Method for green synthesis of silver-cerium oxide-attapulgite multifunctional composite nano-enzyme through plant mediation

Abstract

The invention discloses a method for green synthesis of a silver-cerium oxide-attapulgite multifunctional composite nano-enzyme by plant mediation, which comprises the following steps: mixing cerous nitrate and silver nitrate with attapulgite suspension, adding a honeysuckle extract, adjusting the pH, heating for reaction, centrifugally washing and drying to obtain the plant-mediated green synthesis silver-cerium oxide-attapulgite multifunctional composite nano-enzyme. And calcining in a nitrogen atmosphere to obtain the silver-cerium oxide-attapulgite multifunctional composite nano-enzyme. Cerium ions and active ingredients in the honeysuckle flower extract form a complex, the complex is combined with attapulgite through hydrogen bonds, cerium oxide nano particles are obtained after calcination, and silver ions are reduced by the honeysuckle flower extract to obtain silver nano particles. The preparation process is green and environment-friendly, the process is simple, operation is easy, amplification is easy, and the prepared nano composite material has pH-responsive antibacterial and antioxidant multienzyme activity and can be used as a drug-resistant bacterium resisting nano material to be applied to the fields of biological medicine and the like.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterials, and in particular relates to a method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanoenzyme. Background Art

[0002] Nano-cerium dioxide has reversible Ce 3+ / Ce 4+ Redox pairs, which exhibit a variety of enzyme-like catalytic properties, are considered to be a promising class of nanozymes with potential for disease diagnosis and treatment. The surface chemistry of ceria nanoparticles is crucial for their enzymatic activity, and the abundant surface defects and large surface area can provide sufficient surface active sites, thereby providing obvious peroxidase-like activity. It has been reported that nanoceria with low levels of oxygen vacancy structural defects produce poor activity due to their weak affinity for substrates and low activation ability to produce reactive oxygen species. In general, the abundance of oxygen vacancy structural defects shows a clear size dependence, in which ceria nanoparticles with smaller sizes always introduce more oxygen vacancies. However, small-sized nanoceria are prone to agglomeration due to their high specific surface energy.

[0003] Studies have shown that carrier-type composite nanomaterials can regulate the physicochemical properties of metal oxide nanoparticles by overcoming the agglomeration of the nanoparticles. At the same time, the interface interaction between the carrier and the metal oxide can enhance the reactivity and stability of the nanozymes. Attapulgite is a natural one-dimensional clay mineral with a unique structure. It has a unique pore structure and surface silanol groups. On the one hand, it can provide nucleation sites for nanoparticles, and on the other hand, it can provide a highly concentrated substrate microenvironment for nanozymes, which is conducive to the adsorption and transport of substrates. It can be used as a carrier to regulate the activity of nano-cerium oxide enzymes. In addition, metal doping is also an important strategy to optimize the enzyme activity of cerium oxide nanoparticles. Nanosilver is one of the more mature antibacterial materials. Silver as a dopant can usually regulate the surface properties and catalytic properties of metal oxides. However, due to the huge difference in the redox potential of cerium ions and silver ions, the preparation method of silver and cerium oxide composite materials is complicated and inefficient, and needs to be further improved.

[0004] Plant-mediated green synthesis methods have the advantages of being clean, environmentally friendly, and producing green and safe products, and have attracted much attention in recent years. Plant extracts usually contain active biomolecules with rich functional groups (such as hydroxyl, carboxyl, amino, etc.), such as flavonoids, polyphenols, etc. These active ingredients can not only form complexes with metal ions, but also play a role in reducing and stabilizing nanoparticles. They have advantages in the preparation of multi-metal or metal oxide composite nanoparticles, and can achieve one-pot synthesis of multi-metal-based nanoparticles, greatly simplifying the synthesis process, avoiding complex post-processing operations in the intermediate links, and greatly improving production efficiency. The natural plant honeysuckle is an evergreen winding shrub with a wide distribution. Honeysuckle is a commonly used traditional Chinese medicine with a long history. It contains rich pharmacologically active ingredients such as chlorogenic acid, isochlorogenic acid and luteolin, and has antibacterial, anti-inflammatory, antioxidant, and heat-clearing and detoxifying effects. The phenolic acids and flavonoids of honeysuckle have rich phenolic hydroxyl structures, which can chelate and reduce metal ions and can be used as natural reagents to synthesize metal and metal oxide nanoparticles in a green way. Summary of the invention

