Application of high-entropy alloy nanozymes in antibiotic degradation and antibacterial activity

Abstract

This invention discloses the application of a high-entropy alloy nanozyme in antibiotic degradation and antibacterial activity. By adjusting the composition of the high-entropy alloy nanozyme, its peroxidase-like activity is enhanced. The high-entropy alloy nanozyme generates reactive oxygen species such as hydroxyl radicals, thereby degrading antibiotics and inhibiting bacteria. This invention achieves nearly 100% degradation of tetracycline antibiotics (tetracycline hydrochloride, chlortetracycline hydrochloride, oxytetracycline hydrochloride) within 20 minutes. Under neutral conditions, it exhibits strong inhibitory effects on Staphylococcus aureus and Escherichia coli, with an inhibition rate of over 80% against Staphylococcus aureus. The beneficial effects of this method are: increasing the copper content significantly enhances the peroxidase-like catalytic activity of the high-entropy alloy nanozyme, resulting in excellent antibacterial properties and catalytic oxidation of dyes. The porous nature and high specific surface area of ​​the carbon support are beneficial for increasing the contact area between the high-entropy alloy nanozyme and the substrate, thereby accelerating the reaction rate.

Description

Technical Field

[0001] This invention belongs to the field of catalytic removal of pollutants in water, specifically relating to the application of a porous carbon-supported high-entropy alloy nanoparticle composite material as a nanoenzyme for antibiotic degradation and antibacterial purposes. Background Technology

[0002] Infections caused by microorganisms pose a serious threat to public health, leading to severe health problems. Antibiotics are widely used in humans and animals to prevent bacterial infections; however, the human metabolic system cannot completely absorb antibiotics, and over 75% of antibiotics are digested in feces and enter water systems. If pollutant treatment technologies are inadequate, this can pose a potential threat to ecosystems and human health. Furthermore, the increasing resistance of pathogens to antibiotics makes bacterial resistance one of the most serious threats to modern medicine. Advances in nanotechnology and materials science have made it possible to produce new substances with intrinsic antibacterial activity for new applications in various technological fields, addressing the challenges of modern society. Developing nanomaterials that are both highly degradable and possess high antibacterial activity is one feasible solution to address drug pollution and bacterial resistance in water bodies.

[0003] Since the first report of peroxide-like (POD) enzyme activity in iron(III) oxide in 2007, single-component or multi-component nanozymes have emerged and been widely applied in biosensing, antibacterial applications, cancer diagnosis and treatment, environmental monitoring, and wastewater treatment. Nanozymes possess advantages such as high stability, low cost, and ease of modification; however, their limited catalytic efficiency and poor selectivity have restricted their development. Recently, multimetal nanozymes have attracted considerable attention due to their unique cocktail effect, where electronic interactions between metal components can lead to unexpected properties and nonlinear enhancements of inherent performance. However, most conventional design concepts involving doping, heterojunctions, or a combination of both are trial-and-error strategies. Furthermore, limited component space and a lack of theoretical guidance hinder the diversification of nanozymes and improvements in catalytic performance.

[0004] Since the landmark study on high-entropy alloys (HEAs) was published in 2004, HEAs have attracted significant attention from both academia and industry. HEAs are alloys formed from five or more equal or approximately equal amounts of metals. Recent research indicates that the atomic concentration of each element in HEAs ranges from 5% to 35%. Compared to single metals, HEAs exhibit enhanced chemical stability while maintaining excellent performance due to their diverse surface sites. To address these challenges, we introduce the concept of high entropy into the development of high-performance nanozymes that integrate state-of-the-art high-entropy alloys with intrinsic enzyme-like active sites. HEAs offer an effective countermeasure to key catalytic challenges due to their high tunability and flexibility derived from multidimensional compositional space. In high-entropy alloys, the enthalpy of compound formation is overcome by a sharp increase in configurational entropy caused by the mixing of multiple components, thus promoting the formation of stable single-phase solid solutions rather than intermetallic compound high-entropy alloys. Theoretically, confining atoms with different properties and sizes within the same lattice leads to significant lattice distortion and synergistic effects, resulting in a well-defined structure-property relationship for the catalyst. The rich diversity of atomic sites in high-entropy alloys optimizes the geometry and electronic configuration of the reaction surface, thereby affecting the adsorption energy of the substrate or intermediate and ultimately influencing catalytic activity. Furthermore, the strategy of adapting HEAs to nanostructures can amplify the size effect, thus improving their catalytic performance. Fine-tuning HEAs can maximize catalytic activity and achieve ideal reaction kinetic rates.

