Copper monatomic nano-enzyme as well as preparation method and marine anti-fouling application thereof

Abstract

The invention discloses a preparation method of a copper monatomic nano-enzyme and an application of the copper monatomic nano-enzyme in marine fouling bacteria, and particularly relates to an application of the copper monatomic nano-enzyme in inhibition of Pseudoalteromonas atlantica and Halomonas aquiamarin.According to the preparation method of the copper monatomic nano-enzyme, copper ions are loaded on degummed and purified silk, and the silk is carbonized, washed and dried to obtain the copper monatomic nano-enzyme. The obtained copper monatomic nano-enzyme has excellent photo-thermal performance and good stability, under the synergistic effect of H2O2 and Br, the copper monatomic nano-enzyme generates HOBr through an HPO-like mechanism, cell structures and biological membrane structures of two typical marine fouling bacteria P.atlantica and H.aequamarina can be effectively destroyed, and the biological membrane structure of the two typical marine fouling bacteria P.atlantica and H.aequamarina can be effectively degraded. The compound shows relatively strong antibacterial activity and biological membrane removal capacity, remarkably inhibits growth of marine fouling bacteria, and has a wide application prospect in the field of marine antifouling.

Description

Technical Field

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

[0002] Marine biofouling refers to the damage of underwater facilities caused by the attachment of marine fouling organisms. The main fouling organisms include fouling microorganisms, fouling animals such as barnacles, and fouling plants such as algae. Biofouling increases the navigation resistance of ships, increases fuel consumption, and increases greenhouse gas emissions; it clogs pipelines and cables, endangers the production safety of offshore platforms, coastal factories, and coastal power plants, and accelerates the aging and corrosion of metals; it clogs fishing gear and aquaculture cages in the sea, reduces the water exchange and dissolved oxygen concentration in the nets, resulting in a reduction in catch and aquaculture production; and the attachment of fouling organisms on ships also causes the invasion of alien organisms.

[0003] The formation process of marine biofouling is as follows: Fouling microorganisms form microbial film fouling on the underwater surface, making the smooth surface rough and facilitating the attachment of large fouling organisms. Within 24 hours, small fouling plants such as microalgae and the larvae of fouling animals attach to the microbial fouling surface; after 2-3 weeks, a mature and stable fouling ecological community is formed. Research shows that large fouling organisms such as barnacles, bryozoans, oysters, and ascidians can only form biofouling on immersed equipment with a microbial fouling surface. Therefore, inhibiting microbial fouling can reduce the attachment of large fouling organisms and inhibit marine biofouling at the initial stage of biofouling formation.

[0004] As a newly emerging catalytic antibacterial material, copper single-atom nanozyme has received extensive attention in the antibacterial field due to its unique structure and high catalytic performance. By stably loading copper atoms on a carrier material with a high surface energy, copper single-atom nanozyme can simulate the functional characteristics of natural enzymes. Compared with traditional antibacterial agents, copper single-atom nanozyme has higher catalytic activity, stability, and environmental friendliness.

[0005] In current research, no application of copper single-atom nanozyme as an antibacterial material in the prevention and control 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, a preparation method thereof, and an application. As a HPO-like nanozyme, the copper single-atom nanozyme has excellent antibacterial and biofilm removal performance against marine fouling bacteria, has a significant bactericidal effect on marine fouling bacteria, and can be used as an environmentally friendly, low-cost, and highly efficient marine antifouling material for application in marine antifouling.

[0007] The above object of the present invention is achieved by the following technical solutions: The first aspect of the present invention is to provide a preparation method of copper single-atom nanozyme, comprising the following steps: (1) Take silk and place it in an aqueous sodium carbonate solution, boil it in water for degumming, purify the obtained degummed silk and then dry it; (2) Prepare a salt solution containing copper ions, soak the purified degummed silk in the salt solution containing copper ions to obtain silk with copper ions on its surface, dry the silk with copper ions on its surface and then grind it; (3) Place the powder obtained after grinding in step (2) into a tubular furnace, raise the temperature to the reaction temperature, carry out carbonization treatment under an argon gas flow to obtain a carbonized solid, grind the carbonized solid into a powder, wash it first with a nitric acid solution, and then wash it with deionized water, and dry it under vacuum to obtain copper single-atom nanozyme.

