Facile Fabrication of a Novel Copper Nanozyme for Efficient Dye Degradation.

In this study, a novel copper nanozyme (CNZ) was synthesized by a mild way and characterized by scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR). The as-fabricated CNZ exhibited typical peroxidase activity toward 2, 2'-azinodi-(3-ethylbenzthiazoline)-6-sulfonate. We successfully applied CNZ for the degradation of methyl orange pollutants. Under the optimum conditions (pH, 3.0; T, 60 °C; H2O2 concentration, 200 mM; dosage of CNZ, 8 mg), 93% of the degradation rate could be obtained in less than 10 min. Furthermore, the nanozyme exhibited excellent reusability and storage stability. All these experimental results suggested that CNZ is a powerful catalyst for industrial wastewater treatment.

Introduction

class="Species">Horseradish class="Chemical">pan class="Gene">peroxidase (HRP) can be applied for treating dye wastewater.[1−5] However, it is expensive and cannot work well under harsh conditions.[6] In recent years, some artificial nanozymes, possessing a nanostructure and similar enzyme activity, have been successfully designed to overcome the disadvantages. The concept of a nanozyme has been first proposed by Gao et al. in 2007. They reported a novel modified Fe3O4 nanoparticle, which retains the original magnetic properties but obtains peroxidase-like activity.[7] Chen et al. reported that bentonite-supported nanoscale zero-valent iron can remove methyl orange (MO) from aqueous solutions and the actual fading rate of the dye in wastewater reaches about 99%.[8] In 2018, an iron nanoparticle prepared by a reduction method was reported to have a significant effect on the degradation of the mono azo dye.[9] In fact, all the above nanozymes are designed on the basis of the catalytic ability of iron. The hydroxyl radical (OH•) can be released from hydrogen peroxide (H2O2) in the presence of the Fe(II) catalyst. This is a typical Fenton oxidation, which has attracted much attention of chemical researchers because the released OH• can remove the dye pollutant with high efficiency and economic benefits.[10−12] However, the Fe(II) catalyst is not very stable, and Fenton reaction must be carried out in a narrow pH range (2.5–4).[13] Replacing the iron ion with the copper ion, which was named Fenton-like reaction,[14−16] can overcome the shortcomings. However, the existence of copper ions may lead to secondary heavy metal pollution. Fabricating copper nanozymes (CNZs) can not only prevent this risk but also improve its catalytic performance. For example, copper nanoparticles supported on montmorillonite clay have been successfully synthesized and exhibited excellent catalytic properties for the degradation of methylene blue in wastewater.[17] In 2016, a Cu nanohybrid catalyst-loaded CS–CMM was prepared, which can be applied for the decomposition of nitroaromatic compounds and cresol blue dyes.[18] In 2018, a new copper nanoparticle was synthesized through aqueous chemical reduction and was proved to have high photocatalytic ability for degradation of several dyes, including methyl red, Congo red, and methylene blue.[19] In addition to the advantages of the nanozyme in dye degradation, it is also prominent as a tool for detection and diagnosis tools. The Cu/H3BTC MOF with laccase-like activity can successfully degrade Amido Black 10B and be used for the detection of phenolic pollutants.[20] The peroxidase-like activity of gold nanoparticles has been used to detect mercury ions in the solution.[21] Compared with natural enzymes, the excellent catalytic performance under harsh conditions and the lower cost of the nanozymes receive enormous interest.[22,23]

In this study, we prepared a novel class="Chemical">CNZ through a mild way for achieving the degradation of dye wasteclass="Chemical">pan class="Chemical">water. MO, a typical dye in industrial wastewater, was selected as a model dye pollutant because of its difficult degradation.[24,25] Our fabricated nanozyme was characterized first, and then, the conditions for MO degradation (the dosage of the nanozyme, H2O2 concentration, pH, and temperature) were optimized. Finally, we evaluated the catalytic stability, reusability, and storage stability.

