Synthesis of Porous CoFe2O4 and Its Application as a Peroxidase ...

nanomaterials Article

Synthesis of Porous CoFe2O4 and Its Application as a Peroxidase Mimetic for Colorimetric Detection of H2O2 and Organic Pollutant Degradation Lihong Wu 1 , Gengping Wan 1 , Na Hu 2 , Zhengyi He 1 , Shaohua Shi 1 , Yourui Suo 2 , Kan Wang 1 , Xuefei Xu 1 , Yulin Tang 1 and Guizhen Wang 1, * 1

2

*

Key Laboratory of Advanced Materials of Tropical Island Resources (Hainan University), Ministry of Education, Haikou 570228, China; [email protected] (L.W.); [email protected] (Ge.W.); [email protected] (Z.H.); [email protected] (S.S.); [email protected] (K.W.); [email protected] (X.X.); [email protected] (Y.T.) Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; [email protected] (N.H.); [email protected] (Y.S.) Correspondence: [email protected] or [email protected]; Tel.: +86-0898-66268172

Received: 10 May 2018; Accepted: 14 June 2018; Published: 21 June 2018

Abstract: Porous CoFe2 O4 was prepared via a simple and controllable method to develop a low-cost, high-efficiency, and good-stability nanozyme. The morphology and microstructure of the obtained CoFe2 O4 was investigated by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), high-resolution TEM (HRTEM), specific surface area and pore analysis, and Raman spectroscopy. The results show that the annealing temperature has an important effect on the crystallinity, grain size, and specific surface area of CoFe2 O4 . CoFe2 O4 obtained at 300 ◦ C (CF300) exhibits the largest surface area (up to 204.1 m2 g−1 ) and the smallest grain size. The peroxidase-like activity of CoFe2 O4 was further verified based on the oxidation of peroxidase substrate 3,30 ,5,50 -tetramethylbenzidine (TMB) in the presence of H2 O2 . The best peroxidase-like activity for CF300 should be ascribed to its largest surface area and smallest grain size. On this basis, an effective method of colorimetric detection H2 O2 was established. In addition, the porous CoFe2 O4 was also used for the catalytic oxidation of methylene blue (MB), indicating potential applications in pollutant removal and water treatment. Keywords: CoFe2 O4 ; peroxidase-like activity; artificial enzyme mimetics; hydrogen peroxide

1. Introduction Peroxidases are a kind of efficient redox enzymes that can catalyze the oxidation of enzyme substrate in the presence of H2 O2 . Owing to strong selectivity and high catalytic efficiency, they have been widely applied to various fields, such as biotechnology, chemical industry, biosensors, and immunoassays [1–3]. Unfortunately, there still exist some intrinsic drawbacks, such as being easy to denature in extreme environmental conditions, and the high cost of preparation and purification [4]. Therefore, the construction of novel and efficient artificial enzymes is very essential and has become an increasingly important focus. Over the past few decades, a vast variety of artificial enzymes have been explored to mimic the functions of natural enzyme, such as cyclodextrins, nanomaterials, DNA complexes, and Schiff base complex [5–8]. Among them, nanomaterials have attracted intensive attention because of their ease of preparation, low price, and good stability [9–13]. Additionally, the large specific surface and more activation centers of nanomaterials also greatly extend their application range [14].

Nanomaterials 2018, 8, 451; doi:10.3390/nano8070451

www.mdpi.com/journal/nanomaterials

Nanomaterials 2018, 8, 451

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To date, a large number of nanomaterials have been found to possess unexpected peroxidase-like activity (mimicking the functions of peroxidase), including Pt nanoclusters [15], AgVO3 nanobelts [16], MnO2 nanoparticles (NPs) [17–19], WOx quantum dots [20], Ag NPs [21], V2 O5 nanowires [22], and Fe3 O4 magnetic nanoparticles (MNPs) [23–25]. Additionally, a series of hybrid complexes based on metal nanomaterials have been investigated as peroxidase mimetics, such as GQDs/Ag NPs [26], Au nanocomposites [27,28], and FeSe-Pt@SiO2 nanospheres [29]. Furthermore, carbon-based nanomaterials, e.g., single-walled carbon nanotube [30], graphene oxide (GO) [6], C@Fe3 O4 NPs [31], GQDs/CuO nanocomposites [32] were also explored to mimic the functions of naturally-occurring enzymes. In particular, magnetic nanomaterials have been widely applied in biology, medicine and environmental fields because of their low cost, better stability, ease of separation, and recyclability. For example, Wei and his co-workers reported that the Fe3 O4 MNPs could be a highly-efficient peroxidase-like catalyst towards H2 O2 reduction for biosensing applications [23]. Chen’s group found that magnetic carbon nitride nanocomposites possessed the superior peroxidase-like catalytic activity toward H2 O2 and glucose and can be used as an enzyme-free biosensor [33]. Feng et al., reported the controllable synthesis of monodisperse CoFe2 O4 nanoparticles by the phase transfer method and applied them in the degradation of methylene blue (MB) with H2 O2 [34]. As a kind of high stability ferromagnetism nanomaterials, CoFe2 O4 have been widely applied in lithium batteries, immunoassays, environmental science, and food chemistry [35–37]. Owing to the magnetic property for easy recyclability and high catalytic performance, CoFe2 O4 is expected to be a good candidate for mimicking peroxidase [38,39]. In this study, we established a simple method to prepare porous CoFe2 O4 with the controllable size of nanoparticles and specific surface area. The porous CoFe2 O4 exhibited the excellent peroxidase-like catalytic activity and could be used to detect H2 O2 and degrade MB. Benefiting from simple operation and high efficiency, CoFe2 O4 materials have great potential applications in environmental protection and biotechnology. 2. Materials and Methods 2.1. Reagents 3,30 ,5,50 -Tetramethylbenzidine (TMB) was purchased from Macklin (Shanghai, China). CoCl2 ·6H2 O, FeSO4 ·7H2 O, oxalic acid, methylene blue (MB), ethanol, H2 O2 (30 wt %), isopropanol alcohol (IPA, a scavenger of hydroxyl radicals •OH) and other chemicals were all of analytical reagent grade and were obtained from Guangzhou Chemical Reagent factory (Guangzhou, China). All aqueous solutions were prepared with Milli-Q water (18.2 MΩ cm). 2.2. Preparation of CoFe2 O4 CoFe2 O4 samples were synthesized via a facile method. In a typical procedure, 2.5 g of CoCl2 ·6H2 O and 5.6 g of FeSO4 ·7H2 O were dissolved in 80 mL of deionized water and then transferred into oil bath heating at 80 ◦ C, under vigorous stirring for 1 h. Subsequently, 30 mL 1 M oxalic acid solution was heated to boiling with magnetic stirring, and then added into the above solution slowly under constant stirring to form a final black precipitation, and then cooled by ice-water mixture. The black precipitates were collected by centrifugation and washed several times with water and ethanol, and further dried at 60 ◦ C under vacuum for 12 h. Then, the precipitates were transferred to a tube furnace, heated to 300 ◦ C, and maintained for 1 h. The temperature was raised at a heating rate of 1 ◦ C min−1 [40]. The final product was denoted as CF300. Similarly, other CoFe2 O4 samples prepared at 400 ◦ C, 500 ◦ C, 600 ◦ C, and 700 ◦ C were denoted as CF400, CF500, CF600, and CF700, respectively. 2.3. Characterization of CoFe2 O4 The X-ray diffraction (XRD) patterns were collected on a Bruker D8 Advance X-ray diffractometer (Bruker Inc., Karlsruhe, Germany) with Cu Kα radiation (λ = 0.154056 nm). Scanning electron microscopy (SEM) images were observed by used a Hitachi S-4800N microscope (Hitachi Inc.,