[0008] In an alternative embodiment, in step (1), the silk is tussah silk; the molar concentration of the aqueous sodium carbonate solution is 0.1 - 0.3 M; the conditions for boiling in water are a temperature of 90 - 100 °C, a time of 2 - 5 h, and stirring every 10 - 15 min; the drying temperature is 75 - 85 °C.

[0009] In an alternative embodiment, in step (2), the soaking time of the degummed silk is 24 - 30 h, and the drying temperature is 75 - 85 °C.

[0010] In an alternative embodiment, in step (2), the molar concentration of the salt solution is 0.0035 - 0.0065 M.

[0011] In an alternative embodiment, in step (3), the volume percentage concentration of the nitric acid solution is 1% - 3%; the conditions for carbonization treatment are: heating from room temperature to 900 °C at a heating rate of 3.5 °C / min, and maintaining at this temperature for 1 - 3 h, and then naturally cooling to room temperature; the vacuum drying temperature is 55 - 65 °C.

[0012] The second aspect of the present invention is to provide a copper single-atom nanozyme prepared by the above preparation method.

[0013] The third aspect of the present invention is to provide the application of copper single-atom nanozyme in the preparation of marine fouling organism antifouling agents.

[0014] In an alternative embodiment, the marine fouling organism antifouling agent is P. atlantica antifouling agent and / or H. aquamarina antifouling agent.

[0015] In an alternative embodiment, the marine fouling organism antifouling agent is a marine antifouling coating.

[0016] In an alternative embodiment, the marine antifouling coating comprises copper single-atom nanozyme and a photocatalytic hydrogen peroxide-producing material.

[0017] Compared with the prior art, the technical solution of the present invention has the following advantages: The preparation process of the present invention is relatively simple. Using agricultural waste tussah cocoons as carriers, resources are effectively utilized and waste is reduced. Copper ions are loaded on silk, and the copper in the obtained nanozyme coexists 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 a HPO-like mechanism, which can effectively destroy the cell structures and biofilm structures of two marine fouling bacteria, Pseudomonas pseudoalcaligenes ([[]] P. atlantica ) and Halomonas marina ([[]] H. aquamarina ), showing strong antibacterial activity and biofilm removal ability, significantly inhibiting the growth of marine bacteria, and having broad application prospects in the field of marine antifouling. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 are the X-ray diffraction (XRD) patterns of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3; Figure 2 are the scanning electron microscope (SEM) images of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3; Figure 3 are the transmission electron microscope (TEM) images and (HRTEM) images of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3; Figure 4 are the XPS full spectra of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3; Figure 5 is the ultraviolet-visible absorption curve of the phenol red bromination process catalyzed by Cu-SA-BC-2; Figure 6 is the inhibition effect diagram of Cu-SA-BC-2 on marine fouling bacteria P. aquamarina ; Figure 7 is the inhibition effect diagram of Cu-SA-BC-2 on marine fouling bacteria H. aquamarina ; Figure 8SEM images of the influence of Cu-SA-BC-2 under different treatment conditions on P. aquamarina and H. aquamarina ; Figure 9 SEM images of the removal effect of Cu-SA-BC-2 under different treatment conditions on P. aquamarina and H. aquamarina biofilm. Detailed implementation manners

[0019] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0020] Example 1 A preparation method of copper single-atom nanozyme, comprising the following steps: (1) Weigh 60 g of waste tussah cocoons, place them in an aqueous solution of 0.2 M Na2CO3, boil them in water at 95 °C for 3 h, and stir every 10 min to promote degumming. After degumming is completed, wash the residual substances thoroughly with deionized water to obtain purified tussah silk, and dry it in an oven at 80 °C for later use.

