Preparation, Characterization and Mimetic Activities of Fe 2 O 3 and Fe 3 O 4 Nanoparticles as Catalase and Peroxidase

Different iron oxide nanoparticles (Fe 3 O 4 and Fe 2 O 3 ) were prepared by the sol-gel method (titration). The prepared nanoparticles were heated at 90 and 400°C. The morphology surface and structures were characterized by Fourier Transform Infra-Red (FT-IR) and Ultraviolet/Visible (Uv/Visible) measurements, X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and Atomic Force Microscope (AFM). The enzyme mimetic activities of these nanoparticles (Fe 3 O 4 and Fe 2 O 3 ) such as two enzymes (Catalase (CAT), and Peroxidase (Pxase)) were measured. The results showed the iron oxides (Fe 2 O 3 ) heated at 90°C, have the maximum activity (189.99 K.min -1 ) as catalase (CAT). While the iron oxides (Fe 3 O 4 ) heated at 90°C, have the maximum activity (3.044 U.min -1 ) as peroxidase (Pxase), and there is a decrease in the activity for both nanoparticles when annealed at 400°C. Despite the average grain size decrease in both samples, however, the mimetic activity decrease that is mean the average grain size is not affected in both mimetic activities as catalase and peroxidase.

separation and detection, tissue repair, magnetic hyperthermia, drug delivery, and therapy for apoptotic gene expression in tumors, and transfection of pro-BDNF into neural stem cells [9,10]. Iron oxide lowers medication concentration, toxicity, and other negative consequences while increasing the efficacy of iron oxide-based treatment aside from its antibacterial effectiveness against a wide spectrum of pathogens, the toxicity of the iron oxide utilized to build the nanosystems is also addressed [11]. Nanozymes are a subset of nanoparticle-based catalysts that catalyze enzyme-like activities at room temperature nanozymes are intriguing not just because of their high stability and low cost, but also because they may be utilized to investigate fundamental processes at nanoscale surfaces [8]. Many nanoparticles, such as gold, graphene oxide, and other metal oxides, contain oxidase, peroxidase, and/or catalase-like activity. It's worth mentioning that, whereas many peroxidase nanozymes (those that employ H2O2 as a substrate) have been reported, only a few have oxidase activity [12]. Catalase (CAT) is an oxygen-consuming metalloprotein found in all organisms. Because this enzyme is soluble in erythrocytes and human erythrocytes are usually abundant in catalase, catalase activity in blood is almost entirely attributed to erythrocytes [13]. Peroxidase (Pxae) is a heme-containing enzyme that uses hydrogen peroxide to oxidize a variety of organic and inorganic compounds [14]. In this work, we prepared nano-oxides (Fe2O3 and Fe3O4) in the sol-gel method and measured their enzymatic activity (Catalase and peroxidase).

Preparation of iron oxide nanoparticles (Fe2O3 NPs)
Iron oxide (Fe2O3) nanoparticles were produced using a sol-gel technique, as described in the reference [15], with some modifications. This may be accomplished by using iron (III) chloride (FeCl3), cetyltrimethylammonium bromide (CTAB), and ammonium hydroxide (NH4OH) as described below. To begin, dissolve 3.0 gm. of FeCl3 in distilled water. CTAB (0.5 gm.) in distilled water is dissolved and added to the FeCl3 solution. Then, dilute the ammonium hydroxide in water (1:10) and begin titration until the reaction is complete (pH ≈ 8). The dark precipitate was washed more than 5 times in distilled water before being dried for 60 min at 90 °C. Then, it is annealed for 120 min at 400 °C. The reactions are explained by the equations:

Preparation of magnetite (Fe3O4) nanoparticles
Magnetite Fe3O4 nanoparticles were prepared using sol-gel methods as reference [16], with some modifications such as using a tight cover of parafilm to avoid oxidation and using FeSO4.7H2O, FeCl3, CTAB, and NH4OH, beginning with dissolving (0.695gm) of FeSO4.7H2O in distilled water (40 ml). In addition to dissolving (0.41 gm) of FeCl3 in distilled water and mixing it, dissolve (0.1 gm) of CTAB in distilled water and add to it. Then, dilute 10 mL of NH4OH in 100 mL of distilled water and begin titration on the magnetic stirrer until the reaction is complete (pH ≈ 8