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Tokyo, Japan). The transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were taken on a JEOL JEM-2100 microscope instrument (JEOL Inc., Tokyo, Japan) at an acceleration voltage of 200 kV. The chemical composition and elemental maps of the as-prepared samples were analyzed by energy-dispersive X-ray (EDX) spectroscopy using an EDX attachment to the SEM instrument. The X-ray photoelectron spectroscopy (XPS) data were acquired using AXIS SUPRA (Shimadzu Inc., Kyoto, Japan). The UV-vis absorbance spectra were recorded using a Lambda 750 s UV-vis-NIR absorption spectrophotometer (PerkinElmer Inc., Waltham, MA, USA). The hysteresis loop of the samples was obtained using the vibrating sample magnetometer (VSM, Lakeshore 7400, Westerville, OH, USA). The specific surface areas of the CoFe2 O4 were recorded based on Brunauer-Emmett-Teller (BET) equation by an automatic nitrogen adsorption pore size distribution and specific surface analyzer (ASAP 2460, Atlanta, GA, USA). Raman spectroscopy was performed on a Renishaw inVia Reflex Raman microscope (Renishaw Inc., Gloucestershire, UK) using 532 nm green laser excitation. 2.4. Peroxidase-Like Activity Assay To investigate the peroxidase-like activity of the prepared CoFe2 O4 materials, a typical catalytic oxidation experiment was performed at room temperature with 100 µg/mL CF400 in 3 mL of phosphate-buffered saline (PBS, 20.0 mM, pH 3.5), using 0.8 mM TMB, and 4.0 mM H2 O2 as the substrate. All the reactions were monitored in time scan mode at 652 nm, right after all of the reagents were added and mixed. Control groups included CF400 (100 µg/mL) with TMB (0.8 mM), CF400 (100 µg/mL) with H2 O2 (4.0 mM), and TMB (0.8 mM) with H2 O2 (4.0 mM) in 20.0 mM PBS (pH 3.0), respectively. 2.5. Optimal Conditions for H2 O2 Detection The effect of substrate H2 O2 concentration (0–0.1 M), CF300 concentration (0–200 µg/mL), temperature (20–40 ◦ C), and pH (2–8) on the peroxidase-like activity of CF300 were also investigated to ascertain the optimal conditions. 2.6. Analysis of Active Species The active species generated in the reaction were detected by adding scavengers (IPA) into the reaction solutions [16]. The specific procedure was the same as the CF300 peroxidase-like activity assay experiments. 2.7. Steady-State Kinetic Study A steady-state kinetic experiment was performed in a 3.0 mL reaction solution (20.0 mM PBS, pH = 3.0, 25 ◦ C) with 20 µg/mL CF300. TMB and H2 O2 were involved in the reaction as substrates. The steady-state kinetic data were collected by varying the concentration of one substrate while keeping the other substrate concentration constant, and the kinetic value was measured in time course mode at 652 nm [32,41]. The Michaelis-Menten constant was calculated by using the Lineweaver-Burk double reciprocal according to the equation: 1/v = (Km /V max ) (1/[S]) + 1/V max [42,43], where v is the initial velocity, Km is the Michaelis constant, V max is the maximal reaction velocity, and [S] represents the concentration of the substrate. 2.8. Methylene Blue Degradation The catalytic activity of CoFe2 O4 samples was demonstrated by degrading MB in aqueous solution. In a typical reaction, 0.015 mg of CF300 was added to 50 mL MB aqueous solution (10 mg/L), then 10 mL of aqueous H2 O2 (30 wt %) was added in the reaction mixture. At various time intervals, a small quantity of the mixture solution was separated quickly by centrifugation. Finally, the supernatants


