1 INTRODUCTION

Biofouling is a global issue that can cause increased hydrodynamic drag, fuel consumption, and maintenance costs for marine and industrial equipment (Banerjee et al., 2011; Zhu et al., 2019). However, most of the current antifouling materials would damage the environment through metal leaching and bacteria resistance that may be restricted for the application. Nanozyme has a huge application prospect for environmental and effective antifouling (Natalio et al., 2012; Herget et al., 2017).

Ceria (CeO_2-x) nanoparticles have been demonstrated having strong haloperoxidase mimicry against the biofouling in natural environment (Herget et al., 2017). In addition, nanoceria has attracted widespread attention for their good catalytic properties in recent years (Valente et al., 2011; Seftel et al., 2015; Nayak and Parida, 2016; Zhou et al., 2020). Their different catalytic properties depend on difference of nanoceria themselves (e.g., sizes, morphologies, crystal structures and ratios of Ce³⁺/Ce⁴⁺) and the reaction condition (e.g., pH and so on) (Deshpande et al., 2005; Peng et al., 2011; Nicolini et al., 2015). Although most of studies focus on how to enhance the catalytic properties and optimize the synthetic method (Hong et al., 2017; Lebedeva et al., 2019; He et al., 2020), the uncontrolled tendency of nanoceria aggregation weakens the performance. Therefore, system to overcome the agglomeration of nanoceria nanozymes remains a huge challenge (Lin et al., 2014).

Mixed metal oxides (MMOs) are interesting choices resulting from their large surface area, phase purity, and structural stability (Genty et al., 2016; Hao et al., 2017; Smalenskaite et al., 2017). In our previous work, layered double hydroxide (LDH) was demonstrated as a precursor for preparing ultrathin 2D Ni-V MMO nanosheets with an intrinsic peroxidase (POx)-like catalysis activity (Chen et al., 2019). In this system, the agglomeration of active phase can be avoided effectively and thus the stability is improved. This pioneering work inspires us that Ce containing MMOs will be potential high activity nanozyme materials for marine antifouling. However, there is no report about this topic so far.

Considering that the ionic radius Ce³⁺ (102 pm) is larger than that of most used M³⁺ to form LDH layer with Co²⁺ (74 pm), a fully isomorphic substitution of M³⁺ by Ce³⁺ can induce a lattice distortion and lead to imperfections in the LDH layers (Wang and Zhang, 2016). Thus, in this paper, Co-Al-Ce LDH is chosen as the precursor to prepare Ce containing MMOs. Herein, we demonstrate the development of Co-Al-Ce MMOs composited of Co₃O₄ and CoAl₂O₄ and CeO₂ as antibacterial nanozyme materials for the first time (Fig. 1). First, Co-Al-Ce LDH precursor has been synthesized via a hydrothermal route. After calcination, Co-Al-Ce MMOs show attracted performance of intrinsic enzyme-like activities under optimized conditions. Importantly, the enzyme-like activity of Co-Al-Ce MMOs can be tailored by adjusting the heating temperature and molar ratio of Ce. Owing to its high dispersion properties and high specific area, Co-Al-Ce MMOs with 2.5% Ce at 200℃ exhibited excellent catalytic antibacterial activity in near-neutral pH solution. This strategy will open a new route for the well-dispersed CeO₂ nanoparticle decorated MMO matrix as nanozyme in marine antifouling.

2 EXPERIMENTAL AND METHOD 2.1 Material

3, 3', 5, 5'-tetramethylbenzidine (TMB), 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA), propidium iodide (PI), and SYTO 9 were purchased from Sigma-Aldrich (USA). Hydroxyphenyl fluorescein (HPF) and dihydroethidium (DHE) were purchased from APExBIO (USA) and AAT Bioquest (USA). 30 wt% H₂O₂ aqueous solution, Co(NO₃)₂·6H₂O, Al(NO₃)₃·9H₂O, Ce(NO₃)₃·6H₂O, urea, isopropanol (IPA), p-benzoquinone (BQ), and other chemicals were of analytical grade and bought from Sinopharm Chemical Reagent Co., Ltd. (China). Milli-Q water (Millipore, USA) was used for preparing all aqueous solutions.

