Properties Comparison of Fe₃O₄ Particles with Different Morphologies as Mimetic Enzyme

Abstract

In this work, four different magnetic Fe₃O₄ nanoparticles are prepared via solvothermal method. According to the morphology, the products can be divided into flower-like Fe₃O₄ (F-Fe₃O₄), solid spherical Fe₃O₄ (S-Fe₃O₄), hollow spherical Fe₃O₄ (HO-Fe₃O₄), and hexahedral Fe₃O₄ (HE-Fe₃O₄). A set of measurements is performed to confirm the structure, composition, and pore properties of the obtained materials. The catalytic activities of the prepared materials are examined and compared. The results prove that the four materials have an intrinsic catalytic property. HO-Fe₃O₄ ranks first in the catalytic activity mainly due to its large surface area and reasonable element composition. The maximum specific saturation magnetization and specific surface area of HO-Fe₃O₄ are 72.94 emu/g and 42.60 m²/g. Fe²⁺/Fe³⁺ in HO-Fe₃O₄ is 51.5%. It is found that HO-Fe₃O₄ possesses fantastic stability and perfect reproducibility as it is used as a catalyst several times without significant loss in its activity.

1. Introduction

Natural enzymes have perfect properties such as high substrate specificity and high catalytic efficiency under mild conditions. So, they can be widely applied in the field of catalytic reactions. But unfortunately, environmental conditions, including acidity, temperature, and inhibitors, can easily affect their catalytic activity. In other words, their stability and activity decreased in tough environmental changes such as pH, temperature, and proteases. Moreover, their production, purification, and storage are costly. As a result, their application became constrained. That is why great attention has been paid recently to designing new materials that can play the same role as natural enzymes without previous flaws [1]. Compared with natural enzymes, nanomaterials possess high stability against the tough environmental changes, low preparation and storage costs, elasticity in structure design, and they have abnormal catalytic activities [2]. So, they represent promising candidates for mimetic enzymes. Nanoparticles (NPs), which have the same catalytic properties as natural enzymes, are called mimic enzymes (nanozymes). It was reported that Fe₃O₄ NPs, MnO₂ nanosheets, CeO₂ NPs, V₂O₅ nanowires, BiFeO₃ NPs, positively charged gold NPs, and NiO₂ NPs possessed peroxidase-like activity [3,4,5]. There are other enzyme mimetics such as hemin, hematin, porphyrin, etc. It is well-known that hydrogen peroxide (H₂O₂) is a reactive molecule that has a basic role in several applications, including chemical, biological, and environmental [6,7]. But the previous studies reported that long-term exposure to H₂O₂ is very dangerous as it causes serious harm to a person’s health, such as respiratory inflammation, poisoning, Parkinson’s disease, Alzheimer’s disease, and cancer [8,9]. So, it was urgent to develop sensitive methods for the detection of H₂O₂. For this aim, there are numerous methods such as electrochemistry, chemiluminescence, spectrometry, high-performance liquid chromatography (HPLC), and colorimetric assays [10,11,12]. In spite of HPLC, chemiluminescence and electrochemistry methods are very sensitive and their processes are very complex. Spectrometry is very simple and cheap, but its sensitivity is much lower, so it was rarely used. On the other hand, besides the simplicity of colorimetric methods, it displayed abnormal sensitivity. Furthermore, its results can be noticed by the naked eye without using any instruments.

The colorimetric assay mainly depended on horseradish peroxidase (HRP) as a catalyst, but as a natural enzyme, it cannot be used due to the mentioned reasons above. So, recently, most of the research has focused on developing an artificial enzyme that mimics this natural enzyme, such as Fe₃O₄. Fe₃O₄ NPs have an intrinsic enzyme mimetic activity similar to that of natural peroxidases such as horseradish peroxidase (HRP) [13]. The most remarkable features of Fe₃O₄ NPs are simplicity and the feasibility of their preparation, as they can be separated easily and rapidly from the solution by using an external magnetic field [14,15,16]. Also, its synthesis is non-toxic, and it has economical and efficient properties. Due to these advantages, it can be used for potential applications and fundamental research [17,18]. Decreasing the particle sizes of Fe₃O₄ NPs leads to an increase in their peroxidase-like activity, where Fe₃O₄ NPs with different sizes showed an increasing activity in the order of 300 nm < 150 nm < 30 nm. Many methods can be used for the design of nanostructured magnetic materials, including hydrothermal synthesis, thermal decomposition of organometallic compounds, co-precipitation, sol–gel method, etc. [19,20,21].