[0021] (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. Put the completely soaked tussah silk into an oven at 80 °C to dry thoroughly, grind it and put it into a quartz boat.

[0022] (3) Place the powder obtained after grinding in a tube furnace and carry out carbonization treatment under high-purity argon. Among them, the carbonization conditions are: raise the temperature from room temperature to 900 °C at a heating rate of 3.5 °C / min, and keep it at this temperature for 2 h, and then cool it naturally to room temperature. The obtained carbonized solid is ground thoroughly with a mortar into a uniform powder, washed with 1% HNO3 solution for 30 min, and washed 3 times repeatedly to remove the unreacted cobalt species and impurities, and then washed with deionized water 3 times. Collect the powder by centrifugation and dry it in a vacuum drying oven at 60 °C to obtain copper single-atom nanozyme, named Cu-SA-BC-2.

[0023] Example 2

[0024] The preparation method of the copper single-atom nanozyme in this example is different from that in Example 1 in terms of the molar concentration of the copper chloride solution in step (2). The molar concentration of the copper chloride solution in this example is 0.0035 M, and the rest is the same as in Example 1.

[0025] Example 3

[0026] The preparation method of the copper single-atom nanozyme in this example is different from that in Example 1 in terms of the molar concentration of the copper chloride solution in step (2). The molar concentration of the copper chloride solution in this example is 0.0065 M, and the rest is the same as in Example 1.

[0027] Comparative Example 1 The difference between this comparative example and Example 1 is that tussah silk is soaked in deionized water, and the rest is the same as in Example 1. The obtained product is named BC.

[0028] Comparative Example 2 The preparation method of the copper single-atom nanozyme in this comparative example is different from that in Example 1 in terms of the molar concentration of the copper chloride solution in step (2). The molar concentration of the copper chloride solution in this comparative example is 0.0025 M, and the rest is the same as in Example 1. The obtained product is named Cu-SA-BC-1.

[0029] Comparative Example 3 The preparation method of the copper single-atom nanozyme in this comparative example is different from that in Example 1 in terms of the molar concentration of the copper chloride solution in step (2). The molar concentration of the copper chloride solution in this comparative example is 0.01 M, and the rest is the same as in Example 1. The obtained product is named Cu-SA-BC-3.

[0030] Effect verification I. Characterization of copper single-atom nanozyme

[0031] (I) X-ray diffraction test of Cu-SA-BC 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, then pressed into tablets and placed on the sample stage. The test target was a copper target. A CT tomographic X-ray diffraction system (Empyrean) X-ray diffractometer (XRD, Bruker D8 Advance, Germany) was used to measure the structure of the samples 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. Jade software was used to analyze the data. The results are as Figure 1 shown.

[0032] From Figure 1As can be seen, a significant diffraction peak appeared at 2θ = 26.4° for the BC sample, corresponding to the (002) crystal plane of graphite carbon (C, PDF#41-1487). With the increase in copper loading, the intensity of this diffraction peak decreased significantly, indicating that the introduction of copper had a significant inhibitory effect on the formation of the graphite structure in the carbon matrix and promoted the formation of amorphous carbon. The XRD spectrum further revealed the phase changes of copper species in different samples. For the 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 °, 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 characteristic diffraction peaks increased significantly, indicating that the crystallinity of copper in the biochar matrix increased continuously with the increase in loading. This trend indicates 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 are prone to agglomeration during pyrolysis, forming 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 had high purity and good structural uniformity. The copper loading not only significantly affected its distribution state in the carbon matrix but also had a profound impact on the biochar structure and its catalytic performance. At low loadings, the highly dispersed state of copper single atoms helped to increase the active sites of the nanozyme, thereby improving the catalytic efficiency; at medium loadings, copper coexisted in the form of single atoms and small nanoclusters, providing more excellent catalytic activity; however, when the loading was too high, copper agglomerated to form larger nanoparticles, which not only reduced the number of effective active sites but also might inhibit the formation of single-atom sites, thus having a negative impact on the catalytic performance. Therefore, an appropriate copper loading could not only improve the catalytic activity but also prevent excessive copper agglomeration, thus achieving the best balance between the nanozyme structure and performance.

