Reticular-vein-like Cu@Cu₂O/reduced graphene oxide nanocomposites for a non-enzymatic glucose sensor

Abstract

In this work, reticular-vein-like Cu@Cu₂O/rGO nanocomposites have been synthesized by direct redox reaction of Cu and graphene oxide (GO) through a hydrothermal method where the macropore Cu sheets served as the precursor of reticular-vein-like Cu₂O as well as the reducing agent of GO. FESEM and TEM were employed to characterize the morphology of the as-prepared samples. The results reveal that the reticular-vein-like Cu@Cu₂O nanocomposites are homogeneously anchored onto rGO and act like the skeleton supporting the rGO sheets to avoid its aggregation or stacking. Electrochemical tests show that the Cu@Cu₂O/rGO modified glassy carbon electrode (GCE) exhibits remarkable electrocatalytic activity towards glucose oxidation in both alkaline medium and human serum, including a wide linear range (0.005–7 mM), a low detection limit (0.5 μM), a rapid response (<2 s) as well as good stability and repeatability. More importantly, the interference from the commonly interfering species such as lactose, fructose, ascorbic acid (AA) and uric acid (UA) can be effectively avoided. All these results indicate this novel nanostructured material is a promising candidate for non-enzymatic glucose sensors.

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1. Introduction

Since fast and reliable determination of glucose is of tremendous importance in many areas such as clinical diagnostics, biotechnology and the food industry, the development of electrochemical glucose sensors has attracted extensive attention.^1–3 Considerable efforts have been devoted to develop various glucose sensors over the past decades since Clark and Updike reported the first enzyme electrode in the 1960s.^4,5 In spite that enzyme-based biosensors show good selectivity and high sensitivity, they suffer from the poor stability because the glucose oxidase can be easily affected by pH, humidity, temperature, ionic detergents and other types of interference.^6–9 To address these problems, non-enzymatic sensors based on the direct oxidation of glucose have been explored. Recently, various nanostructured materials have been synthesized and applied to construct non-enzymatic glucose sensors, such as noble metals (Au, Pd, Pt) and their alloys (Au–Pt, Pt–Pd), transition metal (Ni, Cu) and their oxide (NiO, CuO, Cu₂O).^10–17 Among these materials, copper and its oxide show an excellent electrocatalytic activity for glucose oxidation, resulting from the multi-electron oxidation mediated by the surface oxide layer.¹⁸ Especially, cuprous oxide (Cu₂O), a typical p-type transition-metal oxide, has attracted extensive studies in non-enzymatic glucose sensors due to its high electrocatalytic activity, proper redox potentials, non-toxic and low cost. Therefore, Cu₂O crystals with various morphologies have been successfully synthesized to improve its electrochemical properties in the past decades, including nanowires,¹⁹ nanocubes,²⁰ nanospheres,²¹ hollow structures,²² urchins²³ and flowers.²⁴ Unfortunately, the inherent conductivity of Cu₂O is very poor, which is not favorable for the charge transfer.²⁵ Therefore, it seriously affects the performance of Cu₂O for glucose sensing.

Graphene, a new class of two-dimensional (2D) nanomaterial consisting of a single layer of sp² network of carbon atoms, has stimulated wide interests recently owing to its high electrical conductivity, large surface-to-volume ratio and ultra-thin thickness.^26–28 Therefore, various metal oxides have combined with graphene in order to produce synergistic effect, which not only improved conductivity, enhanced catalytic activity and heightened stability of the metal oxides but also prevented the aggregation of graphene sheets for keeping the surface area and pore volume at a high level. Up to now, many approaches have been demonstrated to synthetize metal oxides/graphene sheets hybrids.^27,29–33 Among them, chemical reduction by metal powers is considered to be an efficient and promising method because it is eco-friendly, low-cost and high yield. Recently, metal powers of Fe, Sn, Co, Mn and Zn were chosen to reduce graphene oxide (GO).^32–35 For example, Sarkar et al. successfully synthetized ZnO/rGO hybrids from the commercial metal zinc and GO using hydrothermal technique without adding further reducing agent for photocatalysis.³² Zhao et al. prepared transition metal oxide (Mn₃O₄, Fe₂O₃, Co₃O₄ and ZnO)/rGO composites for lithium ion batteries via a hydrothermal method using the direct reaction between the corresponding commercial metal powders and GO.³³ Chen et al. developed a facile approach to prepare SnO₂/rGO hybrid nanoparticles by a direct redox reaction between GO and commercial tin powder for humidity sensing.³⁵ However, there is still no report on the preparation of cuprous oxide/rGO composites by direct redox of Cu and graphene oxide (GO). Meanwhile, the size of the commercial metal powders is usually large and their morphology is atypical, which show limited effects on the properties of metal oxide/rGO composites. Therefore, using nanostructured metals as the reducing agent to prepare metal oxide/rGO composites can not only effectively avoid the aggregation of graphene because the special structure of metal can act as the skeleton of the graphene but also greatly improve the properties of the composites because of its superior structure. In addition, nano-sized metals also can display unique capabilities to enhance mass transport and increase surface area.³⁶

