Talanta 132 (2015) 871–876

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Synthesis of graphene supported graphene-like C3N4 metal-free layered nanosheets for enhanced electrochemical performance and their biosensing for biomolecules Hui Gu a, Tianshu Zhou b, Guoyue Shi a,n a b

Department of Chemistry, East China Normal University, 500 Dongchuan Road, Shanghai 200241, PR China School of Ecological and Environmental Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 27 July 2014 Received in revised form 15 September 2014 Accepted 18 September 2014 Available online 31 October 2014

A new strategy for the assembly of graphene-like C3N4 on graphene is reported. Transmission electron microscopy (TEM), thermogravimetric analysis (TGA), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) clearly demonstrated that g-C3N4 was successful in the reduction of GO and the immobilization of g-C3N4 on the graphene surface. Investigation of their electrochemical behaviour for several redox systems was conducted, which indicated the unique electron structure endows the G–g-C3N4 with faster transfer and proper amount of g-C3N4 could make G–g-C3N4 advantageous in terms of improving the redox current and promoting electron transfer. Finally, several electroactive biomolecules, such as uric acid (UA), norepinephrine (NE), tyrosine (Tyr), tryptophan (Trp), acetaminophen (APAP) and rutin, were used to probe the biosensing capacity of G–g-C3N4 films by using the cyclic voltammetric method. & 2014 Elsevier B.V. All rights reserved.

Keywords: C3N4 Graphene Electrochemical performance Metal-free catalyst Biosensing

1. Introduction The development of nanoscience and nanotechnology has inspired scientists to continuously explore new electrode materials for constructing an enhanced electrochemical platform. Exploring materials combining good characteristics of electrochemistry (e.g., high sensitivity, low cost, and good compatibility) with unique properties of nanomaterials (e.g., electronic, optical, magnetic, mechanical, and catalytic) has become one of the most exciting topics [1]. Recently, metal-free graphene-like carbon nitride polymer, referred to as g-C3N4, is a rising star in the areas of photocatalytic hydrogen production [2,3], metal-free heterogeneous catalysis in various organic systems [4], and oxygen reduction for fuel cells [5], due to its peculiar thermal stability, appropriate electronic structure and low cost of preparation [6,7]. However, reports of study on its electrochemical performance are rare. Based on previous theory [8,9] a relatively high nitrogen doping can enhance the electron-donor property of the carbon matrix, which greatly improves the interaction between carbon and guest molecules, thus resulting in high and stable catalytic activity. g-C3N4, because of its high nitrogen content and facile synthesis procedure, may provide relatively significant catalytic activity. Nevertheless, the poor conductivity of g-C3N4 is a serious problem strongly restricting the electron transportation and limiting the

n

Corresponding author. Tel.: þ 86 21 54340043. E-mail address: [email protected] (G. Shi).

http://dx.doi.org/10.1016/j.talanta.2014.09.042 0039-9140/& 2014 Elsevier B.V. All rights reserved.

electrocatalytic activity [10]. Graphene, a two dimensional Л carbon structure material with unique electronic properties, large theoretical specific surface area and environmental benignity, has been recognized as a superior candidate for the construction of catalysts [11,12]. Therefore, we incorporated the g-C3N4 onto the surface of graphene as G–g-C3N4 to promote the electron transfer ability of g-C3N4 and increase the active sites of graphene [13]. Here, we took a facile step-by-step metal-free casting method. In brief, after impregnating graphene oxide with the liquid precursor (cyanamide, CN–NH2) 1–3 times, the resulting composites were calcined at 823 K in an inert Ar atmosphere for 4 h to synthesize g-C3N4 in situ on the surface of graphene oxide. Consequently, the obtained G–g-C3N4@1#, G–gC3N4@2#, and G–g-C3N4@3# showed good electrochemical performance as expected. The synthesis route for the development of promising electrocatalysts proposed here is facile, of low cost and environment friendly.

2. Materials and methods 2.1. Chemicals Dopamine (DA), uric acid (UA), norepinephrine (NE), Hexaammineruthenium (III) chloride (Ru(NH3)6Cl3) and cyanamide solution (50 wt%) were all purchased from Sigma Co. L-tryptophan (Trp), L-tyrosine (Tyr), rutin trihydrate (RT), and acetaminophen (APAP) were all purchased from Sinopharm Chemical Reagent Co. Ltd. (NH4)2FeSO4,

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H. Gu et al. / Talanta 132 (2015) 871–876

and K3Fe(CN)6 were purchased from Shanghai Chemical Reagent Company (Shanghai, China). H2O2 (30 wt%) was from Beijing Chemical Co. (Beijing, China). The supporting electrolyte was 0.1 M phosphate buffer solution (PBS) prepared with 0.1 M Na2HPO4 and 0.1 M KH2PO4 and the pH value was adjusted to 7.4 with NaOH. Double-distilled water was used for preparation of all solutions and for washing.

