N-Doped Carbon/CeO₂ Composite as a Biomimetic Catalyst for Antibacterial Application

Abstract

Exploring new and high efficiency mimic enzymes is a vital and novel strategy for antibacterial application. Haloperoxidase-like enzymes have attracted wide attention thanks to their amazing catalytic property for hypohalous acid generation from hydrogen peroxide and halides. However, few materials have displayed halogenating catalytic performance until now. Herein, we synthesized N-doped C/CeO₂ (N-C/CeO₂) composite materials by a combination of the liquid and solid-state method. N-C/CeO₂ can possess haloperoxidase-like catalytic activity by catalyzing the bromination of organic signaling compounds (phenol red) with H₂O₂ at a wide range of temperatures (20 °C to 55 °C), with a solution color changing from yellow to blue. Meanwhile, it exhibits high catalytic stability/recyclability in the catalytic reaction. The synthesized N-C/CeO₂ composite can effectively catalyze the oxidation of Br⁻ with H₂O₂ to produce HBrO without the presence of phenol red. The produced HBrO can resist typical marine bacteria like Pseudomonas aeruginosa. This study provides an efficient biomimetic haloperoxidase and a novel sustainable method for antibacterial application.

1. Introduction

Microbiological contamination constitutes one of the fatal worldwide issues facing both environmental sustainability and public healthcare [1,2]. Various antibacterial methods have been developed to limit microbial growth, such as the addition of excess chlorine dioxide [3] or antibiotics [4,5], even new types of antibacterial materials, such as nano silver [6,7]. However, the toxicity of disinfection byproducts and the bacterial resistance lead to a quest for novel and effective methods [8,9]. Therefore, it is necessary to exploit novel and effective environment-friendly and nontoxic antibacterial materials and antibacterial technology. Different conventional antibacterial agents and biomimetic catalyst emulate nature enzymes to produce intermediates such as halogenated metabolite, which target specific bacterial signaling and regulatory systems for preventing bacterial colonization or biofilm development [10].

In nature, some marine algae can effectively prevent the attachment of microorganisms by self-secreting a haloperoxidase [11,12]. This kind of haloperoxidase can catalyze the oxidation of halides (Cl⁻, Br⁻, I⁻) with H₂O₂ to the corresponding hypohalous acid [13]. Inspired by this phenomenon, natural haloperoxidase and functional recombinant haloperoxidase, especially vanadium haloperoxidases (V-HPOs), are applied to paint as an additive, which can effectively prevent the growth and attachment of microorganisms [14,15]. However, these natural and functional recombinant enzymes were restricted to large-scale application because of their high production costs, short-term stability, and specific reaction conditions (pH and temperature) [16]. Exploring high-performance artificial V-HPOs mimic enzymes is a useful strategy to replicate natural enzymes.

Attempts to mimic haloperoxidases reactions with synthetic enzymes have been successful in catalytic activity [17]. Research on functional enzyme mimics has seen an upsurge in recent years [10]. Several vanadium complexes [18,19] or V₂O₅ nanoparticles [16] have been reported as mimicking V-HPOs, which display catalytic efficiency and selectivity in oxidative halogenation reactions [20,21]. However, the vanadium compounds are mutagenic, carcinogenic, and teratogenic [17]. It is an urgent need to develop efficient and non-toxic materials to replace vanadium-based complexes. Inspired by catalyzing oxidation/halogenation reaction of cerium oxide in organic synthesis, cerium-based materials were reported to have haloperoxidase-like activity [22,23,24,25,26]. For example, cerium oxide nanorods as haloperoxidase mimic have been used in antimicrobial membranes [23,24,25]. Cerium oxides present good catalytic performance, which is attributed to the self-structural properties and environmental compatibility [25]. However, the extreme low abundance of rare-earth metallic cerium on Earth limits its large-scale application. Doping is an efficient strategy to reduce the usage amount of cerium and increase the utilization of cerium. In our previous report, compared with cerium oxide, the same amount carbon-doped cerium oxide exhibited better haloperoxidase mimicry for antimicrobial [26]. Therefore, specific doping and complex can effectively reduce resource utilization and facilitate the widespread application of cerium-based materials.

