A copper single-atom nanozyme, its preparation method, and marine anti-fouling application

Abstract

The present invention discloses a method for preparing a copper single-atom nanozyme and its application in marine fouling bacteria, especially in the treatment of Pseudoalteromonas atlanticus ( Pseudoalteromonas Atlantica ) and Halomonas maritima ( Halomonas aquamarina ) inhibition, the present invention loads copper ions on degummed and purified silk, and then obtains copper single-atom nanozymes after carbonization, washing, and drying. The copper single-atom nanozymes obtained by the present invention have excellent photothermal performance and good stability. Under the synergistic action of H2O2 and Br¯, the copper single-atom nanozymes generate HOBr through an HPO-like mechanism, which can effectively destroy P.atlantica and H.aquamarina The cell structure and biofilm structure of these two typical marine fouling bacteria show strong antibacterial activity and biofilm removal ability, significantly inhibiting the growth of marine fouling bacteria, and have broad application prospects in the field of marine anti-fouling.

Description

Technical Field

[0001] The present invention belongs to the technical field of single-atom nanomaterials, and specifically relates to a copper single-atom nanozyme, a preparation method thereof, and an application thereof in marine anti-bacterial fouling. Background Art

[0002] Marine biofouling refers to damage to underwater facilities caused by the attachment of marine fouling organisms. Major fouling organisms include fouling microorganisms, fouling animals such as barnacles, and fouling plants such as algae. Biofouling increases ship resistance, fuel consumption, and greenhouse gas emissions; it clogs pipes and cables, endangering the safety of offshore platforms, coastal factories, and power stations, and accelerates metal aging and corrosion. It also clogs fishing nets and aquaculture cages, reducing water exchange and dissolved oxygen concentration within the nets, leading to reduced fish catches and aquaculture output. Furthermore, biofouling organisms attaching to ships can cause invasive species.

[0003] The formation process of marine biofouling is as follows: fouling microorganisms form biofilms on underwater surfaces, roughening smooth surfaces and facilitating the attachment of larger fouling organisms. Within 24 hours, small fouling plants such as microalgae and larvae of fouling animals attach to the biofouled surfaces. After 2-3 weeks, a mature and stable biofouling ecosystem is established. Research has shown that large fouling organisms such as barnacles, bryozoans, oysters, and ascidians can only form biofouling on submerged equipment with biofouling surfaces. Therefore, inhibiting microbial fouling can reduce the attachment of larger fouling organisms and suppress marine biofouling in its initial stages.

[0004] As an emerging catalytic antibacterial material, copper single-atom nanozymes have attracted widespread attention in the antibacterial field due to their unique structure and efficient catalytic performance. Copper single-atom nanozymes can simulate the functional properties of natural enzymes by stably loading copper atoms on carrier materials with high surface energy. Compared with traditional antibacterial agents, copper single-atom nanozymes have higher catalytic activity, stability and environmental friendliness.

[0005] In current research, no application of copper single-atom nanozymes as antibacterial materials in the prevention and treatment of marine fouling microorganisms has been found. Summary of the Invention

[0006] Therefore, the purpose of the present invention is to provide a copper single-atom nanozyme and its preparation method and application. The copper single-atom nanozyme, as an HPO-like nanozyme, has excellent antibacterial and biofilm removal properties against marine fouling bacteria, and has a significant bactericidal effect on marine fouling bacteria. The copper single-atom nanozyme can be used as an environmentally friendly, low-cost and efficient marine anti-fouling material in marine anti-fouling.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention is to provide a method for preparing a copper single-atom nanozyme, comprising the following steps:

[0009] (1) placing silk in a sodium carbonate aqueous solution and boiling it for degumming, purifying the degummed silk and then drying it;

[0010] (2) preparing a salt solution containing copper ions, immersing the purified degummed silk in the salt solution containing copper ions to obtain silk with copper ions on its surface, drying the silk with copper ions on its surface and then grinding it;

[0011] (3) The powder obtained after grinding in step (2) is placed in a tube furnace, heated to the reaction temperature, and carbonized under an argon gas flow to obtain a carbonized solid. The carbonized solid is ground into powder and then washed with a nitric acid solution and then with deionized water, and vacuum dried to obtain a copper single-atom nanozyme.