2.2 Preparation of Co-Al-Ce MMOs

The Co-Al-Ce LDH precursor was synthesized using a urea hydrolysis method (Zhang et al., 2017). Co(NO₃)₂·6H₂O (12 mmol), Al(NO₃)₃·9H₂O (4–16×x mmol, x=0%, 1%, 2.5%, and 5%, respectively), Ce(NO₃)₃·6H₂O (16×x mmol), and urea (40 mmol) were dissolved in 70 mL of water vigorous stirring for 20 min. Precursors with different Ce doping contents were denoted as 0CoAlCe, 1CoAlCe, 2.5CoAlCe, and 5CoAlCe. The solution was incubated in a 100-mL Teflon-lined autoclave at 110℃ for 12 h. After hydrothermal treatment, the Co-Al-Ce LDH precipitate was centrifuged and washed with water and anhydrous ethanol three times separately, and dried over 12 h under vacuum at 60℃. The precursors were heated at 200, 300, 400, 500, 600, and 800℃ in air for 2 h with a heating rate of 5℃/min, following cooled to room temperature naturally. Finally, Co-Al-Ce MMOs were formed and denoted as nCoAlCe-200, nCoAlCe-300, nCoAlCe-400, nCoAlCe-500, nCoAlCe-600, and nCoAlCe-800 (n=0, 1, 2.5, and 5), respectively.

2.3 Apparatus and characterization

The as-prepared samples were characterized with the 2θ ranging from 5° to 80° using Cu K radiation (λ=0.154 18 nm) at 40 kV and 40 mA by X-ray diffractometer (XRD) (Ultima IV X-ray diffractometer, Rigaku, Japan). Transmission electron microscopy (TEM), high-resolution TEM (HRTEM) and selected area electron diffraction (SAED) were captured to analyze the morphological features of the sample by a JEM-2100 transmittance electron microscopy (JEOL, Japan). Energy-dispersive X-ray spectroscopy (EDS) was carried on image captured to investigate the chemical compositions and their content. Fourier transform infrared (FT-IR) spectra were carried out through the standard KBr disk method by a Nicolet iS10 Fourier transform spectrometer (Thermo Fisher Scientific, USA). The Brunauer-Emmett-Teller (BET) measurement was performed on a Micromeritics TriStar II 3020. The surface electronic state was investigated by X-ray photoelectron spectroscopy (XPS) spectrometer (ESCALAB 250, Thermo Fisher Scientific Inc., USA).

2.4 Enzyme-like activity

Standard TMB liquid substrate system was used to evaluate enzyme-like activities of Co-Al-Ce MMOs at room temperature. An amount of 40 μg/mL Co-Al-Ce MMO suspension was added into 1 mL of NaAc-HAc buffer solution (pH=4.0) consisting of 1 mmol/L H₂O₂ and 0.4 mmol/L TMB as the substrate. The color changes can be observed with naked eyes immediately, as the substrate added. After 3 min reactions, the absorbance value at 652 nm of the mixed solution was monitored by an Infinite M1000Pro Tecan microplate spectrophotometer (Switzerland). All the samples were carried out in three parallels and the average values were given.