In light of the above, it was reported that Fe₃O₄ NPs have an intrinsic enzyme mimetic activity similar to that of a natural enzyme. Moreover, its preparation is simple and nontoxic. Therefore, in this work, flower-like Fe₃O₄ (F-Fe₃O₄), solid spherical Fe₃O₄ (S-Fe₃O₄), hollow spherical Fe₃O₄ (HO-Fe₃O₄), and hexahedral Fe₃O₄ (HE-Fe₃O₄) are prepared via the solvothermal method. The structure, composition, morphology, and pore properties of the above four types of Fe₃O₄ NPs are characterized systematically. The catalytic activity of the obtained Fe₃O₄ NPs is compared. The main factors affecting the catalytic performance of the materials have been revealed.

2. Materials and Methods

2.1. Materials

Hydrated iron chloride (FeCl₃·6H₂O), ferrous sulfate (FeSO₄·7H₂O), urea (CH₄N₂O), H₂O₂ (30%), and ethylene glycol (EG) were bought from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Sodium polyacrylate (PAAs), polyethylene glycol 400 (PEG-400), tetrabutylammonium bromide (TBAB), hydrazine hydrate (N₂H₄·H₂O), and o-phenylenediamine (OPD) were obtained from Adamas Reagent Co., Ltd., Shanghai, China. Sodium ethanoate trihydrate (CH₃COONa·3H₂O) and trisodium citrate dihydrate (C₆H₅Na₃O₇·2H₂O) were purchased from Tianjin Dongli Industry and Trade Co., Ltd., Tianjin, China. The pure water used was deionized.

2.2. Preparation of Fe₃O₄ Particles

Four different Fe₃O₄ magnetic nanoparticles (MNPs) were synthesized through the solvothermal method as follows [22,23].

F-Fe₃O₄: Typically, 0.5 g of FeCl₃·6H₂O, 4.2 g of TBAB, and 1.75 g of CH₄N₂O were completely dissolved into 75 mL EG. The above solution was transferred to a 100 mL Teflon-lined stainless-steel autoclave. The synthesis process was carried out at 180 °C for 1 h. The product was then collected and washed with pure water. The obtained NPs were dried and calcined in a mixed atmosphere of 5% H₂/Ar at 300 °C for 1 h. The product was F-Fe₃O₄.

S-Fe₃O₄: In total, 2.70 g of FeCl₃·6H₂O, 7.26 g of CH₃COONa·3H₂O, and 4 mL of PEG-400 were dissolved in 72 mL of ethylene glycol (EG) by magnetic stirring. The obtained solution was transferred into a 100 mL Teflon-lined stainless-steel autoclave and treated at 200 °C for 10 h. The black suspension was separated and washed with water several times under a magnetic field. After freeze-drying, solid Fe₃O₄ was obtained and marked as S-Fe₃O₄.

HO-Fe₃O₄: In total, 6.76 g of FeCl₃·6H₂O, 5 g of urea, 16.2 g of C₆H₅Na₃O₇·2H₂O, and 3 g of PAAs were dissolved in 400 mL of water by magnetic stirring at 90 °C. The obtained solution was cooled and transferred into a 500 mL Teflon-lined stainless-steel autoclave and treated at 200 °C for 10 h. The black suspension was separated with a magnet. The products were washed with water several times and freeze-dried. The prepared magnetic nanoparticles showed hollow morphology and were marked as HO-Fe₃O₄.

HE-Fe₃O₄: In total, 1.5 g of Fe₂(SO₄)₃·7H₂O was dissolved in 45 mL of H₂O. Then, 0.6 g of NaOH and 20 mL of N₂H₄·H₂O were added to the solution under vigorous stirring in turn. Then, the solution was transferred into a 100 mL Teflon-lined stainless-steel autoclave. The reaction was maintained at 180 °C for 12 h. After washing with pure water three times and freeze-drying, HE-Fe₃O₄ was obtained.