Herein, the macropore Cu sheets were used as the precursor of Cu₂O and reducing agent of GO to synthesize reticular-vein-like Cu@Cu₂O/rGO nanocomposites through hydrothermal method. In the hydrothermal process, GO was successfully reduced to rGO and Cu was partly oxidized into Cu₂O by GO at the same time. The results show that the reticular-vein-like Cu@Cu₂O nanocomposites are homogeneously anchored onto rGO, which like as the skeleton supporting the rGO sheets to avoid its aggregation or stacking. On the other hand, the residual Cu metal and rGO have enhanced the conductivity of Cu₂O to a great extent. Consequently, the synthetic effect of good conductivity of Cu as well as rGO and the outstanding catalytic ability of Cu₂O makes the as-prepared Cu@Cu₂O/rGO nanocomposites promising for non-enzymatic glucose detection.

2. Experimental section

2.1 Materials

Graphite powder (99.95%, 8000 mesh) was purchased from Aladdin. CuSO₄·5H₂O, cetyltrimethylammonium bromide (CTAB), β-D-glucose, lactose, fructose, ascorbic acid (AA), uric acid (UA) and other reagents were all of analytical grade and used without further purification. Ultrapure water (18.2 MΩ cm⁻¹) generated by an Aike water system was used throughout the work.

2.2 Synthesis of macropore Cu sheets

The macropore Cu sheets were fabricated according to the procedures described in literature with minor modification.³⁷ In a typical synthesis, 0.4 g of CuSO₄·5H₂O was dissolved in 20 mL deionized (DI) water under vigorous stirring to obtain a transparent solution. A piece of leaf (0.1 g) from holly was rinsed with DI water, ethanol, respectively, and dried in air. Then the resulting solution and leaf were transferred into a 50 mL Teflon-lined stainless steel autoclave and kept at 200 °C for 24 h. After that, the obtained Cu samples were thoroughly washed with DI water and ethanol to remove ions possibly remaining in the final products, and dried in air.

2.3 Preparation of reticular-vein-like Cu@Cu₂O/rGO nanocomposites

Graphene oxide (GO) was first prepared from graphite powder according to a modified Hummers' method.³⁸ 50 mg GO was dispersed in 50 mL DI water and ultrasonicated for 2 h to form a homogeneous dispersion. Then, 4 mL of GO aqueous dispersion, 40 mg macropore Cu sheets, 40 mg cetyltrimethylammonium bromide (CTAB) and 30 mL DI water were mixed by vigorous stirring for 30 min. The mixture was sealed into a 50 mL Teflon-lined autoclave and maintained at 200 °C for 10 h. After it was cooled down to room temperature, the precipitation was purified by filtering, washed with DI water, and then dried in vacuum at 70 °C for 12 h. The synthetic process is illustrated in Scheme 1. Moreover, the Cu sample was synthesized with the same process except the addition of GO for comparison.

Scheme 1 Schematic illustration of the preparation process of the Cu@Cu₂O/rGO nanocomposites.