2.5 Steady-state kinetic analysis

The influence of pH and catalyst concentration was carried out to probe the dependence of the POx mimic catalytic activity of Co-Al-Ce MMOs. The steady-state kinetic analysis was performed under the optimized system of pH=4.0 and the catalyst suspension concentration of 50 μg/mL at room temperature. The kinetic analysis of 2.5CoAlCe-200 was carried out by varying concentrations of H₂O₂ at a fixed concentration of TMB or vice versa. The concentration of oxidation products derived by TMB were calculated following all obtained absorbance at 652 nm, through the Beer-Lambert' law (A=ɛbC). In the equation, b is the path length, 0.323 7 cm and ɛ is the molar absorption coefficient (39 000 M-1/cm). Then, kinetic parameters were calculated following the Michaelis-Menten formalism:

in which v₀ is the initial conversion rate reached by the initial slope of absorbance changes with time, [S] is the substrate concentration, K_m is the Michaelis constant, and v_max is the maximum conversion rate. Three parallel tests were conducted to ensure the accuracy of the results.

2.6 Bacteria culture and antibacterial activity test

Escherichia coli (E. coli) (ATCC 6538) was from CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences. After incubated in Luria-Bertani broth medium, the logarithmic phase bacteria were diluted to ~106 CFU/mL and then added to 1 mL of NaAc-HAc buffer solution (pH=4.0) with various concentrations of H₂O₂ and the 2.5CoAlCe-200 sample for 2 h. Then, the bacterial strains were diluted to 103 CFU/mL after treatment and cultured on agar plate for 36 h at 30℃. Three parallel plates corresponding to a particular sample were done to obtain the average for antibacterial efficiency.

2.7 Live/dead fluorescent staining

After treatment with H₂O₂ and 2.5CoAlCe-200, E. coli bacteria were cultured with SYTO 9 and PI for 15 min at room temperature in the dark. Then 10 μL of the stained bacterial suspension was added on the microscope slide covered with a coverslip. The live/ dead bacterial cells were then visualized by Olympus BX53M fluorescence microscopy (Japan).

2.8 Measurement of intracellular ROS

The intracellular reactive oxygen species (ROS) level was assessed by the oxidant-sensitive dye DCFH-DA. Then resuspended bacterial cells with 10 μmol/L DCFH-DA were indicated in the dark at room temperature for 30 min. Before detection, the stained bacterial solution was washed with PBS once. Fluorescent probes of DHE and HPF (10 μmol/L) were further used to investigate the specific type of free radicals, ∙O₂ˉ and ∙OH. The intracellular ROS level was observed using Olympus BX53M fluorescence microscopy (Japan).

3 RESULT AND DISCUSSION 3.1 Synthesis and characterizations of Co-Al-Ce MMOs

The structural information of Co-Al-Ce LDH precursor with different Ce molar ratios is given by X-ray diffraction patterns. As shown in Fig. 2, all the diffraction peaks can be indexed at 2θ=11.6°, 23.5°, 34.6°, 39.2°, 46.7°, 60.2°, and 61.6°, which correspond to the (003), (006), (012), (015), (018), (110), and (113) reflections of the hexagonal hydrotalcite-like structure of Co-Al LDHs (JCPDS card No. 51-0045, R-3m(166)) (Genty et al., 2019). The d₀₀₃ value is 0.758 nm, indicating that the major phase is attributed to the carbonate anion intercalated LDHs. Meanwhile, diffraction characteristic peaks of Co-Al LDHs created with Ce molar ratio of 1.0%, 2.5%, and 5.0% match well with Co-Al LDHs, demonstrating that the LDHs phase is well crystallized in these composites with doping Ce³⁺ less than 5%. This uniform composite structure has a great advantage to construct MMOs. The FT-IR spectra also further confirms the successful composition of Co-Al-Ce LDHs. As showing in Fig. 3, the peak assigned to the stretching vibration of the O-H bond was observed at 3 423/cm due to the metal-OH layer and/or water in the crystal. The peaks observed at 1 552, 1 359, 836, 800 are assigned to v(OCO₂), C-O, δ(Co-OH), and δ(CO₃), respectively (Zhu et al., 2013; Zhou et al., 2015). The peaks in the skeletal region (613, 548 and 426/cm) are typical M-O bonds of Co-Al LDHs. These results suggest that the precursor is Co-Al-Ce LDHs.

