Cobalt-doped graphitic carbon nitride with enhanced peroxidase-like activity for wastewater treatment

Abstract

Cobalt-doped graphitic carbon nitride (Co-g-C₃N₄) materials with different cobalt contents were prepared by a facile one-pot thermal condensation method with urea and cobalt chloride. The morphology and composition of the synthesized products were characterized by different techniques. X-ray photoelectron spectroscopy and elemental mappings revealed that cobalt element is highly dispersed in the framework of g-C₃N₄. For the first time, Co-g-C₃N₄ was demonstrated to own dramatically enhanced peroxidase-like activity, compared with pure g-C₃N₄. Its activity is dependent on the content of cobalt doping, and the optimal content is 2.5% (Co-g-C₃N₄-2) based on the result of inductively coupled plasma atomic emission spectroscopy. The catalytic kinetics reveals that Co-g-C₃N₄-2 has a higher affinity for the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) and similarly efficient performance in comparison to that of natural peroxidase. The proposed mechanism demonstrates that cobalt dopant facilitates the electron transfer of Co-g-C₃N₄ from TMB to H₂O₂, thus enhances its peroxidase-like activity. Based on the greatly enhanced peroxidase-like activity, Co-g-C₃N₄-2 is firstly used for wastewater treatment. With rhodamine B as a model, Co-g-C₃N₄-2 exhibits 11 times higher degradation rate than that of pure g-C₃N₄. This research will be helpful to improve the development of efficient artificial peroxidase mimic for removing contaminants from wastewater.

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Introduction

Compared with natural enzymes, artificial enzymes have many advantages, such as easy preparation, low cost, high stability and so on. Therefore, artificial enzymes have attracted intensive attention over the past few decades.¹ Recently, due to the merging of nanotechnology with biology, researchers have also spent much effort on designing functional nanomaterial-based artificial enzymes called nanozymes,² because of their distinct properties including more catalytic sites on their surface than their bulk counterparts, unique morphology and composition. A variety of nanomaterials, such as cerium oxide nanoparticles,³ Fe₃O₄ nanoparticles,⁴ Co₃O₄ nanoparticles,⁵ graphene oxide⁶ and Au nanomaterials,⁷ have been discovered to exhibit unexpected enzyme-like activity. To date, these reported nanomaterial-based artificial enzymes include peroxidase, catalase, oxidase and superoxide dismutase mimetic enzymes, which belong to redox-based nanozymes.² Among these nanozymes, researchers mainly focus on peroxidase mimics. Peroxidase have already found wide applications in numerous fields, for example biosensing,⁸ pollutant removal,⁹ cancer diagnostics¹⁰ and therapy.¹¹

Graphite carbon nitride (g-C₃N₄) as an organic semiconductor material has attracted much attention because of its excellent performance in photocatalysis¹² and fluorescence property.¹³ As an analogue of graphite, g-C₃N₄ has a stacked 2D structure and belong to the most stable allotrope of carbon nitride under ambient conditions.¹⁴ It consists of carbon and nitrogen, which belong to the most abundant elements in earth, and is thus environmentally friendly. It can be easily synthesized via one-step polymerization of nitrogen-rich precursors with low cost.¹⁵ Therefore, g-C₃N₄ possess many advantages such as good physicochemical stability, abundant and inexpensive, as well as an appealing electronic structure combined with a medium band gap (2.7 eV).¹⁶ These unique properties allow its mainly use in sustainable chemistry as a multifunctional heterogeneous metal-free catalyst, for example water splitting¹⁷ and oxidation of hydrocarbons.¹⁸ Very recently, g-C₃N₄ has been also found to exhibit the peroxidase-like activity. With this unique activity, it was used as a nanosensor for the detection of H₂O₂ and glucose.^19,20 However, compared with natural peroxidase, the catalytic efficiency of g-C₃N₄ was still lower. Therefore, further investigations are indeed essential to construct highly efficient g-C₃N₄-based peroxidase mimics. So far, g-C₃N₄ as peroxidase mimics has only been used in the field of sensor. In this respect, future work need to focus on the exploitation of their new applications.