[0033] (II) Microscopic Morphology Analysis of Cu-SA-BC 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 ground thoroughly, and a small amount of the ground powder was directly adhered to the conductive adhesive, and then the surface morphology of the samples was observed by scanning electron microscopy (SEM, TESCAN MIRA LMS, Czech Republic) and energy-dispersive spectroscopy mapping was performed.

[0034] The results are as Figure 2 shown in Figure 2Among them, Figure a is the surface morphology diagram of the sample of Comparative Example 1 (BC); Figure b is the surface morphology diagram of the sample of Comparative Example 2 (Cu-SA-BC-1), Figure c is the surface morphology diagram of the sample of Example 1 (Cu-SA-BC-2); Figure d is the sample of Comparative Example 3 (Cu-SA-BC-3).

[0035] As can be seen from Figure a, the BC sample presents a smooth plate-like surface structure with a small amount of irregular particles, which is due to the partial collapse of the spatial structure of tussah silk during the high-temperature pyrolysis process. In contrast, a large number of fine spherical particles are distributed on the surfaces of the Cu-SA-BC-2 (Figure c), Cu-SA-BC-1 (Figure b) and Cu-SA-BC-3 (Figure d) samples. Among them, the copper particles of the Cu-SA-BC-1 and Cu-SA-BC-2 samples can be well dispersed on the surface of the biochar, forming uniformly distributed active sites. This uniform distribution of copper particles helps to 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 obvious agglomeration phenomena gradually appear on the surface. This indicates that too high a copper loading will cause copper particles to aggregate on the surface of the carbon matrix, forming larger nanoparticle clusters, thereby reducing the density of surface active sites of the nanozyme, and thus having an adverse effect on the catalytic performance. In summary, 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 the condition of moderate copper loading, copper particles can be evenly dispersed, optimizing the distribution of active sites, thereby improving the catalytic performance of the nanozyme.

[0036] Select a copper mesh with a microgrid carbon support film and remove the static electricity on the copper mesh on a plasma cleaner. Weigh 5 mg of the powder sample, disperse it in DMSO, and use a cell disruptor to probe ultrasonically for 5 min to fully disperse the sample in the solvent. Take 10 μL of the sample and drop it onto the front side of the copper mesh, dry it overnight at room temperature, and observe the sample morphology and perform energy spectrum surface scanning under an accelerating voltage of 120 kV in a transmission electron microscope (TEM, FEI Talos F200x, USA).

[0037] The results are as Figure 3 shown, Figure 3 Figures a and e in show the TEM and HRTEM diagrams of the BC sample respectively, Figure 3 Figures b and f in show the TEM and HRTEM diagrams of the Cu-SA-BC-1 sample respectively, Figure 3 Figures c and g in show the TEM and HRTEM diagrams of the Cu-SA-BC-2 sample respectively, Figure 3 Figures d and h in show the TEM and HRTEM diagrams of the Cu-SA-BC-3 sample respectively, Figure 3The i-n diagram in