Results and Discussion

Scanning Electron Microscopy

Many nanoparticles could be observed in Figure a. High-magnification scanning electron microscopy (SEM) (Figure b) indicated that the prepared class="Chemical">CNZ class="Chemical">presents a beautiful flower-like aclass="Chemical">pclass="Chemical">pearance. Its shaclass="Chemical">pe is similar to that of the Endless Summer flower (the inset of Figure b) with a diameter in the range of 15–20 μm. The as-class="Chemical">preclass="Chemical">pared class="Chemical">pan class="Chemical">CNZ was constructed with plenty of nanosheets and possessed high surface-to-volume ratios. A possible formation mechanism of CNZ is proposed and exhibited in Scheme . The SEM–EDX analysis (Figure c) demonstrated that CNZ is mainly composed of C, Cu, P, and O, while the Cu/P ratio was close to 3:2. When CNZ was fabricated without adding tryptophan (Trp), only disordered copper(II) phosphate precipitation could be obtained (Figure d), which suggested that Trp plays a crucial role for fabricating the flower-like CNZ. The zeta potential approaches 0, which might indicate the favorable long-term reliability and dispersibility of CNZ (Figure S1).

Figure 1

(a) Morphology of CNZ; (b) high-magnification SEM of CNZ; (c) SEM–EDX of the prepared CNZ. (d) Morphology of copper phosphate precipitations; the inset of (b) is the image of the Endless Summer flower in nature.

Scheme 1

Possible Formation Mechanism of the Copper Nanozyme

(a) Morphology of class="Chemical">CNZ; (b) high-magnification SEM of class="Chemical">pan class="Chemical">CNZ; (c) SEM–EDX of the prepared CNZ. (d) Morphology of copper phosphate precipitations; the inset of (b) is the image of the Endless Summer flower in nature.

Fourier Transform Infrared Spectroscopy

The Fourier transform infrared (FTIR) spectra of class="Chemical">copper phosphate, class="Chemical">pan class="Chemical">tryptophan, and CNZ were recorded with the wavelength from 400 to 4000 cm–1 (Figure ). The peaks at 1047 and 559 cm–1 (in curves a and c) are caused by the asymmetric stretching vibration and bending vibration of PO43–, respectively.[26] The sharp absorption peak at 3404 cm–1 is the result of the vibration of N–H in the indole ring.[27] The two poor peaks at 3045 and 1666 cm–1 are triggered by the stretching vibration and asymmetric bending of N–H (−NH3+). The peak at 1591 cm–1 is produced from COO– of Trp, and the peak of 744 cm–1 is attributed to the ortho-disubstituted group of the benzene ring.[28,29] These observed typical peaks in curve b and curve c verified the presence of tryptophan in the as-fabricated CNZ.

Figure 2

(a) FTIR of the Cu3(PO4)2 matrixes; (b) Trp; (c) Trp-incorporated CNZ.

(a) FTIR of the class="Chemical">Cu3(PO4)2 matrixes; (b) class="Chemical">pan class="Chemical">Trp; (c) Trp-incorporated CNZ.

Peroxidase-like Activity of CNZ

As a typical class="Gene">peroxidase substrate, 2, 2′-azinodi-(3-ethylbenzthiazoline)-6-sulfonate (class="Chemical">pan class="Chemical">ABTS) was selected to evaluate the catalytic activity of CNZ (Figure a). It could be found that CNZ oxidizes ABTS in the presence of H2O2 (black) and produces a green solution. However, ABTS cannot be oxidized by CNZ without H2O2 (pink). The experimental phenomenon suggested that the as-fabricated CNZ possesses the mimic peroxidase activity. We proposed a possible mechanism (shown in Scheme ). When the copper ion and hydrogen peroxide (H2O2) coexist, a copper-redox cycle might be formed[30−34] and release the free hydroxyl radical (OH•), which can oxidize ABTS into its corresponding color product.[35] Additional experiments demonstrated that CNZ (yellow) and ABTS–H2O2 solution (green) did not interfere with the absorption spectrum. We also determined the peroxidase-like activity of copper phosphate precipitation, CuSO4, and CuSO4 with tryptophan. Our results showed that CNZ has much higher catalytic activity than copper phosphate precipitation and CuSO4. Also, the presence of tryptophan cannot further enhance the catalytic activity of CuSO4. The nanoporous structure of CNZ might adsorb and aggregate ABTS. Then, the abundant copper ions on CNZ’s nanosheet can easily catalyze the oxidation of ABTS.

