1. Introduction:

    Due to their distinctive electrical, magnetic, and architectural properties as well as their wide range of biological and industrial applications, spinel ferrite nanoparticles (SFNs) have recently obtained significant attention in both basic and applied research [1]. It is critical to have a comprehensive understanding that the crystal structure of these materials is strongly influenced by the production technique, which in turn affects their chemical and physical qualities. In most nanomaterials, spinel ferrites with the chemical formula MFe₂O₄ (where M = Mn, Fe, Ni, Cu, Co, or Zn) frequently exhibit superparamagnetic behavior when their diameter is approximately 20 nm or less [1-2]. Both tetrahedral (A), and octahedral (B) sites have a distribution of metal cations. The stability energy, cationic radius, interstitial site size, synthesis technique, and reaction conditions all affect this distribution [1-2]. Magnetic nanoparticles possess unique properties that improve their performance compared to solid materials, owing to their substantial surface area per unit volume. Superparamagnetic, magnetic spin deflection, high-field irreversibility, dislocations, and surface anisotropies are among these features [3]. Moreover, through material design, these characteristics make them optimal for particular uses [4]. Cobalt ferrite, a cubic spinel type, has attracted a great deal of study interest among the many magnetic nanoparticles because of its outstanding characteristics, which include medium saturation magnetization (Ms) and strong coercivity (Hc) [5, 6] In addition, it exhibits strong magnetic anisotropy (380 kJ m^-3) [7], high magnetic permeability [8], enhanced mechanical strength, excellent chemical stability, and low production cost [9].

These advantages make cobalt ferrite a key choice for developing new substances used in cell freezing. ceramic materials, devices that use magneto-optics [10], sensors for measuring torque without contact [11], various types of sensors, rods used for antennas [10], devices that create movement [12], materials that respond to light and magnetism [13], as well as uses in healthcare, such as heat treatment [5] and absorbers for microwaves [9], imaging methods like magnetic resonance imaging (MRI) and medication delivery [14]. Particle size, shape, purity, and crystalline magneto-crystalline anisotropy are some of the variables that affect the magnetization of CoFe₂O₄ nanoparticles [15]. One of the most important magnetic phenomena observed in ferrite nanoparticles is superparamagnetic behavior. When a magnetic field is applied to CoFe₂O₄ nanoparticles, the coercivity decreases to zero, indicating that the nanoparticles become superparamagnetic. Upon the removal of the applied magnetic field, the nanoparticles return to a non-magnetic condition [16]. There are various methods for manufacturing nanoparticles, and among these, co-precipitation is the most efficient method for producing nano ferrites in large quantities because it yields particles with a uniform structure. Co-precipitation is a simple and inexpensive method, as the size and dispersion of the nanoparticles can be controlled by maintaining their formation and growth rates during the process [16,17]. 

In this context, Dippong et al. reported the synthesis of the Co_xFe_3-xO₄ oxidic system via a redox process sing various Fe/Co ratios, leading to the acquisition of cobalt ferrite nanocrystallites. In samples with abundant iron, cobalt ferrite and iron oxides (Fe₂O₃) were produced, but in samples with excess cobalt, cobalt oxides (CoO) emerged as a secondary phase and the synthesized nanomaterials exhibited particle sizes below 100 nm. [18]. Furthermore, Gheidari et al. showed that synthesized nanoparticles inhibit bacterial growth against E. coli and S. aureus. While increasing the nanoparticle concentration improves their antimicrobial properties, resulting in a larger inhibition zone (18- 13 mm) [19]. Similarly, Bhushan et al. investigated the antibacterial efficacy of innovative iron oxide and cobalt oxide nanoparticles against S. aureus and E. coli. The enhanced activity of the iron oxide/cobalt oxide nanoparticle complex resulted from the synergistic effect of these nanoparticles, showing an inhibitory zone diameter of 10–11 mm [20].