Herein, N-C/CeO₂ composite was prepared and studied as haloperoxidase mimicry for antibacterial, as shown in Scheme 1. N-C/CeO₂ composite as a biomimetic catalyst possesses haloperoxidase-like catalytic activity by catalyzing the bromination of phenol red in the presence of H₂O₂ with a solution color changing from yellow to blue. Meanwhile, it can possess antibacterial application by catalyzing the oxidation of Br⁻ with H₂O₂ (without phenol red) to produce HBrO. The haloperoxidase activity of prepared material and the factors affecting the mimicry activity, such as temperature and concentration of catalysts, were studied. The kinetics of the catalytic reaction were investigated by varying the concentration of one reactant while keeping the concentration of others constant. Consequently, the stability and recyclability of N-C/CeO₂ composite were proved through the reutilization test. It can effectively catalyze the oxidative bromination of Br⁻ and H₂O₂ to produce HBrO. The produced HBrO with a strong antibacterial activity was used to resist microorganisms such as Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Staphylococcus aureus (S. aureus). The proposed high efficiency N-C/CeO₂ artificial enzyme mimic may represent a novel strategy to emulate a natural defense system for restraining biofilm growth and bacterial colonization.

2. Results and Discussion

2.1. Characterization

The morphology and crystal structure of the synthesized composites were investigated by SEM, TEM, and XRD. Figure 1A is the SEM image of the prepared N-C/CeO₂ composite. N-C/CeO₂ composite presents a kind of sheet morphology, and the sheets branch off from each other. The high-resolution TEM (HRTEM) image (Figure 1B) of N-C/CeO₂ shows clear lattice fringes with an interlayer distance of 0.32 nm, which can be indexed to the (002) plane of CeO₂. As shown in Figure 1C, the XRD pattern of N-C/CeO₂ composite shows typical peaks at around 28°, 33°, 47°, and 56°, corresponding to the (111), (200), (220), and (311) planes of the cubic CeO₂ (PDF#34-0394), respectively. The inset of Figure 1C displays the crystal structure illustration of CeO₂ with cubic space group (Fm3m). The diffraction peaks are strong and sharp, implying that the N-C/CeO₂ sample maintains good crystallinity. In addition, XRD patterns of the other N-C/CeO₂ composites are shown in Figure S1. These results suggest that all of the XRD patterns of N-C/CeO₂ composites are nearly identical to the pure CeO₂. Moreover, the loading of N-doped carbon did not change the crystalline phase of the composites. As displayed in Figure 1D, the STEM elemental mapping images reveal a uniform distribution of N, C, O, and Ce. This indicates the high homogeneity of the synthesized N-C/CeO₂ composite. To summarize, the doping modification in the current study has not changed the morphology and crystal structure of the N-C/CeO₂ composites.

XPS was further employed to elucidate the electronic structure and chemical state of N-C/CeO₂. As shown in Figure 1E, the refined Ce 3d XPS spectrum is composed of multiple couples of peaks, corresponding to a mixture of Ce³⁺ and Ce⁴⁺ oxidation states. Generally, cerium switches reversibly between its Ce(Ⅲ) and Ce(Ⅳ) states owing to the non-stoichiometric nature and multiple d-splitting of Ce element [27]. The Ce 3d XPS peaks located at 885/904 eV can be assigned to Ce³⁺ states, while the peaks at 883/901 eV, 889/908 eV, and 898/916 eV are attributed to Ce⁴⁺ states [28]. According to the integrated area, the N-C/CeO₂ composite consisted of a major amount of Ce(Ⅳ) oxide (85.93%) and small amount of Ce(Ⅲ) oxide (14.08%), which is related to the lattice strain induced by Ce³⁺ and the presence of oxygen vacancies [29]. The O 1s XPS spectrum is given in Figure S2. The O1s XPS spectrum displays typical peaks at 529.5 and 531.6 eV, which can be assigned to the Ce⁴⁺-O and Ce³⁺-O bond, respectively. After high-temperature calcination, the final N-C/CeO₂ product also contains abundant nitrogen-doped carbon components. The N 1s XPS spectrum (Figure 1F) of the N-C/CeO₂ composite can be de-convoluted into three species, corresponding to pyridinic N, pyrrolic N, and oxidized N, respectively [30]. Additionally, the C 1s XPS spectrum (Figure 1G) exhibits significant signals at 284.5, 285, and 286 eV, which can be identified as C-C/C=O, C-N, and O-C=O/N-C=O functional groups, respectively [31]. In Raman spectra (Figure S3), the pure CeO₂ delivers typical peaks at 460 cm⁻¹ and 600/1170 cm⁻¹, which can be assigned to the F_2g vibration model of the CeO₂ and oxygen defects, respectively [32]. However, the N-C/CeO₂ displays additional Raman spectrum peaks at around 1680 and 2900 cm⁻¹, corresponding to the C-N band in the composite [33]. Meanwhile, N-C does not change the crystalline phase of the composites, which is consistent with the results of XRD. The above results indicate that the N-C/CeO₂ composite contains ceric oxide and abundant N (O and C-)-functional groups. These are crucial to the high homogeneity of the prepared composites.