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were pipetted into a quartz cell and analyzed with UV-vis spectrophotometer to evaluate the catalytic Nanomaterials 2018, x FOR PEER REVIEW 4 of 15 degradation MB8,[41,44]. Results 3.3.Results 3.1.Characterization Characterization of of the CoFe22O 3.1. O44 Thecrystal crystal structure structure and and purity byby XRD. Figure 1A 1A displays The purity of ofthe theproducts productswere weremeasured measured XRD. Figure displays XRD patterns of CoFe 2O4 prepared at different temperatures. All characteristic peaks of samples XRD patterns of CoFe2 O4 prepared at different temperatures. All characteristic peaks of samples matchvery very well well with structure with the the lattice parameters of a =of8.377 and Å c =and match with the theinverse inversespinel spinel structure with lattice parameters a = Å8.377 8.377 Å , which is consistent with the reported data (JCPDS file no. 03-0864). The synthesized c = 8.377 Å, which is consistent with the reported data (JCPDS file no. 03-0864). The synthesized temperature has a significant effect on the crystallinity of CoFe2O4. With the increase of the temperature has a significant effect on the crystallinity of CoFe2 O4 . With the increase of the prepared prepared temperature, the crystallization of samples increases obviously. Moreover, no other temperature, the crystallization of samples increases obviously. Moreover, no other characteristic characteristic peaks of impurities are detected, indicating a pure phase of CoFe2O4 can be obtained peaks of impurities are detected, indicating a pure phase of CoFe2 O4 can be obtained using the present using the present method. Figure 1B shows the Raman spectra of the as-synthesized samples. The method. Figure 1B shows the Raman spectra of the as-synthesized samples. The low-frequency low-frequency vibrations−1(below 600 cm−1) are belonged to the motion of oxygen around the vibrations (below 600 cm ) are belonged to the motion of oxygen around the octahedral lattice site octahedral lattice site whereas the higher frequencies can be attributed to oxygen around whereas the higher frequencies can bethe attributed to oxygen tetrahedral [45,46].site. In this tetrahedral sites [45,46]. In this work, mode at 682 cm−1 is aaround characteristic of thesites tetrahedral − 1 − work, the mode 682 cm300iscm a−1characteristic site. [47]. The bands at 470 cm 1 and −1 and 2+ attetrahedral The bands at 470atcm correspond toof Cothe octahedral sites − 1 2+ 300 cm correspond to Co at octahedral sites [47].

Figure1.1.(A) (A)XRD XRD patterns patterns and 2O4O prepared at different temperatures. Figure and (B) (B) Raman Ramanspectra spectraofofCoFe CoFe 2 4 prepared at different temperatures.

Themorphology morphology of of as-prepared as-prepared CoFe 4 isis byby SEM image. As As shown in Figure 2A, 2A, The CoFe2O presented SEM image. shown in Figure 2O 4 presented CF300 show the rhombus-like polyhedron morphology with some fragments and cracks. They are are CF300 show the rhombus-like polyhedron morphology with some fragments and cracks. They severalmicrometers micrometers in in size. size. Other at at different temperature alsoalso exhibit several Other CoFe CoFe22OO4 4samples samplesobtained obtained different temperature exhibit similar structure and morphology in Figure S1 (Supplementary Materials). The EDX elemental similar structure and morphology in Figure S1 (Supplementary Materials). The EDX elemental mappings(Figure (Figure 2B) 2B) and and EDX the existence Co,Co, O, O, andand Fe elements. mappings EDX spectrum spectrum(Figure (FigureS2) S2)reveal reveal the existence Fe elements. All three elements can maintain a uniform rhombus-like morphology, further demonstrating that Co, All three elements can maintain a uniform rhombus-like morphology, further demonstrating that Co, O, and Fe are homogeneously distributed in the sample. O, and Fe are homogeneously distributed in the sample.

Figure 2. (A) SEM image of CF300 and (B) EDX elemental mappings.

CF300 show the rhombus-like polyhedron morphology with some fragments and cracks. They are several micrometers in size. Other CoFe2O4 samples obtained at different temperature also exhibit similar structure and morphology in Figure S1 (Supplementary Materials). The EDX elemental mappings (Figure 2B) and EDX spectrum (Figure S2) reveal the existence Co, O, and Fe elements. All three elements can maintain a uniform rhombus-like morphology, further demonstrating that Nanomaterials 2018, 8, 451 5 of 16 Co, O, and Fe are homogeneously distributed in the sample.

Nanomaterials 2018, 8, x FOR PEER REVIEW

Figure (B) EDX EDXelemental elementalmappings. mappings. Figure2.2.(A) (A)SEM SEMimage imageof of CF300 CF300 and (B)

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The microstructure and morphology of the synthesized samples at different reaction The microstructure and morphology of the synthesized samples at different reaction temperature temperature were further investigated by TEM. As observed from the TEM images in Figure 3A–E, were further investigated by TEM. As observed from the TEM images in Figure 3A–E, all the samples all the samples exhibit a homogeneous mesoporous structure. When the synthesized temperature is exhibit a homogeneous mesoporous structure. When the synthesized temperature is increased from increased from 300 °C to 700 °C, the pore size gradually enlarged and the pore density also ◦ C to 700 ◦ C, the pore size gradually enlarged and the pore density also decreased owing to 300 decreased owing to the further crystallization and crystal growth of CoFe 2O4 particles. In this work, thethe further crystallization and growth of CoFe In amount this work, the pores 2 O4 particles. pores are located among thecrystal particles and mainly derived from a large of gases slowlyare located among the particles and mainly derived from a large amount of gases slowly release from release from oxalate particles during thermal decompositions [48]. Figure 3F displays the HRTEM oxalate during thermal decompositions [48]. Figure displays HRTEMofimage CF300. imageparticles of CF300. The image highlights the crystalline lattice 3F fringes withthe a distance aboutof0.296 The image highlights the crystalline lattice a distance about3F0.296 which can be nm, which can be ascribed to the (220) planefringes of CoFewith 2O4. The inset inof Figure is thenm, corresponding ascribed toarea the (220) plane of CoFe2(SAED) O4 . Thepattern, inset in Figure 3F is that the corresponding area electron selected electron diffraction confirming the sample hasselected a monocrystalline diffraction pattern, confirming that the sample has a monocrystalline diffraction structure.(SAED) The diffraction spots can be indexed as inverse spinel-structuredstructure. CoFe2O4, The which is in spots can be indexed inverse spinel-structured CoFe2 O4 , which is in accordance with the XRD result. accordance with theasXRD result.

Figure 3. 3. Typical (C) CF500; CF500;(D) (D)CF600; CF600;and and(E); (E); CF700 HRTEM Figure Typicalimages imagesofof(A) (A)CF300; CF300; (B) (B) CF400; CF400; (C) CF700 (F);(F); HRTEM image of CF300, and the inset in (F) is the SAED pattern. image of CF300, and the inset in (F) is the SAED pattern.