Prior studies have addressed structural, magnetic, or biological properties separately, but have not clearly demonstrated how phase development, including secondary phases such as (α-Fe₂O₃ and Co₃O₄), directly influences functional performance of the synthesized nanoparticles. The range 0.2 ≤ x ≤ 0.8 was chosen to examine the compositional transition region where the pure spinel phase competes with secondary phases. This regime has not been systematically investigated in prior research. This study investigates the influence of cobalt content on phase evolution and identifies the conditions that stabilize the pure spinel phase (Co_xFe_3-xO₄), relative to secondary phases, as the Co/Fe ratio varies. A systematic analysis of the compositional dependence over the range (0.2 ≤ x ≤ 0.8) yields comprehensive correlations among structure, magnetic properties, and antibacterial activity.  In this study, Co_xFe_3-xO₄-based nanoparticles were produced using the co-precipitation method, and the effect of four different concentrations (0.2 ≤ x ≤ 0.8) was observed. The results of the analyses were examined with respect to crystal size, lattice coefficient, saturation magnetization, retention, and magnetic force. Co_xFe_3-xO₄-based nanoparticles were evaluated for antibacterial activity against particular pathogens using the agar diffusion method.

2. Experimental part:

 2.1. Sample synthesis:

The starting precursors were Co(NO₃)₂·6H₂O, FeCl₃ deionized water, and NH₄OH. CoFe₂O₄ nanoparticles were synthesized by the chemical co-precipitation technique, using the formula Co_xFe_3-xO₄ (0.2 ≤ x ≤ 0.8). Cobalt nitrate and ferric chloride were separately dissolved in 25 mL of deionized water, homogenized via magnetic stirring for 15 minutes, and then combined. Aqueous ammonia was added dropwise to the resultant solution until a stable pH 9 was achieved, forming a brown precipitate. The mixture was continuously agitated for an additional 15 minutes to ensure complete stoichiometric precipitation. The precipitate was dried at 70–90 °C for 2 hours, then thermally treated and calcined in ceramic crucibles at 550 °C for 3 hours. Subsequently, the samples were precisely pulverized in an agate mortar to produce fine powders suitable for characterisation and biological evaluation. Antibacterial assays, adhering to established biosafety protocols, using non-clinical reference strains of E. coli and S. aureus, were conducted in suitable laboratory settings.

2.2 Antibacterial Activity Evaluation:

The antibacterial activity of Co_xFe_3-xO₄, nanoparticles was evaluated against E. coli (ATCC 10336), and Stap. aureus (ATCC 29213), obtained from Al-Razi Cether Research and Production of Medical Diagnostic Kits, using the agar well diffusion method. Bacterial suspensions adjusted to the 0.5 McFarland standard were spread onto Mueller-Hinton Agar plates, and 50 µL of nanoparticle suspension 25 mg mL^-1 was loaded into 6 mm wells punched in the agar. After incubation at 37 °C for 24 h, antibacterial activity was determined by measuring the inhibition zone diameters (mm).

2.3 Characterization:

A collection of four samples was synthesized according to the chemical formula Co_xFe_3-xO₄, with varying cobalt concentrations 0.2 ≤ x ≤ 0.8, using the co-precipitation technique. The structural characteristics were analyzed by X-ray diffraction using monochromatic Cu Kα, radiation (λ=1.540 Å) from Bruker (Germany) operating over a 2θ range of (9–80°). The surface morphologies were investigated by FESEM (FEI Company, USA), while their elemental composition was determined using the attached EDX. Fourier-transform infrared (FTIR) spectroscopy (from Shimadzu, Japan) was employed to determine the chemical bonding and characteristic vibrational patterns of the materials over the range 4000-400 cm^-1. The magnetic properties were measured using a VSM (Lake Shore Cryotronics, USA). The antibacterial efficacy of the produced nanocomposites was assessed using the agar well diffusion technique.

