Temperature annealing dependence of structural, magnetic, and antibacterial properties of silver substituted cobalt ferrite nanoparticles produced by coprecipitation route

In this study, silver-substituted cobalt ferrite (Ag0.02Co0.98Fe2O4) nanoparticles were successfully sensitized by the coprecipitation method. Annealing temperature treatment was used to modify the physical properties, i.e., 200 °C, 300 °C, 400 °C, and 500 °C. XRD analysis showed an increase in the annealing temperature, the crystallite size increased from 19.78 to 24.11 nm, and the grain size increased from 54.75 to 61.39 nm. The FTIR spectrum showed two prominent absorption bands around k ∼577 and k ∼381 cm−1, allowing metal oxide absorption in the tetrahedral and octahedral sites. There is a redistribution of cations which is more significant at the tetrahedral sites than at octahedral sites, toward a perfect spinel structure. An increased annealing temperature increased the saturation magnetization and coercive field from 31.80 to 50.60 emu g−1 and 651 to 1,077 Oe, respectively, attributable to an increase in the magnetocrystalline anisotropy constant. The evaluation of S. aureus and E. coli showed that Ag0.02Co0.98Fe2O4 indicated the zone of inhibition (ZOI) around the disks due to its antibacterial properties. The most significant on S.aureus and E.coli were 12.73 mm (mortality of 88%) and 12.43 mm (mortality of 80%), respectively, for Ag0.02Co0.98Fe2O4 annealed at 200 °C that have the minor grain size materials.


Introduction
Cobalt ferrite nanopowders has received significant attention because it has several magnetic characteristics that make it a suitable material for application in the antibacterial field [1][2][3][4][5]. The physical characteristics of cobalt ferrite include having a particle size on the nanoscale (∼<100 nm), a high coercive field at room temperature (∼3.5 kOe), good chemical stability, and has flexible properties to modify [6][7][8][9]. The rest are able to exhibit paramagnetic phenomena at near room temperature when the crystallite size is down-scale around 15 nm order [10]. In general, the antibacterial activity of metals-CoFe 2 O 4 is supported by several factors, including size, morphology, surface area, oxidation ability to absorb oxygen, diffusion of chemical molecules, and release of metal ions [11,12]. Furthermore, antibacterial activity can be improved in various approaches, e.g., the optimization of doping concentrations [13,14], synthesis methods [15], applying ionizing radiation to materials [16], and solvent alteration [17].
Cobalt ferrite has the chemical formula CoFe 2 O 4 which consists of divalent Co 2+ ions that occupy octahedral sites and trivalent Fe 3+ that occupy both octahedral and tetrahedral sites [18]. Substitution of metal cations can improve the structural and magnetic properties of cobalt ferrite. The choice of doping cations to control their physical and magnetic properties is very important to avoid the formation of non-spinel phases [19]. In addition, the presence of doping cations will support the release of metal ions which affect antibacterial activity. The substitution of divalent cations in cobalt ferrite nanoparticles such as Ag is of particular concern given its biocompatibility, i.e., not toxic to the body as well as stable in aqueous media and, it is highly sought as an antibacterial additive for CoFe 2 O 4 . Sokovnin et al, applied different nanoscale silver coatings to the nanoparticles and were able to influence the antibacterial activity in terms of the percentage of live yeast [20]. However, substituted Ag compensated for a larger crystallite size because the ionic radius of Ag + (1.15 Å) is greater than that of Co 2+ (0.83 Å) and Fe 2+ (0.64 Å) [13,21].
If a small crystallite size is desired, lower annealing temperatures are a straightforward procedure resulting in smaller crystallite sizes [22][23][24]. In the cobalt ferrite nanoparticle system, the annealing temperature determines cations' redistribution. Increasing annealing temperature has been reported to promote redistribution of Fe 3+ cations to octahedral sites, with the resulting saturation magnetization increasing with increasing annealing temperature. Other, the presence of a divalent silver cation with a high thermal conductivity (429 W mK −1 ) compared to both Co (100 W mK −1 )) and Fe (80.4 W mK −1 ) facilitates the wall depinning domain, as a consequence, as in a relatively high coercive field above 1 kOe [25]. Following the green synthesis procedure (using Ocimum sanctum), the coercive field of Ag-substituted cobalt ferrite can be reduced to ∼280 Oe [26], which has challenged superparamagnetic behavior at just above room temperature.
Comparing the antibacterial activities performance of Ag, CoFe 2 O 4 , and Ag-CoFe 2 O 4 materials, the silver substituted cobalt ferrite of the Ag-CoFe 2 O 4 has the highest antibacterial activity [27][28][29]. Here, the antibacterial properties of Ag-CoFe 2 O 4 , which are indicated by the zone of inhibition (ZOI) magnitude, increases with Ag concentration [25]. Ag-CoFe 2 O 4 increases antimicrobial activity compared to pure CoFe2O4 nanoparticles, due to the release of Ag+, which causes cell changes and bacterial death [27]. Thus, Ag-CoFe 2 O 4 fabricated using the green synthesis method from tulsi seed and garlic extracts has also been reported to modify the antibacterial properties [26].
Several metals-CoFe 2 O 4 nanoparticles have been developed with excellent antibacterial properties due to owing small crystallite sizes, resulting in large surface areas [10,30,31]. The effect of the annealing temperature of Ag-CoFe 2 O 4 on the performance of the antibacterial characteristic has not been reported. Annealing temperature should control the crystallite size and modify a surface area, directly affecting the antibacterial activity. This study, tuning the antibacterial properties of coprecipitated Ag-CoFe 2 O 4 magnetic nanoparticles following the modification of structural and magnetic characteristics, with variations in annealing temperature, is presented.