Many metals and nonmetals could bind or intercalate into the matrix of g-C₃N₄ and provide a convenient method of fine-tuning the structure and reactivity.^21–26 Herein, in order to improve its catalytic performance, the cobalt doped g-C₃N₄ was successfully prepared and characterized by different techniques. The effects of doping on its structural property and catalytic performance as peroxidase mimics were discussed in detail, and the catalytic kinetics and mechanism of optimized Co-g-C₃N₄ product was also investigated. With the incorporation of cobalt, the peroxidase-like activity of g-C₃N₄ dramatically increased. Based on the greatly enhanced peroxidase-like activity, optimized Co-g-C₃N₄ material is further employed for catalytic removal of rhodamine B, showing efficient degradation activity, compared with pure g-C₃N₄.

Experimental

Materials

All chemicals were of analytical grade and used as received without further purification. Urea, CoCl₂·6H₂O, Na₂HPO₄·2H₂O, citric acid, H₂O₂ and rhodamine B were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich (St. Louis, USA).

Syntheses of g-C₃N₄ and Co-g-C₃N₄

g-C₃N₄ and Co-g-C₃N₄ samples were prepared by a one-pot thermal condensation method according to the reported procedures but with different precursors.²⁷ Briefly, 4.0 g urea was placed in a alumina crucible with a cover and transferred to a muffle furnace under static air atmosphere, and calcined at 520 °C for 3 h with a rate of 2 °C min⁻¹, then cooling naturally. The pure g-C₃N₄ with pale yellow color was obtained and collected (yield: 2.0%). The synthetic procedure of Co-g-C₃N₄ was as follows: 4 g urea was mixed with 2, 4, 6 or 8 mg CoCl₂·6H₂O in 10 mL water, evaporated under heating condition, and dried overnight. With the same heating procedure, the pale purple products with the yields of 1.9%, 1.8%, 1.2% and 1.1% were obtained, which was denoted as Co-g-C₃N₄-1, Co-g-C₃N₄-2, Co-g-C₃N₄-3 and Co-g-C₃N₄-4, respectively.

Characterization

X-ray diffraction (XRD) patterns were obtained by a D8 ADVANCE X-ray diffractometer (Bruker, Germany) using Cu Kα radiation (λ = 1.5418 Å). UV/Vis diffuse reflectance spectra (DRS) were carried out on a UV-2450 UV-vis spectrophotometer (Shimadzu, Japan) equipped with an integrating sphere assembly. The morphology and elemental mappings of the synthesized products were measured by a Tecnai G2 F30 scanning transmission electron microscope (FEI, USA). N₂ adsorption–desorption isotherms were performed on an ASAP 2020 Physisorption Analyzer (Micromeritics, USA), and the specific surface areas were obtained by the Brunauer–Emmett–Teller (BET) method. X-ray photoelectron spectroscopy (XPS) measurement was carried out on an AXIS Ultra DLD X-ray photoelectron spectroscopy (Shimadzu, Japan). The cobalt content of Co-g-C₃N₄ product was measured by an Optima 8000 ICP-OES inductively coupled plasma atomic emission spectroscopy (ICP-AES) (PerkinElmer, USA). The detailed process for measuring the cobalt content was as follows. 100 mg g-C₃N₄ or Co-g-C₃N₄ materials and 20 mL of HNO₃ : HClO₄ (5 : 1 v/v) were placed in a beaker, heated slowly with a hot-plate until fumes of acids appeared. The solution was evaporated almost completely, transferred and adjusted to 100 mL in a flask with HNO₃ (1% v/v), and measured by ICP-AES with the spectral line of cobalt at 228.62 nm.