XRD patterns of the 2.5CoAlCe precursor heated at different temperatures are given by in Fig. 4. As the heating temperature increases, characteristic peaks of LDHs gradually disappear. Samples heated at 200 and 300℃ become essentially amorphous and the XRD pattern exhibits one set of strong diffraction peaks at 2θ=19.0°, 31.2°, 36.8°, 38.5°, 44.8°, 55.6°, 59.4°, and 65.2°, matching well with the (111), (220), (311), (222), (400), (422), (511), and (440) of cubic Co₃O₄ (JCPDS card No. 43-1003) and cubic CoAl₂O₄ (JCPDS card No. 44-0160). After calcined at a higher temperature, the XRD pattern exhibits two sets of diffraction peaks. The other new weak set is attributed to the (111), (220) and (311) reflection of cubic CeO₂ (JCPDS card No. 34-0394), indicating that Co-Al-Ce MMOs is composited of CeO₂, Co₃O₄, and CoAl₂O₄.

The morphologies and microstructures of 2.5CoAlCe-200 were obtained by TEM. From the TEM morphologies (Fig. 5a & b), it can be seen that 2.5CoAlCe-200 has rough surfaces so that it can offer the high specific surface area. As shown in Fig. 5c, HRTEM was used to investigate the detailed microstructure further. The HRTEM image of 2.5CoAlCe-200 displays clear lattice fringes of Co₃O₄/CoAl₂O₄ and CeO₂. From the FFT pattern corresponding to the HRTEM image, the calculated distance between the adjacent lattice fringes is 0.244 nm. This value can be ascribed to the d spacing of (311) plane of Co₃O₄ and CoAl₂O₄ plane. The distance 0.310 nm agrees with the d spacing of (111) plane of CeO₂. The SAED pattern of 2.5CoAlCe-200 (Fig. 5d) displays discontinuous diffused diffraction rings, confirming that CeO₂ has been embedded in the Co₃O₄/CoAl₂O₄ successfully. The EDS mapping of 2.5CoAlCe-200 further describes the uniformity and high dispersion characteristics of CeO₂ nanoparticles mixed with Co₃O₄/CoAl₂O₄ nanoparticles (Fig. 5e). According to the EDS analysis result (the insert of Fig. 5f), the actually Ce molar ratio in 2.5CoAlCe-200 is ~2.5, which is very similar to that in the precursor solution.

Nitrogen adsorption/desorption experiments were applied to investigate the effect of Ce molar ratios on the specific surface area further. From Fig. 6, when the Ce molar ratios is 2.5%, the specific surface area of 2.5CoAlCe-200 increases to the maximum (150.10±4.95 m²/g), which is higher than that of 0CoAlCe-200 (131.96±4.35 m²/g), 1CoAlCe-200 (98.18±3.09 m²/g), and 5CoAlCe-200 (78.73± 1.17 m²/g).

The surface chemical compositions and chemical state of Co-Al-Ce MMOs were investigated by XPS. As shown in Fig. 7a, the signal of Co, Al, Ce, and O can be detected in the survey spectra, without any other impurities. As shown in Fig. 7b, the multiple peaks of Ce 3d spectrum are observed, which correspond to mixed valence states of Ce³⁺ and Ce⁴⁺ (Venugopal et al., 2013; Herget et al., 2017). The peaks appearing at 902.4 eV (u), 907.8 eV (u″), and 917.0 eV (u‴) are corresponding to Ce⁴⁺ 3d_3/2 and the peaks occurring at 882.6 eV (ν), 888.7 eV (ν″), and 898.3 eV (ν‴) are due to Ce⁴⁺ 3d_5/2. The four peaks namely u′, ν′, u_o, and ν_o (904.7, 885.5, 900.7 and 881.5 eV) are corresponding to Ce³⁺. The peaks at 780.15 eV (2p_3/2) and 795.21 eV (2p_1/2) with two satellites located at 790.05 and 804.76.33 eV in Fig. 7c are characteristic of a Co²⁺ of Co₃O₄/CoAl₂O₄ (Xing et al., 2014). While at 779.16 and 794.89 eV for Co 2p_3/2 and 2p_1/2, transitions are ascribed to Co³⁺ of Co₃O₄ (Soundharrajan et al., 2016). The spin-energy separation between Co 2p_3/2 and Co 2p_1/2 orbits is ~15.2 eV, confirming the presence of Co₃O₄ (Schenck et al., 1983; Oku and Sato, 1992; Varghese et al., 2007). As the O 1s orbital spectrum showing in Fig. 7d, a strong peak at about 532.0 eV is assigned to the high-binding energy peak from chemisorbed and dissociated oxygen species, 531 eV is usually attributed to surface oxygen defect species, whereas 530.4 and 529.8 eV are associated with oxygen in the metal-oxygen bonds (Lü et al., 2014; Wang et al., 2020).