2.2. Haloperoxidase Mimicry Activity

To study the haloperoxidase mimicry activity of N-C/CeO₂ composites, phenol red (PR) was used as the color substrate. N-C/CeO₂ composite, as the haloperoxidase mimicry, can catalyze the bromination and PR in the presence of H₂O₂ with a solution color change from yellow to blue. N-C/CeO₂ composite (6:1) exhibits the best haloperoxidase mimicry activity out of all of the N-C/CeO₂ composites in Figure S4. Thus, this doping composite was used throughout the study. As shown in Figure 2A, the absorbance spectra of the solution were obtained in different reaction systems. No obvious absorption signals are detected in the c blank system with the components of NH₄Br + H₂O + H₂O₂ + c. However, there is an obvious peak at ~430 nm in systems a, b, and d, which belongs to the absorption of PR. System e reveals a distinct absorbance at 590 nm, which is attributed to the product bromophenol blue. The N-C/CeO₂ composite can catalyze Br⁻ and PR in the presence of H₂O₂ to produce bromophenol blue. The corresponding color changes in different systems are shown in Figure 2A (insert). The solution is colorless in system c and yellow in systems a, b, and d. This yellow color comes from the color of dilute PR dye. The solution is blue only in system e. It indicates that the N-C/CeO₂ composite can catalyze Br⁻ and PR in the presence of H₂O₂ to produce a blue-color reaction and exhibit good haloperoxidase-like catalytic activity. In addition, to further test the haloperoxidase-like activity of N-C/CeO₂ composites, the catalytic activities of N-C composites and pure CeO₂ were investigated as controls in Figure 2B. There is a faint peak at 590 nm of the N-C composite, which shows that the N-C composite has a certain catalytic activity. N-C/CeO₂ delivers a significant absorbance peak at 590 nm, and its adsorption peak is higher absorbance than that of pure CeO₂, indicating the N dopants carbon as an electron donor atom can facilitate the catalytic activity of CeO₂ to produce HBrO [10]. These above results indicate that the N-C/CeO₂ composite possesses excellent haloperoxidase-like activity higher than that of N-C composites and pure CeO₂. Therefore, the use of nitrogen-doped carbon as a substrate is beneficial to improve the haloperoxidase-like catalytic activity of the N-C/CeO₂ composite.

In order to study the haloperoxidase-like properties of N-C/CeO₂ composite, the UV–Vis spectra of aqueous reaction were measured every 2 min within a total testing time of 40 min. As shown in Figure 2C, the absorbance at 590 nm increases quickly at the early time, while the increase of absorbance at 590 nm slows down after 30 min and tends to be stable at 40 min. The formation rate of bromination product (Br₄PR) was evaluated by the accurate absorbance intensity of 590 nm, as displayed in Figure S5. These results show that the N-C/CeO₂ composite has the same haloperoxidase-like catalytic activity as natural enzymes [34]. In general, the catalytic activity of artificial mimicry is associated with the working temperature. The optimal temperature of the N-C/CeO₂ composite was investigated from 20 °C to 55 °C. The result shown in Figure 2D indicates that the catalytic activities of the N-C/CeO₂ composite were high at various temperatures. These are only slightly affected by the temperature. Therefore, room temperature was chosen in the following experiments.

