensitive supramolecular recognition (glycine detection limit ≈ 3.0 × 10−8 mol L−1) to six investigated amino acids, the basic protein unit. This new functional assembly can be extensively used as electrochemical sensors for ultrasensitive detection because of its good conductivity. Acknowledgments This work was supported by the National Natural Science Foundation of China (20872121), CQ CSTC 2013jcyjA50026, Research Funds for the Doctoral Program of Higher Education of China (20090182120010), Southwest University Doctoral Fund (SWUB2008075) and PolyU grant (G-U993). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.03.010. References [1] A.K. Geim, K.S. Novoselov, The rise of grapheme, Nat. Mater. 6 (2007) 183–191. [2] C.N.R. Rao, A.K. Sood, K.S. Subrahmanyam, A. Govindaraj, Graphene: the new twodimensional nanomaterial, Angew. Chem. Int. Edit. 48 (2009) 7752–7777. [3] Y.X. Xu, H. Bai, G.W. Lu, C. Li, G.Q. Shi, Flexible graphene films via the filtration of water-soluble noncovalent functionalized graphene sheets, J. Am. Chem. Soc. 130 (2008) 5856–5857. [4] M.M. Liu, W. Chen, Graphene nanosheets-supported Ag nanoparticles for ultrasensitive detection of TNT by surface-enhanced Raman spectroscopy, Biosens. Bioelectron. 46 (2013) 68–73. [5] J.W. Wu, C.H. Wang, Y.C. Wang, J.K. Chang, Ionic-liquid-enhanced glucose sensing ability of non-enzymatic Au/graphene electrodes fabricated using supercritical CO2 fluid, Biosens. Bioelectron. 46 (2013) 30–36. [6] L. Wu, L.Y. Feng, J.S. Ren, X.G. Qu, Electrochemical detection of dopamine using porphyrin-functionalized graphene, Biosens. Bioelectron. 34 (2012) 57–62. [7] L.M. Zhu, L.Q. Luo, Z.X. Wang, DNA electrochemical biosensor based on thionine– graphene nanocomposite, Biosens. Bioelectron. 35 (2012) 507–511. [8] X.L. Li, X.R. Wang, L. Zhang, S.W. Lee, H.J. Dai, Chemically derived, ultrasmooth graphene nanoribbon semiconductors, Science 319 (2008) 1229–1232. [9] S.P. Pang, H.N. Tsao, X.L. Feng, K. Mullen, Patterned graphene electrodes from solution-processed graphite oxide films for organic field-effect transistors, Adv. Mater. 21 (2009) 3488–3491. [10] J.L. Vickery, A.J. Patil, S. Mann, Fabrication of graphene polymer nanocomposites with higher-order three-dimensional architectures, Adv. Mater. 21 (2009) 2180–2184. [11] D.H. Wang, D.W. Choi, J. Li, Z.G. Yang, Z.M. Nie, R. Kou, D.H. Hu, C.M. Wang, L.V. Saraf, J.G. Zhang, I.A. Aksay, J. Liu, Self-assembled TiO2–graphene hybrid nanostructures for enhanced li-ion insertion, ACS Nano 3 (2009) 907–914. [12] L.H. Tang, Y. Wang, Y.M. Li, H.B. Feng, J. Lu, J.H. Li, Preparation, structure, and electrochemical properties of reduced graphene sheet films, Adv. Funct. Mater. 19 (2009) 2782–2789. [13] Y. Si, E.T. Samulski, Synthesis of water soluble graphene, Nano Lett. 8 (2008) 1679–1682. [14] X. Huang, Z.Y. Yin, S.X. Wu, X.Y. Qi, Q.Y. He, Q.C. Zhang, Q.Y. Yan, F. Boey, H. Zhang, Graphene-based materials: synthesis, characterization, properties, and applications, Small 7 (2011) 1876–1902.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Graphene–cyclodextrin–cytochrome c layered assembly with improved electron transfer rate and high supramolecular recognition capability Cheng-Bin Gong 1, Cong-Cong Guo 1, Dan Jiang, Qian Tang ⁎, Chang-Hua Liu, Xue-Bing Ma The Key Laboratory of Applied Chemistry of Chongqing Municipality, College of Chemistry and Chemical Engineering, Southwest University, Chongqing, China
a r t i c l e
i n f o
Article history: Received 10 October 2013 Received in revised form 28 December 2013 Accepted 1 March 2014 Available online 12 March 2014 Keywords: Graphene Cyclodextrins Cytochrome c Graphene–cyclodextrin–cytochrome c Supramolecular recognition