[0005] This invention synthesizes porous carbon-supported high-entropy alloy nanozymes with high peroxidase (POD) activity by adjusting the proportion of high-entropy alloy nanozyme components (HEAzyme), and studies its application in antibiotic degradation and antibacterial activity in water. The results show that the peroxidase activity of FeCoNiMoCu2 (HEA-Cu2) is three times that of FeCoNiMoCu2 (HEA-Cu), and it can rapidly and efficiently degrade three tetracycline antibiotics within 20 minutes with a degradation rate of almost 100%. At the same time, it can inhibit Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with an inhibition rate of over 70% against both bacteria. This invention opens up a new field of high-entropy alloy nanozymes and promotes the development of nanoenzymology. Summary of the Invention

[0006] The purpose of this invention is to develop a high-entropy alloy nanozyme and study its application in antibiotic degradation and antibacterial activity. Utilizing porous carbon as a carrier, high-entropy alloy nanoparticles are loaded onto the nanozyme. The HEA generates reactive oxygen species, thereby achieving near-complete decomposition of antibiotics or inactivation of bacteria. It features green efficiency, simplicity, and safety.

[0007] An application of a high-entropy alloy nanozyme (molar ratio Fe:Co:Ni:Mo:Cu = 1:1:1:1:2) in antibiotic degradation includes the following steps:

[0008] Catalytic degradation system: Degradation concentration is 0.2 g / L. －1 The catalytic degradation performance of high-entropy nanomaterials on tetracycline antibiotics was investigated using a tetracycline antibiotic solution. A 1 mg / L stock solution was prepared in advance and diluted as needed for the experiment. The catalytic reaction conditions were optimized by changing the initial antibiotic concentration, pH, and catalyst dosage. The OD was measured by ultraviolet spectrophotometry. 357 The degradation rate was measured at nm.

[0009]

[0010] An antibacterial degradation application of a high-entropy alloy nanozyme includes the following steps:

[0011] Determination of antibacterial rate: Overnight cultured bacteria were inoculated into fresh culture medium at a ratio of 1:100, and the bacterial solution was diluted with sterile culture medium to a concentration of approximately 10. 6 CFU / mL, after culturing the bacterial culture with different concentrations of materials at 37℃ for 2 h, serially diluted with sterile PBS, 10 μL was evenly spread on TSA solid medium and incubated at 37℃ for 24 h for colony counting, with each group repeated three times.

[0012] A high-entropy alloy nanozyme-like peroxidase catalytic condition is provided, wherein the peroxidase-like catalytic condition is used to evaluate the performance of the high-entropy alloy nanozyme in catalyzing the activation of hydrogen peroxide and promoting substrate oxidation in a solution containing the substrate 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2).

[0013] The antibacterial application involves evaluating the inhibitory effect of high-entropy alloy nanozymes on Staphylococcus aureus and Escherichia coli by counting colony counts in a neutral environment using a plate coating method.

[0014] The aforementioned antibiotic degradation application evaluates the performance of high-entropy alloy nanozymes in degrading antibiotics by observing changes in absorbance before and after the degradation of tetracycline antibiotics such as tetracycline hydrochloride (TH), chlortetracycline hydrochloride (CTC), and oxytetracycline hydrochloride (OH) under acidic conditions.

[0015] The beneficial effects of this invention are:

[0016] Increasing the copper content significantly enhances the peroxidase-like catalytic activity of metal oxide nanozymes.

[0017] The porous nature and high specific surface area of ​​carbon supports are beneficial for increasing the contact area between high-entropy alloy nanozymes and substrates, thereby accelerating the reaction rate.

[0018] Using porous carbon as a carrier to load high-entropy alloy nanoparticles as nanoenzymes, antibiotics can be effectively degraded and bacteria killed by generating reactive oxygen species, without the need for other additional conditions, making it green and efficient.

[0019] In actual antibiotic treatment processes, light has little impact on catalytic efficiency, eliminating the need for additional light source equipment. This reduces the cost of treating antibiotic wastewater and enhances the potential application of photocatalysts in the natural environment.

[0020] The preparation cost is low, the process is simple, the operation is convenient, and the reaction conditions are mild. Attached Figure Description

[0021] Figure 1 XRD patterns of three high-entropy alloy nanozymes

[0022] Figure 2 Field emission scanning electron microscope (FE-SEM) image of HEA-Cu2NPs;

[0023] Figure 3 Transmission electron microscopy (TEM) spectra of HEA-Cu2NPs;

[0024] Figure 4 Pore ​​size distribution diagram of HEA-Cu2NPs;

[0025] Figure 5 Electron spin resonance (EPR) spectra of hydroxyl radicals generated by the decomposition of H2O2 catalyzed by HEA-Cu2NPs were captured using 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO).