2.4 Preparation of the working electrode

Prior to the modification, the glassy carbon electrode (GCE) with a diameter of 3.0 mm was polished with 1.0, 0.3 and 0.05 μm alumina slurry, respectively, and sonicated successively in DI water and ethanol for 2 min, followed drying at room temperature. Subsequently, 4 mg Cu@Cu₂O/rGO nanocomposites were dispersed in 2.0 mL ethanol and sonicated for 5 min to obtain homogeneous dispersed solution. 10 μL suspension solution was cast onto the surface of the pretreated GCE. After drying, 5 μL of Nafion (0.5 wt% in ethanol) was then cast on the layer of Cu@Cu₂O/rGO in order to entrap the active material. We also made the Cu sample modified GCE as comparison in a similar way.

2.5 Characterizations

The X-ray powder diffraction (XRD, a Rigaku D/M ax-2400 diffractometer, Japan; monochromated Cu Kα radiation, k = 1.548 Å; 40.0 kV, 60.0 mA) was used to characterize the crystalline structure of the samples. The morphology of the samples was observed using field-emission scanning electron microscopy (FESEM, JEOL JSM-4800) and high resolution transmission electron microscopy (HRTEM, Tecnai G2 F30, FEI, USA). Raman spectra were recorded on inVia Raman microscopy system with a laser wavelength of 628 nm.

2.6 Electrochemical measurement

All of the electrochemical experiments were carried out on a CHI660D electrochemical workstation using a three-electrode system in 0.5 M NaOH electrolyte. The modified GCE, a platinum plate and standard Hg/HgO electrode were served as the working electrode, counter electrode and reference electrode, respectively.

3. Results and discussions

3.1 Structure and morphology

Fig. 1A shows the XRD patterns of GO sheets and the Cu@Cu₂O/rGO nanocomposites, respectively. For the GO sample, the peak at about 2θ = 7.5°corresponds to the (001) reflection of stacked GO sheets with an interlayer spacing of 1.18 nm, which is larger than that of graphite (0.34 nm)^39,40 This could be ascribed to the introduction of oxygenated functional groups (epoxy, hydroxyl, carboxyl and carbonyl) on carbon sheets. However, this peak disappears for the as-prepared Cu@Cu₂O/rGO nanocomposites, and a new broad peak appears at around 2θ = 24°, corresponding to the diffraction from the (002) plane of graphite, which proves that GO is indeed reduced by Cu. Moreover, the characteristic peaks at 29.6°, 36.5°, 42.4°, 61.5°and 73.7° are in good agreement with the (110), (111), (200), (220) and (311) crystal planes of cubic Cu₂O phase (JCDS 78-2076), respectively. And the peaks located at 43.5°, 50.5°and 74.5° can be assigned to the (111), (200) and (220) planes of the residual metallic copper (JCDS 85-1326). The results from the XRD patterns indicate the formation of Cu@Cu₂O/rGO nanocomposites. In addition, the XRD pattern of the sample which was synthesized without GO is shown in Fig. S1.† It can be noted that all the diffraction peaks of the sample can be indexed to the cubic Cu (JCDS 85-1326) except to a weak diffraction at 2θ = 36.5°, which correspond to the cubic Cu₂O from the partial oxidation of nanostructured Cu in air. Thus, it can be proved the Cu@Cu₂O/rGO nanocomposites were synthesized via a redox reaction between the reductive Cu and oxidative GO, and then in situ formation of Cu₂O and rGO.

Fig. 1 (A) XRD patterns of GO and Cu@Cu₂O/rGO nanocomposites; (B) Raman spectroscopy of GO and Cu@Cu₂O/rGO nanocomposites.

Raman spectroscopy is known as a powerful tool to examine the ordered/disordered crystal structures of carbon-based materials, especially for graphene. Raman spectras of GO and Cu@Cu₂O/rGO nanocomposites are shown in Fig. 1B. Two prominent peaks are observed in both cases, centering at 1325.0 and 1571.5 cm⁻¹ for GO and 1329.6 and 1587.5 cm⁻¹ for Cu@Cu₂O/rGO nanocomposites, which correspond to the well-documented D and G bands of graphene, respectively.³³ The D band is a breathing mode of κ-point phonons of Ag symmetry, while the G band is usually assigned to the E_2g phonon of sp² carbon atoms.^41,42 In general, it is accepted that the value of I_D/I_G (where I_D and I_G are the Raman intensity of the D- and G-peak, respectively) is related to the density of defects in graphene-based materials.⁴³ From the results of calculation, the value of I_D/I_G increases from 1.04 for GO to 1.13 for Cu@Cu₂O/rGO nanocomposites, which gives the evidence that GO was successfully reduced into graphene. Based on the above experimental results, a possible mechanism of the chemical reduction of GO by Cu was proposed. The reductive potential of GO is +0.48 (relative to the standard hydrogen electrode),³⁵ and the standard potentials of the Cu oxidation reaction are given as follows:

Cu²⁺ + 2e⁻ → Cu E^o = 0.34 V

Cu²⁺ + e⁻ → Cu⁺E^o = 0.16 V

Apparently, the reducing potential of GO is more positive than the oxidative potentials of Cu. Hence, the redox reaction can be carried out spontaneously, and Cu is more prone to turn into cuprous ions. Then the cuprous ions tend to hydrolyze, and transform to Cu₂O nanoparticles and load on the rGO finally.

Fig. 2 provides the size and morphology information of the as-synthesized Cu samples. From Fig. 2A, it can be seen that the Cu exhibits macropore sheet structures. The details of Cu sheets can be clearly seen in the Fig. 2B. The Cu sheets with the thickness of a few nanometers possess many macropores, which are formed by many polygonal mini-plates on the sheets.

Fig. 2 FESEM images of the pristine Cu samples at different magnifications.

The detailed morphology and microstructure of the as-prepared samples were also investigated by TEM. As shown in Fig. 3A, it is observed that the GO sheet exhibits a paper-like structure with wrinkles and folds. TEM image of the macropore sheet-like Cu superstructures is shown in Fig. 3B. It can be evidently observed that the many randomly polygonal mini-plates are pieced together to form a porous structure, which is in good agreement with the SEM. However, Fig. 3C reveals that the macropore sheet-like structure becomes reticular-vein-like structure and the polygonal mini-plates turn into the thinner and longer nanowires. The reticular-vein-like structures are uniformly distributed on the transparent rGO and combined well with rGO, which like as the skeleton supporting the ultrathin graphene sheets to avoid the aggregation or stacking of rGO. From Fig. 3D, it can be seen that many small particles of Cu@Cu₂O located on the nanowires and rGO sheets, which may be benefited for the enhanced electrochemical properties.

Fig. 3 TEM images of GO sheet (A), as-prepared Cu samples (B) and the Cu@Cu₂O/rGO nanocomposites (C and D).

3.2 Electrochemical characterization of Cu@Cu₂O/rGO nanocomposites

Fig. 4A displays the cyclic voltammograms (CVs) of Cu and Cu@Cu₂O/rGO modified GCE in 0.5 M NaOH solution in the absence and presence of glucose at a scan rate of 20 mV s⁻¹. In the absence of glucose, a pair of broad reduction peaks with a peak potential of approximately 0.65 V vs. Hg/HgO can be distinctly observed at the two electrodes, which corresponded to the Cu²⁺/Cu³⁺ redox couple according to the previous report.^44,45 Upon addition of 2.0 mM glucose, there is a remarkable increase in the anodic current (about 20 μA) with the onset potential of 0.26 V for the Cu@Cu₂O/rGO modified GCE, which is more negative than that on the Cu modified electrode. The largely negative-shifted onset potential demonstrates the superior kinetic of the Cu@Cu₂O/rGO modified GCE towards glucose oxidation. Besides, the increased anodic current of the Cu@Cu₂O/rGO modified GCE is much higher than that of the Cu modified GCE. The superior electrocatalytic performance of Cu@Cu₂O/rGO modified electrode is attributed to the synergistic effect of graphene and the reticular-vein-like architecture. Here, graphene sheets provide large surface area, high conductivity and the reticular-vein-like architecture provides facile transport pathways for ions and offers unhindered diffusion of glucose molecules during kinetic mass transfer in the electrochemical process. This can be further verified by the electrochemical impedance spectroscopy (EIS) as shown in Fig. S2.† It depicts that the Cu@Cu₂O/rGO electrode has the lower charge transfer resistance (R_ct) than that of the Cu electrode, indicating the fast reaction kinetic process. However, the intrinsic resistance of Cu@Cu₂O/rGO electrode is larger than that of Cu electrode because of the good conductivity of Cu. According to the previous report,^46,47 the mechanism can be put forward as following: first, Cu can be oxidized Cu⁺, then Cu⁺ to Cu²⁺, and eventually to Cu³⁺ in alkaline media. Then, Cu³⁺ can catalyze glucose oxidation to generate gluconic or glucuronic acid according to sufficient previous literatures.^47–50 Here, the Cu³⁺ species are proposed to act as an electron-transfer mediator, simultaneously, arousing the C–C bond cleavage of glucose.^21,51,52