as above in static air as follows: 400 mg of the bulk g-C3N4 was placed in an open ceramic container and was heated at 550 1C for 2 h at a ramp rate of 5 1C/min. A light yellow powder of g-C3N4 nanosheets was finally obtained with a yield of about 5.6%. 2.4. Preparation of G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, g-C3N4 and TGOþ g-C3N4 electrodes

2.2. Synthesis of G–g-C3N4 and TGO Graphene oxide was synthesized from graphite by a modified Hummers method [14,15]. 0.25 mL cyanamide solution (50 wt%) and 0.25 mL H2O were added into 50 mg fresh graphene oxide. After ultrasonicating for 30 min, the mixture was stirred overnight and dried statically at 40 1C in air for 12 h. The same impregnation procedure was repeated once, twice, and three times in order to increase the loading of precursors on the graphene oxide. The dried powder was calcined at 550 1C for 4 h in Ar atmosphere and a heating rate of 2.3 1C/min to get the final G–g-C3N4 product. Additionally, the preparation of the thermal treatment graphene oxide (TGO) was similar to that of G–g-C3N4 nanosheets except that there was no addition of cyanamide solution.

Prior to modification, the glassy carbon (GC) electrode (2  1.5 cm2) used in the electrochemical experiments was polished with alumina paste (0.05 μm) on a micro-cloth and subsequently ultrasonically cleaned thoroughly with acetone, NaOH (1:1), HNO3 (1:1) and doubly distilled water and then dried at room temperature. The G–gC3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, g-C3N4 and TGOþ g-C3N4 electrodes were obtained by casting 3 μL of 1 mg/mL G–gC3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, g-C3N4 and TGOþ gC3N4 suspension, respectively, on the surface of a well-polished GC electrode and dried in air. Finally, the modified electrodes were activated by 20 successive scans at a scan rate of 50 mV/s in phosphate buffer solution (pH 7.4) until a steady voltammogram was obtained.

2.3. Synthesis of g-C3N4

3. Results and discussion

g-C3N4 was synthesized according to a procedure described in a previous literature [16]. In detail, dicyandiamide was heated at 550 1C for 4 h in static air at a ramp rate of 2.3 1C/min; the cooling rate was kept at around 1 1C/min. The resultant yellow agglomerates were milled into powder in a mortar. The nanosheets were prepared by thermal oxidation etching of the bulk g-C3N4 obtained

3.1. Characteristics of G–g-C3N4, g-C3N4 The weight contents of g-C3N4 in G–g-C3N4@1#, G–g-C3N4@2#, and G–g-C3N4@3# composites were tested to be about 37.4, 53.7, and 74.7 wt%, respectively, by thermogravimetric analysis (TGA) under flowing N2 atmosphere (see Fig. 1a). Here we selected

120

TGO

80 60

1#

40

2#

Intensity / a. u.

Weight / %

100

2#

TGO

3#

20

GO

C3N4 0

0

200

400

600

800

400 800 1200 1600 2000 2400 2800 3200

o

-1

Raman shift / cm

2# g-C3N4 GO TGO

5

10 15 20 25 30 35 40 45 50

2 Theta / degree

GO

Transmittance / a.u.

Intensity / a.u.

Temperature / C

TGO 2# g-C3N4

500 1000 1500 2000 2500 3000 3500 4000 -1

Wavenumber / cm

Fig. 1. (a) TGA curves of TGO, G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3# and g-C3N4; (b) representative Raman spectra of GO, TGO and G–g-C3N4@2#; (c) typical XRD patterns of GO, TGO, G–g-C3N4@2# and g-C3N4 and (d) FTIR spectra of GO, TGO, G–g-C3N4@2# and g-C3N4.