[0012] In an optional embodiment, in step (1), the silk is tussah silk; the molar concentration of the sodium carbonate aqueous solution is 0.1-0.3 M; the boiling conditions are a temperature of 90-100°C, a time of 2-5 h, and stirring every 10-15 minutes; and the drying temperature is 75-85°C.

[0013] In an optional embodiment, in step (2), the degummed silk is soaked for 24 to 30 hours and the drying temperature is 75 to 85°C.

[0014] In an optional embodiment, in step (2), the molar concentration of the salt solution is 0.0035 to 0.0065 M.

[0015] In an optional embodiment, in step (3), the volume percentage concentration of the nitric acid solution is 1% to 3%; the carbonization treatment conditions are: heating from room temperature to 900°C at a heating rate of 3.5°C / min, maintaining at this temperature for 1 to 3 hours, and then naturally cooling to room temperature; the vacuum drying temperature is 55 to 65°C.

[0016] The second aspect of the present invention is to provide a copper single-atom nanozyme prepared using the above-mentioned preparation method.

[0017] The third aspect of the present invention provides the use of copper single-atom nanozymes in the preparation of marine fouling biofouling antifouling agents.

[0018] In an alternative embodiment, the marine antifouling agent is P. atlantica Antifouling agents and / or H. aquamarina Antifouling agent.

[0019] In an alternative embodiment, the marine antifouling agent is a marine antifouling paint.

[0020] In an optional embodiment, the marine antifouling coating comprises copper single-atom nanozymes and photocatalytic hydrogen peroxide-generating materials.

[0021] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0022] The preparation process of the present invention is relatively simple. It uses agricultural waste tussah cocoons as a carrier, effectively utilizing resources and reducing waste. Copper ions are loaded on silk. The resulting nanozyme contains copper in the form of single atoms and small nanoclusters, providing more excellent catalytic activity, excellent photothermal performance and good stability. Under the synergistic action of H2O2 and Br¯, the copper single-atom nanozyme generates HOBr through an HPO-like mechanism, which can effectively destroy Pseudoalteromonas atlanticus ( P. atlantica ) and Halomonas maritima ( H. aquamarina ) The cell structure and biofilm structure of these two marine fouling bacteria showed strong antibacterial activity and biofilm removal ability, significantly inhibiting the growth of marine bacteria, and have broad application prospects in the field of marine anti-fouling. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 X-ray diffraction (XRD) patterns of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3;

[0024] Figure 2 Scanning electron microscope (SEM) images of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3;

[0025] Figure 3 Transmission electron microscopy (TEM) images and (HRTEM) images of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3;

[0026] Figure 4 The full XPS spectra of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3;

[0027] Figure 5 This is the UV-visible absorption curve of the bromination process of phenol red catalyzed by Cu-SA-BC-2;

[0028] Figure 6 Cu-SA-BC-2 against marine fouling bacteria P. aquamarinaThe inhibitory effect diagram;

[0029] Figure 7 Cu-SA-BC-2 against marine fouling bacteria H. aquamarina The inhibitory effect diagram;

[0030] Figure 8 The effects of Cu-SA-BC-2 on the P. aquamarina and H. aquamarina SEM images of the impact;

[0031] Figure 9 The effects of Cu-SA-BC-2 on the P. aquamarina and H. aquamarina Biofilm removal effect. DETAILED DESCRIPTION

[0032] In order to help those skilled in the art better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0033] Example 1

[0034] A method for preparing a copper single-atom nanozyme comprises the following steps:

[0035] (1) Weigh 60 g of discarded tussah cocoons and place them in a 0.2 M Na2CO3 aqueous solution. Boil them at 95 °C for 3 h and stir them every 10 min to promote degumming. After degumming, wash the remaining material with deionized water to obtain purified tussah silk, which is then dried in an oven at 80 °C for later use.

[0036] (2) Prepare a copper chloride solution with a molar concentration of 0.005 M, soak the purified degummed tussah silk in the copper chloride solution for 24 h to ensure that the copper ions in the solution are fully adsorbed on the surface of the tussah silk. Place the completely soaked tussah silk in an oven at 80°C to fully dry, grind it, and then place it in a quartz boat.