2.3. Kinetics Constant Determination

The reaction kinetics of the N-C/CeO₂ composite were further studied. The Michaelis–Menten constant of substrates (H₂O₂, NH₄Br, and PR) was measured by changing the concentration of one substrate, while keeping all other concentrations constant. Then, kinetics graphs (Figure 3) were obtained by the calculated initial velocity rates using kinetic data according to Equations (2) and (3). For the N-C/CeO₂ composite, Figure 3A (left) shows the kinetic function as the concentration of the N-C/CeO₂ composite, when the concentrations of other substrates are fixed. The kinetic values are fitted nonlinearly (blue line) according to the Michaelis–Menten equation, and the partial kinetic values are fitted linearly (dark dashed line). The results show that the kinetic data fit perfectly with the nonlinear fitting line based on the Michaelis–Menten equation. Thus, the N-C/CeO₂ composite complies with the Michaelis–Menten kinetic of natural enzymes. The middle graph and the right graph in Figure 3A show the corresponding Lineweaver–Burk linearizations and logarithmic correlations, respectively.

The Michaelis–Menten constants (K_m) and the maximal reaction rates (v_max) values of all substrates (H₂O₂, NH₄Br, and PR) calculated are shown in

Table 1. The Michaelis–Menten constant (K_m) and maximal reaction rate (v_max) of N-C/CeO₂ composites.

Substrates	Km (μ·mol·L−1)	vmax (μmol·L−1·min−1)
H2O2 (mmol·L−1)	0.246	0.669
NH4Br (mmol·L−1)	173	1.11
PR (mmol·L−1)	0.0130	2.48

. The kinetic function, corresponding Lineweaver–Burk linearizations, and logarithmic correlations of other substrates, including H₂O₂, NH₄Br, and PR, are treated with the similar method to that of the N-C/CeO₂ substrate (Figure 3B–D). The above results show that the N-C/CeO₂ composite as haloperoxidase mimicry matches with the catalytic reaction kinetics of natural enzymes.

The Michaelis–Menten constant of the N-C/CeO₂ composite and vanadium bromoperoxidase (V-BPO) in the previous reports are summarized in

Table 2. Comparison of the apparent Michaelis–Menten constant and maximal reaction rate between the N-C/CeO₂ composite and V-BPO.

Materials	Substrates	Km (μmol·L−1)
N-C/CeO2 composites	H2O2	0.246
	Br−	173
vanadium bromoperoxidase (V-BPO)	H2O2	22.0
	Br−	18.1

. Compared with the V-BPO biological sample, the prepared N-C/CeO₂ composite delivers an obviously smaller K_m of H₂O₂ and bigger K_m of bromide (NH₄Br) [16,34]. Generally, K_m indicates the affinity between the substrate and catalyst. The lower K_m value of H₂O₂ suggests that H₂O₂ has a higher affinity for the surface of the N-C/CeO₂ composite than V-BPO. The higher K_m value of Br⁻ indicates that Br⁻ has a lower affinity for the surface of the N-C/CeO₂ composite.

To evaluate the effect of bromide source on the catalytic reaction, KBr and NaBr are used as control samples. Figure 4A shows the absorbance of the solution at 590 nm in the presence of NH₄Br, KBr, and NaBr, respectively. The solution absorbances are almost identical for different bromide sources, meaning that the reaction is independent of the bromide source. As the stability of the catalyst is essential for real applications, reutilization tests of N-C/CeO₂ composite are performed with the same concentration of PR, NH₄Br, and H₂O₂ at room temperature. After each reaction cycle, the N-C/CeO₂ composite is separated by centrifugation at 3020× g and washed with ultrapure water. Then, the obtained N-C/CeO₂ composite is treated again with PR, NH₄Br, and H₂O₂ under identical experimental conditions. As shown in Figure 4B, the absorbance at 590 nm stays almost constant through ten cycles. This clearly illustrates that the activity of the N-C/CeO₂ composite has not decreased. The above results suggest that the catalytic activity of the N-C/CeO₂ composite is independent of the bromide source, and it also exhibits high catalytic stability.