2.3. Catalytic Activity Studies

The catalytic properties of the obtained MNPs were examined through the following colorimetric assay [24]. Four 10 mL EP tubes were chosen as the reactor. A total of 1 mL of aqueous solution of OPD (80 mg/L) was poured into 1 mL of 0.4 M H₂O₂ plus 2 mL of purified water. Then, 1 mL of Ho-Fe₃O₄ (1 mg/mL), which was well dispersed in purified water under ultrasonic treatment, was transferred to the first tube. The same dosage of S-Fe₃O₄, F-Fe₃O_4, and HE-Fe₃O₄ was added to the other three tubes. The mixture of all samples was incubated at 30 °C for one hour and separated by centrifugation to isolate the magnetic nanoparticles. The supernatant for each sample was determined by UV/vis absorption, with the wavelength ranging from 300 nm to 600 nm or at 425 nm.

2.4. Characterization

To verify the successful preparation of these four MNPs, a series of measurements was used involving a scanning electron microscope (SEM, JEOL JSM-6700F, Tokyo, Japan) and transmission electron microscope (TEM, JEOL JEM-3010, Tokyo, Japan) to describe the morphology of the products, and powder X-ray diffraction (XRD, Shimadzu XRD-6000, Kyoto, Japan) to determine their crystal structures. The surface chemical compositions of the samples were evaluated by X-ray photoelectron spectroscopy (XPS, Thermo Fisher ESCALAB 250Xi, Waltham, MA, USA). Fourier transform infrared (FTIR, Bruker TENSOR27, Bremen, Germany) spectroscopy enabled us to identify the different functional groups in our samples. A nitrogen adsorption–desorption isotherm experiment was conducted to study the porous features of the prepared samples using a N₂ adsorption instrument (Tristar3020, Micromeritics, Norcross, GA, USA) and BET (Brunauer–Emmet–Teller method). The ultraviolet spectrum (UV, BlueStar, LabTech, Beijing, China) was used to determine the catalytic activity of our products. A vibrating sample magnetometer (VSM, Lake Shore 7307, Westerville, OH, USA) was used for characterizing the magnetic properties of MNPs.

3. Results and Discussion

3.1. Component Analysis

The crystalline structures of the four prepared MNPs (F-Fe₃O₄, S-Fe₃O₄, HO-Fe₃O_4, and HE-Fe₃O₄) were examined by X-ray diffraction (XRD) measurements. As shown in XRD patterns in Figure 1, the main observed diffraction peaks for the synthesized materials occur at 2θ = 30.14°, 35.5°, 43.06°, 53.56°, 57.08°, 62.52°, and 74.08°, which were assigned to (220), (311), (400), (422), (511), (440), and (533) crystal planes, respectively. The resulting diffraction peaks agree with the reported phase of Fe₃O₄ (JCPDS 75-1609) [25]. The above results indicated that all four morphologies of metal oxides prepared were Fe₃O₄.

3.2. Morphologies of Fe₃O₄ Particles

SEM and TEM were used to characterize the morphology of the obtained materials. The results showed that F-Fe₃O₄ NPs had a flower shape with an average size of 1.9 μm, as shown in Figure 1a–c. The particles were made up of stacked layers. The layers present a radial pattern, forming relatively large pores. S-Fe₃O₄ NPs showed a spherical shape (Figure 2d), and the uniform mass thickness contrast reflected their solid structure (Figure 2e). The average particle size of S-Fe₃O₄ NPs was 156 nm (Figure 2f). For HO-Fe₃O₄ NPs, as Figure 2g–i presents, it possesses a hollow structure. The microspheres were composed of small nanocrystals. The average size of HO-Fe₃O₄ NPs was 327 nm. In addition, the results shown in Figure 2j–l proved that HE-Fe₃O₄ consisted of a hexagonal shape with a mean size of 200 nm. The particles exhibited a solid and regular structure. The differences in the morphology of these materials can be ascribed to the different metal source and preparation processes. Compared with the reported Fe₃O₄ NPs, our results show diversified morphologies and potential functionalities.