a b s t r a c t This study aimed to develop a new graphene-based layered assembly, named graphene–cyclodextrin–cytochrome c with improved electron transfer rate. This assembly has combined high conductivity of graphene nanosheets (GNs), selectively binding properties and electronegativity of cyclodextrins (CDs), as well as electropositivity of cytochrome c (Cyt c). This assembly can also mimic the confined environments of the intermembrane space of mitochondria. A β-cyclodextrin (β-CD) functionalized GN (GN–CD) assembly was initially prepared by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide with hydrazine hydrate in the presence of β-CD. Cyt c was then intercalated to the GN–CD assembly to form a layered self-assembled structure, GN–CD–Cyt c, through electrostatic interaction. Compared with GNs and GN–CD, GN–CD–Cyt c assembly displayed improved electron transfer rate and high supramolecular recognition capability toward six probe molecules. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Graphene is a two-dimensional sheet of hexagonally arrayed sp2hybridized carbon atoms packed into a honeycomb lattice structure. Graphene has the advantages of high conductivity, large area surface, excellent chemical stability, and high mechanical flexibility [1–3]. Graphene nanosheets (GNs) are gaining attention as materials with wide-ranging applications, such as in sensors [4–7], nanoelectronics [8,9], nanomaterials [10], catalysis [11], and batteries [12]. However, GNs tend to form irreversible agglomerates due to the π–π interaction between individual GNs and cannot be well dispersed in aqueous solution [13]. This shortcoming can be overcome in two ways: First is to hybridize GNs with surfactants or polymers [14], and second is to chemically modify graphene [15,16]. Cyclodextrins (CDs) [17,18] are oligosaccharides composed of six, seven, or eight glucose units (α-, β-, or γ-CD, respectively). They have a hydrophobic inner cavity and a hydrophilic exterior. The hydrophobic inner cavity makes them suitable for the selective binding of various organic, inorganic, and biological molecules (guest) as the main body (host) to form stable host–guest inclusion assemblies or nanostructured supramolecular assemblies. Meanwhile, the hydrophilic exterior improves the solubility and stability of the host–guest inclusion assembly or the nanostructured supramolecular assemblies. Therefore, CDs ⁎ Corresponding author. Tel.: +86 23 68252360. E-mail address: [email protected] (Q. Tang). 1 These two authors equally contributed to this work.
http://dx.doi.org/10.1016/j.msec.2014.03.010 0928-4931/© 2014 Elsevier B.V. All rights reserved.
have been extensively used to functionalize GNs to improve electrochemical performance and molecular recognition [19–25]. Cytochrome c (Cyt c) is confined in the intermembrane space of mitochondria as an electronic transfer protein in the aerobic respiratory chain [26,27]. Belikova et al. [28] reported that the conformation and peroxidase activity of positively charged Cyt c are significantly influenced by the anionic phospholipid membrane mainly through electrostatic interaction. In recent years, nanoparticles [29–32] have been widely used for the fixation of Cyt c to investigate the structural and surface chemistry effects of these nanoparticles on the properties of Cyt c. However, reports on the intercalation of Cyt c into layered/ laminar structures that mimic the confined environments in the intermembrane space of mitochondria are limited [33,34]. To the best of our knowledge, GN–CD–Cyt c assemblies have not been reported. The resulting electron transfer property of Cyt c after it is intercalated into the layered GN–CD assembly to form a tertiary assembly is unknown. The use of this assembly as an electrochemical sensor has also not yet been studied. In this work, a β-CD functionalized GN assembly (GN–CD) was synthesized by a simple wet-chemical strategy, i.e., in situ thermal reduction of graphene oxide (GO) with hydrazine hydrate in the presence of β-CD. Then, Cyt c was intercalated to the GN–CD layered structure to form the layered self-assembly structure (GN–CD–Cyt c). This tertiary assembly had the following four advantages: (1) GNs with a unique two-dimensional structure, extraordinary conductivity, and large surface area that are suitable for constructing an electrochemically layered assembly; (2) the negatively charged CD–GN surface
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2.2. Apparatus
Fig. 1. UV–vis absorption spectra of GO dispersions within the reaction period (0–80 min). The inset (upper right corner) shows the relationship between the wavelength at the maximum absorption and reaction time.