[0005] The purpose of the present invention is to provide a method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme. The plant-mediated green synthesis technology is adopted, attapulgite is used as a carrier, the enzyme-like catalytic activity of cerium oxide nanoparticles is synergistically regulated by the carrier interface effect and silver nanoparticles, and the reaction conditions are regulated to synthesize the silver-cerium oxide-attapulgite multifunctional composite nanozyme in one pot. The preparation process is clean and environmentally friendly, simple and easy to operate, the product is highly safe, and has pH-responsive efficient bactericidal ability and free radical scavenging activity.

[0006] 1. Preparation and characterization of silver-cerium oxide-attapulgite multifunctional composite nanozyme The present invention provides a method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme, comprising the following steps: The fresh honeysuckle is cleaned, dried in a cool place and then ground into powder, and extracted with water at 55-75°C in a water bath for 1-2 hours, and filtered to obtain the honeysuckle extract; the attapulgite powder is ultrasonically dispersed in deionized water to obtain a attapulgite suspension, cerium nitrate and silver nitrate are added, and the mixture is stirred evenly and then added with the honeysuckle extract to obtain a precursor mixture; sodium hydroxide solution is slowly added to the precursor mixture to adjust the pH value, and then heated to 55-80°C for reaction for 2-5 hours, the obtained product is centrifuged, washed three times with deionized water, vacuum dried, and ground through a 200-mesh sieve; the product is transferred to a tubular furnace and calcined at 300-500°C in a nitrogen atmosphere for 1-3 hours to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0007] The solid-liquid ratio of the honeysuckle powder to water is 1:10-1:100 (g / mL). The solid-liquid ratio of the attapulgite suspension is 1:6-1:60 (g / mL). The volume ratio of the honeysuckle extract to the attapulgite suspension is 1:1-5:1.

[0008] The mass ratio of cerium ions in the cerium nitrate to attapulgite is 1:2.5-1:12; the molar ratio of silver ions in silver nitrate to cerium ions in cerium nitrate is 1:1-1:30.

[0009] The pH regulator is 0.5-5 mol / L sodium hydroxide, and the pH of the reaction system is 8-11. The vacuum drying is carried out at room temperature for 20-25 hours.

[0010] The synthesis mechanism of the present invention is: using phenolic acid and flavonoids in honeysuckle extract as reducing agents and stabilizers, attapulgite as a carrier, and green synthesis of silver-cerium oxide-attapulgite composite nanozyme. The redox potential of silver is +0.8V, so silver ions can be directly reduced by phenolic acid and flavonoids, and trivalent cerium ions are easy to complex with phenolic acid to form cerium complexes, which can generate cerium oxide after thermal decomposition. In addition, the present invention coordinates the enzyme-like catalytic activity of cerium oxide nanoparticles through the interface effect of attapulgite nanorod crystals and silver nanoparticles, so that the prepared ternary composite nanozyme has pH-responsive peroxidase activity and superoxide dismutase activity.

[0011] Figure 1 The XRD spectrum of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1 of the present invention, except for the characteristic diffraction peak of attapulgite (2 θ = 8.5°, 13.9°, 19.8°, 20.7°, etc.), in 2 θ The diffraction peaks at 28.5°, 33.1°, 47.4° and 56.3° correspond to the (111), (200), (220) and (311) crystal planes of cerium oxide, respectively. θ The diffraction peaks at 38.1°, 44.2°, 64.4° and 77.5° correspond to the (111), (200), (220) and (311) crystal planes of silver, respectively, indicating that cerium oxide and silver nanoparticles were successfully loaded on the surface of attapulgite nanorod crystals.