Peroxidase-like activity

The peroxidase-like catalytic activities of the Co-g-C₃N₄ materials were examined in phosphate–citric buffer (3 mL, 100 mM, pH 4.5) with addition of Co-g-C₃N₄ materials and the substrates (H₂O₂ and TMB). The reaction systems were monitored in time-drive mode or wavelength-scan mode using a Cary 300 UV-Vis spectrophotometer (Varian, USA). The apparent steady-state reaction rates were obtained according to the initial linear range of the kinetic curves and the molar absorption coefficient of 39 000 M⁻¹ cm⁻¹ at 652 nm for TMB-derived oxidation products. And these reaction rates were fitted to the Michaelis–Menten equation to calculate the kinetic constants: v = V_max × [S]/(K_m + [S]), where v is the reaction velocity, V_max is the maximal reaction velocity, [S] is the concentration of substrate (H₂O₂ or TMB) and K_m is the Michaelis constant.

Electrochemistry experiments

The glassy carbon electrodes (GCE, 3.0 mm in diameter) were firstly polished with 0.3 and 0.05 mm alumina slurry, and then cleaned ultrasonically in dilute nitric acid, ethanol and ultrapure water successively. The g-C₃N₄ (30 mg) was dispersed ultrasonically into distilled water (10 mL) to obtain a suspension (3 mg mL⁻¹). 6 μL colloidal solution was dropped on the pretreated GCE surface, and allowed to dry at 70 °C. Then, 2 μL Nafion solution (0.5 wt%) was dropped on the layer of g-C₃N₄ and dried at 70 °C to obtain the g-C₃N₄ modified electrode. The Co-g-C₃N₄-2 modified electrode was also prepared with the similar procedure. Cyclic voltammetry (CV) measurements were performed on Epsilon Electrochemical Workstation (BAS, USA). A three-electrode system comprising a platinum wire as auxiliary, a Ag/AgCl electrode as reference and the modified electrode as working electrode was used for the electrochemical experiments.

Degradation of rhodamine B

Firstly, the solution contained Co-g-C₃N₄-2 and rhodamine B was stirred in the dark at 30 °C for 30 min until the adsorption–desorption equilibrium was obtained. At the end of the course, the initial concentration of rhodamine B was measured by UV-Vis spectrometer. Then, the degradation of rhodamine B initiated with addition of H₂O₂. At given time intervals, 3 mL aliquot was withdrawn and centrifuged. The concentration of residual rhodamine B was then analyzed. The calculation of rhodamine B conversion was performed as follows (eqn (1)):²⁸

X = (C₀ − C_t)/C₀ (1)

where C_t is the concentration of rhodamine B at time t (min) and C₀ is the initial rhodamine B concentration after the adsorption–desorption equilibrium.