2.4. Antibacterial Test

N-C/CeO₂ composites have good haloperoxidase mimicry activity by catalyzing the bromination of organic signaling compounds. Therefore, N-C/CeO₂ composites can catalyze the reaction of H₂O₂ and Br⁻ to produce HBrO. In order to investigate the antibacterial property of N-C/CeO₂ composites as haloperoxidase mimicry, N-C/CeO₂ composites are applied onto the titanium plates’ surfaces and antibacterial tests are conducted. As shown in Figure 5A, the bare titanium plate/N-C/CeO₂ composites modified titanium plates are exposed to P. aeruginosa suspensions at 37 °C for 4 h. Bacterial cell density and adhesion is further evaluated by fluorescence microscopy. As a control, a dense P. aeruginosa population is observed on the bare titanium plates surfaces in the absence of N-C/CeO₂ composites in the medium without H₂O₂ and Br⁻ (Figure 5A, left column “Blank”). The same experimental set up is conducted without adding the substrates H₂O₂ and Br⁻ in P. aeruginosa suspensions. In this case, high P. aeruginosa adhesion/density is also observed on the N-C/CeO₂ modified titanium plates (Figure 5A, middle column). In contrast, the absence of P. aeruginosa adhesion is detected on the N-C/CeO₂ composites modified titanium plates in the presence of substrates H₂O₂ and Br⁻ (Figure 5A right column “N-C/CeO₂ + Br⁻ + H₂O₂”). The above results indicate that the system of “N-C/CeO₂ + Br⁻ + H₂O₂” exhibits the best antibacterial adhesion property. As shown in Figure S6, the proposed catalytic reaction with the prepared N-C/CeO₂ catalyst can also work to suppress the microbial adhesion of E. coli and S. aureus. As shown in Figure 5B, the blank sample without adding N-C/CeO₂ catalyst and Br⁻ reveals abundant P. aeruginosa colonies on the entire plate. Figure 5C displays the plate treated with only N-C/CeO₂ catalyst (without Br⁻ and H₂O₂), and Figure 5D reveals the plate photo treated with all components of the catalytic condition (with N-C/CeO₂, Br⁻, and H₂O₂). The sample with the addition of only the N-C/CeO₂ catalyst exhibits a reduced number of P. aeruginosa colonies, implying that the N-C/CeO₂ composite itself has weak antibacterial activity (Figure 5C). However, almost no P. aeruginosa colonies are detected on the plate of Figure 5D because of the generation of HBrO. These above results demonstrate that the N-C/CeO₂ composite itself has weak antibacterial activity, while the N-C/CeO₂ composites can catalyze the reaction of Br⁻ and H₂O₂ to produce HBrO, which plays a major role in antibacterial properties. In addition, the antibacterial properties of N-C/CeO₂ as haloperoxidase mimicry were compared with the previously reported CeO₂-based materials, as shown in Table S1. N-C/CeO₂ presents lower bacterial attachment on the titanium plates than other CeO₂-based materials for E. coli, which indicates that they have good antibacterial adhesion properties. This provides a novel way to prevent biofouling and attachment to marine facilities. Therefore, N-C/CeO₂ composites as haloperoxidase mimics have excellent bromination activity, and the produced hypobromous acid exhibits superb antibacterial activity.

3. Methods

3.1. Reagents and Apparatus

Cerium(III) nitrate hexahydrate (Ce(NO₃)₃•6H₂O) was purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Phenol red (PR), melamine, ammonium bromide, NaCl, KCl, Na₂HPO₄, KH₂PO₄, acetic acid, and hydrogen peroxide solution (30%) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Cell staining kit (K2081) was purchased from APExBIO (Houston, TX, USA). All the reagents and chemicals were used without further purification. All aqueous solutions were prepared with ultra-pure water (18.2 MΩ·cm) throughout this experiment. Phosphate buffered saline (PBS, 0.1 mmol·L⁻¹) was prepared with 8.0 g·L⁻¹ NaCl, 0.2 g·L⁻¹ KCl, 1.44 g·L⁻¹ Na₂HPO₄, and 0.44 g·L⁻¹ KH₂PO₄ in ultra-pure water. Then, the pH of the solution is regulated to 7.0 by NaOH solution. PBS (0.1 mmol·L⁻¹, 7.0) was used in the whole experiment. The aqueous standard solutions of H₂O₂ were stored in the dark because of their photosensitivity.