3.2 The POx mimic catalytic activity of Co-Al-Ce MMOs

The natural POx can catalyze colorless TMB substrate into blue oxTMB rapidly with H₂O₂. The absorbance changes of oxTMB product at 652 nm can be monitored following the subsequent color reaction. Therefore, the standard TMB liquid substrate system was performed to evaluate the nanozyme-like activity of Co-Al-Ce MMOs further at room temperature. From Fig. 8, no obvious color changes are presented in the H₂O₂+TMB (a) and H₂O₂+2.5CoAlCe-200 (c) system. For other two TMB+2.5CoAlCe-200 (b) and H₂O₂+TMB+2.5CoAlCe-200 (d) systems, color changes can be observed. The color reaction indicates that 2.5CoAlCe-200 has both the POx-like activity and the oxidase (ODs)-like activity. The results of the POx mimic activity of Co-Al-Ce MMOs depending on the various molar ratio of Ce and the heating temperature was evaluated in Fig. 9. This indicates that Co-Al-Ce MMO samples obtained by calcination at 200℃ have the best POx mimic catalytic performance for the precursor samples with any Ce molar ratio, then the activity deteriorates as the calcination temperature increases. As poor crystallinity can result in more transportation channels, this can be favorable for improving the catalytic activity of material (Liu et al., 2014). Among the four samples of 0CoAlCe-200, 1CoAlCe-200, 2.5CoAlCe-200, and 5CoAlCe-200, the POx mimic catalytic performance of 2.5CoAlCe-200 is the best. Its good catalytic activity can be attributed to the good dispersion of catalytically active components and high specific surface area.

3.3 Steady-state kinetic assay and catalytic mechanism of Co-Al-Ce MMOs

According to the enzyme kinetic theory and method, the POx mimic catalytic mechanism and kinetic parameters of 2.5CoAlCe-200 were further investigated on the optimized condition. The results in Fig. 10 indicate that the oxidation reaction of TMB catalyzed by 2.5CoAlCe-200 is dependent on pH and the concentrations of the catalyst. Figure 11 shows the double reciprocal plots of POx mimic catalytic activity of 2.5CoAlCe-200. As shown, the parallel lines indicate that the slopes are similar and demonstrate that the catalytic mechanism of 2.5CoAlCe-200 follows the ping-pang mechanism. This is similar with natural horseradish peroxidase (HRP). The K_m value is calculated using the MichaelisMenten equation. The K_m value of 2.5CoAlCe-200 with TMB as the substrate is 0.273 mmol/L, which suggests that 2.5CoAlCe-200 has a higher affinity for TMB compared with HRP (0.434 mmol/L). On the other hand, the K_m value for 2.5CoAlCe-200 with H₂O₂ as the substrate is 32.9 mmol/L, higher than that of 3.7 mmol/L for HRP (Gao et al., 2007).