Result and discussion

Characterization of Co-g-C₃N₄

The composition of synthesized products was characterized by XRD. As shown in Fig. 1a, the strong diffraction peak at 27.4° is indexed for graphitic materials as the (002) peak and attributed well to the interlayer d-spacing (0.336 nm) of the g-C₃N₄, while the other weak one at 13.0° can be assigned to (100) in-planar ordering of tris-triazine units with a period of 0.675 nm. The two diffraction peaks are in good agreement with the g-C₃N₄ materials reported in the literature.¹⁵ Compared with pure g-C₃N₄, the (002) peaks of the four Co-g-C₃N₄ materials decrease significantly and become broaden, showing an inhibition of polymeric condensation by cobalt and decreased crystalline size. Average crystalline sizes calculated from the major diffraction peak (002) are 5.1, 3.8, 3.1, 3.8 and 4.1 nm for the pure g-C₃N₄, Co-g-C₃N₄-1, Co-g-C₃N₄-2, Co-g-C₃N₄-3 and Co-g-C₃N₄-4 materials, respectively. Besides, the peak of cobalt, cobalt oxide, cobalt nitrides, and cobalt carbides is not observed in the patterns of Co-g-C₃N₄. The XRD results indicate that cobalt may be homogeneously incorporated in the framework of g-C₃N₄. The electronic structures of g-C₃N₄ and Co-g-C₃N₄ products were identified by the UV-vis diffuse reflectance spectra. The g-C₃N₄ reveals light absorption from UV to visible light with an absorption edge of 460 nm (Fig. 1b), which is character of the intrinsic band gap of g-C₃N₄. After doping with cobalt, an obvious enhanced optical absorption with peak at 572 nm is found in the visible light spectrum range, which is attributed to the interaction between doped cobalt and g-C₃N₄.²⁹ The absorbance intensity increases as the cobalt doping increases. The observations obtained from UV-vis analysis indicate that the cobalt-doped strategy strengthen the charge carrier transfer in the conjugated system of aromatic CN heterocycles. The cobalt contents of the four Co-g-C₃N₄ materials were 1.0%, 2.5%, 5.2% and 7.2% for Co-g-C₃N₄-1, Co-g-C₃N₄-2, Co-g-C₃N₄-3 and Co-g-C₃N₄-4 determined by ICP-AES.

Fig. 1 XRD patterns (a) and UV-Vis DRS (b) of g-C₃N₄ and Co-g-C₃N₄ materials. (1) g-C₃N₄; (2) Co-g-C₃N₄-1; (3) Co-g-C₃N₄-2, (4) Co-g-C₃N₄-3; (5) Co-g-C₃N₄-4.

As shown in Fig. 2a, the morphology of g-C₃N₄ is a two-dimensional structure consisting of flat sheets with wrinkles and irregular shape. However the crystal planes were not observed in HRTEM image (Fig. 2b), possibly due to the amorphous structure.³⁰ The similar typical graphitic stacking structures can be checked for the four Co-g-C₃N₄ materials (Fig. 2c–f), indicating that the basic morphology does not change after cobalt doping. Compared with g-C₃N₄, there are smaller sheets and more porous sites in the Co-g-C₃N₄ products with the increase of cobalt doping. No obvious accumulation of particles on the surfaces of the Co-g-C₃N₄ materials indicates that atomic cobalt may be incorporated into the structure of Co-g-C₃N₄, rather than the formation of cobalt oxide or cobalt particles. This can be further proved by the elemental mapping characterizations demonstrated in Fig. 3. The elemental mapping pictures demonstrate that atomic cobalt is indeed highly dispersed in the framework of C₃N₄. In order to obtain their specific surface areas, the nitrogen adsorption and desorption isotherms were measured (Fig. S1†). The BET surface areas are calculated to be 106.0, 200.9, 217.2, 246.1 and 187.2 m² g⁻¹ for g-C₃N₄, Co-g-C₃N₄-1, Co-g-C₃N₄-2, Co-g-C₃N₄-3 and Co-g-C₃N₄-4, respectively. The surface areas of Co-g-C₃N₄ are much higher than that of reported material synthesized with dicyandiamide and cobalt acetate,³¹ illustrating that employing urea and cobalt chloride as precursors in this work can introduce cobalt-doped graphitic carbon nitride with high surface area. The highly enlarged surface of Co-g-C₃N₄ can supply more catalytic sites, and then enhance adsorption, desorption, and diffusion of the reactants and products, which may promote its catalytic performance.

Fig. 2 TEM images of g-C₃N₄ and Co-g-C₃N₄. (a and b) g-C₃N₄; (c) Co-g-C₃N₄-1; (d) Co-g-C₃N₄-2; (e) Co-g-C₃N₄-3; (f) Co-g-C₃N₄-4.

Fig. 3 TEM in dark (a) and corresponding C, N and Co elemental mappings (b–d) of the Co-g-C₃N₄-2.

