Preparation method of antibacterial TPN-CuAg nano-cluster

Abstract

The invention discloses a preparation method of an antibacterial TPN-CuAg nano-cluster, and belongs to the technical field of metal materials. According to the method, sulfydryl in tiopronin (TPN) is complexed with Cu < 2 + > and Ag < + >, and a net-shaped structure nano-cluster is formed through step-by-step self-assembly under the condition of normal temperature. The method specifically comprises the following steps: 1) accurately preparing and calibrating tiopronin, copper nitrate and silver nitrate standard solutions; and 2) sequentially adding the copper nitrate solution and the silver nitrate solution into the tiopronin solution, controlling the reaction condition (20-30 DEG C) through magnetic stirring, and finally centrifuging, washing and freeze-drying to obtain the TPN-CuAg nano-cluster. High temperature or reducing agents are not needed, and the process is mild and efficient. Results show that the minimal inhibitory concentrations (MIC) of TPN-CuAg to escherichia coli and staphylococcus aureus are respectively 128 mu g / mL and 512 mu g / mL, the bacteriostasis rate exceeds 90%, and the TPN-CuAg realizes sterilization by destroying the cell membrane structure of bacteria. Meanwhile, the scavenging rate of TPN-CuAg to DPPH free radicals under the concentration of 0.13 mg / mL reaches 90%, and the TPN-CuAg has remarkable antioxidant activity. The nano-cluster is simple in preparation process, excellent in antibacterial performance, environment-friendly and suitable for development of antibacterial materials in the fields of food, medical treatment and the like.

Description

Technical Field

[0001] The present invention relates to the field of metal materials, and particularly to a preparation method of antibacterial TPN-CuAg nanoclusters. Background Art

[0002] At present, bacteria can cause infectious diseases, leading to a series of health problems. Especially in terms of food safety, the spread of Escherichia coli and Staphylococcus aureus can contaminate food and cause diseases. Infection with Escherichia coli can cause gastrointestinal infections, urinary tract infections, etc. Similarly, Staphylococcus aureus is highly toxic, and being infected can cause food poisoning or even septicemia. Therefore, the preparation of antibacterial agents is particularly important.

[0003] Metal antibacterial agents have received extensive attention due to their physical and chemical properties that can effectively inhibit most bacteria. Most of the metals required in antibacterial agents are transition elements such as silver, gold, copper, iron, zinc, etc. They have redox activity due to the filling of d orbitals, are more likely to form nanoparticles, and their binding ability to R-SH is stronger.

[0004] Especially silver-copper metal nanoparticles. Silver nanoparticles can cause changes in the membrane potential of bacteria, increasing the permeability of the cell wall and entering the bacteria to cause damage to the bacteria. Copper is an essential trace element in the human body. Similarly, single copper nanoparticles can release copper ions to damage the cell membrane of bacteria. Metal antibacterial agents can not only use a single metal alone but also use multiple metals in combination.

[0005] In the preparation methods of metal materials disclosed in patents such as CN115254109B, CN119243063A, CN117160459B, etc., the synthesis methods are relatively complex, the synthesis process is cumbersome, some involve high temperatures, etc., and there is a lack of mild preparation methods.

[0006] In the present invention, we report the synthesis of TPN-CuAg by complexing copper ions and silver ions with the sulfhydryl group in tiopronin. The inhibitory activities against Escherichia coli and Staphylococcus aureus are demonstrated by the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), inhibition-concentration curve, and inhibition rate. In addition, scanning electron microscopy (SEM) is used to explore the antibacterial mechanism. Finally, the DPPH radical scavenging rate of TPN-CuAg is also tested to discuss the antioxidant activities of the three. Summary of the Invention

[0007] Object of the Invention: The present invention aims to provide a preparation method of antibacterial TPN-CuAg nanoclusters synthesized by a mild method.