3.3. Magnetic Property Characterization

The hysteresis curves of the Fe₃O₄ products were examined with a vibrating sample magnetometer (VSM), as shown in Figure 3. The magnetism of ferromagnetic materials can be characterized mainly by three important parameters: the saturation magnetization (Ms), the remanent magnetization (Mr), and the coercivity (Hc). The results showed that S-Fe₃O₄ has the highest saturation magnetization value of 78.53 emu/g (Figure 3b), followed by a value of 72.94 emu/g, corresponding to HO-Fe₃O₄ (Figure 3c). Then HE-Fe₃O₄ has a value of 59.96 emu/g (Figure 3d). Finally, F-Fe₃O₄ possessed the lowest saturation magnetization value of 34.53 emu/g as shown in Figure 3a. The saturation magnetization value relies on the spin numbers of Fe²⁺/Fe³⁺ ions, especially since the orbital moments of Fe²⁺ ions give the main contribution to the magnetization. S-Fe₃O₄ possesses the largest Fe²⁺/Fe³⁺ ratio (see the XPS results), which means S-Fe₃O₄ probably shows the largest orbital contribution; therefore, it has the highest saturation magnetization value. The order of the magnetization values for other Fe₃O₄ NPs also follow the Fe²⁺/Fe³⁺ ratio order (see below), which is comparable with the reported results. These products can be easily separated with an external magnetic field without using centrifugation methods. Due to these excellent magnetic properties of the synthesized materials, they have great applicability to reclamation and recycling.

3.4. Surface Performances of Fe₃O₄ Particles

To characterize the structure and the components of the prepared products, FT-IR analysis was conducted, and the results were displayed in Figure 4. In the spectrum of F-Fe₃O₄ (Figure 4a), the observed peaks at 3427 cm⁻¹ and 1637 cm⁻¹ indicated the existence of OH and C=O groups, respectively. In addition, the peak at 578.6 cm⁻¹ proved the existence of Fe-o in Fe₃O₄. Similar peaks appeared in S-Fe₃O₄ (Figure 4b) and HO-Fe₃O₄ (Figure 4c), which indicates that they have the same functional groups. On the other hand, HE-Fe₃O₄ (Figure 4d) only has one clear peak, which appeared at 578.6 cm⁻¹, related to Fe-o from the structure of Fe₃O₄. The absence of clear peaks in the HE-Fe₃O₄ spectrum referred to the increase in biological and organic matter [26].

A nitrogen adsorption–desorption isotherm experiment was conducted to study the porous features of the prepared samples. As shown in Figure 5, the adsorption–desorption isotherms of the obtained products belonged to type IV with small hysteresis loops noticed at a relative pressure of 0.8–1.0, which confirms that the prepared samples had a mesoporous and macroporous structure, as seen in Figure 5.

Table 1. Pore performance data of the obtained products.

Samples	BET (m2/g)	Average Pore Size (nm)	Pore Volume (cm3/g)
F-Fe3O4	30.19	18.16	0.1367
S-Fe3O4	9.53	17.44	0.0387
HO-Fe3O4	42.60	11.16	0.1553
HE-Fe3O4	12.52	26.26	0.0346

shows the BET surface area and pore volume for the synthesized materials. The surface area and pore volume are 30.19, 9.53, 42.60, 12.52 m²/g and 18.16, 17.44, 11.16, 26.26 nm for F-Fe₃O₄, S-Fe₃O₄, HO-Fe₃O_4, and HE-Fe₃O₄, respectively, which are comparable with the reported results [27]. These results were calculated based on the Barrett–Joyner–Halenda (BJH) method. The extra peak that appeared in Figure 5h is due to the hollow structure of HO-Fe₃O₄ nanoparticles. That is why it had the largest specific area, about 42.60 m²/g.

3.5. X-Ray Photoelectron Spectroscopy

The composition and surface oxidation states of Fe were explored with X-ray photoelectron spectroscopy (XPS) measurements. The survey spectra (Figure 6) indicate the presence of Fe components in the as-prepared materials. To further confirm the oxidation ratio of these materials, high-resolution Fe 2p(F) XPS spectra were compared as shown in Figure 6a–d. The two types of Fe species are fitted. Two peaks centered at 724.6 and 711.4 eV can be assigned to 2p_3/2 and 2p_1/2 for Fe²⁺, and two peaks centered at 721.6 and 708.5 eV can be assigned to 2p_3/2 and 2p_1/2 for Fe³⁺, indicating the oxidation state of Fe₃O₄ [28] and further conforming that the nanoparticles were Fe₃O₄ NPs. The calculated Fe²⁺/Fe³⁺ ratio of as-prepared samples from the XPS spectra follows the order of S-Fe₃O₄ > HO-Fe₃O₄ > HE-Fe₃O₄ > F-Fe₃O₄ (

Table 2. Calculated Fe²⁺/Fe³⁺ ratio of as-prepared samples from the XPS spectra.