[0026] Figure 6 Comparison of POD-like enzyme activities of three high-entropy alloy nanozymes in solutions containing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) (the factor of increase in POD-like activity = the rate of increase in absorbance of HEA-Cu2NPs at 652 nm / the rate of increase in absorbance of HEA-CuNPs at 652 nm).

[0027] Figure 7 HEA-Cu2NPs' POD-like catalytic activity at different pH levels;

[0028] Figure 8 Degradation catalytic activity of HEA-Cu2NPs at different material concentrations;

[0029] Figure 9 Degradation catalytic activity of HEA-Cu2NPs at different pH values;

[0030] Figure 10Degradation catalytic activity of HEA-Cu2NPs under different TH values;

[0031] Figure 11 The degradation catalytic activity of HEA-Cu2NPs under different light irradiation conditions;

[0032] Figure 12 Time-tracking CT of the catalytic oxidative degradation of tetracycline hydrochloride by HEA-Cu2NPs t / C0 variation curve, where C t C0 represents the concentration of tetracycline hydrochloride at a fixed time point, and C0 represents the initial concentration of tetracycline hydrochloride.

[0033] Figure 13 Time-tracking CT of HEA-Cu2NPs-catalyzed oxidative degradation of chlortetracycline hydrochloride t / C0 variation curve, where C t C represents the concentration of chlortetracycline hydrochloride at a fixed time point, and C0 represents the initial concentration of tetracycline hydrochloride.

[0034] Figure 14 Time-tracking CT of HEA-Cu2NPs-catalyzed oxidative degradation of chlortetracycline hydrochloride t / C0 variation curve, where C t C represents the concentration of chlortetracycline hydrochloride at a fixed time point, and C0 represents the initial concentration of tetracycline hydrochloride.

[0035] Figure 15 (a) Plate images of Staphylococcus aureus treated under different conditions; (b) Relative survival rates under the corresponding experimental conditions;

[0036] Figure 16 (a) Plate images of Escherichia coli treated under different conditions; (b) Relative survival rates under the corresponding experimental conditions; Detailed Implementation

[0037] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0038] Example 1

[0039] After ultrasonically dissolving acetylacetone and the surfactant, equimolar amounts of iron acetylacetone, cobalt acetylacetone, nickel acetylacetone, molybdenum acetylacetone, and copper acetylacetone were added sequentially and mixed thoroughly. Simultaneously, an appropriate amount of NaOH was added to the reaction system, and the mixture was stirred until it became a paste. After standing at room temperature for two weeks and drying, it was mixed with molybdenum hexacarbonyl, calcined, washed, filtered, and dried. The amount of copper acetylacetone added was varied, and HEA-Cu1.5 and HEA-Cu2NPs were synthesized following the above synthesis steps.

[0040] Example 2

[0041] Test conditions for the POD-like activity of high-entropy alloy nanozyme materials: In a solution containing the reducing substrates TMB and H2O2, HEAzyme decomposes to generate reactive oxygen species. The absorbance of the single-electron oxidation product obtained from the catalytic oxidation of the reducing substrate changes over time at 652 nm. The effect of copper content on the POD-like activity of HEAzyme is evaluated by comparing the rate of increase in absorbance of the product under the catalysis of these three materials. The specific conditions for detecting the POD-like catalytic activity are as follows: At 25℃, TMB (1 mM), H2O2 (0.5 mM), and HEA (100 μg / mL) are added to a 2 mL centrifuge tube containing 0.1 M acetate-sodium acetate (HAc-NaAC) buffer (pH = 3.5), and the absorbance value of the above mixed solution at 652 nm is monitored by an enzyme-linked immunosorbent assay (ELISA) reader.