The chemical composition and surface electronic state of the as-prepared CoFe samples were 2 O42O The chemical composition and surface electronic state of the as-prepared CoFe 4 samples further studiedstudied by XPS.by It XPS. can be seenbeinseen the XPS spectrum (Figure 4A) that mainly were further It can in thesurvey XPS survey spectrum (Figure 4A)the thatsample the sample contains elements: Co, Fe, Co, andFe, O. and The O. C 1s around 284.8 284.8 eV could correspond to the mainly three contains three elements: Thepeak C 1sat peak at around eV could correspond signal from carbon contained in the instrument as calibration. In the Co 2p spectrum (Figure to the signal from carbon contained in the instrument as calibration. In the Co 2p spectrum (Figure4B), thelocated peak located at about eV corresponds to2p Co3/22pwith 3/2 with a satellite peak 786.58 the4B), peak at about 780.58780.58 eV corresponds to Co a satellite peak at at 786.58 eVeVand theat peak at 795.78 eV is ascribed the2p Co 2p 1/2. Figure 4C presents the Fe 2p orbital displaying theand peak 795.78 eV is ascribed to thetoCo . Figure 4C presents the Fe 2p orbital displaying the 1/2 the splitting peaks at binding energies of around 724.58 eV and 711.28 eV, which can be assigned to splitting peaks at binding energies of around 724.58 eV and 711.28 eV, which can be assigned to the the Fe 2p1/2 orbital and Fe 2p3/2 orbital, respectively. These results are well in agreement with many other reports [49,50]. Figure 4D shows the high-resolution XPS spectra of the O 1s. Based on the previous reports, the signal peak located at 530.94 eV could be assigned to typical lattice oxygen in CoFe2O4. The peak located at 531.4 eV is assigned to C–O bonds. The XPS spectrum is in good agreement with the literature [51].


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Fe 2p1/2 orbital and Fe 2p3/2 orbital, respectively. These results are well in agreement with many other reports [49,50]. Figure 4D shows the high-resolution XPS spectra of the O 1s. Based on the previous reports, the signal peak located at 530.94 eV could be assigned to typical lattice oxygen in CoFe2 O4 . The peak located at 531.4 eV is assigned to C–O bonds. The XPS spectrum is in good agreement with theNanomaterials literature [51]. 2018, 8, x FOR PEER REVIEW 6 of 15

Figure 4. XPS spectra ofof CF300: resolutionXPS XPSspectra spectraofof(B) (B)CoCo 2p, Figure 4. XPS spectra CF300:(A) (A)survey surveyspectrum, spectrum, high high resolution 2p, (C)(C) Fe 2p, andand (D)(D) O 1s. Fe 2p, O 1s.

N2 N adsorption/desorption of the the samples samplesare aredepicted depictedininFigure Figure5A. 5A.Typical Typicaltype type 2 adsorption/desorption isotherms isotherms of IVIV adsorption isotherms observedfor forall allsamples samplesexcept except CF700, adsorption isothermswith withananH3-type H3-typehysteresis hysteresis loop loop are observed CF700, indicating thethe mesoporous O nanostructures. Figure 5B shows the relevant indicating mesoporousfeature featureofofthe theCoFe CoFe 2 O 4 nanostructures. Figure 5B shows the relevant 2 4 Barrett-Joyner-Halenda of the theporous porousCoFe CoFe which there exists Barrett-Joyner-Halenda(BJH) (BJH)pore poresize size distribution distribution of 2O , in which there exists a 24O 4 , in sharp maximum maximum at indicating the the material has ahas mesoporous structure. The a sharp at 3.5–20.6 3.5–20.6nm, nm,further further indicating material a mesoporous structure. Theporous poroustrait traitwill willgreatly greatlyenhance enhancethe thesurface surfacearea areaand andactive activesites, sites,and andthus thusimprove improvethe thecatalytic catalytic performance [52]. The specific surface, pore diameter, and pore volume of these samples are shown performance [52]. The specific surface, pore diameter, and pore volume of these samples are shown in in Table We seeasthat, the preparing temperature increases, the obvious decrease is found Table 1. We 1. can seecan that, the as preparing temperature increases, the obvious decrease is found for the 3 2 3 2 forvolume the pore volume (0.228cm to 0.019 cm specific /g) and specific 18 m /g).magnetism The magnetism of pore (0.228 to 0.019 /g) and surface surface (204 to (204 18 mto/g). The of CF300 CF300 sample was determined by the hysteresis loop on VSM. It can be seen in Figure S3 that sample was determined by the hysteresis loop on VSM. It can be seen in Figure S3 that CF300 displays CF300 displays typical ferromagnetism room temperature. The coercivity (Hc), saturation typical ferromagnetism at room temperature.atThe coercivity (Hc), saturation magnetization (Ms) and magnetization (Ms) and residual magnetization (Mr) are about 12 Oe, 8.372 emu/g and 0.064 emu/g residual magnetization (Mr) are about 12 Oe, 8.372 emu/g and 0.064 emu/g respectively. respectively. Table 1. Specific surface, pore diameter, and pore volume of synthesized CoFe2 O4 samples. Table 1. Specific surface, pore diameter, and pore volume of synthesized CoFe2O4 samples. Sample Sample

CF300 CF300 CF400 CF400 CF500 CF500 CF600 CF600 CF700 CF700

Specific Surface Specific Surface(m (m22/g) /g) 204.10 204.10 112.17 58.44 58.44 21.78 21.78 18.25 18.25

3 /g) 3/g) PoreDiameter Diameter(nm) (nm) Pore Pore Volume (cm Pore Volume (cm 4.47 0.228 4.47 0.228 8.84 0.248 8.84 0.248 14.29 0.209 14.29 0.209 5.56 0.030 5.56 0.030 4.18 0.019 4.18 0.019

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Figure 5. (A) N2 adsorption/desorption isotherms and (B) pore size distribution of the porous Figure 5. (A) N2 adsorption/desorption isotherms and (B) pore size distribution of the porous CoFe2 O4 . CoFe2O4.