Measuring enzymes 3.1. Measuring catalase (CAT) mimetic activity
Catalase (CAT) mimetic activity was measured using absorbance by the Uv/Visible spectrometer technique at a wavelength of 240 nm [18] as follows: three (50 mL) containers labeled (T, C, and B) for each nanoparticle (Fe2O3 and Fe3O4). Both the T and B containers received (0.001gm) and (0.002 gm) of (Fe2O3 and Fe3O4), respectively. In the C, B, and T containers, two and one milliliters of buffer phosphate (50 mM, pH = 7) were added, accordingly. T and C each received one milliliter of hydrogen peroxide (2.8 mM) containers. On the shaker, all containers were shaken for 5 min. The absorbance of each container is measured using a Uv/Visible spectrometer at a wavelength of 240 nm. Based on the first-order reaction, the following equation was utilized to calculate catalase mimetic activity [19].
Where, (t) denotes the response time in minutes. The concentrations of H2O2 in the cell reaction before and after the reaction is denoted by Co and C, respectively. C is the same as (C1 -T1).

Measuring peroxidase mimetic activity
The Uv/Visible spectrometer technique was used to assess the peroxidase mimetic activity of Fe2O3 and Fe3O4 at a wavelength of 292 nm as follows: three 50-mL containers labeled (T, C, and B) for each nanoparticle (Fe2O3 and Fe3O4). Both the T and B containers received a weight (0.012 gm) of (Fe2O3 and Fe3O4). T, C, and B containers received 4.95 mL and 5 mL of buffer phosphate (50 mM, pH = 7.2) accordingly. In the C and T containers, 0.05 ml of orthophenylene diamine (OPD) was added. Then, in each container, one milliliter of hydrogen peroxide (2.8 mM) was added. On the shaker, all containers were shaken for 5 min. The absorbance of each container is measured using a Uv/Visible spectrometer at a wavelength of 292 nm. The peroxidase mimetic activity was calculated using the equation below [20].
Where B = blank, C = control, T = test, Vol = total volume of reaction solution, and Wt = weight of nanoparticles in container. t represents the response time after 45 min.

Optical Properties of the Nanoparticles Solutions
The optical characteristics of nanoparticle solutions were done by dissolving nanoparticles in ethanol (approximately 1×10 -5 M), the optical properties (transmittance) were created of nanoparticles at different temperatures (90 and 400°C) ranging from 250 to about 550 nm.

The spectra of Fe3O4 Nanoparticles
The spectra of Fe3O4, Figure 1-a, show the transmittance edge is shifted to a higher wavelength (redshift) from 478 to 502 nm when increasing annealing temperature to 400 °C, resulting in higher optical transmittance and attributed to structural homogeneity and particle crystallization. The following equation can be used to calculate the value of the energy gap [21].
Where 1240 is the factor used to convert nm to eV and λ max is the maximum transmittance in nm. According to Eq. (9), the energy gap of Fe3O4 as-prepared is 2.59 eV, and that of annealing at 400 °C is 2.47 eV, this result agrees with the references [16,17]

The spectra of Fe2O3 nanoparticles
The spectra of Fe2O3, Figure 1-b, illustrate the transmittance edge that has moved to a higher wavelength (redshift) from 476 to 492 nm with a rise in annealing temperatures to 400°C. As a result and Eq. (9) the energy gap of Fe2O3 as-prepared is 2.6 eV, while that of Fe2O3 annealing at 400°C is 2.52 eV this result agrees with the reference [22,23].