Electrochemical measurements were carried out using CHI 660A Electrochemical Workstation (Shanghai Chenhua Instruments Co., Ltd., Shanghai, China): A three-compartment electrochemical cell containing a modified glassy carbon electrode (GCE; φ = 4 mm) as a working electrode, a platinum wire as an auxiliary electrode and a saturated calomel electrode as a reference electrode. Electrochemical impedance spectroscopy (EIS) data were collected for the 100 kHz to 0.1 Hz frequency range with an AC amplitude of 10 mV peak to peak. The surface morphologies of GNs, GN–CD, and GN–CD–Cyt c were identified by scanning electron microscopy (SEM; S-4800, Hitachi, Tokyo, Japan) and atomic force microscopy (AFM; Veeco Instruments Inc., USA). Ultraviolet–visible (UV–vis) absorption spectra were recorded on a UV-4802 spectrophotometer (Shanghai UNICO Instruments Co., Ltd., Shanghai, China). Fourier-transform infrared (FT-IR) spectra were recorded on a PerkinElmer Model GX Spectrometer using a KBr pellet method with polystyrene as a standard. Zeta potential was measured by dynamic light scattering (DLS) (VEBA/LMU, UK). All experiments were carried out at room temperature. 2.3. Preparation of GNs and GN–CD
promotes the self-assembly of positively charged Cyt c through electrostatic interaction; (3) β-CD has a hydrophobic inner cavity that can enable to accomplish host–guest recognition and enrichment; and (4) the intercalated structure similar to the phospholipid membrane confers onto Cyt c excellent electrical properties. These made the GN– CD–Cyt c assembly particularly suitable for ultrasensitive determination as electrochemical sensors.
2. Experimental 2.1. Reagents and materials Graphite oxide (GO) was purchased from Xian Feng Nano Co., Ltd. (Nanjing, China). Cyt c was purchased from Shanghai Ruiqi Biological Co., Ltd. (Shanghai, China). β-CD and hydrazine hydrate solution (50 wt.%) were obtained from Shanghai Aladdin Reagent Co., Ltd. (Shanghai, China). Phosphate-buffered saline (PBS) with various pH values was prepared using stock solution of 0.1 M Na2HPO4, and 0.1 M NaH2PO4 , and the supporting electrolyte was 0.1 M KCl. All chemicals used were of analytical grade and used as received without further purification. Double-distilled water was used throughout the experiment.
GN–CD hybrid nanosheets were prepared according to a previous method with minor modification [15]. In a typical procedure, 30.0 mL of homogeneous GO dispersion (0.5 mg mL−1) and 30.0 mL (3 wt.%) of β-CD aqueous solution were mixed in a 100 mL three-necked flask. The resultant mixture was vigorously stirred overnight at 50 °C and then cooled to room temperature. About 175.0 μL of ammonia solution (25–28 wt.%) and 20.0 μL of hydrazine hydrate solution were introduced, followed by stirring for 5 min. The resultant mixture was heated to 95 °C for 1.5 h, and a stable black dispersion was obtained. The dispersion was filtered through a microporous membrane (0.22 μm), and the solid was throughly washed with double-distilled water, finally the organic–inorganic hybrid nanosheets, GN–CD, were collected. These nanosheets can be readily dispersed in water (0.25 mg mL−1) by ultrasonication. GNs were prepared in an identical fashion except that no β-CD was added. 2.4. Preparation of GN–CD–Cyt c About 20 μ L (0.5 mg mL−1) of Cyt c PBS (pH = 7.0) was introduced to 100 μ L (0.25 mg mL−1) of GN–CD solution. The mixture was placed in a refrigerated chamber for 24 h. A black GN–CD–Cyt c suspension was obtained after decanting the supernatant. 2.5. Preparation of GCE modified GO, GNs, GN–CD, and GN–CD–Cyt c The GCE was polished with 0.3 and 0.05 μm alumina slurry to mirror-like quality, sonicated in ethanol and double-distilled water for several minutes, and dried in N2. About 10 μL (0.25 mg mL−1) of Table 1 Zeta potential of GO, GNs, GN–CD and GN–CD–Cytc c. No
Zeta(mV)
Average value (mV)
GO
−50.4 −52.0 −51.7 −33.9 −34.1 −33.0 −45.6 −46.0 −45.0 −29.5 −28.1 −30.0
−51.4
GNs
GN–CD
GN–CD–Cytc c Fig. 2. FT-IR spectra of GO, GNs, and GN–CD.