[0037] (3) The powder obtained after grinding was placed in a tubular furnace and carbonized under high-purity argon. The carbonization conditions were as follows: heating from room temperature to 900 °C at a heating rate of 3.5 °C / min and maintaining at this temperature for 2 h, and then naturally cooling to room temperature. The obtained carbonized solid was fully ground into a uniform powder using a mortar, washed with 1% HNO3 solution for 30 min, and repeated washing three times to remove cobalt species and impurities that did not participate in the reaction. It was then washed three times with deionized water. The powder was collected by centrifugation and dried in a vacuum drying oven at 60 °C to obtain a copper single-atom nanozyme, named Cu-SA-BC-2.

[0038] Example 2

[0039] The preparation method of the copper single-atom nanozyme in this embodiment is different from that in Example 1 in that the molar concentration of the copper chloride solution in step (2) is 0.0035 M, and the rest is the same as in Example 1.

[0040] Example 3

[0041] The preparation method of the copper single-atom nanozyme in this embodiment is different from that in Example 1 in that the molar concentration of the copper chloride solution in step (2) is 0.0065 M, and the rest is the same as in Example 1.

[0042] Comparative Example 1

[0043] The difference between this comparative example and Example 1 is that the tussah silk is soaked in deionized water. The rest is the same as Example 1. The obtained product is named BC.

[0044] Comparative Example 2

[0045] The preparation method of the copper single-atom nanozyme in this comparative example is different from that in Example 1 in that the molar concentration of the copper chloride solution in step (2) is 0.0025 M. The rest is the same as in Example 1, and the obtained product is named Cu-SA-BC-1.

[0046] Comparative Example 3

[0047] The preparation method of the copper single-atom nanozyme in this comparative example is different from that in Example 1 in that the molar concentration of the copper chloride solution in step (2) is 0.01 M. The rest is the same as in Example 1, and the obtained product is named Cu-SA-BC-3.

[0048] Effect verification

[0049] 1. Characterization of Copper Single-Atom Nanozymes

[0050] (1) X-ray diffraction test of Cu-SA-BC

[0051] After fully grinding the samples prepared in Example 1 (Cu-SA-BC-2), Comparative Example 1 (BC), Comparative Example 2 (Cu-SA-BC-1), and Comparative Example 3 (Cu-SA-BC-3), the samples were pressed into tablets and placed on a sample table. The test target was a copper target. The structure of the samples was measured using a CT tomography X-ray diffraction system (Empyrean) and an X-ray diffractometer (XRD, Bruker D8 Advance, Germany) with a Cu Kα radiation source (45 kV, 40 mA). The scanning range was 2θ = 5 ~ 90°, and the scanning speed was 5° min-1. The data was analyzed using Jade software. The results are shown in Figure 2. Figure 1 shown.

[0052] from Figure 1It can be seen that the BC sample has a significant diffraction peak at 2θ = 26.4°, corresponding to the (002) crystal plane of graphitic carbon (C, PDF#41-1487). With the increase of copper loading, the intensity of this diffraction peak weakened significantly, indicating that the introduction of copper significantly inhibited the formation of graphite structure in the carbon matrix and promoted the formation of amorphous carbon. XRD spectra also further revealed the crystal phase changes of copper species in different samples. For Cu-SA-BC-1, Cu-SA-BC-2 and Cu-SA-BC-3 samples, characteristic diffraction peaks of copper (Cu, PDF#04-0836) were detected at 2θ = 43.3 °, 50.4 ° and 74.1 °, respectively, corresponding to the (111), (200) and (220) crystal planes of copper, respectively. As the copper loading gradually increased from 0.0025 M to 0.01 M, the intensity of the above-mentioned characteristic diffraction peaks increased significantly, indicating that the crystallinity of copper in the biochar matrix continued to increase with the increase in copper loading. This trend shows that at low loadings, copper species mainly exist in the form of highly dispersed single atoms and small-sized nanoclusters; while at higher loadings, copper species tend to agglomerate during pyrolysis to form larger nanoparticles, resulting in stronger XRD diffraction peaks. In addition, no diffraction peaks of other impurity phases were detected in the XRD spectrum, indicating that the prepared nanozyme has high purity and good structural uniformity. The copper loading not only significantly affects its distribution state in the carbon matrix, but also has a profound impact on the biochar structure and its catalytic performance. At low loadings, the highly dispersed state of copper single atoms helps increase the active sites of the nanozyme, thereby improving catalytic efficiency. At medium loadings, copper coexists in the form of single atoms and small nanoclusters, providing even better catalytic activity. However, when the loading is too high, copper aggregates to form larger nanoparticles, which not only reduces the number of effective active sites but may also inhibit the formation of single-atom sites, thus negatively affecting catalytic performance. Therefore, an appropriate copper loading can enhance catalytic activity while preventing excessive copper aggregation, thereby achieving an optimal balance between nanozyme structure and performance.