H. Gu et al. / Talanta 132 (2015) 871–876

G–g-C3N4@2# obtained by two times precursor impregnation as an example. For better understanding of the structure and properties of the as-prepared G–g-C3N4, TGO and g-C3N4 were taken as references. From the Raman spectra (see Fig. 1b) the D band (1351 cm  1) corresponding to defects in the curved graphene sheets, the staging disorder, and the G band (1597 cm  1) related to the sp2 hybridized carbon of graphene were observed for both samples. In agreement with previous reports for chemically reduced GO [17,18] the D/G intensity ratio increased from 0.92 (GO) to 1.07 (TGO) and 1.02 (G–g-C3N4@2#), which illustrated the good reduction of TGO and G–g-C3N4 from GO. The results also indicate more disorder of the TGO than G–g-C3N4@2#, which may be due to the CN–NH2 creates a layered g-C3N4 template for better reduction of GO [19]. From the X-ray diffraction (XRD) patterns (see Fig. 1c) the interplanar distance of G–g-C3N4@2#, based on the diffraction peak, is between those of TGO and g-C3N4, indicating that crystalline g-C3N4 layers were separated by graphene sheets [20]. The typical Fourier transform infrared spectroscopy (FTIR) spectrum (see Fig. 1d) also implied the successful immobilization of g-C3N4 on the graphene surface [21] and XPS examinations confirmed this result as well (see ESI, Fig. S1†). Fig. 2 gives representative transmission electron microscopy (TEM) overviews of these samples. TGO displays crumpled multilayer sheets and the g-C3N4 on graphene obtains a layered film G–g-C3N4 which is relatively flat. From G–g-C3N4@1#, we can also clearly identify the crumpled curve from graphene. To clearly confirm the layer structure, high magnification TEM of TGO, G–gC3N4@1#, G–g-C3N4@2#, and G–g-C3N4@3# was further examined (see ESI, Fig. S2†). With another one or two more casting procedures, the transparency decreases obviously because of the g-C3N4 films covered on graphene.

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3.2. Electrochemical behaviour for different redox species of G–g-C3N4 Several redox systems with different electrode kinetics were selected to firstly study the electrochemical properties of G–gC3N4. The cyclic voltammograms (CVs) of various redox species of 3  =4  3 þ =2 þ FeðCNÞ6 , RuðNH3 Þ6 , Fe2 þ =3 þ , and dopamine at diverse modified electrodes are illustrated in Fig. 3 and their cyclic voltammetric data and electroactive surface areas are summarized in Table S1. All the CVs data were obtained out and recorded for 5 scans and the reported CVs here are for the third scan, which is rather stable and more or less of the same CVs pattern as that of 3 þ =2 þ the later scans. RuðNH3 Þ6 , involving simple electron transfer on most electrodes including diamond and sp2 carbon, is relatively insensitive to surface microstructure, surface oxides and adsorbed monolayers on sp2 carbon electrodes [22]. Therefore, we took this redox system as an example. G–g-C3N4 composites, showing enhanced peak currents and relatively lower ΔEp values, displayed 3 þ =2 þ faster and more reversible behaviour of the RuðNH3 Þ6 couple. Moreover, we found that the CVs were rather stable, and the repeatability and long-term cyclic voltammograms (CVs) of the above six compounds at G–g-C3N4@1#, G–g-C3N4@2#, G–gC3N4@3#, TGO, and mixed TGO stability were also quite good (data not shown) even 1 or 2 years later. Considering the 3 þ =2 þ reversible redox reaction of RuðNH3 Þ6 on the G–g-C3N4 electrodes, the density of the electronic states should be sufficient 3 þ =2 þ to support the rapid electron transfer of RuðNH3 Þ6 . This redox system also selected for further investigation on the effect of scan rate on the peak current (see ESI, Fig. S3†). For scan rates in the range from 0.02 to 0.35 mV/s, the oxidative and reduction peak currents of the G–g-C3N4@1# electrode increase linearly with the

Fig. 2. Typical TEM images of (a) TGO, (b) G–g-C3N4@1#, (c) G–g-C3N4@2#, and (d) G–g-C3N4@3#.

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Fig. 3. Cyclic voltammograms for four kinds of redox systems at G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, mixed TGOþ g-C3N4, and g-C3N4 (up to down) electrodes 3  =4  3 þ =2 þ deoxygenated with N2: (a) 1.0 mM FeðCNÞ6 in 0.2 mM KCl, (b) 1.0 mM RuðNH3 Þ6 in 0.2 mM KCl, (c) 1.0 mM Fe2 þ /3 þ in 0.1 M HClO4 and (d) 1.0 mM dopamine in 0.1 M HClO4. Data shown are for the third scan. Scan rate 100 mV s  1.

square root of the scan rate, implying that the reaction is controlled by semi-infinite linear diffusion. Furthermore, the specific electroactive surface areas for these electrodes are calculated in the ESI according to the Randless–Sevcik equation [23,24] and summarized in Table S1. Compared with other materials, the G–gC3N4 composites displayed remarkably higher electroactive surface areas. In the four different redox systems the results shown in Table S1 are in keeping with each other, which suggests that the unique electron structure endows the G–g-C3N4 with faster transfer. Especially G–g-C3N4@1# shows the highest peak current and largest electroactive surface area, indicating that too much g-C3N4 on graphene could make them disadvantageous in terms of improving the redox current and promoting electron transfer. Nevertheless, impregnating graphene oxide with less CN–NH2 cannot produce gC3N4, thus restricting obtaining of good composites. 3.3. Electrochemical behaviour for different electroactive biomolecules of G–g-C3N4 The important characteristics described above enable G–g-C3N4 to be a good material for electrochemical sensing and biosensing. Here the electrochemical behaviours of two biomolecules [uric acid (UA) and norepinephrine (NE)], two amino acids [tyrosine (Tyr), tryptophan (Trp)] and two drugs [acetaminophen (APAP), rutin], which are all electroactive, were investigated. Fig. 4 shows cyclic voltammograms (CVs) of the above six compounds at G–gC3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, mixed TGOþ gC3N4, and g-C3N4 electrodes. We used bare GC electrode to compare the electrochemical performance here. All the oxidation