Material and methods
Ag 0.02 Co 0.98 Fe 2 O 4 magnetic NPs were synthesized using the coprecipitation method [32]. The number of stoichiometric chemicals, i.e., Fe (NO 3 ) 3 ·9(H 2 O), Co (NO 3 ) 2 ·6(H 2 O), and AgNO 3 dissolved with double distilled water under magnetic stirring of 250 rpm. After the obtained homogenous solutions, it is wised dropped by NaOH solution at 95°C under a rotational speed of 250 rpm. While the titration process was finished, coprecipitated products were washed using double distilled water several times. Then, the obtained samples were put in an oven for the drying process at a temperature of 100°C for 12 h. To modify the physical properties of the Ag 0.02 Co 0.98 Fe 2 O 4 were annealed at atmospheric conditions of 200°C, 300°C, 400°C, and 500°C for 4 h. After grinding for approximately 1 h, the final product was ready for physical characterization.
The crystalline structure of the nanoparticles magnetic samples was performed using x-ray diffraction (XRD) PanAnalytical X'pert Pro (with x-rays of Cu-Kα = 1.54 nm) of 20°-80°with a step size of 0.02°. The changes in the crystalline structure parameters of Ag 0.02 Co 0.98 Fe 2 O 4 , including the crystallite size (D), lattice parameter (a), sample density (d x ), and lattice strain (ε), are summarized in table 1. The crystallite size (D) was calculated using the Scherrer equation at the strongest peak of hkl (311) [33][34][35], where λ means the wavelength of the Cu-Kα 1 source, β denotes the full width at half maximum at the most substantial peak (311), and θ means the Bragg diffraction angle. The lattice parameter (a) is calculated as follows where d denotes the distance between the planes by the Bragg equation (d = λ/2sinθ) and h, k, and l are Miller indices. The lattice strain was calculated as follows The mass density (r x ) [36] and specific surface area (saa) calculate by using equation [37] where 8 denotes the atomic number per cubic unit cell, M is the molecular weight, a 3 denotes the cubic unit cell's volume, G is the grain size diameter SEM analysis, and N A is Avogadro's number. Morphology was observed using desktop scanning electron microscopy (SEM) Phenom ProX-G6 equipped with an energy-dispersive spectrometer (EDS) magnification of 150,000×. From the SEM image, 100 granules will be selected, so the grain size distribution was obtained. It is followed by EDS analysis to check the correctness of the inclusion of elements in materials.
Fourier transform infrared (FTIR) spectroscopy was performed with Shimadzu IF Prestige 21 in a range of 350-4000 cm −1 . From the absorption peak of the FTIR spectrum, information about the bonds that occur in the material will be obtained. The shift in the band's position in both tetrahedral and octahedral sites changes the constant force value [38,39].
where M denotes the molecular weight of cations at the tetrahedral and octahedral sites. Vibrating sample magnetometer (VSM) characterization was performed using OXFORD VSM 1.2H from −10 to + 10 kOe at room temperature. Magnetic parameters extracted from the magnetic hysteresis curve. The magnetic moment η B and magnetocrystalline anisotropy K are, respectively, calculated using equations (8) and where M denotes the molecular weight, M s denotes the saturation magnetization, and 5,585 is N × β (Avogadro's number × conversion factor of the magnetic moment per atom in a Bohr magneton). This explanation of the net preference of the material magnetic moment (m) in the spinel structure [42] å å where Σm B-sites and Σm A-sites denote the magnetic moments at the octahedral and tetrahedral sites, respectively.
The dominant cations at the tetrahedral sites increase the net magnetic moment and saturation magnetization. Antibacterial activity was assessed by the well diffusion method and total plate count. There are the diameter of ZOI around disks and the number of bacteria growing (NBG). Comparing the ZOI diameter between the samples and positive control shows the strength of inhibiting the growth of bacteria for the 24-h incubation period. Comparing the difference in the number of bacteria that still survive after between the samples and positive control shows the value of the mortality rate (M) of bacteria during the 24-h incubation.