Figure 3

(a) Absorption spectra of the CNZ–ABTS–H2O2 system (black), Cu3(PO4)2 precipitation–ABTS–H2O2 (red), CuSO4–ABTS–H2O2 (mazarine), CuSO4–Trp–ABTS–H2O2 (light green), ABTS–H2O2 (green), CNZ + ABTS (pink), and CNZ (yellow); (b) verification for peroxidase-like activity of the supernatant and CNZ before and after treatment.

Scheme 2

Catalytic Mechanism of CNZ

(a) Absorption spectra of the class="Chemical">CNZ–class="Chemical">pan class="Chemical">ABTS–H2O2 system (black), Cu3(PO4)2 precipitation–ABTS–H2O2 (red), CuSO4–ABTS–H2O2 (mazarine), CuSO4–Trp–ABTS–H2O2 (light green), ABTS–H2O2 (green), CNZ + ABTS (pink), and CNZ (yellow); (b) verification for peroxidase-like activity of the supernatant and CNZ before and after treatment.

Besides, we designed another experiment to exclude the possibility that the mimic class="Gene">peroxidase activity of class="Chemical">pan class="Chemical">CNZ is due to the overflow of Cu2+ from the copper nanoparticles. We incubated CNZ in the phosphate-buffered saline (PBS) buffer (pH 3) for 20 min. After centrifugation, the precipitate and liquid supernatant were used to catalyze the reaction. The processed CNZ has almost no loss of activity, and the supernatant exhibited no activity (Figure b). These results suggested that the intrinsic peroxidase-like activity is caused by the intact CNZs. Some other metal nanoparticles (Ca2+, Mn2+, and Zn2+) have been fabricated to compare their mimic peroxidase activities with that of CNZ (Figure S2). We found that the nanozyme containing copper ions showed the best mimic enzyme activity. Therefore, we use CNZ for further applications.

The kinetic parameters of class="Chemical">CNZ were detected by the Michaelis–Menten model (Figures S3–S6), and the results are listed in Table . class="Chemical">pan class="Chemical">CNZ exhibited higher Km than HRP, implying that CNZ has less affinity for the substrate due to its rigid structure. In contrast, Vmax of CNZ is much higher than that of HRP. This should be attributed to the enormous number of catalytic active sites (Cu2+) exposed on the surface of CNZ’s abundant nanopetals.

Table 1

Comparison of the Kinetic Parameters between CNZ and HRP

substrate	enzyme	Vmax (μM/min)	Km (Mm)
H2O2	HRP	1.47	0.29
	CNZ	2.32	25
ABTS	HRP	1.35	0.22
	CNZ	1.74	36

K is the Michaelis constant, and Vmax is the maximal reaction rate.

Effect of Parameters on MO Degradation

The class="Gene">peroxidase-like activity makes class="Chemical">pan class="Chemical">CNZ a potential catalyst for a wide range of applications, such as dye removal, biotechnological and biomedical tools, and so forth. In this study, we applied the Trp-incorporated CNZ for the degradation of MO. We investigated the effect of different parameters on the degradation rate of MO, including CNZ dosage, H2O2 concentration, temperature, and pH (Figure ).

Figure 4

Effect of reaction parameters on MO degradation catalyzed by CNZ. (a) Effect of CNZ dosage; (b) effect of H2O2 concentration; (c) effect of temperature; (d) effect of pH.

Efclass="Chemical">fect of reaction class="Chemical">parameters on MO degradation catalyzed by class="Chemical">pan class="Chemical">CNZ. (a) Effect of CNZ dosage; (b) effect of H2O2 concentration; (c) effect of temperature; (d) effect of pH.