3.2. Peroxidase-Like Activity of CoFe2 O4 3.2. Peroxidase-Like Activity of CoFe2O4 To examine the peroxidase-like activity of the synthesized CoFe2 O4 , the catalytic oxidation of To examine the peroxidase-like activity of the synthesized CoFe2O4, the catalytic oxidation of the typical peroxidase substrate TMB in the presence or absence of H2 O2 was carried out (Figure 6A). the typical peroxidase substrate TMB in the presence or absence of H 2O2 was carried out (Figure Color changes of different systems can be observed from Figure 6B. The H2 O2 + CF400 system and the 6A). Color changes of different systems can be observed from Figure 6B. The H2O2 + CF400 system Hand + TMB show no absorption, while the TMB + H2 O2++HCF400 system shows an obvious 2 O2 the H2Osystem 2 + TMB system show no absorption, while the TMB 2O2 + CF400 system shows an absorption peak at 652 nm, which indicates CF400 could act as a peroxidase to oxidize TMB produce obvious absorption peak at 652 nm, which indicates CF400 could act as a peroxidase to to oxidize a TMB blue color in the presence of H O . Interesting, the TMB + CF400 system shows a weak absorption, to produce a blue color in 2the2presence of H2O2. Interesting, the TMB + CF400 system shows a indicating that CF400 could catalyze thecould oxidation of TMB in the absence to produce 2 O2absence weak absorption, indicating that CF400 catalyze the oxidation of TMBofinHthe of H 2aOblue 2 dye. The oxidase-like activity for TMB oxidation may originate from their catalytic ability to reduce to produce a blue dye. The oxidase-like activity for TMB oxidation may originate from their dissolved Additionally, peroxidase mimetic catalytic activitiesmimetic of CoFecatalytic catalytic oxygen ability to[4]. reduce dissolved the oxygen [4]. Additionally, the peroxidase 2 O4 at the different were tested and compared through the catalytic oxidation of TMB activitiessynthesized of CoFe2O4temperatures at the different synthesized temperatures were tested and compared through the from presence of H 2. It CF300 can beexhibits seen from 6C that CF300 inthe thecatalytic presenceoxidation of H2 O2 . of It TMB can beinseen Figure 6C2Othat the Figure best peroxidase mimetic exhibitsactivity the bestamong peroxidase mimetic catalytic amongThe the synthesized CoFe O4 samples. The catalytic the synthesized CoFeactivity color changes of 2TMB with different 2 O4 samples. color of are TMB with different 4 samples arethat shown in Figure It can be found that CoFe shown in FigureCoFe 6D. It2Ocan be found all the CoFe2 O6D. prepared under 2 Ochanges 4 samples 4 samples all the temperature CoFe2O4 samples prepared under different temperature possesses different possesses distinct peroxidase mimetic catalytic activities.distinct CF300 peroxidase system shows mimetic catalyticblue activities. system shows the most obvious blueCF600, color inand comparison with the most obvious color CF300 in comparison with that of CF400, CF500, CF700. The most that of CF400, CF500, CF600, and CF700. The most superior peroxidase mimetic activity of CF300 superior peroxidase mimetic activity of CF300 could be mainly owing to the rich catalytic activity could be mainly owing to the rich catalytic activity provided by the largest specific surface sites provided by the largest specific surface area. Assites shown in Scheme 1, H 2 O2 reduction first takes area. As shown in Scheme 1, H2O2 reduction first takes place in the presence of chromogenic place in the presence of chromogenic electron donor TMB, and then accelerates by partial electron electron donor TMB, and then accelerates by partial electron transferring to the surface of CF300. transferring to the surface of CF300. Subsequently, the TMB is oxidized through one electron transfer Subsequently, the TMB is oxidized through one electron transfer and the color of the solution and the color of the solution changes to blue. changes to blue.


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Figure 6. (A) UV-vis absorption spectra and (B) colorchanges changesinin indifferent differentreaction reaction systems (a. TMB Figure UV-vis absorption spectra and (B) color changes different reactionsystems systems(a. (a.TMB TMB+ 6. (A) UV-vis absorption spectra and (B) color + CF400; b. H 2O2 + CF400; c. H2O2 + TMB; d. TMB + H2O2 + CF400); (C) UV-vis absorption spectra +CF400; CF400; + CF400; c. 2H + TMB; TMB H22O+2 CF400); + CF400); UV-vis absorption spectra b. b. H2H O22O+2 CF400; c. H O22O+2 TMB; d. d. TMB + H+2 O (C)(C) UV-vis absorption spectra and color changes in presence different CoFe 2O4 samples (a. CF300; b. CF400; c. CF500; d. andand (D)(D) color changes thethe presence ofofdifferent CoFe 2 O 4 samples (a. CF300; b. CF400; c. CF500; d. (D) color changes in theinpresence of different CoFe2 O samples (a. CF300; b. CF400; c. CF500; d. CF600; 4 CF600; e. CF700). e. CF700). CF600; e. CF700).

Scheme 1. Schematic illustration of peroxidase-like activity for CF300. Scheme 1. Schematic illustration of peroxidase-like activity for CF300.

Scheme 1. Schematic illustration of peroxidase-like activity for CF300.

3.3.3.3. Optimal Condition forfor H2HO2O 2 2Detection Optimal Condition Detection

3.3. Optimal Condition for H2the O2 Detection In order to investigate intrinsic catalytic activities of CF300, further experiments were carried In order to investigate the intrinsic catalytic activities of CF300, further experiments were outcarried with various CF300 andCF300 H2 O2and concentrations. Figure 7A shows that the that peroxidase-like catalytic out with various H 2O2 concentrations. Figure 7A shows the peroxidase-like In order to investigate the intrinsic catalytic activities of CF300, further experiments were catalytic rate increases with theconcentrations elevating concentrations For the convenience of reaction ratereaction increases with the elevating of CF300. of ForCF300. the convenience of operation, carried out with various CF300 and H2O2 concentrations. Figure 7A shows that the peroxidase-like operation, the concentration of CF300, 20 μg/mL, is used in the following experiments. Previous the concentration of CF300, 20 µg/mL, is used in the following experiments. Previous studies showed catalytic reaction rate increases with the elevating concentrations of CF300. For the convenience of showed that theofcatalytic activities of horseradish peroxidase (HRP)atcould beconcentrations inhibited at thatstudies the catalytic activities horseradish peroxidase (HRP) could be inhibited excess operation, the concentration of CF300, 20 μg/mL, is used in the following experiments. Previous excess concentrations of H 2O2the [53]. Figure 7B depicts the time course-dependent absorbance change of H O [53]. Figure 7B depicts time course-dependent absorbance change at 652 nm different 2 2showed that the catalytic activities of horseradish peroxidase (HRP) could be for studies inhibited at at 652 nm for different concentrations of H 2 O 2 . The catalytic reaction rates increase with the increase concentrations of H2 O2 . The catalytic reaction rates increase with the increase of H2 O2 concentration, excess concentrations of H2O2 [53]. Figure 7B depicts the time course-dependent absorbance change of H 2O2 concentration, and no inhibition is found in the catalytic reaction at high H 2O2 at 652 nm for different concentrations of H2O2. The catalytic reaction rates increase with the increase of H2O2 concentration, and no inhibition is found in the catalytic reaction at high H 2O2