The C 1s spectra show mainly two carbon species with binding energies at 284.8 and 288.1 eV (Fig. 4a). The peak at 284.8 eV belongs to C–C coordination, which is determined as the standard carbon. The major carbon peak at 288.1 eV correspond to sp² bonded carbon in C–N–C coordination.²⁸ The N 1s spectra of Co-g-C₃N₄-2 display four peaks locating at different binding energies, including 398.7, 400.2, 401.2 and 404.5 eV, respectively (Fig. 4b). The dominant peak at 398.7 eV is attributed to sp² bonded nitrogen in the form of C–N=C in triazine rings, while the medium peaks at 400.2 and 401.2 eV is assigned to bridging N atoms in N–(C)₃ and N bonded with H atoms.³¹ The weak peak at 404.5 eV is attributed to the charging effects or positive charge localization in the cyano-group and heterocycles.²⁸ The Fig. 4c shows a main peak related to Co 2p_3/2 at 781.2 eV, which is higher than cobalt oxide (∼780 eV) and metallic Co (∼779 eV), thus excluding the presence of cobalt oxide and metallic Co in Co-g-C₃N₄-2.³¹ The binding energy of 781.2 eV can be ascribed to the cobalt center coordinated to nitrogen.²⁷ Despite different cobalt content of four Co-g-C₃N₄ materials, the main peaks of Co 2p_3/2 do not shift, and the intensity increases as the cobalt content increases (Fig. 4d). This once again illustrates that cobalt element successfully incorporated into the framework of g-C₃N₄ homogeneously.

Fig. 4 XPS spectra of C 1s (a), N 1s (b) and Co 2p (c) of Co-g-C₃N₄-2 and Co 2p comparison (d) of different Co-g-C₃N₄ materials.

Peroxidase-like properties of Co-g-C₃N₄

The peroxidase can catalyze some amines and phenols as the organic substrates in presence of H₂O₂.³² The peroxidase-like activities of g-C₃N₄ and Co-g-C₃N₄ were investigated by the UV-vis absorption spectra. As shown in Fig. 5a, H₂O₂ can't oxidize TMB in absence of g-C₃N₄ catalyst. With addition of g-C₃N₄, the oxidation of TMB develops a blue color with absorbance maxima at 652 nm, similar to the phenomena of horseradish peroxidase (HRP).⁴ However, the absorbance intensity of oxidation product catalyzed by Co-g-C₃N₄ is much bigger than that catalyzed by g-C₃N₄, which is attributed to their different catalytic activities. The time-dependent catalytic curves of g-C₃N₄ and Co-g-C₃N₄ under the standard reaction conditions also show that the catalytic reaction rate of Co-g-C₃N₄ is significantly higher than that of g-C₃N₄. The above results reveal that the strategy of cobalt incorporation into g-C₃N₄ evidently enhance the peroxidase-like activity. The enhanced peroxidase-like activity may be related with the porous morphology and enlarged surface area of Co-g-C₃N₄, which enrich more catalytic sites than that of g-C₃N₄. The effect of cobalt content on the catalytic activity was investigated with g-C₃N₄ and the four Co-g-C₃N₄ materials. Compared with pure g-C₃N₄, a small amount of cobalt doping dramatically increase the peroxidase-like activity of Co-g-C₃N₄, and then the catalytic activity decrease at high content of cobalt doping (Fig. 5b). The results show that the peroxidase-like activity of Co-g-C₃N₄ is dependent on the content of cobalt doping. The optimal cobalt doping is 2.5%, therefore Co-g-C₃N₄-2 with the highest catalytic activity was adopt as the object of following research. The effect of Co-g-C₃N₄-2 amount on the peroxidase-like activity was studied (Fig. 5c), indicating a linear relationship between the amount of catalyst and its catalytic activity.

