CuFe / OKCN nanoszyme, and preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a CuFe / OKCN nano-enzyme as well as a preparation method and application thereof. The CuFe / OKCN nano-enzyme is successfully prepared through one-step thermal polycondensation and in-situ deposition, and the preparation method is simple, low in cost and more safe and environmentally friendly. The prepared nano-enzyme can activate H2O2 to generate ·OH, so that colorless TMB is changed into blue TMB in the oxidized state, and has excellent peroxidase-like activity. Meanwhile, the peroxidase-like activity generates ·OH active species, and has a photocatalytic inactivation effect. The CuFe / OKCN nano-enzyme has the advantages of high stability, low influence on the environment and reusability, can be used for photocatalytic inactivation of drug-resistant bacteria in wastewater, has good application potential in the fields of environmental monitoring and wastewater treatment, and has a wide market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a CuFe / OKCN nanozyme, its preparation method, and its application. Background Technology

[0002] Antibiotic-resistant bacteria have been detected in various environments, including surface water, groundwater, sediments, soil, and air. This poses a potential threat to ecological balance and human well-being. Numerous studies have confirmed that human activities, including livestock farming, disease treatment, and industrial production, significantly contribute to the widespread occurrence and spread of antibiotic-resistant bacteria in the environment. Wastewater treatment plants serve as a link between human activities and the natural environment, collecting sewage and wastewater from all aspects of human society. The discharge of wastewater from these plants into rivers, lakes, oceans, and other aquatic environments leads to the spread of antibiotic-resistant bacteria. These bacteria can also be transmitted to humans through drinking water, the food chain, or direct contact, enabling certain human pathogens to acquire antibiotic resistance characteristics and endanger public health.

[0003] In wastewater treatment, chemical and physical disinfection methods are commonly used to inactivate pathogenic microorganisms in the wastewater. Commonly used chemical disinfection methods include chlorination and ozone disinfection; commonly used physical disinfection methods include ultraviolet disinfection, heat sterilization, and microwave technology. These traditional methods all have drawbacks such as poor killing effect, incomplete sterilization (easily leading to bacterial reactivation), and low sterilization efficiency. Some technologies also produce disinfection byproducts. Therefore, there is an urgent need to find a more promising sterilization technology.

[0004] Natural enzymes possess excellent catalytic efficiency and substrate specificity, but their relatively high cost and lack of stability significantly hinder their practical application in wastewater treatment. Nanozymes, artificial nanomaterials with enzyme-like catalytic properties, have the potential to replace natural enzymes. Furthermore, nanozymes offer advantages in wastewater treatment, including low cost, ease of preparation, high stability, low environmental impact, and reusability, making them a promising alternative to natural enzymes in environmental monitoring and wastewater treatment. However, they currently lack successful applications, particularly in the research of CuFe / OKCN nanozymes, where reports are scarce. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a CuFe / OKCN nanozyme, its preparation method, and its applications. The CuFe / OKCN nanozyme exhibits peroxidase-like activity, generating reactive oxygen species (ROS), which can be used for photocatalytic inactivation of drug-resistant bacteria in wastewater, achieving unexpected technical effects. It also possesses the advantages of being inexpensive, simple, and reusable.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution;

[0007] A method for preparing CuFe / OKCN nanozymes includes the following steps:

[0008] S1: Mix urea, formic acid and potassium nitrate, add ultrapure water and dissolve in a water bath; then dry, collect the dried product and calcine it. After the calcine reaction is complete, a solid product is obtained. Cool it naturally to room temperature and take out the cooled solid product, which is OKCN.

[0009] S2: Dissolve the OKCN prepared in S1 in anhydrous ethanol, sonicate, add ferric chloride and stir until dissolved, then add copper chloride and stir until dissolved, and finally add ammonium bicarbonate and continue stirring at room temperature. After the reaction, allow to stand, filter, wash and dry to obtain the final product, denoted as CuFe / OKCN nanozyme.

[0010] Preferably, the mass ratio of urea, formic acid, and potassium nitrate in S1 is 40000:1:200; the ratio of urea to ultrapure water is 20g:5mL.

[0011] Preferably, the calcination reaction temperature in S1 is 400-600℃ and the reaction time is 1-3h; more specifically, the calcination reaction temperature is 550-600℃ and the reaction time is 1-2h.

[0012] Preferably, the temperature of the water bath in S1 is 60°C;

[0013] Preferably, the drying temperature in S1 is 60-65℃, and the drying time is 6-12h.