[0053] (2) Micromorphology analysis of Cu-SA-BC

[0054] The samples prepared in Example 1 (Cu-SA-BC-2), Comparative Example 1 (BC), Comparative Example 2 (Cu-SA-BC-1), and Comparative Example 3 (Cu-SA-BC-3) were respectively ground thoroughly. A small amount of the ground powder was directly adhered to the conductive adhesive. The surface morphology of the samples was then observed using a scanning electron microscope (SEM, TESCAN MIRA LMS, Czech Republic) and an energy spectrum scan.

[0055] The results are as follows Figure 2 As shown, in Figure 2In the figure, Figure a is the surface morphology of the sample of comparative example 1 (BC); Figure b is the surface morphology of the sample of comparative example 2 (Cu-SA-BC-1); Figure c is the surface morphology of the sample of embodiment 1 (Cu-SA-BC-2); and Figure d is the surface morphology of the sample of comparative example 3 (Cu-SA-BC-3).

[0056] As shown in Figure a, the BC sample exhibits a smooth, plate-like surface structure with a small number of irregular particles, which is due to the partial collapse of the tussah silk spatial structure during high-temperature pyrolysis. In contrast, the surfaces of the Cu-SA-BC-2 (Figure c), Cu-SA-BC-1 (Figure b), and Cu-SA-BC-3 (Figure d) samples are distributed with a large number of fine spherical particles. The copper particles in the Cu-SA-BC-1 and Cu-SA-BC-2 samples are well dispersed on the biochar surface, forming uniformly distributed active sites. This uniform distribution of copper particles helps improve the surface activity and catalytic performance of the nanozyme. However, when the copper loading is further increased (Cu-SA-BC-3), the copper particles increase significantly, and significant surface agglomeration gradually appears. This suggests that excessive copper loading causes copper particles to aggregate on the carbon matrix surface, forming larger nanoparticle clusters, which reduces the surface active site density of the nanozyme and adversely affects its catalytic performance. Taken together, the SEM analysis results show that the copper loading has a significant impact on the surface microstructure and particle distribution of Cu-SA-BC. Under moderate copper loading conditions, copper particles can be evenly dispersed, optimizing the distribution of active sites and thus improving the catalytic performance of nanozymes.

[0057] A copper grid with a microgrid carbon support film was selected and static electricity removed in a plasma cleaner. A 5 mg powder sample was weighed and dispersed in DMSO. A cell disruptor was used for 5 minutes to ensure thorough dispersion. A 10 μL sample was dripped onto the front of the grid and dried overnight at room temperature. The sample morphology was observed using a transmission electron microscope (TEM, FEI Talos F200x, USA) at an accelerating voltage of 120 kV and an energy spectrum scan.

[0058] The results are as follows Figure 3 As shown, Figure 3 Figures a and e in the figure represent the TEM and HRTEM images of the BC sample, respectively. Figure 3 Figures b and f show the TEM and HRTEM images of the Cu-SA-BC-1 sample, respectively. Figure 3 Figures c and g in the figure represent the TEM and HRTEM images of the Cu-SA-BC-2 sample, respectively. Figure 3 Figures d and h in the figure represent the TEM and HRTEM images of the Cu-SA-BC-3 sample, respectively. Figure 3Figure in shows the HAADF-STEM image and EDX mapping image of the Cu-SA-BC-1 sample.

