2.1. Materials

Copper(II) chloride dihydrate (CuCl₂·2H₂O), with a copper content of 99%, and sodium hydroxide (NaOH) were purchased from Shanghai Aladdin Reagent Co., Ltd. Iron(III) chloride hexahydrate (FeCl₃·6H₂O), with 99% purity, was obtained from American Sigma Reagent Co., Ltd. Absolute ethanol was purchased from Sinopharm Group Chemical Reagent Co., Ltd. Potassium dihydrogen phosphate (KH₂PO₄) and disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O), both with a purity of 99%, were purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. All reagents mentioned above were of analytical grade and were used without further purification. Luria–Bertani (LB) medium and nutrient agar were purchased from Beijing Auboxing Biological Reagent Co., Ltd.

2.2. Cu-Modified Fe₃O₄ Catalyst Synthesis

Three mmol of FeCl₃·6H₂O (4.915 g) were dissolved in 10 mL of absolute ethanol under magnetic stirring. Then, 0.3 mmol of CuCl₂·2H₂O (0.307 g) was added to this solution, followed by stirring for 10 min; this solution was labeled solution A. Separately, 9.6 mmol of NaOH (2.375 g) were dissolved in 30 mL of absolute ethanol and vigorously stirred until complete dissolution; this solution was labeled solution B. Solution B was then added to solution A drop by drop, and the mixture was stirred for another 10 min; the resulting solution was labeled solution C. A total of 20 mL of ethylene glycol was added to solution C and mixed; this solution was labeled solution D. Finally, solution D was transferred into a 100 mL reaction kettle and incubated at 200°C for 10 h. The resulting product was cooled to room temperature and washed three times with deionized water and once with ethanol. After drying in an oven at 60°C for 12 h, the final product was ground into a fine powder.

2.3. Cu-Modified Fe₃O₄ Catalyst Characterization

The crystal structure of the material was studied using XRD (D8 Advance). A transmission electron microscope (JEM-2100) was used to observe the material’s morphology and high-resolution structure at an accelerating voltage of 200 kV. The micromorphology of the composite material was observed using a scanning electron microscope (JSM-7001F). XPS (ESCALAB-250) was used to further confirm the elemental composition of the samples. The free radicals generated during the reaction of the material were measured using an electron paramagnetic resonance spectrometer (EPR, Escan). The concentration of copper ions in aqueous solution was measured by ICP-MS (ELAN-9000).

2.4. Fenton-Like Bactericidal Reaction Performance Testing

E. coli was used for the bactericidal experiments and cultured in LB medium. The dosage of H₂O₂ used in the Fenton-like reaction sterilization experiment was 5 mM, the concentration of the material used was 1 mg/mL, and the colony-forming unit per mL (CFU/mL) of E. coli was 2 × 10⁹.

A total of 9.9 mL of phosphate buffered saline (PBS) was added into a Petri dish with a 60 mm diameter. Then, 10 mg Cu²⁺-Fe₃O₄ nanocomposite was added to the PBS solution and ultrasonically dispersed for 10 min. After ultrasound-based treatment, 5 mM H₂O₂ and 100 μL of the bacterial suspension were added to the solution and then mixed using a pipette. The initial bacterial concentration was 2 × 10⁷ CFU/mL. At regular intervals, 100 μL of the mixture was collected for serial dilutions and spread onto LB agar plates. Finally, the plates were incubated overnight at 37°C, and the bacterial survival rate was calculated. This experiment was repeated three times.

3.1. Material Characterization

From Figure 1, it can be clearly seen that the 2θ values of Fe₃O₄ at 30.1°, 35.5°, 37.0°, 43.1°, 47.2°, 53.5°, 57.0°, 62.6°, 65.8°, 70.1°, 74.0°, 75.0°, and 79.0° correspond to the characteristic diffraction peaks (JCPDS number 88–0315) of Fe₃O₄ [24]. No obvious copper diffraction peaks can be found in Figure 1(a). From Figure 1(b), it can be seen that the position of the main diffraction peak deviates to the left and the linewidth becomes broader as the copper content rises [25], which indicates that copper ions may have replaced some of the iron in the samples.

The morphology and microstructure of the 10% Cu²⁺-Fe₃O₄ nanocomposite were observed by transmission electron microscopy (TEM). Figure 2 shows the TEM images of the 10% Cu²⁺-Fe₃O₄ nanocomposite. From Figure 2(a), it can be clearly observed that the particles of Cu²⁺-Fe₃O₄ nanocomposites are relatively homogeneous, with diameters around 10 nm. The Fe₃O₄ sample is distributed in the shape of clusters, which is consistent with the findings of Marchianò et al. [26]. Due to the high surface energy and magnetic dipole interactions, the nanomaterials mainly consist of aggregated spherical nanoparticles. In Figure 2(b), a lattice spacing of d = 0.25 nm, corresponding to the characteristic diffraction peaks of the Fe₃O₄ crystal planes, can be observed.

Figure 4 shows the magnetic properties of Cu²⁺-Fe₃O₄ nanocomposites with different copper contents. The nanocomposites have similar ferromagnetic properties and the saturation magnetization values gradually increase along with the doping ratio. This may be caused by the cation rearrangement following doping, and the literature shows that Cu²⁺ can occupy either the tetrahedral or octahedral sites within the spinel structure [33]. According to Tripathy et al. [34], if Cu²⁺ ions migrate to tetrahedral sites, the number of Fe³⁺ ions at the octahedral sites increases, while the number of the same ions at the tetrahedral sites decreases, leading to an imbalance in spin distribution on the two sublattices and resulting in enhanced net magnetization. In addition, no remanence and coercivity is observed, indicating that the magnetic particles exhibit superparamagnetic properties [35, 36]. From Figure 4, the maximum saturation magnetization of the nanocomposites reaches 65.5 emu/g. At the same time, when the saturation magnetization reaches 16.3 emu/g, magnetic separation becomes possible with conventional magnets [37]. This indicates that the nanocomposites can be efficiently separated by magnets. As confirmed by the inset in Figure 4, Cu²⁺-Fe₃O₄ nanocomposites, uniformly dispersed in water, can be easily separated by magnets in just 1 min. Therefore, the catalyst used in this study can be easily recovered using an external magnetic field after the reaction in aqueous solution, making it ready for reuse. This is an important advantage of Fe₃O₄ nanocomposites for practical applications.