[0038] As shown in Figure 3 Figure a, the BC sample exhibits a smooth microstructure, which is mainly attributed to the graphitization of the carbon material during the high-temperature pyrolysis process. In contrast, no obvious copper nanoclusters or particles are found in the Cu-SA-BC-1 sample, indicating good dispersion of copper. It is speculated that copper is 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, thus significantly improving the catalytic performance of the nanozyme. With the increase of copper loading, gradually increasing nanoclusters and nanoparticles are observed in the Cu-SA-BC-2 and Cu-SA-BC-3 samples, respectively (Figures c and d). As shown in Figure 3From the e-h figures and their illustrations, the HRTEM images clearly show the existence forms and distribution characteristics of copper species under different copper loadings. The HRTEM image of the BC sample (Figure e) shows a lattice spacing of 0.34 nm in the edge region, which matches the (002) crystal plane of graphite carbon, further confirming the formation of the graphene structure. In the Cu-SA-BC-1 sample (Figure f), 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 (Figure g) and Cu-SA-BC-3 (Figure h) samples, not only the existence of copper single atoms is detected, but also nanoclusters and nanoparticles with a lattice spacing of 0.21 nm are observed, which matches the lattice spacing of the (111) crystal plane of copper clusters. The lattice structures of these copper species were further accurately analyzed using FFT. The interlayer spacing of the copper nanoclusters and nanoparticles is 0.21 nm, which corresponds to the XRD analysis results. In addition, graphitized carbon layers corresponding to a lattice spacing of 0.34 nm were also 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 graphite carbon layers. The carbon layer coating structure can not only protect the copper particles but also promote the rapid transfer of electrons between the copper and the graphite carbon layer, thereby enhancing the electron conductivity and catalytic performance. Next, the HAADF-STEM image of Cu-SA-BC-2 reveals the distribution of copper single atoms and nanoclusters in the carbon-based material (Figure g). The corresponding EDX elemental mapping shows that the elements C, O, N, S, and Cu are evenly distributed in the Cu-SA-BC-1 sample (Figures i-n). Among them, nitrogen may act as a coordinating atom in Cu-SA-BC-2 to form a stable Cu-N structure with copper atoms, thereby enhancing the stability and activity of the nanozyme. In Cu-SA-BC-2, the introduction of nitrogen not only helps to improve the dispersion of copper but also can regulate the electron density and increase the rate of the catalytic reaction. In addition, the presence of sulfur may be trace impurities, but its weak coordination with copper may have a certain effect on adjusting the electronic structure.

[0039] In summary, Cu-SA-BC exhibits significantly different microstructures and elemental distribution characteristics under different copper loadings. A small amount of copper loading (Cu-SA-BC-1 sample) results in a relatively dispersed state of copper single atoms, but the number of copper atoms is small. Although it has certain catalytic activity, the activity is low. An appropriate amount of copper loading (Cu-SA-BC-2 sample) enables the co-generation of copper single atoms and copper nanoclusters. The latter (copper nanoclusters) are uniformly coated by a graphitized carbon layer, enhancing the stability and catalytic activity of the nanozyme. Integrating different-sized sites such as copper single atoms, copper particles, and nanoclusters into a system can utilize the electron transfer and synergy between different sites to optimize the catalytic performance by regulating the electronic structure of the active sites, and its catalytic performance is good. However, for an excessive amount of copper loading (Cu-SA-BC-3 sample), although it also contains copper single atoms, copper particles, and nanoclusters, due to the too-large copper nanoclusters and too-small copper single atoms, the dispersion degree of copper single atoms is significantly reduced, and some active sites may be passivated due to aggregation, thus weakening the catalytic performance and leading to a significant reduction in its catalytic performance.

[0040] (III) X-ray photoelectron spectroscopy analysis of Cu-SA-BC The samples were tested by full-spectrum and fine-spectrum using an X-ray photoelectron spectrometer (Thermo Scientific K-Alpha, USA). The X-ray source was Al Kα (1486.6 eV), and the binding energy of C 1s at 284.8 eV of adsorbed carbon was used as the standard for calibration. The surface chemical composition and element valence states of BC, Cu-SA-BC-1, Cu-SA-BC-2, and Cu-SA-BC-3 were characterized by XPS technology. The results are as Figure 4 shown.

[0041] Figure 4 It shows that in addition to the BC sample, elements C, O, N, and Cu were detected on the surfaces of other nanozymes, indicating that copper species were successfully loaded onto the biochar matrix by the impregnation pyrolysis method.