[0012] Figure 2 This is a TEM image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1. It can be clearly seen from the figure that It can be seen that the surface of the attapulgite nanorod crystals is loaded with uniformly dispersed nanoparticles; high-resolution image analysis shows that the particle size of cerium oxide nanoparticles is about 3nm, and the particle size of silver nanoparticles is about 5nm. The particles are evenly distributed and have good dispersion. This result further demonstrates the successful preparation of the silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0013] Figure 3 This is the FTIR spectrum of the silver-cerium oxide-attapulgite multifunctional composite nanozyme synthesized in Example 1. -1 and 2853.7cm -1 The absorption peaks of methyl and hydroxyl groups appeared at 486 cm, indicating that the plant active ingredients still existed on the surface of the nanoparticles after calcination, which was beneficial to the stability of the nanoparticles. −1 There is a strong absorption band corresponding to the metal-oxygen bond, indicating the formation of cerium oxide.

[0014] 2. Evaluation of the enzyme activity of silver-cerium oxide-attapulgite multifunctional composite nanozyme TMB was used to evaluate peroxidase activity. Briefly, a certain amount of sample (3 mg / mL) was mixed with TMB (0.5 mmol / L) and H in acetic acid-sodium acetate buffer. 2 O 2 (0.5mmol / L) was incubated at room temperature (27°C), and the absorbance (652nm) of the resulting mixture was detected at different time points. 2 O 2 The kinetic parameters were collected at the concentration of . The superoxide dismutase activity detection kit was used according to the instructions to evaluate the inhibitory effect of nanozymes on superoxide anions. Figure 4 This is the evaluation result of the peroxidase activity of the silver-cerium oxide-attapulgite multifunctional composite nanozyme. As can be seen from the figure, the nanozyme has excellent peroxidase activity, and the activity is strongest when the pH is less than 4; Figure 5 To show that the nanozyme can produce abundant hydroxyl radicals. Figure 6 The results of superoxide dismutase activity evaluation of silver-cerium oxide-attapulgite multifunctional composite nanozyme indicate that the nanozyme exhibits pH-dependent superoxide dismutase activity and has optimal enzyme activity when the pH is greater than 6.

[0015] 3. Antibacterial activity of Ag-Cerium oxide-attapulgite multifunctional composite nanozyme The antibacterial properties of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in the example were investigated by using methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase-producing Escherichia coli (ESBLs- E. coli ) is the test strain, and the plate count method is used for evaluation. The test method is: 1) ESBLs revived on the culture plate- E. coli 0.5 McF bacterial suspension was prepared with PBS for MRSA. The concentration of bacterial suspension was about 1.5×10 8 CFU / mL; dilute the bacterial suspension 100 times to make the final concentration of the bacterial suspension about 1×10 6 CFU / mL; 2) Add 20 µL of the sample dispersion and 50 mL of buffer (pH 3.7) to each well of a 96-well plate, and add 10 µL of H 2 O 2 (H 2 O 2 The final concentration is 100µM), add 20µL of bacterial suspension; after gently mixing, place in a 37°C incubator for 2h; after the incubation, take 20 µL of the co-culture solution and spread it on the nutrient agar medium, place in a 37°C incubator for 18~24h, and count. 3) After the incubation, take 20µL of the co-culture solution and spread it on the nutrient agar medium, place in a 37°C incubator for 18~24h, and count the colonies. The antibacterial performance evaluation results of the silver-cerium oxide-attapulgite multifunctional composite nanozyme in Examples 1-5 are shown in Table 1. It can be seen that the composite nanozyme prepared by the present invention is effective against Gram-positive MRSA and Gram-negative ESBLs- E. coli The inhibition rates of the two drug-resistant bacteria were both above 90%, indicating that the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared by the present invention has an effective inhibitory effect on ESBLs- E. coli and MRSA have high antibacterial activity.