[0059] from Figure 3 As shown in Figure a, the BC sample exhibits a smooth micromorphology, which is mainly attributed to the graphitization of carbon materials during high-temperature pyrolysis. In contrast, no obvious copper nanoclusters or particles were found in the Cu-SA-BC-1 sample, indicating good dispersion of copper, which is speculated to be mainly anchored in the carbon-based structure in the form of single atoms (Figure b). This single-atom dispersion state can provide highly active catalytic sites, thereby significantly improving the catalytic performance of the nanozyme. With the increase of copper loading, gradually larger nanoclusters and nanoparticles were observed in Cu-SA-BC-2 and Cu-SA-BC-3 samples, respectively (Figures c and d). From Figure 3From the eh diagram and its illustration in Figure 1, the HRTEM images clearly show the existence form and distribution characteristics of copper species under different copper loading conditions. The HRTEM image of the BC sample (e) shows a lattice spacing of 0.34 nm in the edge area, which matches the (002) crystal plane of graphitic carbon, further confirming the formation of graphene structure. In the Cu-SA-BC-1 (f) sample, the HRTEM image shows that copper exists in the form of single atoms, and no significant nanoclusters or particles are observed. In the Cu-SA-BC-2 (g) and Cu-SA-BC-3 (h) samples, not only the presence of copper single atoms was detected, but also nanoclusters and nanoparticles with a lattice spacing of 0.21 nm were observed, which matches the lattice spacing of the copper cluster (111) crystal plane. The lattice structure of these copper species was further accurately analyzed using FFT. The interlayer spacing of copper nanoclusters and nanoparticles was 0.21 nm, which corresponds to the XRD analysis results. Furthermore, graphitized carbon layers corresponding to a 0.34 nm lattice spacing were observed at the edges of the Cu-SA-BC-2 and Cu-SA-BC-3 samples, indicating that the copper nanoclusters and particles are uniformly encapsulated by the graphitic carbon layers. This carbon coating not only protects the copper particles but also facilitates rapid electron transfer between the copper and graphitic carbon layers, thereby improving electronic conductivity and catalytic performance. HAADF-STEM images of Cu-SA-BC-2 reveal the distribution of copper single atoms and nanoclusters within the carbon-based material (Figure g). The corresponding EDX elemental mapping shows that C, O, N, S, and Cu are uniformly distributed in the Cu-SA-BC-1 sample (Figure in). Nitrogen in Cu-SA-BC-2 likely acts as a coordinating atom, forming a stable Cu-N structure with copper atoms, thereby enhancing the stability and activity of the nanozyme. The introduction of nitrogen into Cu-SA-BC-2 not only improves copper dispersion but also modulates electron density, thereby increasing the rate of the catalytic reaction. In addition, the presence of sulfur may be a trace impurity, but it may have a certain adjustment effect on the electronic structure through its weak coordination with copper.

[0060] In summary, Cu-SA-BC exhibits significantly different microstructures and elemental distribution characteristics under different copper loading conditions. A small amount of copper loading (Cu-SA-BC-1 sample) causes copper atoms to be distributed in a more dispersed state, but the number of copper atoms is small. Although there is a certain catalytic activity, the activity is low. An appropriate amount of copper loading (Cu-SA-BC-2 sample) can lead to the synergistic generation of copper atoms and copper nanoclusters. The latter (copper nanoclusters) are uniformly coated with a graphitized carbon layer, which improves the stability and catalytic activity of the nanozyme. The integration of different-sized sites such as copper atoms, copper particles, and nanoclusters into a single system can utilize electron transfer and synergistic effects between different sites to optimize catalytic performance by regulating the electronic structure of the active site. Its catalytic performance is better. Although the excessive copper loading (Cu-SA-BC-3 sample) also contains copper atoms, copper particles, and nanoclusters, the copper nanoclusters are too large and the copper atoms are too few, resulting in a significant decrease in the dispersion of copper atoms. Some active sites may be passivated due to agglomeration, thereby weakening the catalytic performance and greatly reducing its catalytic performance.

[0061] (3) X-ray photoelectron spectroscopy analysis of Cu-SA-BC

[0062] The samples were subjected to full spectrum and fine spectrum testing using an X-ray photoelectron spectrometer (Thermo Scientific K-Alpha, USA). The radiation source was Al Kα (1486.6 eV) and the adsorbed carbon C 1s 284.8 eV was used as the standard for calibration. The surface chemical composition and elemental valence state of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3 were characterized by XPS technology. The results are shown in Figure 2. Figure 4 shown.