Results and discussion
3.1. Scanning electron microscophy (SEM) and energy dispersive spectroscopy (EDS) analysis Figure 1 shows  [13]. The presence of 2% was confirmed by an analysis test using the desktop SEM Phenom ProX-G6 equipped with an EDS, which confirmed that the range of Ag substituted in CoFe 2 O 4 magnetic NPs was 0.255%-0.283%, which comparably fit the stoichiometric calculation. Some spectra generated from the EDS analysis are shown in figure 2, and summaries for individual elements are provided in table 1.   (311) showed that the intensity increased slightly with annealing temperature, indicating an increase in crystallinity, according to [43].

X-Ray diffraction (XRD) analysis
The most substantial peak (311) shifted toward smaller 2θ [ figure 3(b)], i.e., the shift was closer to the ICDD data (35.44°), indicating that a fine crystalline structure was attained as the annealing temperature increased.
The calculation of grain size (G), crystallite size (D), lattice parameter (a), density (ρ x ), specific surface area (ssa), and lattice strain (ε) from the XRD data are summarized in table 2. It is clearly observed that the calculations of G from the SEM results and D from the XRD data are significantly different. From the point of view of solid-state theory, several crystallites develop a single grain, so the grain size is larger than that of the crystallites. However, for the case of nanoparticles, some papers describe comparable grain size and crystallite size.  [13], and the sample density ρ x is in the range of 5.275-5.361 g cm −3 according to Ashour et al,(2018) and Shyamaldas et al, (2020) [44,45].
From the point of view of solid-state theory, several crystallites develop a single grain, so the grain size is larger than that of the crystallites. However, for the case of nanoparticles, some papers describe comparable grain size and crystallite size.
The grain size increase due to annealing will cause the material's density in column 5 to decrease, as well as the surface area of the particles. Smaller NPs have larger specific surface areas, resulting in a higher probability of being in touch with and passing through the bacterial cell membrane than larger NPs [46]. An annealing   temperature of 200°C produces the largest ssa, so it should show the best antibacterial characteristics. The results of the antibacterial test will be presented in the next section. An increase in annealing temperature showed a decrease in lattice strain (ε) from 5.72 × 10 −3 at 200°C to 4.72 × 10 −3 at 500°C. The annealing process caused the Ag 0.02 Co 0.98 Fe 2 O 4 material to become more ductile, and some stress was removed, resulting in reduced strain [47]. At 200°C, Ag 0.02 Co 0.98 Fe 2 O 4 had low crystallinity and high lattice strain due to the large surface-to-volume ratio or specific surface areas of the nanocrystalline material, which yielded a significant broadening of the spectral peaks [35].
The decrease in lattice strain at a higher annealing temperature of Ag 0.02 Co 0.98 Fe 2 O 4 is attributable to the cation distribution at the tetrahedral and octahedral sites [42,48]. The intensity ratio of I 220 /I 222 is related to the cation distribution at the tetrahedral sites. In contrast, the ratio of I 422 /I 222 is associated with the cation distribution at the octahedral sites, which are peak intensities corresponding to the (220), (222), and (422) planes of the Miller indices [23,39]. The ratios of I 220 /I 222 for the annealing temperatures of 200°C, 300°C, 400°C, and 500°C were 2.79, 2.89, 3.18, and 3.85, respectively. Further, the ratios of I 422 /I 222 were 0.95, 1.11, 0.82, and 1.10. The cation distribution at the tetrahedral sites experienced a more significant change, whereas the cation distribution at the octahedral sites did not show a specific pattern.

Vibrating sample magnetometer (VSM) analysis
Hysteresis curves produced by the VSM of Ag 0.02 Co 0.98 Fe 2 O 4 at various annealing temperatures are shown in figure 5. Magnetic parameters, such as saturation magnetization M s , coercive field H c , remanent magnetization M r , ratio squareness M r /M s , magnetic moment η B , and magnetocrystalline anisotropy K, are presented in table 4.
The saturation magnetization M s increased with annealing temperature, indicating that cation redistribution occurred at the tetrahedral sites where increasing annealing temperature caused Fe 3+ ions to migrate to the octahedral sites [23].
The remanent magnetization M r also undergoes a similar change to the saturation magnetization. A sligth lattice strain influences the changes that occur; the effect of internal stress decreases, and the magnetostriction coefficient approaches zero [47]. Further, the annealing temperature changes the coercive field, H c ; at the annealing temperature of 200°C, H c was 651 Oe; afterward, it was 502, 691, and 1,077 Oe for the annealing temperatures of 300°C, 400°C, and 500°C, respectively. The characteristic of magnetocrystalline anisotropy increases with the annealing temperature [25]. Increasing the coercive field with crystallite sizes smaller than   70 nm indicates a single-domain configuration [48,51]. The magnetic moment η B increased from 1.42 to 2.26 μ B as the annealing temperature increased, closely related to changes in saturation magnetization. In addition, the highest annealing temperature (500°C) revealed the most significant value of magnetocrystalline anisotropy.