[0016] 2. Antioxidant properties of silver-cerium oxide-attapulgite multifunctional composite nanozyme The antioxidant properties of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in the example were evaluated by the salicylic acid method. Specifically, 1 mL of 6 mmol / L ferric sulfate heptahydrate, 1 mL of 9 mmol / L salicylic acid ethanol solution, and 1 mL of samples of different concentrations (0.1-5 mg / mL) were added to a 10 mL test tube, and then 0.1 mL of hydrogen peroxide (0.3%) and 1 mL of ultrapure water were added to start the reaction. The test tube was then placed in a 37 ° C water bath for 30 min, and the absorbance was measured at 510 nm. Ultrapure water was used instead of the sample as a blank group, and 0.1 mL of ultrapure water was used instead of H 2 O 2 As a control group, each group was tested three times. Figure 7 It shows that the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared by the present invention has excellent hydroxyl radical scavenging activity.

[0017] In summary, the plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme in the present invention has the following advantages: 1. Using natural plant extracts as reducing agents and stabilizers, the one-pot preparation of silver and cerium oxide composite nanoparticles is achieved. This method is clean and environmentally friendly, has a simple process, and the product has high safety and good stability. It is a clean and green preparation method.

[0018] 2. The present invention synergistically regulates the enzyme-like catalytic activity of cerium oxide nanoparticles through the interfacial effect of attapulgite nanorod crystals and silver nanoparticles, effectively enhancing the enzyme-like catalytic performance of cerium oxide, and the prepared nanozyme has pH-responsive antibacterial and antioxidant dual effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the XRD pattern of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 2 This is a TEM image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 3 This is the FTIR image of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 4 The peroxidase activity evaluation result of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 5 The test result of hydroxyl radical generation by the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 6 This is the evaluation result of superoxide dismutase activity of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; Figure 7 The evaluation results of the hydroxyl radical scavenging activity of the silver-cerium oxide-attapulgite multifunctional composite nanozyme prepared in Example 3 of the present invention; DETAILED DESCRIPTION

[0020] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0021] Example 1 Fresh honeysuckle was cleaned, dried in a cool shade, and then ground into powder. 7 g was weighed and added into 100 mL of deionized water, extracted in a water bath at 60°C for 1.5 h, and filtered to obtain a honeysuckle extract. 5 g of attapulgite powder was ultrasonically dispersed in 30 mL of deionized water to obtain a attapulgite suspension, 1.26 g of cerium nitrate and 0.4 g of silver nitrate were added, and after stirring, 90 mL of honeysuckle extract was added to obtain a precursor mixture. 1 M sodium hydroxide was slowly added to the precursor mixture to adjust the pH to 9, and then heated to 80°C for reaction for 2 h. The obtained product was centrifuged, washed three times with deionized water, vacuum dried at room temperature for 24 h, and then ground through a 200-mesh sieve. The product was transferred into a tubular furnace and calcined at 500°C for 2 h in a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0022] Embodiment 2: Fresh honeysuckle was cleaned, dried in a cool shade and then ground into powder. 5 g was weighed and added into 100 mL of deionized water, extracted in a water bath at 55°C for 2 h, and filtered to obtain a honeysuckle extract. 0.5 g of attapulgite powder was ultrasonically dispersed into 30 mL of deionized water to obtain a attapulgite suspension, 0.63 g of cerium nitrate and 0.008 g of silver nitrate were added, and after stirring, 60 mL of honeysuckle extract was added to obtain a precursor mixture. 0.5 M sodium hydroxide was slowly added to the precursor mixture to adjust the pH to 8, and then heated to 55°C for reaction for 5 h. The obtained product was centrifuged, washed three times with deionized water, dried in vacuo at room temperature for 24 h, and then ground through a 200-mesh sieve. The product was transferred into a tubular furnace and calcined at 500°C for 2 h in a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0023] Embodiment 3: Fresh honeysuckle was cleaned, dried in a cool shade, and then ground into powder. 3 g was weighed and added into 100 mL of deionized water, extracted in a water bath at 60°C for 1.5 h, and filtered to obtain a honeysuckle extract; 2 g of attapulgite powder was ultrasonically dispersed in 30 mL of deionized water to obtain a attapulgite suspension, 1 g of cerium nitrate and 0.095 g of silver nitrate were added, and after stirring, 30 mL of honeysuckle extract was added to obtain a precursor mixture; 2 M sodium hydroxide was slowly added to the precursor mixture to adjust the pH to 9, and then heated to 60°C for reaction for 3 h. The obtained product was centrifuged, washed three times with deionized water, dried in vacuo at room temperature for 24 h, and then ground through a 200-mesh sieve; the product was transferred into a tubular furnace and calcined at 500°C for 2 h in a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0024] Embodiment 4: Wash fresh honeysuckle, dry it in a cool place, grind it into powder, weigh 9g, add it into 100mL deionized water, extract it in a water bath at 70°C for 1h, and filter it to get the honeysuckle extract; ultrasonically disperse 3g of attapulgite powder into 30mL of deionized water to get an attapulgite suspension, add 3g Cerium nitrate and 0.19g silver nitrate were stirred evenly, and then 120mL honeysuckle extract was added to obtain a precursor mixture; 3M sodium hydroxide was slowly added to the above precursor mixture to adjust the pH to 10, and then heated to 70°C for 2h. The obtained product was centrifuged and washed three times with deionized water. After vacuum drying at room temperature for 24h, it was ground through a 200-mesh sieve; the product was transferred into a tubular furnace and calcined at 400°C for 2h in a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