−33.7
−45.5
−29.2
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Fig. 3. AFM image and section analysis of GNs (A) on mica. AFM image (2-D (left) and 3-D (right)) of Cyt c on mica (B). SEM image of GN–CD (C) and GN–CD–Cyt c (D).
GN–CD–Cyt c aqueous suspension was dropped onto the clean and bright surface of the GCE and dried in air at room temperature to fabricate an electrochemical sensor (GN–CD–Cyt c/GCE). For comparison, GO/GCE, GNs/GCE, and GN–CD/GCE were also prepared in the same way. 3. Results and discussions 3.1. Structural and morphologic characterization The reaction process for the reduction of GO was monitored by UV–vis absorption spectroscopy. The reaction mixture was sampled at an interval of 10 min. Fig. 1 shows that at the beginning (at 0 min), the maximum absorption at 231 nm and the shoulder absorption at 300 nm can be attributed to the π–π* transition of aromatic C_C and the n–π* transition of the C_O bond of GO, respectively [20,35]. With increased reaction time, the color of the dispersion changed from light yellow to dark brown and finally to black. The absorption peak of GO at 231 nm gradually shifted to 267 nm, and the shoulder absorption at 300 nm gradually decreased until it disappeared. Moreover, the absorbance throughout the entire spectral region increased with an increased reaction time,
demonstrating that the oxygen-containing groups (e.g., \COOH, \OH) were reduced by hydrate hydrazine and the electronic conjugation within GNs was restored. After 70 min, the system reached a photostationary state, indicating the completion of reduction reaction. FT-IR spectra of GO, GNs and GN–CD are shown in Fig. 2. After reduction of GO to GNs, the adsorption peaks of O\H (ν O\H at 2900–3700 cm− 1) and C_O in carboxylic acid and carbonyl moieties (νC_O at 1720 cm−1) significantly decreased [13]. After functionalization with β-CD, the FT-IR spectrum of GN–CD displayed the typical CD absorption peaks of ring vibrations at 585, 875 and 950 cm−1, the coupled C\O\C stretching/O\H bending vibrations at 1120 cm−1, the CH2 stretching vibrations at 2990 cm−1, C\H/O\H bending vibrations at 1400 cm−1, and O\H stretching vibrations at 3477 cm−1 [20]. The redshift of O\H stretching vibrations from 3700 cm−1 (free OH) to 3477 cm−1 demonstrated the formation of hydrogen bond between CD molecules and some oxygen-containing groups of GNs [36]. The electronegativity of GO, GNs, GN–CD and GN–CD–Cyt c was detected by DLS, and the results are shown in Table 1. GO's zeta potential (−51.4 mV) was markedly lower than −30 mV, so it stably existed in aqueous solution [37]. The zeta potential of GNs (−33.7 mV) increased
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because of the hydrophilic exterior of β-CDs. This negative zeta potential (more negative than −30 mV) stabilized the GN–CD nanosheets from aggregation in solution. The negative charges on the surface of GN–CD facilitated the assembly process with Cyt c. The zeta potential increased to − 29.2 mV after the combination of GN–CD with the positively charged Cyt c through electrostatic interaction. Fig. 3A shows a typical AFM image of GNs. The average thickness of GNs measured from the height profile of the AFM image was about 0.79 nm, which was close to the theoretical values of two graphene layers [3]. The AFM image of Cyt c is shown in Fig. 3B, which showed the typical morphology of globular proteins. After functionalization with β-CD (Fig. 3C), small CDs adsorbed on both sides of GNs were clearly observed. After self-assembly with Cyt c through electrostatic interaction (Fig. 3D), graphene became thicker because of the agglomeration of GN–CD–Cyt c, which was due to the increase in zeta potential, and the surface was covered by Cyt c. 3.2. Electrochemical characterization
Fig. 4. (A) Cyclic voltammograms of GNs/GCE (curve a), GN–CD/GCE (curve b), and GNs– aqueous solution. (B) EIS plot of GNs (curve a), CD–Cyt c/GCE (curve c) in Fe(CN)3−/4− 6 aqueous solution: inset is GN–CD (curve b), and GN–CD–Cyt c (curve c) in Fe(CN)3−/4− 6 fitting of impedance spectra for Fe(CN)3−/4− at GN–CD/GCE and GN–CD–Cyt c/GCE. 6 (C) The equivalent circuit.