Fig. 5 Comparison of peroxidase-like activity of g-C₃N₄ and Co-g-C₃N₄-2 (a). (1) TMB + H₂O₂; (2) g-C₃N₄ + TMB + H₂O₂; (3) Co-g-C₃N₄-2 + TMB + H₂O₂. Inserts of right and left parts show the catalytic kinetic curves and color change of different reaction systems, respectively. Conditions: 0.1 mg mL⁻¹ Co-g-C₃N₄-2, 0.5 mM TMB and 100 mM H₂O₂. Effect of cobalt doping content on the peroxidase-like activity of Co-g-C₃N₄ (b). Peroxidase-like activity of Co-g-C₃N₄-2 at different concentrations (c).

The effect of external conditions on the peroxidase-like activity of Co-g-C₃N₄-2 was measured varying the pH from 2 to 6 and the temperature from 15 to 70 °C. As demonstrated in Fig. 6, the catalytic activity of Co-g-C₃N₄-2 is dependent on pH and temperature. The optimal pH is 4.5, showing that TMB oxidation catalyzed by Co-g-C₃N₄-2 occurs easily under weakly acidic conditions, as have also been reported for some inorganic nanomaterials as peroxidase mimics.^4,6 And the optimal temperature is 30 °C, exhibiting the high catalytic ability at room temperature. The catalytic activity of the Co-g-C₃N₄-2 remained 69% at 55 °C, indicating its robustness under harsh conditions.

Fig. 6 Effect of pHs (a) and temperatures (b) on the peroxidase-like activity of Co-g-C₃N₄-2. The reaction conditions are the same as Fig. 5. The maximum point in each curve was set as 100%.

Catalytic kinetics and proposed mechanism of Co-g-C₃N₄

The peroxidase-like kinetics of Co-g-C₃N₄-2 were further investigated using steady-state kinetics. Because Co-g-C₃N₄-2 as mimetic peroxidase catalyzed the bisubstrate reaction containing TMB and H₂O₂, its catalytic kinetics was measured by varying one substrate concentration while keeping the other substrate concentration constant (Fig. 7a and b). From these kinetic catalytic curves at a fixed concentration of one substrate, the typical Michaelis–Menten curves are obtained for Co-g-C₃N₄-2 (Fig. 7c and d).⁴ And the Lineweaver–Burk double reciprocal plots show the good linear relationship (Fig. 7e and f). The results indicate that the reaction catalyzed by Co-g-C₃N₄-2 follows the typical Michaelis–Menten model. The kinetic parameters shown in Table 1 can be obtained by the slopes and intercepts of these lines. K_m values represent the affinity of an enzyme towards the substrates, and smaller K_m values demonstrate a stronger affinity between the enzyme and the substrates. As shown in Table 1, the K_m value of Co-g-C₃N₄-2 with organic substrate TMB is smaller than that of the natural peroxidase, suggesting that Co-g-C₃N₄-2 owns a higher affinity for TMB than that of natural peroxidase. The reason is that there are more active sites on the porous and high surface of Co-g-C₃N₄-2 than that of natural peroxidase, which has only one active center in one natural enzyme molecule. While the K_m value of Co-g-C₃N₄-2 with H₂O₂ is significantly higher than that of natural peroxidase, in agreement with the observation that a higher H₂O₂ concentration is required to achieve maximal activity for the Co-g-C₃N₄-2. The V_max value is the rate of reaction when an enzyme is saturated with the substrate. As the indicator of reaction activity, the V_max value of the Co-g-C₃N₄-2 is very close to the natural enzyme HRP. This indicates that the catalytic activity of Co-g-C₃N₄ is highly efficient, similar to natural peroxidase.

Fig. 7 Steady-state kinetic assay of Co-g-C₃N₄-2. The kinetic curves of Co-g-C₃N₄-2 at different concentrations of H₂O₂ (a) and TMB (b). The Michaelis–Menten curves of Co-g-C₃N₄-2 for H₂O₂ (c) and TMB (d). Double-reciprocal plots of Co-g-C₃N₄-2 for H₂O₂ (e) and TMB (f). The velocity of the reaction was measured using 100 μg mL⁻¹ Co-g-C₃N₄-2 in 100 mM phosphate–citric buffer (pH 4.5). (a) The concentration of TMB was 0.5 mM and the H₂O₂ concentration was varied. (b) The concentration of H₂O₂ was 100 mM and the TMB concentration was varied.