[0025] Embodiment 5: Wash fresh honeysuckle, dry it in a cool place, grind it into powder, weigh 10 g, add it into 100 mL of deionized water, extract it in a water bath at 65°C for 1.5 h, and filter it to obtain the honeysuckle extract; ultrasonically disperse 4 g of attapulgite powder into 30 mL of deionized water to obtain an attapulgite suspension, add 3 g of cerium nitrate and 0.13 g of silver nitrate, stir well, and then add 150 mL of honeysuckle extract to obtain a precursor mixture; add the above precursor mixture to the 5 M sodium hydroxide was slowly added to the liquid to adjust the pH to 11, and then heated to 60°C for reaction for 3 hours. The obtained product was centrifuged, washed three times with deionized water, dried in vacuum at room temperature for 24 hours, and then ground through a 200-mesh sieve. The product was transferred into a tubular furnace and calcined at 300°C for 2 hours under a nitrogen atmosphere to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

Claims

1. A method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme, comprising the following steps: The fresh honeysuckle is cleaned, dried in a cool place and then ground into powder, extracted with water at 55-75°C in a water bath for 1-2 hours, and filtered to obtain the honeysuckle extract; the attapulgite powder is ultrasonically dispersed in deionized water to obtain a attapulgite suspension, cerium nitrate and silver nitrate are added, and the mixture is stirred evenly and then added with the honeysuckle extract to obtain a precursor mixture; sodium hydroxide solution is slowly added to the precursor mixture to adjust the pH value, and then heated to 55-80°C for reaction for 2-5 hours, the obtained product is centrifuged and washed three times with deionized water, vacuum dried, and then ground and sieved; the product is transferred to a tubular furnace and calcined at 300-500°C in a nitrogen atmosphere for 1-3 hours to obtain a silver-cerium oxide-attapulgite multifunctional composite nanozyme.

2. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: The solid-liquid ratio of the honeysuckle powder to water is 1:10-1:

100.

3. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: The solid-to-liquid ratio of the attapulgite suspension is 1:6-1:

60.

4. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: The mass ratio of cerium ions in the cerium nitrate to attapulgite is 1:2.5-1:12; the molar ratio of silver ions in silver nitrate to cerium ions in cerium nitrate is 1:1-1:

30.

5. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: The volume ratio of the honeysuckle extract to the attapulgite suspension is 1:1 to 5:

1.

6. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: The pH regulator is 0.5-5 mol / L sodium hydroxide, and the pH of the reaction system is 8-11.

7. The method for plant-mediated green synthesis of silver-cerium oxide-attapulgite multifunctional composite nanozyme according to claim 1, characterized in that: Vacuum drying is done at room temperature for 20 to 25 hours.

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