Antibacterial activity analysis
The .00-13.14 mm, which offers excellent antibacterial characteristics because getting a 7-mm ZOI diameter represents a good antibacterial activity against the tested bacteria [25,29]. In addition to observing the ZOI formed by the well-diffusion test method, measuring for mortality against bacteria was also carried out by observing the total plate count method. After the MHA media was smeared with the antibacterial agent Ag 0.02 Co 0.98 Fe 2 O 4 , incubated for 24 h at 37°C, the number of colonies that were still growing was counted and compared with the positive antibacterials according to equation (11), the results are shown the number of bacteria growing (NBG) and the mortality (M) of the tables 5 and 6.
Antibacterial activity was detected for both S. aureus and E. coli bacteria which showed that the Ag 0.02 Co 0.98 Fe 2 O 4 nanoparticles were confirmed as antibacterial agent for both gram-positive and negative bacteria [52,53]. There are papers reporting that if nanoparticles have gram-positive antibacterial performance, they do not perform gram-negative antibacterial and otherwise [54]. Furthermore the antibacterial performance of nanoparticles prepared at different annealing temperatures can be confirmed in this paper.
The antibacterial properties of Ag 0.02 Co 0.98 Fe 2 O 4 supported by its relatively small crystallite size (19.78-24.11 nm), allow the interaction of the material with bacteria based on a large surface area [30]. Several scholars have reported that materials with a crystallite size of ∼40 nm can suppress S. aureus and E. coli growth [14,55]. The results of the antibacterial property test are shown in figures 6(a) and (b).
In addition to crystallite size, the resulting saturation magnetization value plays an essential role in antibacterial activity. The increase in saturation magnetization suggests decreasing Ag + ions replacing Co 2+ at the tetrahedral site. This resulted in a decrease in ZOI diameter due to reduced release of Ag + in cobalt ferrite nanoparticles, while an increase in ZOI diameter at 300°C annealing in S. aureus and 500°C annealing in E. coli was probably caused by other factors such as changes in the magnitude of magnetocrystalline anisotropy which leading to structural changes [29]. A high saturation magnetization magnitude may cause the cell membrane to rupture, affecting enzyme degradation and resulting in bacterial death [26]. The possible mechanism for antibacterial activity is the interaction between cobalt ferrite silver nanoparticles and the bacterial cell membrane which causes disruption/change in the bacterial cell membrane [56]. The release of metal ions (Ag) in cobalt The relative error in ZOI was 0.05 mm and in NBG was 10%. ferrite which interact with oxygen species will form reactive oxygen species (ROS) [11,57]. The resulting ROS production will affect the level of antibacterial activity [58]. The balance will be disrupted when there is an increase in ROS in which the role of ROS is to inhibit protein function, cause DNA damage, and affect the level of lipid peroxidation which causes damage to cell membranes [59][60][61]. ROS production is determined by nanoparticles and depends on several factors such as size, morphology, shape, and release of metal ions [62,63]. Furthermore, the comparison between another nanoparticles as antibacterial agent list in the table 7. Table 7 shows several comparisons of the antibacterial activity of the nanoparticles. The results showed that the Ag 0.02 Co 0.98 Fe 2 O 4 in the study with annealing temperature variations also had antibacterial activity performance. The results obtained were not significantly different from those previously reported.

Conclusion
Ag 0.02 Co 0.98 Fe 2 O 4 NPs were successfully synthesized using the coprecipitation method and then modified by annealing temperature treatment at 200°C, 300°C, 400°C, and 500°C for four hours. Ag + ions were substituted in the Co 2 Fe 2 O 4 lattice to form a single crystalline phase. FTIR analysis confirmed metal oxide absorption in the tetrahedral and octahedral sublattices, and there is a cation-sensitive redistribution phenomenon at the tetrahedral sites.
Increasing the annealing temperature increased the crystallite size of Ag 0.02 Co 0.98 Fe 2 O 4 from 19.78 to 24.11 nm, the number of saturation magnetization from 31.80 to 50.60 emu g −1 , and the coercive field from 651 to 1077 Oe. Tests on S. aureus and E. coli showed that Ag 0.02 Co 0.98 Fe 2 O 4 samples showed good antibacterial properties, supported by an almost perfect crystal structure, small crystallite size, and high saturation magnetization. An increase in annealing temperature increased the grain and crystallite sizes, weakening the material's interaction with the bacterial surface. In addition, annealing increased the crystallinity and magnetic properties so that the antibacterial properties of the material could be maintained.