[0014] Preferably, the mass ratio of OKCN, ferric chloride, copper chloride, and ammonium bicarbonate in S2 is 10:6.5:1:7; and the ratio of OKCN to anhydrous ethanol is 0.7g:40mL.

[0015] Preferably, the stirring reaction continues for 6-10 hours in step S2; more specifically, the stirring reaction lasts for 10 hours.

[0016] Preferably, in step S2, the washing involves sequentially washing with water and anhydrous ethanol 3-5 times; the drying temperature is 60-65°C, and the drying time is 6-12 hours.

[0017] The CuFe / OKCN nanozyme described in this invention exhibits excellent catalytic activity; in a reaction system consisting of 100 μg / mL CuFe / OKCN, 500 μmol / L TMB, 200 μmol / L H2O2 and pH=4.0 acetate buffer, the nanozyme exhibits the highest catalytic activity after being reacted at 35°C for 20 min.

[0018] The CuFe / OKCN nanozyme described in this invention is used for inactivating bacteria or for preparing sterilizing drugs.

[0019] Furthermore, the prepared CuFe / OKCN nanozyme has the application of photocatalytic inactivation of drug-resistant bacteria in wastewater; the drug-resistant bacteria include, but are not limited to, Escherichia coli or Staphylococcus aureus.

[0020] The steps are as follows: Place the drug-resistant bacteria in a reaction system of CuFe / OKCN nanozyme, HAc-NaAc buffer and H2O2, and irradiate with visible light for a period of time to achieve the purpose of inactivating the drug-resistant bacteria;

[0021] The concentration of the drug-resistant bacteria was 10. 7 CFU·mL -1 The concentration of CuFe / OKCN nanozyme in the reaction system is 100 μg·mL. -1 The concentration of H2O2 is 200 μmol·L. -1 The HAc-NaAc buffer solution had a concentration of 0.1 M and a pH of 5.5; the visible light source was irradiated with a power of 300 W for 60-75 min.

[0022] The beneficial effects of this invention are:

[0023] The CuFe / OKCN nanozyme disclosed in this invention can be obtained through a simple one-step thermal polycondensation and in-situ deposition method. The preparation method is simple, low-cost, highly stable, has low environmental impact, and is highly reproducible. Furthermore, the amount of raw materials has been optimized, achieving substantial results.

[0024] The CuFe / OKCN nanozyme obtained in this invention exhibits excellent peroxidase-like activity. By activating H₂O₂ to generate reactive oxygen species, it further exerts its photocatalytic inactivation effect on drug-resistant bacteria in wastewater, resulting in high sterilization efficiency and shorter treatment time. The widespread presence of drug-resistant bacteria in the environment poses a greater threat to human health. Therefore, effective inactivation of drug-resistant bacteria in wastewater has good application potential in environmental monitoring and wastewater treatment, with broad market prospects. Attached Figure Description

[0025] Figure 1 The graphs are for CuFe / OKCN; (a) is the XRD pattern of CuFe / OKCN; and (b) is the XPS pattern of CuFe / OKCN.

[0026] Figure 2 The diagram shows the optimized conditions for CuFe / OKCN; where (a) is the concentration of nanozyme; (b) is the pH optimization; (c) is the TMB concentration; (d) is the H2O2 concentration; (e) is the reaction time; and (f) is the reaction temperature.

[0027] Figure 3 This is a schematic diagram of the POD activity of CuFe / OKCN; where (a) is the measurement of absorbance change of oxTMB; (b) is a comparison of absorbance values ​​under light and dark conditions; (c) is the full wavelength of the metal cation; (d) is the full wavelength of the metal anion; (e) is the cycle number; and (f) is the fluorescence spectrum.

[0028] Figure 4 The graph shows the inactivation of CuFe / OKCN; where (a) is the E. coli colony count and (b) is the S. aureus colony count. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] Example 1: Preparation of CuFe / OKCN nanozymes

[0033] OKCN was prepared by a one-step thermal polycondensation method. Urea (20.00 g), formic acid (0.5 mg), and potassium nitrate (100.00 mg) were placed in an alumina crucible, and 5.0 mL of ultrapure water was added to dissolve them completely. The mixture was then placed in a 60°C water bath and stirred until homogeneous. The crucible was then dried in a 65°C oven for 6 hours. After the moisture had evaporated, the alumina crucible was removed from the oven, covered, and transferred to a muffle furnace. It was calcined at 600°C for 1 hour. After calcination, the muffle furnace was allowed to cool to room temperature. The alumina crucible was then removed, and the naturally cooled solid product was crushed into powder and labeled as OKCN.