after reduction of GO, which was probably due to the decrease in oxygen containing groups, making GO less hydrophilic. The zeta potential of GN–CD decreased to −45.5 mV after combination of β-CD, demonstrating that GN–CD possessed numerous negative charges on the surface
The cyclic voltammetric response of GCE modified with GNs, GN–CD redox couple in and GN–CD–Cyt c was examined using an Fe(CN)3−/4− 6 containing 0.1 M KCl (Fig. 4A). neutral solution of standard Fe(CN)3−/4− 6 Compared with the GNs, GN–CD showed slightly better electrochemical response. This result indicated that the rate of electron transfer on the electrode surface was enhanced after the functionalization of GNs with CD. This finding illustrated that the enrichment of β-CD molecules between the electrochemical probe Fe(CN)36 −/4 − and the surface of GCE aided to the conductivity [20]. After the combination of Cyt c with GN–CD through electrostatic interaction, the tertiary assembly, GN–CD–Cyt c showed the highest current, demonstrating that GN–CD–Cyt c possessed a higher electron transfer rate than GNs and GN–CD. EIS was used to evaluate the surface layer kinetics for solution containing 0.1 M KCl as a redox probe. Fig. 4B repFe(CN)3−/4− 6 resents a Nyquist plot for a GNs/GCE (curve a), GN–CD/GCE (curve b) and GN–CD–Cyt c/GCE (curve c). It was found that GNs/GCE displayed a semicircle at high frequency region and a straight line at low frequency region. However, GN–CD/GCE and GN–CD–Cyt c/GCE showed a low frequency straight line with a very small semi-circle at high frequency on region, indicating a diffusion controlled process for the Fe(CN)3−/4− 6 GN–CD/GCE and GN–CD–Cyt c/GCE. The Randles equivalent circuit was used for GNs/GCE analysis (Fig. 4C), where Rs, Rct, Rw, and CPE represent the solution resistance, the charge-transfer resistance, the Warburg resistance, and the constant phase element, respectively. The charge transfer resistance at GN–CD/GCE is higher than that at GN–CD–Cyt c. The much lower Rct value on GN–CD–Cyt c/GCE illustrates that the electron transfer is quasi-reversible. Namely, GN–CD–Cyt c acreaction for Fe(CN)3−/4− 6 celerated the electron transfer between the electrochemical probe and the GCE. This phenomenon can be attributed to the Fe(CN)3−/4− 6 confined environment of Cyt c that was similar to the phospholipid membrane and conferred Cyt c with excellent electrical property. Fig. 5A depicts the cyclic voltammetric responses of GN–CD–Cyt c/GCE in PBS (pH = 7.0) at various scan rates. With increased scanning speed, the redox peak current (Fig. 5B) linearly increased and the redox peak potential slightly moved, indicating a typical adsorptioncontrolled process. 3.3. Optimization of the determination conditions
Fig. 5. Cyclic voltammograms (A) and Imax (B) (inset) of the GN–CD–Cyt c/GCE at different scan rates in 0.1 M PBS (pH = 7.0).
To obtain the best properties, experimental conditions such as the assembly time, pH, detection temperature, and dosage of GN–CD–Cyt c were optimized in PBS solution. Assembly time plays an important role in the assembly layer of GN–CD–Cyt c. Fig. 6A shows that the current value of electrode modified with GNs–CD–Cyt c initially increased and then decreased with increased assembly time, and the maximum value was obtained at 36 h. This phenomenon can be explained as follows. At the first stage (0–36 h),
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Fig. 6. Effect of assembly time (A), pH (B), temperature (C) and amount of GN–CD–Cyt c (D) on the GN–CD–Cyt c/GCE response in 0.1 M PBS. The insets in (B), (C) and (D) show the current versus pH, temperature and volume of GNs–CD–Cyt c solution, respectively.
Fig. 7. Procedure for preparing GNs, GN–CD, GN–CD–Cyt c nanosheets, and sensing guest molecules by an electrochemical method.
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2.0 to 7.0, reaching a maximum at pH 7.0. The possible reason is that Cyt c possesses better redox potential at neutral pH [38]. The influence of experimental temperature (0–55 °C) was investigated in buffer solution at pH 7. Fig. 6C shows that the current peak reached the maximum value between 45 and 50 °C. However, 37 °C (close to the human body temperature) was selected as the optimum temperature to maintain the bioactivity of Cyt c as long as possible. Finally, the optimum dosage of GN–CD–Cyt c was investigated. Fig. 6D shows that the maximum current was obtained when the volume of GNs–CD–Cyt c solution is ≥7 mL. In summary, the optimum experimental conditions for obtaining the best GN–CD–Cyt c properties were assembly duration of 36 h, pH = 7.0, 37 °C, and 7 μL of GNs–CD–Cyt c solution.