Fig. 4 (A) Cyclic voltammograms of Cu modified GCE (a and b) and Cu@Cu₂O/rGO modified GCE (c and d) in 0.5 M NaOH in the absence (a and c) and presence (b and d) of 2 mM glucose at 20 mV s⁻¹. (B) Amperometric response of Cu@Cu₂O/rGO modified GCE with successive addition of different concentrations glucose to 0.5 M NaOH solution at 0.50 V. Inset: a partial magnification of the current response toward a low concentration of glucose solution. (C) The corresponding calibration curve. The error bars indicate the standard deviation of triplicate determinations. (D) Current response of upon successive addition of 1.0 mM glucose, 0.05 mM fructose, 0.05 mM lactose, 0.05 mM UA and 0.05 mM AA in a 0.5 M NaOH solution.

Considering that the applied potential can strongly affect the current response of the sensor, the applied potential was systemically optimized. Fig. S3† shows the amperometric response of the Cu@Cu₂O/rGO modified GCE electrode towards 0.5 mM glucose under different potentials from 0.45 V to 0.65 V in 0.5 M NaOH solution. A maximum response current and a weak noise signals can be observed at 0.50 V. Therefore, 0.50 V (0.42 V vs. Ag/AgCl) is selected as the optimal potential in the subsequent study, which is more negative than that previously reports.^20,47,53,54 Moreover, this optimal potential will have a positive effect on improving the selectivity because most of the interfering species such as uric acid (UA) and ascorbic acid (AA) are not active when the potential is below 0.5 V (vs. Ag/AgCl).⁵⁵ The amperometric response of the Cu@Cu₂O/rGO modified electrode at 0.50 V with successive stepwise change of the glucose concentrations in 0.5 M NaOH electrolyte is shown in Fig. 4B. One can see that after each addition of glucose solution, notable enhancement of current response is obtained and 95% of the steady-state current is achieved within 2 s, indicating a sensitive and rapid response to glucose oxidation. The concentration of glucose and corresponding current responses were used to derive a calibration curve, which is displayed in Fig. 4C. The calibration curve shows excellent linearity between the steady state current and glucose concentration in the range of 0.005–7.0 mM. The linear regression equation is I (μA) = 1.009 + 10.256C (mM) with a correlation coefficient of 0.9978. The sensitivity of the electrode is 145.2 μA mM⁻¹ cm⁻² and the limit of detection is estimated to be 0.5 μM at a signal to noise of 3.0. Table 1 shows a comparison of the as-developed glucose sensor with some of the reported glucose sensors based on Cu₂O. From a comparison, though some electrodes exhibit super sensitivity than our proposed electrode, the narrower linear range will not be workable in the practical application. As a result, our sensor exhibits a satisfactory integrative performance that has the characteristics of larger linear range, lower detection limit, and shorter response time.

Table 1 Comparison of the performances of the as-developed glucose sensor with other reported Cu₂O-based non-enzymatic glucose sensors

Materials	Sensitivity	Linear range (mM)	LOD (μM)	Response time (s)	Ref.
Cu2O/GNs	0.285 mA mM^−1 cm^−2	0.3–3.3	3.3	<9	20
Cu2O@CRG		0.1–1.1	1.2	<3	56
RGOs–Cu2O	185 μA mM^−1	0.01–6.0	0.05	<3	47
Cu2O hollow nanocubes	52.5 μA mM^−1	0.001–1.7	0.87	<3	57
Cu–Cu2O nanoporous NPs	123.8 μA mM^−1 cm^−2	0.01–5.5	0.05	<4	58
Cu@Cu2O/rGO	145.2 μA mM^−1 cm^−2	0.005–7.0	0.5	<2	This work