[0034] Take 0.70 g OKCN and add 40.0 mL of anhydrous ethanol. Sonicate for 30 min and transfer to a round-bottom flask. Add 0.455 g ferric chloride and stir until dissolved. Then add 0.070 g copper chloride and stir rapidly for 10 min. Finally, add 0.490 g ammonium bicarbonate and continue stirring at room temperature for 10 h. After standing, filter to collect the precipitate and wash it three times each with water and anhydrous ethanol to remove excess reagents. Finally, dry at 60 °C to obtain CuFe / OKCN.

[0035] Example 2: Preparation of CuFe / OKCN nanozymes

[0036] OKCN was prepared by a one-step thermal polycondensation method. A mixture of urea (20.00 g), formic acid (0.5 mg), and potassium nitrate (100.00 mg) was placed in an alumina crucible. 5.0 mL of ultrapure water was added to the crucible to dissolve the mixture completely. The crucible was then placed in a 60°C water bath and stirred until homogeneous. The mixture was then dried in a 65°C oven for 6 hours. After the water had evaporated, the alumina crucible was removed from the oven, covered, and transferred to a muffle furnace. It was calcined at 400°C for 3 hours. After calcination, the muffle furnace was allowed to cool to room temperature. The alumina crucible was then removed, and the naturally cooled solid product was crushed into powder, placed in test tubes, and labeled as OKCN.

[0037] Take 0.70 g OKCN and add 40.0 mL of anhydrous ethanol. Sonicate for 30 min and transfer to a round-bottom flask. Add 0.455 g ferric chloride and stir until dissolved. Then add 0.070 g copper chloride and stir rapidly for 10 min. Finally, add 0.490 g ammonium bicarbonate and continue stirring at room temperature for 6 h. After standing, filter to collect the precipitate and wash it three times each with water and anhydrous ethanol to remove excess reagents. Finally, dry at 60 °C to obtain CuFe / OKCN.

[0038] Taking the CuFe / OKCN prepared in Example 1 as an example, subsequent experiments were conducted.

[0039] Example 3: Characterization of CuFe / OKCN

[0040] Figure 1(a) shows the X-ray diffraction (XRD) spectra, which characterized the crystal structures of CN, OKCN, and CuFe / OKCN nanozymes. The figure shows that CN, OKCN, and CuFe / OKCN exhibit typical g-C3N4 crystal diffraction peaks at diffraction angles 2θ≈13.6° and 27.5°, corresponding to the (100) and (002) crystal planes of g-C3N4, respectively. This indicates that the small amount of FeCl3 and CuCl2 doping during preparation did not change the crystal surface structure of g-C3N4. However, the peak intensities decreased significantly after Fe and Cu doping, which may be because the introduction of FeCl3 and CuCl2 inhibited the formation of g-C3N4 crystals during preparation. Meanwhile, no other diffraction peaks were observed in the XRD spectrum of the CuFe / OKCN nanozyme, indicating that the introduction of FeCl3 and CuCl2 did not cause any change in the OKCN crystal structure.

[0041] Figure 1 The X-ray photoelectron spectroscopy (XPS) in (b) clearly shows the presence of elements C, N, O, K, Fe, and Cu, which also proves the successful synthesis of CuFe / OKCN nanozyme.

[0042] Example 4: Condition optimization of CuFe / OKCN nanozyme

[0043] The catalytic activity of CuFe / OKCN nanozymes was investigated by conducting single-factor experiments on six factors: nanozyme concentration, pH, TMB, H2O2, time, and temperature.

[0044] The TMB concentration in the reaction system is 500 μmol·L⁻¹ -1 The H2O2 concentration is 200 μmol·L. -1 Acetate buffer, pH 4.0, with nanozyme concentrations varied to 0, 25, 50, 100, 150, and 200 μg / mL. -1 After the final 10 mL reaction mixture was prepared, it was reacted in a constant-temperature shaker at 35 °C for 20 min. 2 mL of the mixture was then transferred to a centrifuge tube and centrifuged at 15000 rpm. -1 Centrifuge at a certain speed for 1 min, collect the supernatant, and measure the changes in the absorption curve using UV-Vis.

[0045] Similarly, while keeping other experimental conditions constant, the pH (3.5, 4, 4.5, 5, 5.5, 6), TMB (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mM), H2O2 (0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mM), time (0, 5, 10, 15, 20, 30, 45, 60 min), and temperature (15, 25, 35, 45, 55, 65℃) were changed sequentially.