The amounts of the catalyst (class="Chemical">CNZ) and oxidant (class="Chemical">pan class="Chemical">H2O2) were important factors in the degradation of MO. The decolorization rate of MO was determined with varying the amount of CNZ from 4 to 10 mg. As shown in Figure a, the decolorization rate increased gradually with the increase of catalyst inputs. When 8 mg of CNZ was added into the reaction mixture, the decolorization rate reached about 93% for 10 min. More CNZ could not enhance the final degradation rate further. Therefore, 8 mg of CPN was opted for the following experiments. The relative activity of CNZ with various H2O2 concentrations is plotted in Figure b. The maximal relative activity could be obtained when the H2O2 concentration was 200 mM. A higher H2O2 content will reduce the removal rate of MO. Compared with the optimal H2O2 concentration of HRP, CNZ exhibited far more resistance to high H2O2 concentrations, which can greatly promote the decolorization. Temperature and pH can also affect the degradation rate of MO (Figure c,d). The results shown in Figure c indicated that a high temperature could improve the degradation ability of CNZ and the highest degradation rate was obtained when the reaction temperature was 60 °C. As for free HRP, its maximum catalytic efficiency could be observed at about 30 °C, and increasing the temperature will denature the enzyme and decrease its activity. Industrial wastewater is usually discharged at a high temperature. Obviously, the thermoduric CNZ is very suitable for treating the dye sewage with a high temperature. Figure d shows the effect of initial pH on the degradation of MO. The results demonstrated that CNZ can keep its high catalytic efficiency in a wide range of pH. The highest degradation rate could be obtained at a pH level of 3.0, and the decolorization rate will decrease slightly with the increase of the pH value. The experimental phenomenon was in accordance with previous studies.[36,37] The reason may be that the specific pH (pH = 3.0) is beneficial to the release of hydroxyl radicals (•OH) from hydrogen peroxide (H2O2) catalyzed by CNZ. However, higher pH might have a negative effect on the release of •OH and then decrease the degradation rate. As for HRP, its optimal pH was about pH 6. However, it can only exhibit its high catalytic performance in a very narrow range of pH (pH 5–7). All these experimental results suggested that CNZ can exhibit its much higher catalytic performance under harsh conditions compared with natural HRP.

CNZ versus Other Reported Catalysts

When class="Chemical">CNZ has been aclass="Chemical">pclass="Chemical">plied as a catalyst, the absorclass="Chemical">ption sclass="Chemical">pectra of MO at difclass="Chemical">pan class="Chemical">ferent time intervals are recorded. It could be found that the absorption peak decreases rapidly (Figure a) and the color of the reaction mixture faded away in a short time (the inset of Figure a). In this experiment, we compared the degradation efficiencies of CNZ and HRP under their own optimal conditions. As shown in Figure b, the degradation rate of HRP was only about 55% after 20 min and prolonging the reaction time cannot increase its degradation rate greatly. As for CNZ, it needed much less time (lower than 10 min) to obtain its highest degradation rate (about 93%). Obviously, CNZ can show better decolorization efficiency toward MO compared with HRP.

Figure 5

(a) UV–vis spectra of MO degradation by CNZ at different times; (b) degradation effects of CNZ and HRP on MO. (■): [H2O2] = 200 mM; [MO] = 8 mg/L; dosage of CNZ = 8 mg; T = 60 °C; pH 3.0. [○(red)]: [H2O2] = 16 mM; [MO] = 8 mg/L; dosage of HRP = 2 mg; T = 30 °C; pH 6.0. The inset of (a) is the color change during the degradation of MO.

(a) UV–vis spectra of MO degradation by class="Chemical">CNZ at difclass="Chemical">pan class="Chemical">ferent times; (b) degradation effects of CNZ and HRP on MO. (■): [H2O2] = 200 mM; [MO] = 8 mg/L; dosage of CNZ = 8 mg; T = 60 °C; pH 3.0. [○(red)]: [H2O2] = 16 mM; [MO] = 8 mg/L; dosage of HRP = 2 mg; T = 30 °C; pH 6.0. The inset of (a) is the color change during the degradation of MO.