Fourier Transform Infrared Spectroscopy (FTIR)
The results of the Fourier Transform Infrared Spectroscopy (FTIR) tests disclose the absorption profile of the infrared spectra of nanoparticle samples. To describe these nanoparticles, an FTIR wavenumber range of 4000 to 400 cm -1 in the KBr disc was utilized. Figure 2-a shows FTIR spectra for Fe3O4 as-prepared, the first two bands (3444 and 1629 cm -1 ) are referred to as O-H bond vibrations stretching and bending of water, respectively, in iron hydroxide nanoparticles (Fe(OH)3). These findings are consistent with the reference [24]. While the third band at 570 cm -1 is due to Fe-O vibration stretching [25]. In the spectrum of Fe3O4 nanoparticles (Figure 2-b), the stretching and bending vibrations of the O-H bond in water create two bands at 3440 and 1665 cm -1 respectively. The peaks at 532 and 445 cm -1 are caused by Fe-O vibrations. The results were quite similar to [26]. Figure 2-c shows FTIR spectra for Fe2O3 as-prepared, with the shoulder at 3408 cm -1 and peak at 1629 cm -1 associated with OH stretching and bending, respectively. The two peaks of 474 and 437 cm -1 are attributable to Fe-O stretching vibrations; these results are similar to those reported in reference [27].    [29]. The XRD pattern of Fe3O4 nanoparticles follows the typical pattern in the JCPDS: . When annealing at 400 o C, (Figure 3- [30]. The diffraction peaks might be attributed to a cubic structure of the Fe3O4 phase with the lattice value a = 8.39 °A [31]. The following is the Scherer's formula (Eq. (10)) for calculating the size of crystalline particles:

The X-Ray Diffraction for Fe2O3
Nanoparticles X-ray diffraction (XRD) of Fe2O3 was used as prepared measurements to verify the sample's crystal structure and phase composition. The indexed peaks and the recorded diffraction pattern are shown in Figure 4 and 300) planes, respectively, and indicate its usual cubic spinel structure. This is consistent with the peculiar structure of pure α-Fe2O3 crystals, which have a rhombohedral centered hexagonal shape the maximum diffraction peak at 33.17° indicates that the prevalent α-Fe2O3 crystal structure has (104) faces [34]. The lattice constants (a and c) of Fe2O3 nanoparticles were determined according to Eq. (12), as in Table 2.
Where (d) is the interplanar distance, (hkl) are the Miller indices, and (a) and (c) is the lattice constants for the α-Fe2O3 hexagonal structure the computed the crystallite size (D) as well as the lattice constants (a and c) of Fe2O3 [32]. The size of crystalline particles (D) can be calculated using the Shearer's equation (Eq. (10)).

Surface Morphology by Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Analysis (EDX).
The surface morphology image SEM with magnification at (100 K) and its EDX results of the metal oxides (Fe3O4 and Fe2O3) nanoparticles produced by hydrothermal technique and annealed to 400 °C for 120 min. It can be seen that Fe3O4 nanoparticles ( Figure 5-a) have an irregular crystalline form, and the surface morphology analysis demonstrates the agglomeration of many ultrafine particles because of their small size seem as sawdust. While EDX results find the binding energies of O and Fe are shown as peaks in the Fe3O4 EDX spectrum ( Figure  5-b) at 0.55, 0.7, 6.4, and 7.0 keV, respectively. The theoretical percentage calculations of Fe and O in the Fe3O4 nanoparticles are 42.85% and 57.14% respectively, while the experimental percentage calculations are 32.188% and 67.811% respectively. These results are listed in Table 3. About the SEM images of Fe2O3 nanoparticles show is irregularly assembled and agglomerated, very tiny in size, resembling fish eggs. (Figure 5-c). While EDX findings reveal that the binding energies of O and Fe are displayed as peaks in the Fe2O3 EDX spectrum ( Figure 5-d) at 0.55, 0.7, 6.4, and 7.1 keV, respectively. Theoretical percentage estimates for Fe and O in Fe2O3 nanoparticles are 40% and 60%, respectively, while the experimental percentage calculations are 36.93% and 62.28% respectively, Table 3. Besides Cl and Si appearance in the EDX spectrum as process contaminations.

Iron oxide (Fe3O4) Nanoparticles
The distribution and accumulation of Fe3O4 nanoparticles convert from bubbles (Figure 7-a) to uniform shapes (Figure 7-b). This may relate to converting most of the hydroxides (Fe(OH)2 and Fe(OH)3) to oxides form (Fe3O4) when heating at a high temperature (400 °C). The average grain size decreases from 91.36 nm (asprepared) to 69.44 nm (annealing), as in Table 4, the decrease in the size may be due to converting the sample from hydroxide to oxide as the Eq. (6).
A B Figure 6: AFM images at 3D and Granularity accumulation distribution charts of Fe3O4 (a) as-prepared and (b) annealing, and Fe2O3 at 400 °C for 120 min respectively.