[0042] II. Enzyme-like activity test of copper single-atom nanozyme (Cu-SA-BC-2) In this study, the HPO-like activity of Cu-SA-BC-2 was evaluated by ultraviolet-visible absorption spectroscopy. Using the phenol red bromination reaction as the reaction model, time-dependent catalytic tests were carried out under light (AM 0.85 G) and dark conditions, as follows: The bromide oxidation performance of Cu-SA-BC-2 was determined according to the phenol red method. 0.1 mg / mL of the 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 filtering the single-atom nanozyme from the mixture through a filter head. The time-dependent absorption of the recovered solution was monitored using a UV-visible spectrophotometer, scanned in the wavelength range of 300 - 750 nm, measured once every 30 min for a total of 4.5 h, and the change in the absorbance of phenol red over time was observed. The results are as Figure 5 shown.

[0043] Figure 5 Figure showing the change in the UV-visible spectrum with reaction time under illumination conditions. It can be seen from the figure that as the reaction time extends, the absorption peak of phenol red at approximately 450 nm gradually weakens, indicating that the bromination reaction of phenol red continues and phenol red is gradually consumed. It shows that the synergistic effect between copper single-atom sites and H2O2 generates •OH to promote the bromination of phenol red. This result confirms that Cu-SA-BC-2 has excellent HPO-like activity under illumination conditions.

[0044] III. Application of Copper Single-Atom Nanozyme (Cu-SA-BC-2) in Marine Antimicrobial Fouling Tests

[0045] (I) Verification of the Removal Effects on Marine Depleted Bacteria Pseudomonas spp. ( P. atlantica ) and Vibrio spp. ( H. aquamarina ) First, a single colony on a 2216E solid medium was picked with a disposable inoculation loop and placed into a conical flask containing liquid 2216E medium, and cultured at 150 rpm and 30 °C for 12 h. The exponentially growing bacteria were dispersed into the liquid 2216E medium for dilution, and the diluted bacterial concentration (0.2 < OD < 0.8) was detected using a UV-visible spectrophotometer. In the antibacterial system, PBS buffer was used as the diluent (artificial seawater), and the number of bacteria was 10 6 CFU / mL. After being dispersed evenly, it was added to a 24-well plate, 1 mL per well.

[0046] Twenty-four orifice plates were divided into four groups, and the treatment conditions of the four groups were different, namely the treatment group without H2O2 and Br¯ (-H2O2, -Br¯), the treatment group with H2O2 and without Br¯ (+H2O2, -Br¯), the treatment group without H2O2 and with Br¯ (-H2O2, +Br¯), and the treatment group with H2O2 and with Br¯ (+H2O2, +Br¯). Subsequently, the bacterial dispersions in different treatment groups were co-incubated with Cu-SA-BC-2 (0.6 mg / mL) under the conditions of 37 °C and a light intensity of AM 0.85 G with shaking for 1 h. Each treatment group was set with a PBS system as a blank control (the blank control group did not contain Cu-SA-BC-2, and the other conditions were the same as those of the treatment group). After the light exposure, the catalytic antibacterial performance was evaluated using the standard plating technique. After diluting 1000 times with PBS buffer, 100 μL of the bacterial suspension of each sample was spread on a 2216E solid medium and cultured in the dark in a 30 °C incubator for 24 h. The bacterial survival rate was calculated according to the following formula: .

[0047] 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. For P. aquamarina the inhibitory effect is as Figure 6 shown, and for H. aquamarina the inhibitory effect is as Figure 7 shown. Among them, Figure 6 and Figure 7 Figure a in each shows the microscopic images under the microscope; Figure b in each shows the statistical chart of the bacterial survival rate in the experimental group; Figure c in each shows the statistical chart of the bacterial survival rate in the artificial seawater blank control group.