Compared with other relative works[38−42] (Table ), the preparation method of class="Chemical">CNZ is much simclass="Chemical">pler and its fabrication conditions are milder. Moreover, its degradation efficiency is comclass="Chemical">parable to or better than that of the reclass="Chemical">ported class="Chemical">pan class="Chemical">copper nanoparticles.

Table 2

Comparison of the Advantages between CNZ and Other Reported Catalysts

catalyst	preparation conditions	preparation method	time (min)	decolorization rates	reference
CNZ	mild	easy	10	93	this work
Cu/Cu2O nanoporous	heating	hard	50	100	(38)
Cu2O particles	high temperature and pressure	hard	120	96.5	(39)
Cu–BCN	heating	hard	15	95	(40)
Core–shell Cu@Cu2O	mild	easy	100	90	(41)
Cu–TiO2 nanotubes	heating	hard	300	80	(42)

Reusability and Storage Stability

Reusability of catalyst can save cost, which is of great significance to the practicability. In order to investigate the reusability of class="Chemical">CNZ, the reaction solution was centrifuged at 12,000 rclass="Chemical">pm for 20 min and then the collected class="Chemical">preciclass="Chemical">pitate was washed and dried at 28 °C for subsequent recycling. The results show that the degradation rate is still above 90% after 10 reaction cycles comclass="Chemical">pared with the first cycle (Figure a). These results suggested that class="Chemical">pan class="Chemical">CNZ has relatively stable catalytic activity. In addition, we have also evaluated the storage stability of CNZ (shown in Figure b). The results indicated that the activity of CNZ has almost no loss after a week of storage and still shows high degradation efficiency. This indicates that CNZ has excellent storage stability.

Figure 6

(a) Reusability of CNZ; (b) storage stability of CNZ.

(a) Reusability of pan class="Chemical">CNZ; (b) storage stability of class="Chemical">pan class="Chemical">CNZ.

Catalytic Stability of CNZ

For any catalyst, its catalytic stability is always a fatal problem in catalytic stability applications. To further investigate the catalytic stability of class="Chemical">CNZ, we first inclass="Chemical">pan class="Chemical">cubated CNZ and HRP at different pH values (pH 2–9) and a range of temperatures (20–90 °C) for 1 h. Then, the mimic peroxidase activity was measured under the respective optimum conditions. CNZ maintained high activity in a wide range of pH (pH 2–9) (Figure a) and temperatures (20–90 °C) (Figure b). In contrast, the treated HRP has almost no activity at pH less than 5 or temperatures higher than 70 °C. As an inorganic–organic hybrid nanoparticle, the robust nanostructure of CNZ endows its much higher catalytic stability than that of the free natural enzyme.

Figure 7

(a) Enzyme activities of CNZ and HRP treated with different pH (2–9) under their respective optimum conditions; (b) enzyme activities of CNZ and HRP treated with different temperatures (20–90 °C) under their respective optimum conditions.

(a) Enzyme activities of class="Chemical">CNZ and HRclass="Chemical">pan class="Chemical">P treated with different pH (2–9) under their respective optimum conditions; (b) enzyme activities of CNZ and HRP treated with different temperatures (20–90 °C) under their respective optimum conditions.

Conclusions

To conclude, a novel class="Chemical">CNZ is synthesized by a simclass="Chemical">ple way. The as-fabricated nanozyme can exhibit excellent class="Chemical">pan class="Gene">peroxidase-like activity under extreme conditions, especially under high temperatures and high H2O2 concentrations. Furthermore, the nanozyme has satisfied reusability and storage stability. All these excellent qualities make it a potential catalyst for industrial wastewater treatment, especially for treating dye wastewater with high temperatures.

Materials and Methods

Chemicals

HRclass="Chemical">P (300 U/mg), MO, class="Chemical">pan class="Chemical">copper(II) sulfate pentahydrate (CuSO4·5H2O), manganese(II) sulfate tetrahydrate (MnSO4·4H2O), calcium chloride (CaCl2), zinc sulfate heptahydrate (ZnSO4·7H2O), ABTS tryptophan, hydrogen peroxide (H2O2, 30%, w/v), and all other reagents for preparing PBS (NaCl, KCl, Na2HPO4, and KH2PO4) were purchased from Shanghai Chemical Reagents Company (Shanghai, China). All aqueous solutions were prepared with ultrapure water.