Iron oxide (Fe2O3) Nanoparticles
The distribution and accumulation of Fe2O3 nanoparticles convert from random shapes (Figure7-c) into more regular shapes (Figure 7-d). This relates to converting most of the hydroxides (Fe(OH)3) to oxides (Fe2O3) when heating at a high temperature (400 °C). While the average grain size increases from 104.52 nm (as-prepared) to 90.25 nm (annealing), as in Table 5, the increase in the size may be due to the accumulation of the nanoparticles of the sample as the Eq. (2).  annealing at 400 °C for 120 min respectively.

Applications 5.1. Catalase Mimetic Activity
The mimetic activities of our prepared nanoparticles as catalase (CAT) in phosphate buffer solution (50 mM, pH= 7), using spectrophotometric method [19], at the wavelength (λ = 240 nm), The CAT mimetic activity was determined by the Eq. (7). The results show the CAT mimetic activities of the nanoparticles are different according to their types and the degree of heating temperatures. The highest CAT mimetic activity of the nanoparticles (as-prepared) relates to Fe2O3 while the lowest activity relates to Fe3O4. Also, the highest CAT mimetic activity of the nanoparticles (annealing) relates to Fe2O3 while the lowest activity relates to Fe3O4, as in Figure 7 and Table 6. The increased CAT mimetic activity of Fe2O3, when annealing at 400 o C, may be due to converting the sample from a nonmagnetic to a magnetic form. Meanwhile, the high decrease in CAT mimetic activity of Fe3O4 (annealing) compared with its as-prepared (from 129.89 K. min -1 to 56.20 K. min -1 ), may be related to losing its magnetic property.

Peroxidase Mimetic Activity
The mimetic activities of our nanoparticles (Fe3O4 and Fe2O3) as peroxidase (Pxase) in phosphate buffer solution (50 mM, pH= 7.2), utilizing the reference [20], with some modification by employing spectrophotometric technique at a wavelength (λ = 292 nm), including the use of orthophenylenediamine solution as an indicator, The equation was used to calculate the peroxidase mimetic activity (8). The results show the Pxase mimetic activities of nanoparticles were depending on the kind of nanoparticle and the degree of heating. The nanoparticles with the highest Pxase mimetic activities (as prepared) are Fe3O4, while the nanoparticles with the lowest activity are Fe2O3. The nanoparticles with the highest Pxase mimetic activities (annealing at 400 o C) are Fe2O3, while the nanoparticles with the lowest activity are Fe3O4. The simulation activity was arranged in the following order (Fe2O3> Fe3O4) exactly the opposite of the as-prepared, as shown in Figure 8 and Table 7. The high decrease in Pxase mimetic activity of Fe3O4 compared with its as-prepared (from 3.044 U/min -1 to 0.640 U/min -1 ), may be related to losing its magnetic property when annealing at 400 o C. While the increased the Pxase mimetic activity of Fe2O3 (as-prepared) nonmagnetic sample (Fe(OH)3) more than the magnetic sample (Fe2O3) when annealing at 400 o C, despite the average grain size of as-prepared is larger than annealing samples (104.52 to 90.25 nm) respectively, Table 7 this indicates that the average grain size does not affect in Pxase mimetic activity. Despite the decrease in the average grain size in all annealing samples, there are no effects on Pxase mimetic activity.

Conclusions
Iron oxides nanoparticles (Fe3O4 and Fe2O3) have been successfully synthesized by the Sol-Gel. Both nanoparticles were heated at two temperatures (90 and 400 o C), then characterized by different techniques such as FT-IR, Uv/Visible, XRD, SEM, and AFM. The AFM study showed that the grain size of the powders annealing at 400 o C, was 90.25 and 69.44 nm for Fe3O4 and Fe2O3 respectively. Catalase (CAT) and peroxidase (Pxase) mimetic activities were measured against H2O2 and OPD as substrate respectively. The results indicated that the highest mimetic activities as CAT for Fe3O4 were at sample heated at 90 o C, while that for Fe2O3 were at same annealing at 400 o C because both samples have magnetite properties. On the other hand, the mimetic activities as Pxase of iron oxides nanoparticles were found when heated at 90 o C, compared with corresponding their iron oxides heated at 400 o C. Despite the average grain size was decreased with heating in both samples. This means the particle size of these nanoparticles does not affect the mimetic activity of CAT and Pxase.