[0042] Figure 1 These are XRD patterns of high-entropy nanoalloys with different molar amounts of copper. Figure 1 This indicates the formation of a single-phase high-entropy nanoalloy with a typical face-centered cubic structure. The increase in copper content does not affect the crystal form and structure of the high-entropy alloy nanoenzyme. Figure 2 The field emission scanning electron microscope results for HEA-Cu2NPs show that the carbon substrate has porous properties, and the high-entropy alloy nanozymes are uniformly distributed on the porous carbon. Figure 3 The image is a transmission electron microscope (TEM) image of HEA-Cu2NPs, indicating that the size of the high-entropy alloy nanoparticles is approximately 10-15 nm. Figure 4 The transmission electron microscopy (TEM) image of HEA-Cu2NPs shows that the carbon material grown with high-entropy alloy nanoparticles has a large specific surface area and abundant micropores and mesopores. The most accessible pore size is 0.822 nm after DFT calculation. Figure 1-4 This indicates that the patent successfully synthesized porous carbon materials loaded with high-entropy alloy nanoparticles. Figure 5 Data indicate that HEA-Cu2NPs can catalyze the decomposition of H2O2 to generate hydroxyl radicals. Figure 6 The POD-like catalytic data of the materials in the study indicate that they are similar to HEA-Cu and HEA-Cu. 1.5 Compared to HEA-Cu2, the POD-like activity is increased by 3 times. Figure 7 This refers to the POD-like activity of high-entropy alloy nanozymes at different pH levels when other test conditions remain consistent. Figure 7 The data indicate that, with other conditions remaining constant, the material exhibits different POD-like activities at different pH environments. Its enzyme-like activity is highest at pH 3.5.

[0043] Example 3

[0044] Figure 8This indicates that, under otherwise constant conditions, as the material concentration increases from 0 g / L to 0.15 g / L, the degradation rate k of tetracycline hydrochloride increases from 0.00027 min. -1 Increased to 0.0542 min -1 As the material concentration increased from 0.15 g / L to 0.25 g / L, k increased from 0.0542 min. -1 Increased to 0.0867 min -1 As the material concentration increases, the degradation rate of tetracycline increases. However, when the material concentration reaches 0.15 g / L, the effect of increasing the material concentration on the reaction rate becomes smaller, and the cost of increasing the material increases. Considering all factors, a material concentration of 0.15 g / L was selected for subsequent experiments.

[0045] Figure 9 This indicates that, under otherwise constant conditions, tetracycline hydrochloride degradation is most efficient at pH 4 within the pH range of 2-10, with a degradation rate of 96.2% after 20 minutes, indicating almost complete degradation. However, in a strongly alkaline environment at pH 10, the degradation rate of tetracycline hydrochloride is only 40%, suggesting that OH- in a strongly acidic environment... - It inhibits the catalytic activity of the catalyst. Even in a strongly acidic environment with pH 2, tetracycline hydrochloride can still be completely degraded, but the degradation time is prolonged by 10 minutes, indicating that H+ in the acidic environment... + It has little effect on catalytic activity. The effect of pH on the degradation of tetracycline hydrochloride is consistent with the effect on enzyme-like activity.

[0046] Figure 10 This indicates that, under otherwise constant conditions, as the tetracycline hydrochloride concentration increases from 0.005 g / L to 0.01 g / L, the tetracycline hydrochloride degradation rate k increases from 0.0288 min / L. -1 Increased to 0.377 min -1 As the concentration of tetracycline hydrochloride increased from 0.01 g / L to 0.025 g / L, the degradation rate k of tetracycline hydrochloride decreased from 0.377 min. -1 Increased to 0.0264 min -1 When the concentration of tetracycline hydrochloride is 0.015 mg / L, the degradation rate k = 0.159 min. -1 The reaction rate was too fast when the TH concentration was less than 0.01 g / L, and the degradation rate was only 0.0288 when the TH concentration was greater than 0.2 g / L. Therefore, considering all factors, 0.15 g / L tetracycline hydrochloride and a degradation time of 20 min were selected for subsequent experiments.

[0047] Figure 11 This indicates that, under otherwise unchanged conditions, light has little effect on the degradation rate of tetracycline hydrochloride; the degradation rate without light decreases by only 2.8% compared to the rate under light.

[0048] Example 4

[0049] 400 μL of 1 mg / mL tetracycline hydrochloride solution, 3 mL of 1 mg / mL HEA-Cu2, and 16.6 mL of MES buffer solution (pH=4) were added to the solution. Samples were taken at regular intervals, and the absorbance of the liquid samples at 357 nm was measured. The rate of tetracycline hydrochloride degradation catalyzed by commercial HEA-Cu2 was obtained by observing the change in absorbance before and after tetracycline hydrochloride degradation. Figure 12 This indicates that when the reaction time reaches 20 minutes, the degradation rate of tetracycline hydrochloride by HEA-Cu2 is almost 100%.

[0050] Example 5

[0051] 400 μL of 1 mg / mL chlortetracycline hydrochloride solution, 3 mL of 1 mg / mL HEA-Cu2, and 16.6 mL of MES buffer solution (pH=4) were added to the solution. Samples were taken at regular intervals, and the absorbance of the liquid samples at 357 nm was measured. The rate of tetracycline hydrochloride degradation catalyzed by commercial HEA-Cu2 was obtained by observing the change in absorbance before and after the degradation of chlortetracycline hydrochloride. Figure 13 This indicates that when the reaction time reaches 20 minutes, the degradation rate of chlortetracycline hydrochloride by HEA-Cu2 is 83%.