3. Results and Discussions:

 3.1 XRD Analysis:

Figure 1 shows the X-ray diffraction patterns of (Co_xFe_3-xO₄) nanoparticles synthesized with different cobalt concentrations (0.2 ≤ x ≤ 0.8). The sample doped with (x=0.2) exhibits a polycrystalline hematite (α-Fe₂O₃) structure as a dominant phase alongside, a minor CoF₂O₄ phase, confirmed by the presence of distinct peaks related to the crystal planes (012), (104), (110), (113), (024), (116), and (300). These reflections correspond to the rhombohedral structure of the hematite phase according to (JCPDS card 00-024-0072). The dominance of this phase suggests that the concentration of Co²⁺ is insufficient to form the spinel structure; instead, it enters as a partial substitutional impurity within the Fe₂O₃ structure, leading to the formation of the α-Fe₂O₃ phase [21]. As the cobalt content increases to x = 0.4–0.6, a complete phase transformation into a cubic spinel structure is observed. The diffractograms reveal specific peaks at (440), (511), (422), (400), (311), (220), and (222) which perfectly match CoFe₂O₄ phase as noted in (JCPDS:01-086-2267). At these ratios, the peaks characteristic of the hematite phase disappeared, indicating that the prepared powder consisted entirely of phase-pure cobalt ferrite. This transition is attributed to the stoichiometric balance allowing Co²⁺ / Fe³⁺ ratio and its distribution within tetrahedral (A) and octahedral (B) sites of the spinel lattice. These results indicate the phase transition from α-Fe₂O₃ to CoFe₂O₄ as the cobalt content increases. Furthermore, the peaks in sample x = 0.6 were broader than those in x = 0.4, demonstrating that increasing the cobalt content reduces the crystallite size [22, 15]. The average crystallite size for each sample was determined using the Debye–Scherrer equation applied to several prominent diffraction peaks. [23].

D = K λ / β cos θ          (1) 

where Dp is the crystallite size, β is the full width at half maximum (FWHM), λ is the wavelength of CuKα radiation (λ = 1.54A°), and θ is the Bragg angle.  With an increased proportion of Co²⁺, content to (x =0.8), the Co₃O₄ phase becomes dominant. This resulted from an excess of cobalt ions exceeding, the spinel structure's capacity, leading to the separation of the Co₃O₄ phase. Therefore, these results indicate that the formation of the spinel phase depends on the Co/Fe ratio in the initial solution; the CoFe₂O₄ phase is formed when the ratio is between x = 0.4–0.6, whereas at x = 0.8 the structure transforms toward the (Co₃O₄) phase, when at lower ratios the powder consists of hematite or of α-Fe₂O₃ doped with cobalt ions. These findings are consistent with previous reports by L.T. Teixeira et al. [24], in which the coexistence of CoFe₂O₄ and α-Fe₂O₃ phases was found to depend on the composition and synthesis conditions. Similarly, B. J. Rani et al. [25] reported that the formation of the spinel phase is strongly influenced by the synthesis method, particularly in co-precipitation systems. A decrease in the lattice constant was observed for cobalt ferrite (a ≈ 8.210 ± 0.005 Å) up to cobalt oxide (a ≈ 8.056 Å) with increasing cobalt ion concentration. This is due to the substitution of iron (III) ions (Fe³⁺) by cobalt (II) ions (Co²⁺), which have a smaller ionic radius, resulting in lattice stress and the formation of secondary phases that alter the unit-cell dimensions [26]. The corresponding lattice parameter values are presented in Table 1.

Figure 1. X-ray diffraction patterns of the synthesized nanoparticles at different cobalt concentrations: (a) x = 0.2, (b) x = 0.4, (c) x = 0.6, and (d) x = 0.8.

3.2 FTIR Analysis:

The FTIR spectra of the produced Co_xFe_3-xO₄-based nanoparticle samples reveal changes in the vibrational peaks, which correspond to the phase structure shown in Figure 2. Peaks in the range of about 420 to 664 cm^-1 in sample (a) indicate that the Fe–O and (Co, Fe)–O bonds are vibrating. This means that there is an impure spinel phase, and the α-Fe₂O₃ phase may also be present [27]. Sample (b) exhibits two separate peaks at approximately 420–668 cm^-1, corresponding to the vibrations of the metal-oxygen (M–O), bonds at the tetrahedral (A-site) and octahedral (B-site) locations inside the spinel structure. This validates the development of the spinel CoFe₂O₄ phase [28, 29]. In sample (c), the same ferrite peaks were observed; however, they exhibited a slight shift to approximately 807–420 cm^-1. This means there is a cobalt oxide phase without occupying the spinel structure. The bands at 3407 and 1389 cm^-1 are thought to be due to the H-O-H bond in adsorbed water molecules, which are expanding. FTIR spectroscopy indicated that samples (b) and (c) contain the spinel structure of CoFe₂O₄ with varying degrees of purity. These results agree with previous reports [30].

Figure 2. FTIR spectra of nanoparticles with varying compositions: (a) x = 0.2, (b) x = 0.4, (c) x = 0.6, and (d) x = 0.8.