Sample Information	F-Fe3O4	S-Fe3O4	HO-Fe3O4	HE-Fe3O4
Fe2+/Fe3+	46.2%	52.3%	51.5%	50.1%

), which is in good agreement with the VSM results (vide supra).

3.6. Comparison of Catalytic Activity

The prepared magnetic nanomaterials had an intrinsic peroxidase-like activity due to the presence of ferrous ions at their surfaces. These catalytic properties were tested through the colorimetric assay in the presence of OPD and H₂O₂ as substrates. The four kinds of as-obtained nanoparticles were used for catalytic reaction as the mimetic peroxidase. The role of these nanomaterials was to catalyze the decomposition of H₂O₂ to produce a reactive oxygen atom [O], which could oxidize o-Phenylenediamine (OPD). The results showed that the darkest color was seen when HO-Fe₃O₄ was used as a catalyst, which indicated its possession of the highest catalytic properties compared with other materials. HO-Fe₃O₄ had the fastest catalytic reaction rate as shown in Figure 7c. The other materials had lower catalytic properties, where HE-Fe₃O₄> S-Fe₃O₄> F-Fe₃O₄ as shown in Figure 7. The catalysis mechanism is illustrated in Figure 8. The reaction could be dominated by three stages on the Fe₃O₄ NPs surface. The oxidation–reduction process between Fe(III) and Fe(II) is the essence of simulating enzyme catalysis.

3.7. Catalytic Properties of HO-Fe₃O₄ Particles

According to the material’s structure, the materials that have a large surface area possess a lot of active sites, so their absorbance increases. As a result, a wide variety of structures, such as hollow, porous, and foamed, were investigated to obtain high-performance absorption units. It was found that the hollow structure possesses abnormal catalytic properties due to its large surface area. In the present study, HO-Fe₃O₄ possessed a hollow structure, so it was used to oxidize OPD in the colorimetric assay in the presence of H₂O₂. The UV results showed that the catalytic activity of the reaction system depends on the amount of the nanocatalyst (HO-Fe₃O₄).

As shown in Figure 9a, the catalytic activity increased with the increase in HO-Fe₃O₄ concentration, and this increase was drastic when a concentration of 0.1 mL to 0.6 mL was used. After 0.6 mL, the increase became very slow. Also, the amount of H₂O₂ affected the catalytic properties of the reaction system, as the dosage of H₂O₂ led to an increase in the catalytic activity, as seen in Figure 9b. From 0.1 mL to 0.5 mL dosages, the increase was very clear, while at amounts more than 0.5 mL, no considerable change in the absorbance value was observed. So, the optimal added amounts of H₂O₂ and HO-Fe₃O₄ nanoparticles were 0.8 mL and 0.6 mL, respectively. For H₂O₂ and HO-Fe₃O₄, the maximum absorbance was observed at 425 nm.

Reusability and reproducibility are very important for the assessment of materials’ catalytic properties. So, they were tested by performing the experiment 15 times. After each time the HO-Fe₃O₄ was separated using a magnetic field, then washed with ethanol and water. The results proved that a slight loss of activity was noticed even after 15 cycles, as shown in Figure 10a. SEM was tested for HO-Fe₃O₄ as displayed in Figure 10b. After 15 cycles, the result showed little loss and no change in its shape, which demonstrated the abnormal stability of HO-Fe₃O₄ and its perfect reproducibility.

4. Conclusions

In this study, four different magnetic Fe₃O₄ nanoparticles were synthesized by the solvothermal method. To verify the successful preparation of the obtained MNPs, a series of measurements was carried out. A nitrogen adsorption–desorption isotherm experiment was conducted to obtain the surface area and pore-size distribution of the obtained materials. The catalytic properties of the synthesized materials were tested through the colorimetric assay in the presence of OPD and H₂O₂. It was found that HO-Fe₃O₄ had the highest catalytic activity as it gave the maximum absorbance in UV spectra compared with other materials. Meanwhile, the results showed that the catalytic activity of the reaction system depended mainly on the HO-Fe₃O₄ and H₂O₂ amounts, as the optimal added amounts of them were 0.8 mL and 0.6 mL. The maximum absorbance was obtained at 425 nm. Reusability and reproducibility were tested by conducting the experiment 15 times. The results clearly showed that HO-Fe₃O₄ had abnormal stability and perfect reproducibility as it was used as a catalyst 15 times without a great loss in the activity.