The morphology and structure investigation of the synthesized N-C/CeO₂ composites were carried out by scanning electron microscopy (SEM, Reguas, Japan). The phase structures of these electrodeposits were determined using X-ray diffraction (XRD, Rigaku D/max-Ultima IV, Tokyo, Japan). The fine structures of these samples were further investigated by transmission electron microscopy TEM (JEM 2100F, Tokyo, Japan). The heteroatoms and functional groups were determined by X-ray photoelectron spectroscopy XPS (Escalab K-alpha 250Xi). The Raman spectra were collected on Renishaw MZ20-FC Raman microscope. The absorption spectra of UV–Vis and absorbance-time were measured with an UV–Vis spectrophotometer (UV–Vis, U-3900 HITACHI, Tokyo, Japan). Observation of bacteria was performed using a fluorescence microscopy (BX-51 with image software of Cellsens, Olympus, Japan) after staining with K2081 kit, as previously described [35].

3.2. Synthesis of N-C/CeO₂ Composites

Herein, 3.0 g melamine and different amounts of Ce(NO₃)₃•6H₂O were dissolved into 40 mL acetic acid and 40 mL ultra-pure water. The obtained solutions were mixed by ultrasonication for 30 min and transferred into a stainless-steel vessel. The hydrothermal reaction was carried out at 120 °C for 12 h. Thereafter, the solvent was removed from the product by the freezing drying process using vacuum equipment. The resultant materials were annealed at 520 °C for 4 h at a ramp rate of 5 °C·min⁻¹ in the air. Different N-C/CeO₂ composites were obtained by varying the mass ratio of melamine and Ce(NO₃)₃•6H₂O (6:1, 3:1, 2:1, 1:1, 1:2).

3.3. Haloperoxidase-like Activity of N-C/CeO₂ Composites

The haloperoxidase-like activity of the synthesized N-C/CeO₂ composites was analyzed using an optical absorption spectroscopy. The reaction scheme was as follows: N-C/CeO₂ composite catalyzes the oxidative bromination H₂O₂ and Br⁻ in the presence of PR, resulting in the color change from yellow to blue. The 950 μL mixed solutions (containing 28 μmol·L⁻¹ PR, 69.4 mmol·L⁻¹ NH₄Br, 830 μmol·L⁻¹ H₂O₂, and 50 μg·mL⁻¹ N-C/CeO₂ composites) reacted at room temperature for 40 min. Afterwards, the absorption was measured by UV–Vis spectroscopy. As a control, the absorption spectra of mixtures were measured when one of the substrates was absent in all mixtures. The amount of the added reagent was quantified. In addition, the optimal reaction conditions such as temperature, H₂O₂ concentration, and N-C/CeO₂ composite concentration were tested by changing one reaction condition while leaving other conditions unchanged. Three replicate experiments were performed.

3.4. Determination of Kinetic Constant

The kinetic constants were carried out in time course mode of UV–Vis by fixing the wavelength at 590 nm [20]. The absorbance of mixed solutions was measured by changing the concentration of one reactant while keeping others constant in kinetic tests. In order to obtain the optimal concentration of all reactants, each measurement was carried out at 590 nm for 40 min. In addition, kinetic parameters were calculated based on the slopes (dA_590nm/dt), which were kept constant over 5 min. The kinetic constants (Michaelis–Menten constant K_m and the maximum reaction velocity v_max) were obtained using the Linewaver–Burk linearization (Equation (1)) [36,37].

1/v = K_m/v_max[C] + 1/v_max

(1)

where v is the initial velocity and C is the concentration of substrate. In order to evaluate the K_m and v_max, v was calculated. In our experiments, the product Br₄PR was used as a measure of the reaction rate to obtain the initial reaction rate (Equation (2)).

v = d[Br₄PR]/dt

(2)

The Br₄PR concentration was obtained according to the Lambert–Beer law [25] (Equation (3)).