peak currents of these six compounds are increased at G–g-C3N4 electrodes relative to the currents at TGO, mixed TGO þg-C3N4, and g-C3N4 electrodes, indicating a favourable catalytic activity of G–g-C3N4 toward the oxidation of the compounds. At the G–gC3N4@1# electrode, all show a remarkable increase in the peak currents relative to that at the other five electrodes. This illustrates that appropriate amount of g-C3N4 on graphene not only can optimize the structure of the graphene but can also have a synergic effect on each other, which can further enhance electrochemical reactivity of G–g-C3N4@1# electrode. The great electrochemical performance of the G–g-C3N4@1# electrode toward H2O2 and O2 serves as a further verification of the above results (see Fig. 5). As the G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, mixed TGO þg-C3N4, g-C3N4 and GC electrodes can produce different background currents in blank solution, the background current is deducted from the linear sweep voltammetry (LSV). Upon G–g-C3N4@1#, G–g-C3N4@2#, and G–g-C3N4@3# electrodes, both on-set potential and peak potential of the reduction reaction shift to more positive positions with a concomitant increase in the peak currents density than those of mixed TGO þg-C3N4, TGO, and g-C3N4 electrodes (Table 1). However, because of the poor conductivity of g-C3N4, the on-set potential and peak potential of the reduction reaction were just slightly negative to g-C3N4 electrode. Such results clearly indicate a significant improvement in the catalytic activity when g-C3N4 is incorporated onto the graphene due to the enhanced electron transfer efficiency. Much higher current and lower overpotential are found on G–g-C3N4@1# electrode in comparison with other electrodes, which further reveals that G–g-C3N4@1# is an excellent material for

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Fig. 4. CVs of 50 μM (a) UA, (b) NE, (c) Tyr, (d) Trp, (e) APAP, and (f) rutin at G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, mixed TGOþg-C3N4, g-C3N4 and GC electrodes in 0.2 M phosphate buffer (pH 7.4). Scan rate 100 mV s  1.

Fig. 5. Background deducted LSV responses of G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, mixed TGO þg-C3N4, TGO, g-C3N4 and GC electrodes in 0.2 M phosphate buffer (pH 7.4) (a) containing 5 mM H2O2 and (b) saturated with O2.

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Table 1 Summary of reduction performances on G–g-C3N4@1#, G–g-C3N4@2#, G–g-C3N4@3#, TGO, mixed TGOþg-C3N4, g-C3N4 and GC electrodes in 0.2 M phosphate buffer (pH 7.4) containing 5 mM H2O2 or saturated with O2.

Acknowledgements This work was funded by National Natural Science Foundation of China (21175044, 21277048).

On-set potential Peak potential Peak current response (V) (V) (μA) H2O2 G–g-C3N41# G–g-C3N42# G–g-C3N43# TGO TGOþ gC3N4 g-C3N4 GC O2

G–g-C3N41# G–g-C3N42# G–g-C3N43# TGO TGOþ gC3N4 g-C3N4 GC

0

 0.30

82.98

 0.05

 0.39

63.38

 0.10

 0.40

32.78

 0.22  0.21

 0.54  0.50

6.904 11.22

 0.25  0.39

 0.57  0.58

4.168 0.89

 0.05

 0.40

74.44

 0.11

 0.42

68.35

 0.18

 0.45

56.80

 0.22  0.27

 0.65  0.68

45.91 50.04

 0.30  0.36

 0.70  0.74

44.89 35.03

electrochemical sensing. Additionally, excellent reproducibility and its great long-term stability was also tested using G–gC3N4@1# electrode for these compounds after 1 or 2 years resulting in a relative standard deviation of 2.5% (data not shown here).

4. Conclusion In conclusion, layered G–g-C3N4 composites were synthesized through a facile step-by-step casting route and electrocatalytically investigated. Compared with the TGO, mixed TGOþ g-C3N4 and gC3N4, G–g-C3N4 exhibited much higher electrochemical performance. Furthermore, we found a proper amount of g-C3N4 on graphene which could improve the electrocatalytic activities for redox reaction. We believe that the results could bring important applications in sensors.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.09.042.

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