[0046] Considering the accuracy of UV-vis in actual use, combined with Figure 2 Based on the data and comprehensive consideration, the optimal conditions are: 100 μg / mL CuFe / OKCN in 500 μmol / L TMB and 200 μmol / L H2O2 in an acetate buffer solution at pH 4.0, at 35℃ for 20 min, exhibiting the highest catalytic activity and most significant effect of the nanozyme.

[0047] Example 5: Peroxidase-like activity of CuFe / OKCN nanozymes

[0048] Under the optimal conditions for nanozyme established in Example 4, 2 mL was taken into a centrifuge tube and centrifuged at 15000 r·min. -1 Centrifuge at a certain speed for 1 min, collect the supernatant, and measure the changes in the absorption curve using UV-Vis.

[0049] like Figure 3 As shown in (a), in the presence of the prepared CuFe / OKCN nanozyme, TMB and H2O2, there is an absorption peak at 652 nm, and the absorbance value increases under light irradiation. However, in the control group with TMB alone, without H2O2 or CuFe / OKCN nanozyme, there is no obvious color change or characteristic absorption peak.

[0050] Figure 3 Figure (b) shows that by comparing the absorbance values ​​under light and darkness, it is clear that the catalytic performance of the nanozyme is better under light; the above results confirm the POD-like activity of CuFe / OKCN.

[0051] Example 6: Anti-interference ability of CuFe / OKCN nanozymes

[0052] Under the optimal conditions for the nanozyme established in Example 4, 1 mM metal cation (Na₂) was added. + Ca 2+ Mg 2+ Mn + K + ) and 1mM metal anions (SO4) - HCO3 - HPO4 - NO3 - and Cl - Add 2 mL to the reaction system and centrifuge in a centrifuge tube at 15000 r·min. -1 Centrifuge at a certain speed for 1 min, collect the supernatant, and measure the changes in the absorption curve using UV-Vis.

[0053] Figure 3In Figure (c), the full wavelength of the metal cation is shown, and in Figure (d), the full wavelength of the metal anion is shown. It can be seen from Figures (c) and (d) that the enzyme activity of the ions does not change much, indicating that these interfering substances have no effect on the activity of CuFe / OKCN nanozymes, proving that CuFe / OKCN nanozymes have good anti-interference ability.

[0054] Example 7: Reproducibility of CuFe / OKCN Nanozymes

[0055] A concentration of 100 μg·mL -1 The nanozyme solution was prepared under the optimal conditions for nanozymes established in Example 4, at 15000 r·min -1 After centrifugation at a certain speed for 1 min, the absorbance of the supernatant was measured at 652 nm. Simultaneously, the precipitate (CuFe / OKCN nanozyme) was redispersed, and the enzyme activity measurement process was repeated. After 5 cycles, the activity of the CuFe / OKCN nanozyme remained at 85.5% of the maximum enzymatic reaction, indicating high recovery rate, good stability, and suitability for multiple cycles. Results are shown below. Figure 3 Figure (e) above shows that the CuFe / OKCN nanozyme has relatively high reproducibility.

[0056] Example 8: Peroxidase-like activity mechanism of CuFe / OKCN nanozymes

[0057] The generation of ·OH during the CuFe / OKCN nanozyme catalysis was determined by fluorescence method. The final concentrations of TA, CuFe / OKCN, H2O2, and HAc-NaAc buffer were 0.5 mM, 100 μg·mL, and 100 μg·mL, respectively. -1 The concentrations were 0.2 mM and pH = 4. The reaction was carried out at 35 °C for 20 min at an autoclave speed of 15000 r·min. -1 After centrifugation at a certain speed for 1 min, the fluorescence intensity at 435 nm was recorded at an excitation wavelength of 315 nm.

[0058] like Figure 3 In Figure (f), the fluorescence spectra of different combinations are shown. The presence of TA+H₂O₂+CuFe / OKCN exhibits fluorescence changes, with a significant increase in fluorescence intensity under illumination, while other groups show almost no fluorescence. This is similar to the results of CuFe / OKCN nanozymes oxidizing TMB. This indicates that CuFe / OKCN nanozymes possess POD-like activity and photo-enhanced POD-like activity.