[0063] Figure 4 It was shown that, except for the BC sample, C, O, N, and Cu elements were detected on the surface of other nanozymes, indicating that copper species were successfully loaded into the biochar matrix via the impregnation pyrolysis method.

[0064] 2. Enzyme-like activity test of copper single-atom nanozyme (Cu-SA-BC-2)

[0065] In this study, the HPO-like activity of Cu-SA-BC-2 was evaluated by UV-visible absorption spectroscopy. The bromination reaction of phenol red was used as a reaction model. Time-dependent catalytic tests were conducted under light (AM 0.85 G) and dark conditions, as follows:

[0066] The bromide oxidation performance of Cu-SA-BC-2 was determined according to the phenol red method. 0.1 mg / mL of single-atom nanozyme was added to 1 mL of deionized water containing phenol red (50 µM) and NH4Br (25 mM). Under vigorous stirring, H2O2 was added to the solution (800 µM) to promote bromide oxidation. The experiment was carried out under AM 0.85 G illumination (λ≥420 nm). The reaction was terminated by removing the single-atom nanozyme from the mixture by filtration. The time-dependent absorption of the recovered solution was monitored by UV-visible spectrophotometer, scanning in the wavelength range of 300 ~ 750 nm, measuring once every 30 min for a total of 4.5 h, and observing the change of phenol red absorbance over time. The results are shown in Figure 2. Figure 5 shown.

[0067] Figure 5 The following plot shows the UV-visible spectrum of phenol red as a function of reaction time under illumination. As shown, the absorption peak of phenol red at approximately 450 nm gradually weakens with increasing reaction time, indicating that the bromination of phenol red continues and is gradually consumed. This suggests that the synergistic interaction between the copper single atom site and H₂O₂ generates •OH, which promotes the bromination of phenol red. This result confirms the excellent HPO-like activity of Cu-SA-BC-2 under illumination.

[0068] 3. Application of Copper Single Atom Nanozyme (Cu-SA-BC-2) in Marine Antimicrobial Fouling Testing

[0069] (1) Marine loss bacteria Pseudoalteromonas ( P. atlantica ) and Bacillus ( H. aquamarina ) removal effect verification

[0070] First, a single colony on the 2216E solid medium was picked with a disposable inoculation loop and placed in a conical flask containing liquid 2216E medium. The culture was then incubated at 150 rpm and 30 °C for 12 h. The exponentially growing bacteria were dispersed into the liquid 2216E medium for dilution. The bacterial concentration after dilution was measured using a UV-visible spectrophotometer (0.2 < OD < 0.8). In the antibacterial system, PBS buffer was used as the diluent (artificial seawater), and the number of bacteria was 10 6 CFU / mL, disperse evenly and add to a 24-well plate, 1 mL per well.

[0071] Twenty-four well plates were divided into four groups with different treatment conditions: the group without H2O2 and Br¯ (-H2O2, -Br¯), the group with H2O2 and without Br¯ (+H2O2, -Br¯), the group without H2O2 and with Br¯ (-H2O2, +Br¯), and the group with H2O2 and with Br¯ (+H2O2, +Br¯). The bacterial dispersions in the different treatment groups were then incubated with Cu-SA-BC-2 (0.6 mg / mL) at 37 °C under a light intensity of AM 0.85 G for 1 h with shaking. A PBS system was set as a blank control for each treatment group (the blank control group did not contain Cu-SA-BC-2, and the other conditions were the same as those of the treatment groups). After the illumination, the catalytic antibacterial performance was evaluated using a standard plating technique. After diluting 1000 times with PBS buffer, 100 μL of the bacterial suspension of each sample was spread on 2216E solid medium and cultured in a dark incubator at 30 °C for 24 h. The bacterial survival rate was calculated according to the following formula:

[0072] .

[0073] Where Nt is the number of colonies formed in the experimental group, and Nc represents the number of colonies formed in the blank control group. The bacterial survival rate was calculated as 1-A, and all experiments were repeated 3 times. P. aquamarina The inhibitory effect of Figure 6 As shown, H. aquamarina The inhibitory effect of Figure 7 As shown. Among them, Figure 6 and Figure 7 Figure a is a microscopic image; Figure b is a statistical graph of bacterial survival rate in the experimental group; and Figure c is a statistical graph of bacterial survival rate in the artificial seawater blank control group.