3.3 FESEM and EDX analysis: 

Using energy-dispersive X-ray spectroscopy (EDX), Figure 3 shows the chemical and structural analysis of the prepared Co_xFe_3-xO₄-based nanoparticle samples. The cobalt content increases, according to the formula (0.2 ≤ x ≤ 0.8), as the value of x increases, indicating the successful substitution of Fe⁺³ ions with Co⁺² ions in the crystal lattice of the samples. This analysis confirms the presence of the main elements (Fe, Co, and O), consistent with the cobalt spinel ferrite structure. Because the compound is highly effective at absorbing oxygen, it may exhibit a high adsorption capacity [31]. The secondary peaks of (Au), which are created by coating the samples for microscopic study, also include trace amounts of other elements such as (Cl, Ca, and Mn), as shown in Figure 3. Although they are believed to be caused by impurities in the materials used or by the laboratory preparation procedures, these traces do not affect the primary phase [32].

Figure 3. EDX spectra of nanoparticles with varying compositions: (a) x = 0.2, (b) x = 0.4, (c) x = 0.6, and (d) x = 0.8.

Figure 4. FESEM micrographs of Co_xFe_3-xO₄ nanoparticles at different cobalt concentrations (0.2 ≤ x ≤ 0.8) show a semi-spherical shape. Some agglomeration is visible due to magnetic interaction forces. According to the attached graphs (A-D), there is a regular decrease in particle size from (~71 ± 5nm at x=0.2 to ~49 ± 3 nm at x = 0.8), as reported previously [33]. This decrease is physically explained by Co⁺² slowing grain growth and increasing the number of nucleation sites during the reaction [34]. This enhances nucleation, leading to smaller, more homogeneous particles [35]. FESEM particle sizes (49–71 nm), are larger than XRD crystallite sizes (14–21 nm), due to agglomeration of smaller crystallites into larger particles [36]. The transition to smaller sizes increases the effective surface area. The irregular nanoparticle distribution on the surface results from variations in cation distribution between tetrahedral and octahedral sites in the spinel structure, driven by differences in the (Co/Fe) ratio. Preparation conditions, temperature, crystal growth mechanism, and pH influence lead to non-uniform particle distribution [37].

Figure 4. FESEM micrographs of Co_xFe_3-xO₄ nanoparticles at different cobalt concentrations: (a) x = 0.2, (b) x = 0.4, (c) x = 0.6, and (d) x = 0.8. All images were recorded with a scale bar of 200 nm.

3.4 Magnetic Properties:

The VSM was used to analyze the magnetic properties of Co_xFe_3-xO₄-based nanoparticles (0.2 ≤ x ≤ 0.8), as shown in Figure 5. The M-H curves of the prepared samples show that the gradual substitution of Co²⁺ ions in the cobalt-iron ferrite leads to a modification in the magnetic properties. This is mainly due to the increase in crystalline anisotropy associated with the unquenched orbital moment of the Co²⁺ ions located in octahedral positions. The nonlinear behavior (Ms) and (nB) accurately represent how cations are distributed across tetrahedral and octahedral sites, leading to changes in the strength of the A–B superexchange interactions and consequently affecting the magnetic properties of the nanoparticles [38]. The sample with composition X = 0.4 shows the highest values ​​of Mr and K, which indicates the achievement of the best magnetic stability and stronger interaction between molecules. Conversely, the low values of Mr in the other samples indicate relatively softer magnetic behavior owing to nanoscale effects. However, the finite coercive force and remanent magnetization observed in all samples confirm that the nanoparticles remain in a magnetically blocked ferrimagnetic state [39]. The variation in (Hc) values underscores the crucial role of crystalline anisotropy in counteracting size effects. The present results, align with those reported by Sangsuriyonk et al. [40] for Co_xFe_1-xFe₂O₄, nanoparticles synthesized, via co-precipitation. An increase in Co²⁺, content led to elevated Hc, and Mr values confirming ferrimagnetic behavior. This finding is consistent with the magnetically blocked ferrimagnetic state observed in the current study.

The moment (nB) was calculated, using the following equation [41-42].

nB = M × Ms / 5588                 (2)

where μB is the magnetic moment (Bohr magnetons), Mw is the molecular weight (g·mol^-1), Ms is the saturation magnetization (emu·g^-1), and 5585 is a constant. The magnetic moments indicate that all the samples exhibit ferromagnetism. The following equation calculates the K-anisotropy constant [43,44].