[0048] It can be seen from Figure 6 and Figure 7 that in the -H2O2, -Br¯ group, the colony density and bacterial survival rate were similar to those of the control group, indicating that under the conditions of lack of H2O2 and Br¯, the antibacterial effect of Cu-SA-BC-2 on P. aquamarina and H. aquamarina 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 an obvious inhibitory effect on bacterial growth. While in the +H2O2, +Br¯ group, no colonies were formed and the bacterial survival rate was 0, indicating that under the synergistic action of H2O2 and Br¯, Cu-SA-BC-2 generated HOBr through the HPO-like mechanism and had an antibacterial effect on P. aquamarina and H.aquamarina Exhibits strong antibacterial activity and can significantly inhibit the growth of these two bacteria.

[0049] (II) Verification of the effect of removing biofilms of marine-depleted bacteria Add 990 μL of fresh 2216E medium and 10 μL of bacterial solution (10 7 CFU / mL, P. aquamarina or H. aquamarina ) into a 96-well cell culture plate, place it in a constant temperature incubator, change the medium every 24 h, and culture for a total of 48 h. Subsequently, discard the supernatant, gently wash three times with PBS buffer (artificial seawater), and a complete biofilm can be observed to form at the bottom of the wells. Add 200 μL of PBS buffer, Cu-SA-BC-2 (0.6 mg / mL) to each well with a well-developed biofilm, conduct biofilm removal experiments under four conditions (-H2O2, -Br¯; +H2O2, -Br¯; -H2O2, +Br¯; +H2O2, +Br¯), set up a control group, set 3 replicates for each condition, and incubate under AM 0.85 G light for 12 h. Then remove the supernatant and gently wash 3 times with PBS buffer, add crystal violet staining solution to each well, stain for 30 min, wash 3 times with PBS after staining, and blot dry the remaining liquid with filter paper. Next, add absolute ethanol to the wells to dissolve the crystal violet at the bottom of the wells, and measure the absorbance value at 585 nm with an enzyme-linked immunosorbent assay (ELISA) reader to evaluate the removal rate of the biofilm. The results are as Figure 8 and Figure 9 shown.

[0050] Figure 8 respectively show the effects of Cu-SA-BC-2 on the morphological characteristics of two typical marine bacteria under four experimental conditions P. atlantica (Figure a) and H. aquamarina (Figure b), and observe the changes and damage of the bacterial surface structure through scanning electron microscopy (SEM). The experiment includes an artificial seawater control group and a Cu-SA-BC-2 experimental group.

[0051] As can be seen from Figure 8 , in the artificial seawater control group (Artificial seawater), P. atlantica and H. aquamarinaAll presented a complete and smooth cell surface, showing normal cell morphology. In the experimental group (Cu-SA-BC-2), when treated with Cu-SA-BC-2 alone, no obvious damage to cell morphology was observed. Under the condition of adding H2O2 alone, slight damage appeared on the surface of a very small number of cells, but the overall structure remained intact. When Br¯ was added alone, the morphology of bacterial cells remained intact. However, when both H2O2 and Br¯ were present, the structure of bacterial cells was significantly damaged, and the cell wall and cell membrane disintegrated, showing a significant antibacterial effect.

[0052] The above results indicate that Cu-SA-BC-2 exhibits significant antibacterial properties as a mimetic HPO, especially under the synergistic effect of H2O2 and Br¯, its antibacterial effect is particularly prominent. Cu-SA-BC-2 shows high mimetic HPO activity and generates highly oxidizing HOBr through catalytic reactions in the presence of H2O2 and Br¯, which can rapidly oxidize biomacromolecules in the bacterial cell wall and cell membrane, resulting in irreversible damage to the cell structure and effectively destroying the cell structure of marine bacteria.