To investigate the POx-like catalytic mechanism of 2.5CoAlCe-200, IPA, and BQ was used to eliminate ∙OH and ∙O₂ˉ and put into the optimal system with other conditions remained still to determine corresponding radicals produced. As shown in Fig. 12, the addition of BQ can reduce the absorbance at 652 nm significantly. As shown in Fig. 12, the system added IPA shows almost no signal of the absorbance at 652 nm as Control-IPA, and the characteristic signal shows poor reduction by adding 2.5CoAlCe-200 as IPA, suggesting that 2.5CoAlCe-200 possesses the POx mimic ability to decompose H₂O₂ to generate reactive ∙O₂ˉ radicals. As a result, it can be deduced that the strong oxidizing ∙O₂ˉ radicals play major roles in the ODs-like and POx-like catalytic reaction.

3.4 Antibacterial studies

We have revealed that 2.5CoAlCe-200 exhibits ODs and POx like properties by significantly induced H₂O₂ species subsequently to convert into ∙O₂ˉ radicals. As 2.5CoAlCe-200 is highly expected to kill bacteria by inducing the formation of ROS, we evaluated its antibacterial activities. Especially, concentrations of H₂O₂ will cause high toxicity and harm other organisms. The depending on concentrations of H₂O₂ of materials against Gram-negative E. coli bacteria were tested further. As shown in Fig. 13, E. coli in the blank group remain alive well at pH=4 and the inhibition rate of 2.5CoAlCe-200 against E. coli is 87.45% in the absence of H₂O₂. After the addition of H₂O₂, the inhibition rate against E. coli increases gradually. As the concentration of H₂O₂ increasing to 0.3 mmol/L, the inhibition rate of E. coli can arrive at 99.98%. It means that 2.5CoAlCe-200 can kill E. coli with the biologically relevant concentrations of H₂O₂.

Especially, pH is still a key parameter which have an effect on the efficiency of POx activity. To further examine the antibacterial activities of 2.5CoAlCe-200 in pH=6, fluorescence-based Live/Dead assays were performed. As shown in Fig. 14a, the green fluorescence and the lack of red fluorescence indicated that most bacteria in the control group remained alive. Addition of 5 mmol/L of H₂O₂ without 2.5CoAlCe-200 (Fig. 14b) or addition of 2.5CoAlCe-200 without H₂O₂ (Fig. 14c), most bacteria remained alive. While, 2.5CoAlCe-200 as well as 5 mmol/L of H₂O₂ both added in the system (Fig. 14d), the result shows the intensification of the red fluorescence signal, which indicates a perceptible increase in the number of dead cells.

3.5 Mechanism of antibacterial studies

Next, to examine whether the bacterial death is caused by oxidative damage (oxidative stress), the intracellular ROS level was determined. From Fig. 15 the representative fluorescence images in E. coli cells confirm that the antibacterial property is caused by the generation of ROS catalyzed by 2.5CoAlCe-200, which is indicated by the clear green fluorescence signal (Fig. 15d). From Fig. 16a, the clear intracellular red fluorescence signals were detected obviously, which further proves that the generated ∙O₂ˉ promotes the antibacterial property.

4 CONCLUSION

In summary, LDH derived Co-Al-Ce MMOs composed of CeO₂ compounding with Co₃O₄ and CoAl₂O₄ have been successfully synthesized by a rational approach. When the molar ratio of Ce is 2.5% and calcination temperature is 200℃, the obtained 2.5CoAlCe-200 sample exhibits the best POx-like activity and possess dual enzyme-like activities similar to those of ODs and POx. Co-Al-Ce MMOs with dual enzyme-like activities can catalyze H₂O₂ and O₂ to generate ROS, mainly ∙O₂ˉ. Owing to the excellent antibacterial capacity of ∙O₂ˉ, Co-Al-Ce MMOs exhibit antibacterial activity even in near-neutral pH solution. Our results provide strong evidence that Ce containing-MMO can be utilized as the potential green marine antifouling nanozyme material.

5 DATA AVAILABILITY STATEMENT

All data of this project are public. The entire data have been annotated building process in the paper. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.