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concentrations, which suggests a more stable catalytic for CF300 than which that ofsuggests HRP at high and no inhibition is found in the catalytic reaction at highactivity H2 O2 concentrations, a more H2Ocatalytic 2 concentration. 4.0 mM is that chosen as the optimal O2 concentration themM following stable activity The for CF300 than of HRP at high H2 OH22concentration. Thein4.0 is chosen as experiments. the optimal H2 O2 concentration in the following experiments. Similar to HRP and other mimetic enzymes, the catalytic relativeofactivity CF300 is on Similar to HRP and other mimetic enzymes, the catalytic relative activity CF300 isofdependent on the pH Hence, and temperature. Hence, investigated activity the peroxidase-like activity of thedependent pH and temperature. we investigated thewe peroxidase-like of CF300 through varying ◦ ◦ CF300 through varying the pH from 2 to 8 and the temperature from 20 °C to 40 °C. As shown in the pH from 2 to 8 and the temperature from 20 C to 40 C. As shown in Figure 7C, the peroxidase-like Figure 7C, the peroxidase-like activity of CF300 is much higher in weakly acidic solutions than in activity of CF300 is much higher in weakly acidic solutions than in alkaline and neutral solutions. alkaline and neutral solutions. The maximum absorption for CF300 catalytic system appears at pH The maximum absorption for CF300 catalytic system appears at pH 3.0. In addition, the catalytic 3.0. In addition, the catalytic activity of CF300 is significantly affected by the temperature (Figure activity of CF300 is significantly affected by the temperature (Figure 7D). The catalytic activity offers 7D). The catalytic activity offers an upgrade, firstly, with the increasing temperature. Subsequently, an upgrade, firstly, with the increasing temperature. Subsequently, it starts to decline when the it starts to decline when the temperature is higher than 25 °C. Thus, for the convenience of temperature is higher than 25 ◦ C. Thus, for the convenience of operation, the 25 ◦ C is selected as the operation, the 25 °C is selected as the experimental temperature. experimental temperature.

Figure (A)Time-dependence Time-dependenceabsorbance absorbance at at 652 solution in in thethe Figure 7. 7. (A) 652 nm nm of of the the0.08 0.08mM mMTMB TMBreaction reaction solution absence presenceofofdifferent differentdoses dosesof ofCF300 CF300 in in 20 20 mM mM PBS 2O2 at room absence oror presence PBS (pH (pH ==3.0) 3.0)with with0.8 0.8mM mMHH O at room 2 2 temperature. (B) Time-dependence absorbance at 652 nm of the 0.4 mM TMB reaction solution temperature. (B) Time-dependence absorbance at 652 nm of the 0.4 mM TMB reaction solution ininthe the absence or presence of different concentrations of H 2O2 in 20 mM PBS (pH = 3.0) with 20 μg/mL absence or presence of different concentrations of H2 O 2 in 20 mM PBS (pH = 3.0) with 20 µg/mL CF300 CF300 at room temperature. Dependence of peroxidase-like activity pH (C) and at room temperature. Dependence of peroxidase-like activity of CF300 on of pHCF300 (C) andontemperature (D). temperature (D).

3.4. Catalytic Mechanism Study 3.4. Catalytic Mechanism Study In the presence of H2 O2 , CF300 could catalyze H2 O2 to form the oxidized intermediate •OH In the presence of H2O2, CF300 could catalyze H2O2 to form the oxidized intermediate •OH radicals and subsequently oxidize TMB to produce blue dye. In order to confirm the catalytic radicals and subsequently oxidize TMB to produce blue dye. In order to confirm the catalytic mechanism, the •OH radical scavenger IPA was used in the CF300-TMB-H2 O2 system, where IPA mechanism, the •OH radical scavenger IPA was used in the CF300-TMB-H2O2 system, where IPA would easily react with •OH, leading to the decrease of the absorption at 652 nm and color fading of would easily react with •OH, leading to the decrease of the absorption at 652 nm and color fading theofsystem [2]. The corresponding chemical equation was: the system [2]. The corresponding chemical equation was: 3 +3 H CH33CH(OH)CH CH(OH)CH3 3+ +•OH •OH→→CH CH3CH(O)CH +2O H2 O 3 CH(O)CH


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Figure 8 shows an apparent color fading and absorption decrease at 652 nm for the system with Figure 8 shows an apparent color fading and absorption decrease at 652 nm for the system IPA, confirming that CF300 could catalytically activate H2 O2Hto generate •OH radicals, and then with IPA, confirming that CF300 could catalytically activate 2O2 to generate •OH radicals, and oxidize TMB toTMB produce a TMBa oxide with blue Obviously, these results indicate thatthat •OH then oxidize to produce TMB oxide with color. blue color. Obviously, these results indicate radicals play a key role in the catalytic reaction. •OH radicals play a key role in the catalytic reaction.

Figure 8. Time-dependent absorbance at 652 nm of the 0.4 mM TMB reaction solutions in the Figure 8. Time-dependent absorbance at 652 nm of the 0.4 mM TMB reaction solutions in the absence absence or presence of scavengers (IPA) in 20 mM PBS (pH = 3) with 20 μg/mL CF300 and 0.8 mM or presence of scavengers (IPA) in 20 mM PBS (pH = 3) with 20 µg/mL CF300 and 0.8 mM H2 O2 . H2O2.