3.2. Fenton-Like Performance Testing

3.2.1. Bactericidal Performance Test

Figure 5(a) presents the results for 2 × 10⁷ CFU/mL of E. coli and the effects of different ratios of Cu²⁺-Fe₃O₄ nanocomposite materials after 150 min. The results indicate that the inhibitory effect on E. coli is not significant when the concentration of 5 mM H₂O₂ or pure Fe₃O₄ nanomaterials acts on the microorganism separately. The inhibition of E. coli by unmodified Fe₃O₄ material in the Fenton reaction at the same time with H₂O₂ was also negligible. Saravanan et al. [38] synthesized magnetic nanocatalysts of Cu@Fe₃O₄in situ using Moringa oleifera flower extract, demonstrating a significant improvement in the efficiency of degradation of Congo red under visible light. Our modified 10% Cu²⁺-Fe₃O₄ nanocomposites successfully eliminated 2 × 10⁷ CFU/mL of E. coli within 120 min without light irradiation, using only the addition of H₂O₂. Moreover, the bactericidal effect was further enhanced with increasing copper content.

The effect of pH on the survival of E. coli is presented in Figure 5(b). At pH = 7, the bactericidal effect on E. coli was not significant when only H₂O₂ was added, and even at a concentration of 10 mM, the concentration of the bacterial solution was reduced by only two orders of magnitude. Regardless of pH value (5, 6, 7, or 8) and the H₂O₂ concentration (5 or 10 mM), the 10% Cu²⁺-Fe₃O₄ nanocomposites exhibited a remarkable killing effect on E. coli with a CFU/mL equal to 2 × 10⁷ within 130 min, with the most pronounced bactericidal activity on E. coli observed at pH = 6 and H₂O₂ concentration of 10 mM. It was also found that E. coli can be more easily adsorbed at lower pH values, which may be due to the negatively charged bacterial membrane [39].

Stability and reusability are important parameters for evaluating the potential applicability of catalysts [40]. Recyclable antimicrobial nanocomposites are environmentally friendly as they improve material utilization [41]. Figure 6 shows the results for the 10% Cu²⁺-Fe₃O₄ nanocomposite after three cycles of killing E. coli bacteria at 2 × 10⁷ CFU/mL in the Fenton-like reaction. As shown in Figure​ 6, the efficiency of the composite material in killing E. coli remains excellent over three cycles. Although the bactericidal effect decreases slightly through third cycles, the E. coli kill ratio in the third Fenton-like reaction still reach to 94.8%. The results demonstrate the reusability of the Cu²⁺-Fe₃O₄ nanomaterial [42].

3.2.2. Antibacterial Mechanism Test

To investigate the antibacterial mechanism of 10% Cu²⁺-Fe₃O₄ nanocomposites in Fenton-like reaction, EPR experiments were conducted to investigate the reactive oxygen species produced during the killing of E. coli. In this experiment, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as a trapping agent to capture the ^•OH and ^•OOH produced by the Fenton-like reaction. Based on the literature, the 4-fold characteristic peak of DMPO-^•OH adducts with an intensity ratio of 1:2:2:1 [43].

As shown in Figure 7, ^•OH and ^•OOH [44] signal peaks were not detected in the Fe₃O₄ nanomaterials without the addition of co-catalyst H₂O₂ or co-catalyst H₂O₂ was added singly. Even though pure Fe₃O₄ and H₂O₂ coexisted, there were still no signal peaks of ^•OH and ^•OOH. However, the 10% Cu²⁺-Fe₃O₄ nanocomposite showed strong signal peaks of ^•OH and ^•OOH after the addition of H₂O₂, which further fully proves that Cu²⁺-Fe₃O₄ nanocomposites produce ^•OH and ^•OOH in the Fenton-like reaction. This might be due to the introduction of Cu ions providing more active sites, which improves the carrier separation efficiency and the generation of reactive oxygen signals [45]. Due to the strong oxidizing effect of reactive oxygen radicals, when they come into contact with bacteria, they will destroy the barrier effect of E. coli cell walls and cell membranes, increase the permeability of E. coli cell membranes, and lead to leakage of intracellular proteins so that the bacteria will not be able to maintain normal life activities [46]. In addition, reactive oxygen radicals will pass through the pores on the cell surface and enter the cell interior, further oxidizing and degrading intracellular proteins, destroying intracellular protective enzymes, and ultimately leading to bacterial death [47].

Due to its remarkable toxic domino effect at excess amounts [48], the concentration of Cu²⁺ dissolved in the aqueous reaction system was measured by ICP-MS. From Figure 8, the solubility of Cu ions was consistently in the range of 0.09–0.11 mg/L, which is much lower than the detection limit of Cu ions in drinking water (1.0 mg/L). This is in full compliance with the “Sanitation Standards for Drinking Water” (GB5749-2006) promulgated by the Ministry of Health [49]. Therefore, for the Cu-modified Fe₃O₄ nanocomposites synthesized in this work, the dissolved copper ion concentration in water is much lower than the safety threshold, and the impact on the human body and the environment is negligible.