[Br₄PR] = A₅₉₀/dε_Br4PR

(3)

where ε_Br4PR is the extinction coefficient of Br₄PR and its value is 72,200 L·mol⁻¹·cm⁻¹.

In addition, in order to test the dependence on the Br⁻ source, some Br-salts such as KBr, NaBr, and NH₄Br served as the Br⁻ source in the mixture reaction solutions. The reutilization test of N-C/CeO₂ composite was carried out in ten recycles with N-C/CeO₂, PR, NH₄Br, and H₂O₂.

3.5. Bacterial Adhesion Tests

For bacterial adhesion tests, P. aeruginosa (Gram-negative, typical marine bacterium, risk group 2 organism) as a model bacteria was grown in Luria Broth (LB) medium with shaking at 160 rpm and 37 °C for 12 h. The cell concentration of P. aeruginosa in the medium was calculated using the plate colony counting method [38]. Here, 10⁷ colony-forming units (cfu) mL⁻¹  P. aeruginosa were separately obtained by centrifugation. These cells were resuspended in 0.1 mmol·L⁻¹ PBS to obtain a cell concentration of 10⁷ cfu·mL⁻¹. Multiple sets of 10 mL of this PBS cell suspension solutions were placed into 50 mL inoculation tubes and used for later bacterial adhesion tests.

Titanium plates (Ti, 0.1 × 1 × 1 cm³), with/without N-C/CeO₂ composite, were placed into the above bacterial solution and cultivated with agitation at 37 °C for 4 h in different systems: (1) Ti without Br⁻ and H₂O₂ (blank), (2) modified Ti without Br⁻ and H₂O₂ (N-C/CeO₂), and (3) modified Ti with Br⁻ and H₂O₂ (N-C/CeO₂ + Br⁻ + H₂O₂). Three replicate experiments were performed per system. Afterwards, Ti was stained using a staining kit (K2081) for 15 min in the dark. Excess stain was gently removed by sterile PBS. The stained samples were examined by fluorescence microscopy. Bacterial solutions containing the same concentration of N-C/CeO₂ composite, Br⁻, and H₂O₂ were cultured on an agar plate at 37 °C for 24 h. Three parallel agar plates were painted for each bacterial solution. As a control, PBS bacteria solutions containing the N-C/CeO₂ composite were cultured under the same conditions. Three parallel experiments were performed. E. coli (Gram-negative) and S. aureus (Gram-positive) were also studied using the same experimental method. These above results show that the N-C/CeO₂ composite as haloperoxidase mimicry presents good antibacterial activity.

4. Conclusions

In summary, the N-C/CeO₂ composite was successfully synthesized using melamine as a carbon and nitrogen source. The N-C/CeO₂ composite can effectively catalyze the oxidation of H₂O₂ with bromination of organic signaling compounds to produce a blue-color reaction, and presents excellent intrinsic haloperoxidase mimicry activity. The catalytic activity of the N-C/CeO₂ composite is influenced by the substrate concentration and almost not influenced by temperature. The N-C/CeO₂ composite as haloperoxidase mimicry can catalyze the reaction process of H₂O₂, Br⁻, and PR, which complied with the typical Michaelis–Menton kinetics process. The N-C/CeO₂ composite shows good catalytic stability and recyclability in multiple reaction cycles. In the absence of phenol red, the produced HBrO catalyzed by N-C/CeO₂ composites presents good antibacterial activity against the model bacteria, especially P. aeruginosa. The N-doped carbon/CeO₂ composite as a biomimetic catalyst for antibacterial application is a novel and efficient “green” strategy to emulate and utilize a natural defense system for preventing bacterial colonization and biofilm growth. However, the catalytic activity of the N-doped carbon/CeO₂ composite is mainly attributed to the action of CeO₂, and the formation mechanism of the halogenated reactive oxygen species needs to be further improved. This work introduces a stable, green, and environment-friendly biomimetic material for antibacterial applications.