Instrumentation and Characterization

The morphology of the sample was observed using a JSM-IT500A electron microscope (JEOL, Japan) with an acceleration voltage of 10 kV. EDX (JSM-IT500A, JEOL, Japan) and an FT-IR spectrometer (IRpan class="Chemical">Prestige-21, Shimadzu corclass="Chemical">poration, Jaclass="Chemical">pan) have also been used for characterizing the class="Chemical">preclass="Chemical">pared samclass="Chemical">ples. The degradation rate of MO was measured using an ultraviolet (UV) sclass="Chemical">pectroclass="Chemical">photometer [UV–visible (UV–vis), Thermo-Fisher, Thermo Evolution 220, Thermo-Fisher Ltd., Waltham, MA, USA] at 463 nm.

Preparation of the Copper Nanozyme

class="Chemical">CNZ was class="Chemical">preclass="Chemical">pared using a simclass="Chemical">ple method. First, 120 mM class="Chemical">pan class="Chemical">CuSO4 and 1 mg/mL tryptophan solution in deionized water were prepared. Then, 60 μL of tryptophan solution was added into 3 mL of PBS solution (5 mM, pH 7.4) containing 20 μL of CuSO4 solution. The solution was gently shaken for about 10 min, and then, the mixture was incubated at 28 °C for 24 h. The reaction solution was centrifuged at 12,000 rpm for 20 min to obtain the precipitates. The collected blue precipitates were washed with deionized water three times and dried at 28 °C for further characterization and applications.

Determination of the Peroxidase-like Activity

class="Chemical">ABTS was selected as a substrate to investigate the class="Chemical">pan class="Gene">peroxidase-like activity of CNZ in the presence of H2O2. First, 8.5 μL of H2O2 with a mass fraction of 30% and 0.3 mL of ABTS (10 mM) were added in 3 mL of PBS (10 mM, pH 3). Then, 2 mg of CNZ was added into the mixture to start the reaction, which was monitored at 417 nm.

Application for MO Degradation

class="Chemical">CNZ (8 mg) was added in 3 mL of class="Chemical">pan class="Chemical">PBS solution (5 mM, pH 3) containing 8 mg/L MO and 200 mM H2O2 to start the reaction at 60 °C. The reaction solution was measured at 463 nm every 2.5 min. The decolorization rate of MO can be calculated by the following formulawhere IC is the initial concentration of MO and C is the instant concentration of MO at different times.

Parameter Optimization of CNZ on MO Degradation

For degrading MO more economically and efclass="Chemical">fectively, various exclass="Chemical">perimental class="Chemical">parameters should be further oclass="Chemical">ptimized. The efclass="Chemical">pan class="Chemical">fect of dosage on the relative activity of CNZ and HRP was explored at different catalyst dosages (4–10 mg). The effect of H2O2 concentration on the relative activity of CNZ was estimated at different H2O2 concentrations (0–500 mM). For HRP, the H2O2 concentration range of 0–40 mM was selected. The effect of pH on the relative activity of CNZ and HRP was determined in the pH range of 2–9. The effect of temperature on the relative activity of CNZ and HRP was measured at different temperatures (20–90 °C). The catalyst-free reaction under its corresponding conditions was run as the control.

Reusability and Storage Stability of CNZ on MO Degradation

To evaluate the reusability, 8 mg of class="Chemical">CNZ was first added to 3 mL of class="Chemical">pan class="Chemical">PBS solution (5 mM, pH 3) containing 8 mg/L MO and 200 mM H2O2 to start the reaction at 60 °C. After the reaction was completed, CNZ was centrifuged and washed with PBS (5 mM, pH 7.4). A new round of reaction was initiated in a fresh solution. We also explored the long-term stability of CNZ. CNZ was stored at room temperature, and the degradation rate of CNZ at different storage times (1–7 days) was measured. All these experiments were repeated at least three times.