[0008] The present invention is achieved through the following technical solutions: A method for synthesizing antibacterial TPN-CuAg nanoclusters, characterized in that: the composite nano-metal material is formed by the complexation of copper ions and silver ions with the sulfhydryl groups in tiopronin to form extremely small nanoclusters, which form a similar network structure through self-assembly. The synthesis process of the antibacterial material TPN-CuAg includes the following two steps: The first step is the preparation of tiopronin, copper nitrate, and silver nitrate standard solutions: First, weigh 1.0000 - 2.0000 g of tiopronin with an electronic balance with a precision of 0.0001 g, place it in a 150 mL beaker, add 40 - 60 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask, and make up the volume to the calibration line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 tiopronin, and dry KIO 3 in an oven at 105 °C for 2 hours, cool to room temperature, and accurately weigh about 0.0178 g of KIO 3 , place it in a 250 mL iodine flask, add 50 mL of distilled water, shake until completely dissolved, add 5 mL of 6M HCl to make the solution strongly acidic, add 2 g of KI solid, shake well and let stand in the dark for 10 min. I 2 is generated, titrate with the tiopronin solution until the solution turns light yellow, add 2 mL of starch indicator, the solution turns blue, continue titrating until the blue color disappears, record the volume of tiopronin consumed, and calculate the concentration of the tiopronin standard solution; then, weigh 2.0000 - 3.0000 g of copper nitrate trihydrate and place it in a 150 mL beaker, add 30 - 40 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask, and make up the volume to the calibration line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01mol•L -1 copper nitrate, accurately weigh 3.7224 g of disodium EDTA, dissolve it in deionized water, transfer it to a 1 L volumetric flask and make up the volume, shake well and set aside. Accurately measure 25.00 mL of a 0.01 M Cu(NO 3 ) 2In a conical flask, add 10 mL of HAc-NaAc buffer solution with a pH of 5.0. Add 2 - 3 drops of PAN indicator (ethanol solution of 0.1% 1-(2-pyridylazo)-2-naphthol). The solution turns blue. Titrate with the EDTA standard solution until the solution suddenly changes from blue to bright yellow, and record the volume of EDTA consumed. Repeat the titration 2 - 3 times. Take the two results with a volume difference ≤ 0.02 mL and calculate the average value to accurately calculate the concentration of the copper nitrate solution. Finally, weigh 1.0000 - 2.0000 g of silver nitrate and place it in a 150 mL beaker. Add 20 - 30 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask and make up to the mark with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 For silver nitrate, weigh 0.1461 g of dried NaCl. Dissolve the NaCl in deionized water, transfer it to a 100 mL volumetric flask, make up to the mark and shake well to obtain a 0.025 mol•L -1 NaCl standard solution. Use a pipette to accurately transfer 25.00 mL of the NaCl standard solution to a conical flask. Add 0.1 g of calcium carbonate to make the solution pH 6.5 - 10.5 (neutral to weakly alkaline). Add 1 mL of 5% K 2 CrO 4 solution, shake well, and titrate with the prepared 0.01 mol•L -1 to determine AgNO 3 solution. Shake while titrating until the solution shows a brick-red Ag 2 CrO 4 precipitate as the end point, record the consumed volume, perform parallel determinations 3 times, take the average value, and the relative deviation should be ≤ 0.2%. Store in the dark for later use; The second step is the preparation of antibacterial TPN-CuAg nanoclusters: Measure 20 - 40 mL of the above-prepared tiopronin standard solution and add it to a 250 mL three-necked flask. Place the three-necked flask in a magnetic stirring water bath. Then measure 5 - 15 mL of the above-prepared copper nitrate standard solution and add it dropwise to the three-necked flask. React at a speed of 500 - 800 rpm at 20 - 30 °C for 30 - 50 min to obtain a reaction mixture solution. Finally, measure 5 - 15 mL of the above-prepared silver nitrate standard solution and add it to the above reaction mixture solution. React at a speed of 600 - 800 rpm at 20 - 30 °C for 16 - 18 h. The color of the reaction mixture solution gradually changes from green to orange-yellow. Divide the reaction solution equally into two 50 mL centrifuge tubes, centrifuge at a speed of 8000 - 1000 rpm for 10 min to collect the lower-layer precipitate, and wash and centrifuge it repeatedly with deionized water 3 times to obtain antibacterial TPN-CuAg nanoclusters. Freeze-dry them. Finally, put the obtained orange-yellow powder into a refrigerator at 4 °C for storage and standby.

[0009] Advantages compared with the prior art: The TPN-CuAg nanoclusters of the present invention are prepared by a two-step synthesis method to obtain a material with effective antibacterial properties. Antibacterial experiments in vitro show that the MIC values of TPN-CuAg nanoclusters against Escherichia coli and Staphylococcus aureus are 128 μg / mL and 512 μg / mL respectively, and the antibacterial rate against the two bacteria reaches more than 90%; the antioxidant activity of TPN-CuAg is tested experimentally, and the results show that TPN-CuAg nanoclusters have an effect on the antioxidant activity against DPPH, reaching more than 90% at 0.13 mg / mL. Description of the Drawings

[0010] Figure 1 is the antibacterial route of the TPN-CuAg nanosynthesized by the present invention against Escherichia coli and Staphylococcus aureus.

[0011] Figure 2 are the SEM and TEM images of the TPN-CuAg nanosynthesized by the present invention.