[0059] Example 9: Inactivation of bacteria by CuFe / OKCN nanozymes

[0060] The antibacterial activity of the nanozyme was tested using a serial dilution method and a plate count method. 100 μg / mL -1The CuFe / OKCN nanozyme was uniformly dispersed in sterile water, and E. coli or S. aureus suspension (10). 7 CFU·mL -1 HAc-NaAc buffer (0.1M, pH=5.5), final H2O2 concentration 200 μmol·L⁻¹ -1 The total reaction volume was 100 mL. A 300 W Xe lamp (Philips, Netherland) and a UV cutoff filter (λ>400 nm) were used as the visible light source. A magnetic stirrer was turned on, and 100 μL of the reaction solution was aspirated at 0 min. The light source was either turned on or off, and the timer was started. At predetermined time intervals (0, 5, 10, 15, 20, 30, 45, 60, 75 min), 1 mL of the reaction solution was immediately aspirated and serially diluted with sterile water, twice (10-fold each time). 100 μL of the final dilution was evenly spread onto LB solid medium. The medium was inverted and incubated in a constant temperature incubator (37℃, 18 h). After incubation, photographs were taken, and the inactivation efficiency was assessed using the plate count method.

[0061] like Figure 4 As shown in (a), under 75 min of light irradiation, only the control group, H2O2, and CuFe / OKCN showed very low inactivation activity against E. coli, with almost no decrease in colony count, indicating that the cytotoxicity of H2O2 or CuFe / OKCN to E. coli is negligible. E. coli was completely inactivated after 75 min under the simultaneous presence of H2O2 and CuFe / OKCN. Figure 4 Figure (b) shows that *S. aureus* exhibits a similar behavior to *E. coli*, completely inactivating *S. aureus* after 60 minutes of illumination in the presence of both H₂O₂ and CuFe / OKCN. Compared to metal-MOF nanozymes, which require 3 hours to inactivate bacteria, the prepared CuFe / OKCN nanozyme completely inactivates bacteria in just 75 minutes, achieving an unexpectedly significant effect. The CuFe / OKCN nanozyme activates H₂O₂ to generate ·OH, thereby killing bacteria. More ·OH is generated under illumination, consistent with the nanozyme test results.

[0062] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. The use of a CuFe / OKCN nanozyme for inactivating bacteria, characterized in that, The preparation method of CuFe / OKCN nanozyme includes the following steps: S1: Mix urea, formic acid and potassium nitrate, add ultrapure water and dissolve in a water bath; then dry, collect the dried product and calcine it. After the calcine reaction is complete, a solid product is obtained. Cool it naturally to room temperature and take out the cooled solid product, which is OKCN. The mass ratio of urea, formic acid, and potassium nitrate is 40000:1:200; the ratio of urea to ultrapure water is 20g:5mL. S2: Dissolve the OKCN prepared in S1 in anhydrous ethanol, sonicate, add ferric chloride and stir until dissolved, then add copper chloride and stir until dissolved, and finally add ammonium bicarbonate. Continue stirring at room temperature. After the reaction, allow to stand, filter, wash and dry to obtain the final product, denoted as CuFe / OKCN nanozyme; the mass ratio of OKCN, ferric chloride, copper chloride and ammonium bicarbonate is 10:6.5:1:

7. The CuFe / OKCN nanozyme is used for photocatalytic inactivation of drug-resistant bacteria in wastewater; the drug-resistant bacteria include Escherichia coli or Staphylococcus aureus. The application steps are as follows: Place the drug-resistant bacteria in a reaction system of CuFe / OKCN nanozyme, HAc-NaAc buffer and H2O2, and irradiate with visible light for a period of time to achieve the purpose of inactivating the drug-resistant bacteria.

2. The use according to claim 1, characterized in that, The water bath temperature in S1 is 60 ℃; the drying temperature is 60-65 ℃, and the drying time is 6-12 h; the calcination reaction temperature is 400-600 ℃, and the reaction time is 1-3 h.

3. The use according to claim 2, characterized in that, The calcination reaction is carried out at a temperature of 550-600℃ for 1-2 hours.

4. The use according to claim 1, characterized in that, In step S2, the ratio of OKCN to anhydrous ethanol is 0.7 g : 40 mL; the stirring reaction continues for 6-10 h; the washing is performed by washing with water and anhydrous ethanol 3-5 times in sequence; the drying temperature is 60-65℃ and the drying time is 6-12 h.

5. The use according to claim 4, characterized in that, The stirring reaction was carried out over a period of 10 hours.

6. The use according to claim 1, characterized in that, The concentration of the drug-resistant bacteria was 10. 7 CFU·mL -1 The concentration of CuFe / OKCN nanozyme in the reaction system is 100 µg·mL. -1 The concentration of H2O2 is 200 μmol·L. -1 The concentration of HAc-NaAc buffer was 0.1 M, and the pH was 5.5; the power of the visible light source was 300W, and the time was 60-75 min.

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