K = Ms × Hc / 0.98         (3)                                            

The concentration of the substituted ion influences the anisotropy constant. That is, the variation constant increases with cobalt content. Where K is the anisotropy constant, Ms is the saturation magnetization, Hc is the coercivity field, and 0.98 is a constant factor. 

Figure 5. Magnetic hysteresis (M–H) loops of CoₓFe₃₋ₓO₄ nanoparticles with various cobalt concentrations (a) x = 0.2, (b) x = 0.4, (c) x = 0.6, and (d) x = 0.8 

Table 2: Magnetic properties of Co_xFe_3-xO₄ nanoparticle at different cobalt concentrations (x = 0.2, 0.4, 0.6, and 0.8). 

3.5. Antibacterial activities:

The antibacterial activities of the Co_xFe_3-xO₄-based nanoparticles were tested against selected microbes, including E. coli and S. aureus. Antibacterial activity against E. coli and S. aureus was tested using the agar well diffusion method (30 mg.mL^-1). Inhibition zones were measured after 24 hours of incubation at 37°C to ensure reproducibility. These strains were chosen as models of common bacterial pathogens that infect the community and can be ingested through contaminated water. Figure 6 and Table 1 show that a concentration of 30 mg/mL of synthesized COFNPs, CO₃O₄_COFNPs, and CO₃O₄ nanoparticles resulted in strong antibacterial activity against S. aureus and E. coli, with mean inhibition diameters of 24±1mm, consistent with previous studies [45]. All measurements were performed on three independent samples. No significant differences were found between G-negative and G-positive bacteria. This similarity suggests that the inhibition mechanism depends on the nanoparticles' immediate surface effect rather than on differences in cell wall composition [46]. A slight increase in the inhibition zone with increasing cobalt concentration may lead to improved surface catalytic activity of the two-phase system and increased generation of reactive oxygen species (ROS). Moreover, Co²⁺ ions may interfere with cell membrane permeability and inhibit intracellular metabolic processes. The presence of a secondary phase, such as CO₃O₄, additionally enhances the interaction between the substance and the bacteria [21, 47].

Figure 6. Antibacterial activity of as-synthesized materials against E. coli (Negative Strain) and Staph. aureus (Positive Strain).

Table 3. Antibacterial activity results expressed as zone of inhibition (ZOI), diameters (mm) for all prepared samples. 

Table 4. presents an overview of the structural, magnetic, and antibacterial characteristics of the produced Co_xFe_3-xO₄ nanoparticles.

Table 4: Summary of structural, magnetic, and antibacterial properties of  Co_xFe_3-xO₄ nanoparticles.

Conclusions:

In this study, Co_xFe_3-xO₄ nanoparticles were prepared by the co-precipitation method, with the cobalt ratio varied. FTIR and XRD analyses showed that the crystalline phase depends mainly on the Co/Fe ratio, with the (α-Fe₂O₃) phase appearing at low ratios. Spinel CoFe₂O₄ forms at medium ratios, and the Co₃O₄ phase appears at high ratios. It was found that the size of crystals and nanoparticles decreases with increasing wt.Co%, due to the inhibition of grain growth and an increase in nucleation sites. Magnetic properties were also affected by particle size, cation distribution, phase integrity, and the tendency toward superparamagnetic behavior at small nanoscale dimensions. The synthesized nanoparticles demonstrated significant antibacterial activity, likely attributable to their increased surface area, phase heterogeneity, and the potential generation of reactive oxygen species. These findings highlight the multifunctional potential of (Co_xFe_3-xO₄) nanoparticles for biomedical applications, particularly as antibacterial agents, magnetic materials, and targeted drug-delivery agents. Subsequently, research should concentrate on optimising synthesis conditions to improve crystalline purity and on assessing in vivo effectiveness for advanced medicinal applications.

Funding: This investigation is conducted as an independent research initiative without external financial support.

Data Availability Statement:  All of the data supporting the findings of the presented study are available at the corresponding author.

Declarations:

Ethical Approval:  No human participants or animals were involved in this study.

Conflict of Interest: The research team affirms that no conflicts of interest, financial or otherwise.

Author Contributions: Thair Q. Jassim conducted the experiments, analyzed the data, and wrote the manuscript. Q. Noufan Abdullah conceived the study, supervised the research.