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For a glucose sensor, selectivity is another important parameter to affect its electrochemical properties because the interfering species such as ascorbic acid (AA), uric acid (UA), lactose and fructose usually coexist with glucose in real samples. Herein, the electrochemical response of the interfering species was examined at the Cu@Cu₂O/rGO modified electrode. Because the normal physiological level of glucose in human blood is at least 30 times higher than those of interfering species,⁵⁹ the anti-interference effect of the modified electrode was carried out by successive addition of 1.0 mM glucose, followed by additions of 0.05 mM fructose, 0.05 mM lactose, 0.05 mM UA and 0.05 mM AA in a 0.5 M NaOH solution. As shown in Fig. 4D, it is clearly observed that a well-defined current response to 1.0 mM glucose was obtained, while insignificant responses were observed for interfering species compared to that of glucose. The observation proves that the Cu@Cu₂O/rGO nanocomposites modified electrode possesses a good selectivity towards the detection of glucose.

The reproducibility of the Cu@Cu₂O/rGO modified electrode was evaluated. Seven successive amperometric measurements of 1.0 mM glucose on one single modified electrode yielded a reproducible current with the relative standard deviation (R.S.D.) of 2.1%, demonstrating that the electrode was not poisoned by the oxidation product and can be used repeatedly. In addition, five electrodes were made in the same method and the current responses of the electrodes towards 1.0 mM glucose were measured. In this case, the R.S.D. of 3.4% was obtained, confirming that the fabrication method was highly reproducible. Meanwhile, the long-term stability of the modified electrode was performed by measuring its current response to 1.0 mM glucose for two weeks. The modified electrode was stored in ambient condition and its current response was tested every 2 days. As shown in Fig. S4,† the response current of the electrode retains about 95% of its original current after two weeks. The stability of the modified electrode was also tested by measuring the current response to 1.0 mM glucose over a period of 2000 s. As depicted in the inset of the Fig. S4,† 88% of the initial value is maintained. These results suggest the Cu@Cu₂O/rGO modified electrode have a good stability.

As a demonstration for practical applicability, the Cu@Cu₂O/rGO modified electrode was further applied to detection of glucose in human serum sample. The concentration of glucose in human serum sample was calculated using standard glucose solution and the results were presented in Table 2. The good agreement of the results between Cu@Cu₂O/rGO modified electrode and the commercial GOD-based sensor implies the potential application of the Cu@Cu₂O/rGO modified electrode in the glucose detection.

Table 2 Concentration of glucose in human serum sample measured by commercial GOD-based glucose sensor and the as-prepared Cu@Cu₂O/rGO modified electrode

Sample	Commercial sensor (mM)	Our sensor (mM)	R.S.D. (%) (n = 3)
1	6.0	6.19	2.50%
2	15.2	15.42	2.86%
3	6.0	6.17	1.95%

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4. Conclusion

Reticular-vein-like Cu@Cu₂O/rGO nanocomposites have been synthesized by direct redox of Cu and graphene oxide (GO) through hydrothermal method where the macropore Cu sheets served as the precursor of Cu₂O and reducing agent of GO. The results showed that the reticular-vein-like Cu@Cu₂O nanocomposites with rich porosity are homogeneously anchored onto rGO and like as the skeleton to avoid the aggregation or stacking of rGO, which can enhance the conductivity and provide numerous electroactive sites for redox reaction. The Cu@Cu₂O/rGO modified electrode showed remarkable electrocatalytic towards glucose oxidation, including a wide linear range, high sensitivity, good selectivity, low detection limit, good stability and reproducibility, which attributed to the unique reticular-vein-like architecture and enhanced conductivity. In addition, the electrode presented comparable application with a commercial glucose oxidase (GOD)-based sensor. All these results suggest that the reticular-vein-like Cu@Cu₂O/rGO modified electrode could be potentially applied for the construction of a non-enzymatic sensor.

Acknowledgements

This work was supported by grants from the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant no. J1103307), the Basic Scientific Research Business Expenses of the Central University and Open Project of Key Laboratory for Magnetism and Magnetic Materials of the Ministry of Education, Lanzhou University (LZUMMM2013004), and the National College Students' Innovative Entrepreneurial Training Program of Lanzhou University (no. 20130730096).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra02390k

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