[0074] from Figure 6 and Figure 7 It can be seen that in the -H2O2 and -Br¯ groups, the colony density and bacterial survival rate were similar to those of the control group, indicating that in the absence of H2O2 and Br¯, Cu-SA-BC-2 had a significant effect on the bacterial survival rate. P. aquamarina and H. aquamarina The antibacterial effect was poor. In the +H2O2, -Br¯ group, the number of colonies decreased, indicating that the generated •OH had a certain antibacterial effect, but the effect was limited. In the -H2O2, +Br¯ group, the colony density and bacterial survival rate were similar to those of the control group, indicating that this condition did not have a significant inhibitory effect on bacterial growth. In the +H2O2, +Br¯ group, no colonies were generated and the bacterial survival rate was 0, indicating that under the synergistic effect of H2O2 and Br¯, Cu-SA-BC-2 generated HOBr through an HPO-like mechanism, which was effective against P. aquamarina andH. aquamarina It exhibits strong antibacterial activity and can significantly inhibit the growth of these two bacteria.

[0075] (II) Verification of the removal effect of marine bacterial biofilm

[0076] Add 990 μL of fresh 2216E medium and 10 μL of bacterial suspension (10 7 CFU / mL, P. aquamarina or H. aquamarina ) and placed in a constant-temperature incubator, with the medium replaced every 24 hours for a total of 48 hours. Subsequently, the supernatant was discarded and the wells were gently washed three times with PBS buffer (artificial seawater). A complete biofilm was observed at the bottom of the wells. 200 μL of PBS buffer and 0.6 mg / mL of Cu-SA-BC-2 were added to each well containing intact biofilms. Biofilm removal experiments were performed under four conditions (-H2O2, -Br¯; +H2O2, -Br¯; -H2O2, +Br¯; +H2O2, +Br¯), with a control group. Three replicates of each condition were set up. The wells were incubated under AM 0.85 G illumination for 12 hours. The supernatant was then removed and the wells were gently washed three times with PBS buffer. Crystal violet stain was then added to each well and stained for 30 minutes. After staining, the wells were washed three times with PBS and the remaining liquid was blotted dry with filter paper. Next, anhydrous ethanol was added to the wells to dissolve the crystal violet at the bottom of the wells, and the absorbance at 585 nm was measured using a microplate reader to evaluate the removal rate of the biofilm. Figure 8 and Figure 9 shown.

[0077] Figure 8 The results show that Cu-SA-BC-2 can inhibit the growth of two typical marine bacteria under four experimental conditions. P. atlantica (Figure a) and H. aquamarina (B) Effects of morphological characteristics: SEM observation of changes in bacterial surface structure and damage. The experiment included an artificial seawater control group and a Cu-SA-BC-2 experimental group.

[0078] from Figure 8 It can be seen that in the artificial seawater control group (Artificial seawater), P. atlantica and H. aquamarinaAll exhibited intact and smooth cell surfaces, demonstrating normal cell morphology. In the experimental group (Cu-SA-BC-2), treatment with Cu-SA-BC-2 alone showed no significant damage to cell morphology. When H₂O₂ was added alone, a few cells showed minor surface damage, but the overall structure remained intact. When Br¯ was added alone, the bacterial cell morphology remained intact. However, when H₂O₂ and Br¯ were present simultaneously, the bacterial cell structure was significantly damaged, with the cell wall and membrane disintegrating, demonstrating a significant antibacterial effect.

[0079] These results demonstrate that Cu-SA-BC-2, as an HPO-like compound, exhibits significant antibacterial properties, particularly under the synergistic action of H2O2 and Br¯. Cu-SA-BC-2 exhibits highly effective HPO-like activity. In the presence of H2O2 and Br¯, it catalyzes the generation of highly oxidizing HOBr, which rapidly oxidizes biomacromolecules in bacterial cell walls and membranes, causing irreversible damage to the cell structure and effectively destroying the cellular structure of marine bacteria.