Table 1 Apparent Michaelis–Menten constants (K_m) and maximum reaction rates (V_max) for Co-g-C₃N₄-2 and HRP

Catalyst	Substrate	Km/mM	Vmax/10^−8 M s^−1	Ref.
Co-g-C3N4-2	TMB	0.113	8.64	The present work
Co-g-C3N4-2	H2O2	318.58	9.46	The present work
HRP	TMB	0.434	10.00	4
HRP	H2O2	3.70	8.71	4

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The electrochemistry behavior of the Co-g-C₃N₄-2 was measured in order to investigate the mechanism of peroxidase-like catalytic activity. With the addition of 20 mM H₂O₂, no obvious current is found with bare electrode, however the currents of the g-C₃N₄ and Co-g-C₃N₄-2 modified electrodes increase significantly, and the current of the Co-g-C₃N₄-2 is much bigger than that of the g-C₃N₄ (Fig. S2†). This indicates that the g-C₃N₄ and Co-g-C₃N₄-2 materials have the ability of electron transfer between electrode (electron donor) and H₂O₂ (electron acceptor), and the ability of electron transfer of Co-g-C₃N₄-2 is higher than that of g-C₃N₄. The g-C₃N₄ and Co-g-C₃N₄-2 materials may fulfill the electron transfer between TMB and H₂O₂, and thus exhibit the intrinsic peroxidase-like activity. Therefore, the possible mechanism is proposed as follows (Scheme 1). On one hand, TMB is easily absorbed on the surface of Co-g-C₃N₄-2, due to the π–π stacking, and donates the lone-pair electrons of amino groups into the Co-g-C₃N₄-2, making TMB oxidized into TMB˙⁺ (the blue complex with absorbance maxima at 652 nm). On the other hand, the electron donation increases the electron density and mobility in Co-g-C₃N₄-2, which facilitates the electron transfer from Co-g-C₃N₄-2 to H₂O₂,³³ making H₂O₂ reduced into water. The cobalt is incorporated into the framework of g-C₃N₄, thus cobalt doping leads to the crystal surface defects, which increases its conductivity, in accordance with the enhanced charge carrier transfer of its UV-Vis DRS characterization. Therefore, cobalt doping may accelerate the electron transfer from TMB to H₂O₂, in accordance with the above results of electrochemistry, thus increasing the oxidation rate of TMB by H₂O₂ in presence of Co-g-C₃N₄-2. Besides, the larger specific surface area of Co-g-C₃N₄-2 may facilitate its binding of redox substances with low stereohindrance and provide a higher density of catalytically active sites. Moreover, smaller crystalline size of Co-g-C₃N₄-2 means more powerful redox ability because of its quantum-size effect,³⁴ thus accelerate the electron transfer. All in all, it would be reasonable to explain a higher catalytic activity of Co-g-C₃N₄-2 than that of pure g-C₃N₄.

Scheme 1 Proposed mechanism of Co-g-C₃N₄ as peroxidase mimic with reducing substrate TMB and H₂O₂.