3.5. Steady-State Kinetic Assay 3.5. Steady-State Kinetic Assay The catalytic evaluatedby byusing usingTMB TMBand andHH 2 O2 as substrates The catalyticsteady-state steady-statekinetic kineticof of CF300 CF300 was was evaluated 2O2 as substrates (Figure experimentswere wereperformed performed only varying concentration of TMB (Figure9).9).The The kinetic kinetic experiments byby only varying the the concentration of TMB or or HH2O O while keeping the other substrate concentration constant. As illustrated in Figure 9A,B, 2 2 2while keeping the other substrate concentration constant. As illustrated in Figure 9A,B, the theMichaelis–Menten Michaelis–Menten curves were obtained both TMB and a certain concentration range. curves were obtained forfor both TMB and H2H O22 O in2 aincertain concentration range. Accordingly, obtained, and andthe thedouble doublereciprocal reciprocal plots (the Accordingly,a aseries seriesofofinitial initialreaction reaction rates rates were were obtained, plots (the reciprocal initial velocity vs. the reciprocal substrate concentration) were drawn as demonstrated reciprocal initial velocity vs. the reciprocal substrate concentration) were drawn as demonstrated in in Figure 9C,D. TheThe Michaelis–Menten constant (Km )(Kand maximal reaction velocity (V max(V ) for Figure 9C,D. Michaelis–Menten constant m ) and maximal reaction velocity max) this for system this system were calculated using Lineweaver–Burk and the Michaelis-Menten equation. Km of were calculated using Lineweaver–Burk plots andplots the Michaelis-Menten equation. The Km The (TMB) CF300 0.387 is mM, which lower than value the reported HRPsuggesting (0.434 mM), the(TMB) CF300ofisthe 0.387 mM,iswhich lower thanisthe reported of HRPvalue (0.434ofmM), that suggesting that CF300 has higher affinity for TMB than HRP (Table 2). On the other hand, the K CF300 has higher affinity for TMB than HRP (Table 2). On the other hand, the Km (H2 O2 ) of mthe (H2O2is ) of themM, CF300 is 8.89 mM,higher significantly higher that for of 3.7 mM[25], for HRP [25], indicating CF300 8.89 significantly than that of than 3.7 mM HRP indicating that CF300 that CF300 has lower affinity for H 2O2. Additionally, the peroxidase-like catalytic reaction based on has lower affinity for H2 O2 . Additionally, the peroxidase-like catalytic reaction based on CF300 CF300 followed the Michaelis-Menten typical Michaelis-Menten behavior [2,13] towards both substrates: followed the typical behavior [2,13] towards both substrates: TMBTMB and and H2 O2 . H 2O2. Additionally, the double-reciprocal plots (Figure 9C,D) exhibited the characteristic parallel Additionally, the double-reciprocal plots (Figure 9C,D) exhibited the characteristic parallel lines of a ping-pong mechanism, indicating that CF300 bound and reacted with the first substrate of lines a ping-pong mechanism, indicating that CF300 bound and reacted with the first substrate and and then released the first product before reacting with the second substrate, which is similar to then released the first product before reacting with the second substrate, which is similar to that of that of HRP [2,25]. HRP [2,25]. Table 2. Comparison of Km and Vmax between CF300 and HRP for H2O2 and TMB. Table 2. Comparison of Km and V max between CF300 and HRP for H2 O2 and TMB.

Catalysts Catalysts CoFe2O4

CoFe 2O4 CoFe 2 O4 HRP CoFe O 2 4 HRP HRP HRP

Substances Km (mM) Substances Km 8.89 (mM) H2O2 TMB 0.387 H2 O2 8.89 H2O2 3.7 TMB 0.387 H O 3.7 TMB 0.434 2 2 TMB

0.434

Vmax (M/s) References V max× (M/s) References 1.93 10−8 This work −8 − 8 2.90 × 10 This work This work 1.93 × 10 8.71 [10] work This 2.90 × ×10 10−8−8 − 8 −8 [10] 8.71 × 10 10.00 × [10] [10] 10.00 × 10−8


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Figure assays of CF300, the reaction velocity (v) was(v)measured using 20using µg/mL Figure9.9.Steady-state Steady-statekinetic kinetic assays of CF300, the reaction velocity was measured 20 of CF300ofinCF300 20 mM (pHPBS = 3) (pH at room The TMBThe concentration was varied the μg/mL inPBS 20 mM = 3) temperature. at room temperature. TMB concentration wasand varied concentration of H2 O2 was H2 O2(A); concentration varied andwas the concentration of and the concentration of 4HmM 2O2 (A); was the 4 mM the H2O2 was concentration varied and the TMB was 0.4 mM (B); and of peroxidase-like of CF300 with a fixed concentration of TMB wasthe 0.4double-reciprocal mM (B); and the plots double-reciprocal plotsactivity of peroxidase-like activity of concentration one substrate relative varying concentration the otherconcentration substrate (C,D). CF300 with aoffixed concentration of to one substrate relative toofvarying of the other substrate (C,D).

3.6. Detection of H2 O2 and Oxidative Degradation of Methylene Blue 3.6. Detection of H2O 2 and Oxidative Degradation of Methylene Blue On the basis that peroxidase mimics the activity of the CoFe2 O4 materials, we developed a facileOn andthe label-free colorimetric approach H2 O As CoFe shown Figure 10,we a typical H2 O2a basis that peroxidase mimics to thedetect activity of2 . the 2Oin 4 materials, developed concentration response curve under the optical conditions is measured. The detection range for facile and label-free colorimetric approach to detect H 2O2. As shown in Figure 10, a typicalHH2 O 2O 22 is from 0.02 to response 6 mM. The linear regression equation is A652nm = 0.18388 +The 0.0049C (mM), withfor a concentration curve under the optical conditions is measured. detection range correlation coefficient 0.9945. detection limit for H2 Ois2 A is652nm 0.02=mM, suggesting that the optical H2O2 is from 0.02 to 6 of mM. The The linear regression equation 0.18388 + 0.0049C (mM), with a biosensing builtofin0.9945. this study be applicable correlationsystem coefficient The can detection limit fortoHthe 2O2H is2 O 0.02 mM, suggesting that the optical 2 determination. Some studies confirmed could to bethe used remove pollutants from waste biosensing system built in this that studynanozymes can be applicable H2Oto2 determination. waterSome [54–57]. Based on the excellent catalytic activity of CF300, we further investigated MB studies confirmed that nanozymes could be used to remove pollutants from wasteitswater degradation ability. to other enzyme reactions, the maximum UV-vis absorptionits (Amax [54–57]. Based on Similar the excellent catalyticmimic activity of CF300, we further investigated MB) of MB aqueous solution is located at 665 nm [44]. Figure 11A shows the UV-vis absorption spectra of degradation ability. Similar to other enzyme mimic reactions, the maximum UV-vis absorption MB aqueous the presence of CF300 andnm H2[44]. O2 atFigure different reaction A sharpabsorption decrease (Amax ) of MBsolution aqueousinsolution is located at 665 11A showstime. the UV-vis for the absorption intensity of MB in is observed withofthe increasing time, thereaction band at 665 nm spectra of MB aqueous solution the presence CF300 and Hreaction 2O2 at different time. A become very weak and broad, and the removal rate of MB can reach 91.2% at 10 h (Figure 11B), sharp decrease for the absorption intensity of MB is observed with the increasing reaction time, the suggesting MB has nearly completed degradation. band at 665that nmthe become very weak and broad, and the removal rate of MB can reach 91.2% at 10 h (Figure 11B), suggesting that the MB has nearly completed degradation.