[0080] Figure 9 Cu-SA-BC-2 (0.6 mg / mL) was shown to be an HPO-like agent against typical marine bacteria. P. aquamarina (Figure a) and H. aquamarina (Fig. b) Biofilm removal effect. The experiments were conducted in the following groups: the experimental group without H2O2 and Br¯ (Cu-SA-BC-2), the experimental group with only H2O2 (Cu-SA-BC-2+H2O2), the experimental group with only Br¯ (Cu-SA-BC-2+Br¯), and the experimental group with both H2O2 and Br¯ (Cu-SA-BC-2+H2O2+Br¯). The biofilm removal rates of the different experimental groups were calculated and analyzed from the perspective of catalytic antibacterial and antifouling. In the group with both +H2O2 and +Br¯, P. atlantica and H. aquamarinaThe biofilm removal rates for Cu-SA-BC-2 and Cu-SA-BC-2 were 81.83% and 79.72%, respectively. This result indicates that under the combined action of H2O2 and Br¯, Cu-SA-BC-2 generates HOBr through an HPO-like mechanism, effectively disrupting the biofilm structure. In contrast, the removal rates for the +H2O2 and -Br¯ (Cu-SA-BC-2+H2O2) groups were 10.93% and 8.81%, respectively, indicating that •OH generated by the decomposition of H2O2 has some destructive effect on biofilms, but the removal effect is limited. The removal rates for the -H2O2 and +Br¯ (Cu-SA-BC-2+Br¯) groups were 5.34% and 3.79%, respectively, indicating that Br¯ alone is less effective in biofilm removal without the synergistic effect of H2O2. The removal rates for the -H2O2 and -Br¯ (Cu-SA-BC-2) groups were 5.74% and 5.37%, respectively, indicating that Cu-SA-BC-2 alone is less effective in biofilm removal. The PBS group (None) had almost no effect on biofilm removal, verifying the reliability of the experiment. These results indicate that Cu-SA-BC-2 exhibits significant HPO-like activity in the presence of both H2O2 and Br¯, effectively removing marine bacterial biofilms.

[0081] Although the present invention has been described using the above preferred embodiments, they are not intended to limit the scope of protection of the present invention. Any person skilled in the art who makes various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention still fall within the scope of protection of the present invention.

Claims

1. Application of copper single-atom nanozymes in the preparation of antifouling agents for marine fouling organisms, wherein the marine fouling organisms are Pseudoalteromonas atlanticus ( P. atlantica ) and / or Halomonas marinum ( H. aquamarina ); in, The marine antifouling agent is a marine antifouling coating, which comprises a copper single-atom nanozyme, a photocatalytic hydrogen peroxide-generating material, and bromide ions; The copper single-atom nanozyme is prepared by the following preparation method: (1) placing silk in a sodium carbonate aqueous solution and boiling it for degumming, purifying the degummed silk and then drying it; (2) preparing a salt solution containing copper ions, wherein the molar concentration of the salt solution is 0.0035 to 0.0065 M, immersing the purified degummed silk in the salt solution containing copper ions to obtain silk with copper ions on its surface, and drying the silk with copper ions on its surface and then grinding it; (3) The powder obtained after grinding in step (2) is placed in a tube furnace, heated to the reaction temperature, and carbonized under an argon gas flow to obtain a carbonized solid. The carbonized solid is ground into powder and then washed with a nitric acid solution, then washed with deionized water, and vacuum dried to obtain a copper single-atom nanozyme.

2. The use according to claim 1, characterized in that In step (3), the volume percentage concentration of the nitric acid solution is 1% to 3%; the carbonization treatment conditions are: heating from room temperature to 900°C at a heating rate of 3.5°C / min, maintaining at this temperature for 1 to 3 hours, and naturally cooling to room temperature; the vacuum drying temperature is 55 to 65°C.

3. The use according to claim 1, characterized in that In the step (1), the silk is tussah silk; the molar concentration of the sodium carbonate aqueous solution is 0.1-0.3 M; the boiling conditions are a temperature of 90-100° C., a time of 2-5 h, and stirring every 10-15 minutes; and the drying temperature is 75-85° C.

4. The use according to claim 1, characterized in that In the step (2), the degummed silk is soaked for 24 to 30 hours, and the silk is dried at a temperature of 75 to 85°C.

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