Application for wastewater treatment

Intensive industrial activities have led to considerable contamination of water containing some organic pollutants, which are harmful both for the environment and human health. With the catalytic activity, peroxidase can be applicated in the field of wastewater treatment in presence of H₂O₂.³⁵ Because Co-g-C₃N₄-2 owns high peroxidase-like activity, it may be potentially used as a peroxidase mimic for wastewater treatment. As a typical industrial pollutant, rhodamine B is chosen as a model to examine the catalytic performance of Co-g-C₃N₄-2. Since the peroxidase-like activity of Co-g-C₃N₄-2 with TMB as substrate is dependent on pH, the degradation performance of rhodamine B by Co-g-C₃N₄-2 was measured at different pHs. As demonstrated in Fig. 8a, Co-g-C₃N₄-2 has a pronounced removal efficiency for rhodamine B from pH 3 to pH 10 for 30 min. The wide pH range shows the advantage of Co-g-C₃N₄-2 as catalyst for wastewater treatment. The optimal pH for degradation of rhodamine B by Co-g-C₃N₄-2 is 7, indicating that the catalytic performance of Co-g-C₃N₄-2 is very efficient under neutral condition.

Fig. 8 Removal efficiency of rhodamine B catalyzed by Co-g-C₃N₄-2 at different pHs (a) and degradation of rhodamine B under different conditions (b). (1) Co-g-C₃N₄-2 + H₂O₂; (2) g-C₃N₄ + H₂O₂; (3) H₂O₂.

As shown in Fig. 8b, only 1.5% of rhodamine B is degraded in 80 min in presence of H₂O₂ without catalysts. However, the degradation efficiency increases dramatically with addition of g-C₃N₄ or Co-g-C₃N₄-2: 34% and 97% removal rhodamine B is achieved after 40 min, respectively. The cobalt ion solution (25 μg mL⁻¹) has negligible activity (data not shown), which demonstrates that the degradation activity of Co-g-C₃N₄-2 does not result from cobalt leaching, but is due to intact Co-g-C₃N₄-2. The degradation of rhodamine B as a function of time was observed to follow a first-order kinetic reaction:

where r is the degradation rate of rhodamine B, C_t the concentration of rhodamine B, k_app the apparent reaction rate constant, and t the reaction time.³⁶ Eqn (2) can be transform to:

From eqn (3), the apparent reaction rate constants can be obtained by the gradient of the graph of ln(C_t/C₀) versus time (Fig. S3†). The k_app values for H₂O₂ alone, g-C₃N₄ and Co-g-C₃N₄-2 are 0.00021, 0.0077, and 0.084 min⁻¹, respectively. This indicates that Co-g-C₃N₄-2 exhibits 11 times stronger catalytic activity than that of pure g-C₃N₄. The dramatically enhanced degradation activity may be derived from the synergetic effect of cobalt and g-C₃N₄. Taking the above results together, the easy-preparation, low cost, high stability, and efficiently catalytic performance of Co-g-C₃N₄-2 enable it potential in the treatment of industrial wastewater.

Conclusions

Co-g-C₃N₄ materials with different cobalt contents were synthesized by a facile one-pot thermal condensation method, and the cobalt element successfully incorporated into the framework of g-C₃N₄. Cobalt doping can dramatically enhance the peroxidase-like activity of Co-g-C₃N₄. The enhanced activity is dependent on the content of cobalt doping, and the optimal content of cobalt doping is 2.5%. Compared with natural peroxidase, the optimized Co-g-C₃N₄ owns smaller K_m and similar V_max values, which reveal that Co-g-C₃N₄ has a higher affinity for organic substrate (TMB) and similar efficient performance than that of natural peroxidase. The enhanced peroxidase-like activity of Co-g-C₃N₄ originates from its acceleration of electron transfer between reducing substrates and H₂O₂. Based on the highly catalytic ability, Co-g-C₃N₄ was successfully used for wastewater treatment. It exhibits obvious enhancement (11 times) in rhodamine B degradation than that of pure g-C₃N₄. This work will encourage new developments in inorganic material-based enzyme mimics and promote their practical application in environmental remediation.

Acknowledgements

The authors gratefully acknowledge the National Natural Science Foundation of China (Grants 21571140, 21531005 and 21371134), the 973 Program (2014CB845601), the Program for Innovative Research Team in University of Tianjin (TD12-5038) and Doctoral Program Foundation of Tianjin Normal University (52XB1508).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02911f

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