3.4. Supramolecular recognition of GN–CD–Cyt c assembly
Fig. 8. (A) Cyclic voltammograms of 50 μM glycine at GCE (a), GNs/GCE (b), GN–CD– Cyt c/GCE (c), and GN–CD–Cyt c/GCE (d) in 0.1 M phosphate buffer (pH 7.0) at a scan rate of 50 mV/s. (B) Cyclic voltammograms of GN–CD–Cyt c/GCE at different concentrations of glycine. (C) Imax versus glycine concentration. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)
more Cyt c molecules were adsorbed and the number of assembled layers increased. The electrical properties of GN–CD–Cyt c gradually improved and finally reached the maximum value. At the second stage (N36 h), the thickness of GN–CD–Cyt c was too large, which impeded the electron transmission and markedly reduced conductive performance. This finding was similar to that of a cell membrane, whose maximum permeability is obtained only at the optimal thickness [26,27]. The pH markedly influences the bioactivity of protein and enzyme. The influence of pH values on the cyclic voltammetric response of GN–CD–Cyt c was investigated from pH 2.0 to 12.0 in 0.1 M PBS. As shown in Fig. 6B, the peak current increased with increased pH from
As described above, the tertiary assembly GN–CD–Cyt c showed improved electron transfer rate. Furthermore, the good biocompatibility of Cyt c, and non-cytotoxicity of carbon [39] made the layered GN–CD–Cyt c assembly particularly suitable for ultrasensitive determination because of host–guest recognition and enrichment of CD. To verify our assumption (Fig. 7), the electrochemical behaviors of GN–CD–Cyt c toward six kinds of small biomolecules (glycine, alanine, valine, proline, phenylalanine, and tryptophane) including the simplest, aromatic, and heterocyclic amino acids were investigated. Glycine was chosen as a representative analyte, because it is generally used as a food additive to relieve the sour and bitter taste (caused by saccharin), thereby increasing the sweet taste of food. However, excessive intake of glycine breaks the absorption equilibrium of amino acids and affects the absorption of other amino acids for humans, which can result in nutrient imbalance and affect human health. Therefore, the detection or sensing of glycine has important scientific significance and practical value. Fig. 8A shows the cyclic voltammograms of glycine at bare GCE (curve a, black), GNs/GCE (curve b, red), GN–CD/GCE (curve c, green), and GN–CD–Cyt c/GCE (curve d, blue) in 0.1 M PBS (pH = 7.0) at a scan rate of 50 mV/s. The electrochemical response decreased in the order: GN–CD–Cyt c/GCE N GN–CD/GCE N GNs/GCE N blank (GCE), illustrating favorable catalytic activity of GNs, GN–CD, and GN–CD–Cyt c toward the oxidation of glycine. This finding also demonstrated that β-CD molecules on the surface of GNs with high supramolecular recognition capability can enrich the analyte molecules to the detection surface. GN–CD–Cyt c/GCE electrode showed the best electrochemical properties among the four, demonstrating that the layered GN–CD– Cyt c assembly had all the merits of three constituents like the high conductivity and high surface area of GNs, the host–guest recognition and enrichment of β-CD molecules, as well as the high conductivity of Cyt c. Fig. 8B shows that the cyclic voltammograms of GN–CD–Cyt c modified GCE toward different glycine concentrations. The current increased with an increased glycine concentration. Fig. 8C displays the relationship between Imax and glycine concentration. GNs–CD–Cyt c/GCE had two concentration ranges that linearly responded to glycine. From 0.1 μM to 7.0 μM, the regression equation was ip (mA) = 1.5263 C (μM) + 24.588, and R2 = 0.9941; from 7.0 μM to 20.0 μM, the regression equation was ip (mA) = 0.5418 C (μM) + 31.256, and R2 = 0.9983. The limit of detection for glycine was estimated to be 3.0 × 10− 8 mol L− 1 (S/N = 3). These data further indicated that GCE modified with GN–CD–Cyt c exhibited very high electrochemical performance toward the target molecules. A similar electrochemical behavior of GN– CD–Cyt c toward the other five amino acids (alanine, valine, proline, phenylalanine, and tryptophane) was observed (see Supporting information). These data indicated that using GN–CD–Cyt c/GCE was a simple and reliable electrochemical technique that can be applied to the development of sensitive electrochemical sensors. These sensors can be used to determine a wide variety of electro-active compounds.
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