[0053] Figure 9 showed the removal effect of Cu-SA-BC-2 (0.6 mg / mL) as a mimetic HPO on typical marine bacteria P. aquamarina (Figure a) and H. aquamarina Figure b) of biofilms. The experiments were 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¯). By calculating the biofilm clearance rates of different experimental groups, the analysis was carried out from the perspective of catalytic antibacterial antifouling. In the group with both +H2O2 and +Br¯, P. atlantica and H. aquamarinaThe biofilm removal rates 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 the HPO-like mechanism and can effectively destroy the biofilm structure. In contrast, the removal rates of the +H2O2, -Br¯ (Cu-SA-BC-2 + H2O2) group were 10.93% and 8.81% respectively, indicating that •OH generated by the decomposition of H2O2 has a certain destructive effect on the biofilm, but the removal effect is limited. The removal rates of the -H2O2, +Br¯ (Cu-SA-BC-2 + Br¯) group were 5.34% and 3.79% respectively, indicating that the removal effect of Br¯ alone on the biofilm is poor in the absence of H2O2 coordination. The removal rates of the -H2O2, -Br¯ (Cu-SA-BC-2) group were 5.74% and 5.37% respectively, indicating that Cu-SA-BC-2 itself has a poor removal effect on the biofilm. The PBS group (None) had almost no removal effect on the biofilm, verifying the reliability of the experiment. The above results show that Cu-SA-BC-2 exhibits significant HPO-like activity under the conditions of containing both H2O2 and Br¯ and can effectively remove marine bacterial biofilms.

[0054] Although the present invention has been described by using the above preferred embodiments, it is not intended to limit the protection scope of the present invention. Any person skilled in the art can make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, and these still belong to the protection scope of the present invention.

Claims

1. Application of copper single-atom nanozyme in the preparation of antifouling agents for marine fouling organisms, where the marine fouling organisms are P. atlantica and / or H. aquamarina.

2. The application according to claim 1, characterized in that The antifouling agent for marine fouling organisms is a marine antifouling coating.

3. The application according to claim 2, wherein The marine antifouling coating comprises a copper single-atom nanozyme and a photocatalytic hydrogen peroxide-producing material.

4. The application according to claim 1, wherein The copper single-atom nanozyme is prepared by the following preparation method: (1) Take silk and place it in an aqueous sodium carbonate solution, boil it in water for degumming, purify the obtained degummed silk and then dry it; (2) Prepare a salt solution containing copper ions, soak the purified degummed silk in the salt solution containing copper ions to obtain silk with copper ions on its surface, dry the silk with copper ions on its surface and then grind it; (3) Place the powder obtained after grinding in step (2) into a tubular furnace, raise the temperature to the reaction temperature, carry out carbonization treatment under an argon gas flow to obtain a carbonized solid, grind the carbonized solid into a powder, wash it first with a nitric acid solution and then with deionized water, and dry it under vacuum to obtain a copper single-atom nanozyme.

5. The preparation method of the copper single-atom nanozyme according to claim 4, wherein In step (3), the volume percentage concentration of the nitric acid solution is 1% - 3%; the conditions for carbonization treatment are: raise the temperature from room temperature to 900 °C at a heating rate of 3.5 °C / min, keep it at this temperature for 1 - 3 h, and then naturally cool to room temperature; the temperature for vacuum drying is 55 - 65 °C.

6. The preparation method of the copper single-atom nanozyme according to claim 4, characterized in that, In step (1), the silk is tussah silk; the molar concentration of the aqueous sodium carbonate solution is 0.1 - 0.3 M; the conditions for boiling in water are a temperature of 90 - 100 °C, a time of 2 - 5 h, and stir once every 10 - 15 min; the drying temperature is 75 - 85 °C.

7. The preparation method of the copper single-atom nanozyme according to claim 4, characterized in that, In step (2), the soaking time of the degummed silk is 24 - 30 h, and the drying temperature is 75 - 85 °C.

8. The preparation method of the copper single-atom nanozyme according to claim 4, characterized in that, In step (2), the molar concentration of the salt solution is 0.0035 - 0.0065 M.

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