[0052] Example 6

[0053] 400 μL of 1 mg / mL oxytetracycline hydrochloride solution, 3 mL of 1 mg / mL HEA-Cu2, and 16.6 mL of MES buffer solution (pH=4) were added to the solution. Samples were taken at regular intervals, and the absorbance of the liquid samples at 357 nm was measured. The rate of tetracycline hydrochloride degradation catalyzed by commercial HEA-Cu2 was obtained by observing the change in absorbance before and after the degradation of oxytetracycline hydrochloride. Figure 14 This indicates that when the reaction time reaches 20 minutes, HEA-Cu2 achieves a degradation rate of 96% for oxytetracycline hydrochloride.

[0054] Example 7

[0055] Evaluation of the in vitro bactericidal performance of high-entropy nanoalloys against Staphylococcus aureus. First, Staphylococcus aureus glycerol bacteria (S. aures) were cultured overnight in TSB liquid medium (200 rpm, 37℃). Subsequently, the bacteria were activated at a 1:100 concentration for 2 h (200 rpm, 37℃), and the activated bacteria in the logarithmic growth phase were used for antibacterial experiments. Next, S. aures (1×10⁻⁶) were... 6CFU / mL was incubated with different types of high-entropy alloy nanozymes (final concentration 600ug / mL) for 2 hours. After serial dilution with sterile PBS, 10μL was evenly spread on TSA solid medium and incubated at 37℃ for 24 hours for colony counting. Each group was repeated three times. Survival percentage = number of colonies in experimental group / number of colonies in control group.

[0056] Evaluation of the in vitro bactericidal properties of *Escherichia coli*. First, *E. coli* glycerol bacterium was cultured overnight in TSB liquid medium (200 rpm, 37°C). Then, the bacteria were activated at a 1:100 concentration for 2 hours (200 rpm, 37°C), and the activated bacteria in the logarithmic growth phase were used for the antibacterial experiment. Next, *E. coli* (1×10⁻⁶) was... 6 CFU / mL was used to incubate different types of high-entropy alloy nanozymes (final concentration 600 μg / mL) for 2 hours. After serial dilution with sterile PBS, 10 μL was evenly spread on TSA solid medium and incubated at 37°C for 24 hours for colony counting. Each group was repeated three times. Survival percentage = number of colonies in experimental group / number of colonies in control group.

[0057] Figure 15 , 16 The results showed that HEA-Cu2 inhibited the growth of Gram-positive Staphylococcus aureus by 81% and the growth of Gram-negative bacilli by 65%. Compared with the control group, HEA-Cu2 showed significantly better inhibitory effects on both Gram-positive Staphylococcus aureus and Gram-negative bacilli than HEA-Cu and HEA-Cu2. 1.5 The higher inhibition rate of HEA-Cu2 against Staphylococcus aureus compared to Escherichia coli may be due to the fact that Gram-positive bacteria are not protected by the outer cell membrane.

Claims

1. An application of a high-entropy alloy nanoenzyme, characterized in that: The application of porous carbon-supported high-entropy alloy nanoparticle FeCoNiMoCu2 composite material as a nanozyme in the catalytic degradation of antibiotics is as follows: high-entropy alloy nanozyme and tetracycline antibiotic solution are added to a Mes buffer solution containing a concentration of pH=4.

2. The application according to claim 1, characterized in that, The antibiotics include one or more of tetracycline hydrochloride, chlortetracycline hydrochloride, and oxytetracycline hydrochloride.

3. The application according to claim 1, characterized in that, The degradation reaction conditions include: pH value of 2-10, temperature of 10-30℃, and time of 10-60 min.

4. An application of a high-entropy alloy nanoenzyme, characterized in that: The application of porous carbon-supported high-entropy alloy nanoparticles FeCoNiMoCu2 composite material as nanozymes in the catalytic antibacterial action of antibiotics is as follows: porous carbon-supported high-entropy alloy nanoparticles FeCoNiMoCu2 composite material are added to bacterial suspension; the porous carbon-supported high-entropy alloy nanoparticles FeCoNiMoCu2 composite material catalyzes the generation of reactive oxygen species.

5. The application according to claim 4, characterized in that, The bacteria in question are bacteria.

6. The application according to claim 5, characterized in that, The bacteria are Staphylococcus aureus and / or Escherichia coli.

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