Nanomaterials 2018, 8, x FOR PEER REVIEW Nanomaterials Nanomaterials2018, 2018,8,8,451 x FOR PEER REVIEW

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Figure concentration response curve detection.Inset: Inset:the the linear calibration plot. Figure AAconcentration response curve HH O2O detection. calibration plot. 2O 2 2detection. Figure 10. 10. A10. concentration response curve H Inset: thelinear linear calibration plot.

Figure 11. (A) UV-vis spectra obtained during the MB oxidative degradation over CF300; and (B) the MB degradation rate at various reaction time. Figure 11. (A) UV-vis spectraobtained obtained during oxidative degradation over CF300; and (B) and the (B) Figure 11. (A) UV-vis spectra duringthe theMB MB oxidative degradation over CF300; MBdegradation degradation rate reaction time. 4. Conclusions the MB rateatatvarious various reaction time.

In summary, we developed a simple and controllable method for porous CoFe2O4 preparation. 4. Conclusions The specific surface area of CoFe2O4 materials was dependent on the annealing temperature and the In summary, we developed a simple and controllable method for porous CoFe2 O4 preparation. CF300 exhibited much higher specific and surface area (204.1method m2 g−1) than other CoFe 2O42O samples. The In summary, weadeveloped a simple controllable for porous CoFe 4 preparation. The specific surface area of CoFe2 O4 materials was dependent on the annealing temperature and large specific surface area of CF300 endowed them more actives which resulted in the enhanced The specific surface areaa much of CoFe 2O4 materials was dependent on temperature and the −1 ) annealing the CF300 exhibited higher specific surface area (204.1 m2 gthe than other CoFe O4 samples. intrinsic peroxidase mimics activity. Additionally, based on2 the peculiarity, CF300 2had excellent −1 CF300 exhibited a much higher area (204.1 g ) than CoFe O4 enhanced samples. The The large specific surface area specific of CF300surface endowed them morem actives whichother resulted in 2the catalytic activity for the degradation of MB. Owing to the facile preparation, low cost, large specific largeintrinsic specificperoxidase surface area of CF300 endowed them based moreon actives which resulted in the enhanced mimics activity. Additionally, the peculiarity, CF300 had excellent surface area, and magnetic properties, the present CoFe2O4 showed great potential for applications catalytic activity for the degradation of MB. Owing to the facile preparation, low cost, large specific intrinsic peroxidase mimics activity. Additionally, based on the peculiarity, CF300 had excellent in environmental protection, biomedical analysis, and other related areas. surface area, and properties, present CoFe O4 showed great potential forcost, applications in catalytic activity for magnetic the degradation of the MB. Owing to 2the facile preparation, low large specific environmental protection, biomedical analysis, and other related areas. Supplementary Materials:properties, The followingthe are present available online surface area, and magnetic CoFe2atOwww.mdpi.com/xxx/s1. 4 showed great potential for applications

4. Conclusions

in environmental protection, biomedical and other related areas. Author Contributions: G.W. (Guizhen conceived and the experiments; L.W., G.W. (Gengping Supplementary Materials: The followingWang) areanalysis, available online atdesigned http://www.mdpi.com/2079-4991/8/7/451/s1. Wan), and S.S. performed the experiments; L.W., K.W., Z.H., X.X., Y.T., N.H., and Y.S. analyzed the data; G.W. Author Contributions: G.W. (Guizhen Wang) conceived and designed the experiments; L.W., G.W. (Gengping

Supplementary Materials: The are L.W., available online attools; www.mdpi.com/xxx/s1. (Guizhen Wang) contributed reagents, materials, and analysis and G.W.and (Guizhen Wang) and L.W. G.W. wrote Wan), and S.S. performed thefollowing experiments; K.W., Z.H., X.X., Y.T., N.H., Y.S. analyzed the data; the paper. (Guizhen Wang) contributed reagents, materials, and analysis tools; and G.W. (Guizhen Wang) and L.W. wrote

Author G.W. (Guizhen Wang) conceived and designed the experiments; L.W., G.W. (Gengping theContributions: paper. Acknowledgments: was financially by X.X., the National Natural Foundation China Wan), and S.S. performedThis thework experiments; L.W.,supported K.W., Z.H., Y.T., N.H., andScience Y.S. analyzed theofdata; G.W. Acknowledgments: This work was financially supported by the National Natural Science(514207, Foundation of China (11564011, 21706046, 51362010) and the Natural Science Foundation of Hainan Province 514212). (Guizhen Wang) contributed reagents, materials, and analysis tools; and G.W. (Guizhen Wang) and L.W. wrote (11564011, 21706046, 51362010) and the Natural Science Foundation of Hainan Province (514207, 514212). the paper. Conflicts of Interest: The authors declare no conflict of interest. Acknowledgments: This work was financially supported by the National Natural Science Foundation of China (11564011, 21706046, 51362010) and the Natural Science Foundation of Hainan Province (514207, 514212). Conflicts of Interest: The authors declare no conflict of interest.


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Conflicts of Interest: The authors declare no conflict of interest.

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