[0012] Figure 3 are the ultraviolet-visible light spectrum (A) and infrared spectrum (B) of the TPN-CuAg nanosynthesized by the present invention.

[0013] Figure 4 is the MIC graph of the TPN-CuAg nanosynthesized by the present invention against Escherichia coli and Staphylococcus aureus.

[0014] Figure 5 is the MBC graph of the TPN-CuAg nanosynthesized by the present invention against Escherichia coli and Staphylococcus aureus.

[0015] Figure 6 SEM images of the effects of the TPN-CuAg nanoparticles synthesized in the present invention on Escherichia coli and Staphylococcus aureus.

[0016] Figure 7 Growth curves of the TPN-CuAg nanoparticles synthesized in the present invention against Escherichia coli and Staphylococcus aureus.

[0017] Figure 8 Bacteriostatic rate graphs of the TPN-CuAg nanoparticles synthesized in the present invention against Escherichia coli and Staphylococcus aureus at the same concentration.

[0018] Figure 9 DPPH radical scavenging rate graph of the TPN-CuAg nanoparticles synthesized in the present invention.

[0019] Figure 1 The preparation of TPN-CuAg is achieved by the complexation of the sulfhydryl group in tiopronin (TPN) with metal ions Cu 2+ and Ag + without using a reducing agent in a greenhouse environment. Figure 1 It shows the synthesis process of TPN-CuAg and exhibits bacteriostatic activity against Escherichia coli and Staphylococcus aureus. After co-incubation with bacteria, TPN-CuAg can release metal ions, causing damage to the bacterial cell membrane, lysing into the interior of the bacteria for destruction, and resulting in the inactivation and death of the bacteria.

[0020] Figure 2 shows the SEM and TEM images of this example. The pictures (A) and (B) of TPN-CuAg taken by SEM resemble an aggregated network structure, and through the observation of TEM (C) and (D), TPN-CuAg is composed of smaller nanoparticles aggregated together.

[0021] Figure 3 shows the ultraviolet-visible spectrum (A) and infrared spectrum (B) of this example. TPN-CuAg has a broad ultraviolet absorption peak at 250 - 350 nm and 400 - 500 nm respectively. This may be due to the addition of silver resulting in the appearance of a new ultraviolet absorption peak. Separate tiopronin (TPN) has a stretching vibration peak at 2529 cm -1 indicating the presence of an S-H group in this substance. After the reaction between TPN and Cu / Ag is completed, the peak at 2529 cm -1 in the infrared spectrum of the generated TPN-CuAg disappears. This is because the interaction between the sulfhydryl group and metal Cu / Ag cleaves the S-H bond to form a new S-Cu(Ag) bond. At the same time, in the figure, it can also be observed that due to the stretching vibrations of the amino and carbonyl groups of amides, the sample has peaks at approximately 3500 cm -1 and 1635 cm-1 Characteristic peaks appear everywhere, indicating that the material is generated by the complexation reaction between the mercapto group in tiopronin and the metal.

[0022] Figure 4 This is the MIC graph of TPN-CuAg synthesized in this example against Escherichia coli and Staphylococcus aureus. The 96-well plate method was used to determine the MIC values of each material against Escherichia coli and Staphylococcus aureus. It can be observed with the naked eye that after a certain concentration and as the concentration increases, there is no precipitation in the wells, indicating that this concentration has a certain inhibitory effect on the bacteria. The MIC values of TPN-CuAg against Escherichia coli and Staphylococcus aureus are 128 μg / mL and 512 μg / mL, respectively.

[0023] Figure 5 This is the MBC graph of TPN-CuAg synthesized in this example against Escherichia coli and Staphylococcus aureus. (A) is Escherichia coli, and (B) is Staphylococcus aureus. The plate pictures show that Escherichia coli and Staphylococcus aureus were observed in the culture dishes at the MIC concentration of TPN-CuAg, while no bacteria were produced in the culture dishes incubated at 256 μg / mL and 1024 μg / mL. This also indicates that this concentration is the MBC value of TPN-CuAg against this bacterium.

[0024] Figure 6 This is the electron microscopy graph of the action of TPN-CuAg synthesized in this example against Escherichia coli and Staphylococcus aureus. Figures (A) and (C) are the electron microscopy graphs of untreated bacteria. It can be seen from the figures that Staphylococcus aureus is spherical, and Escherichia coli presents a rod-shaped morphology, with smooth, complete and plump surfaces. After co-incubation with TPN-CuAg, as shown in Figures (B) and (D), the bacterial cells shrink and lyse, accompanied by the leakage of internal substances. It can be seen from this that TPN-CuAg has a damaging effect on the overall morphology of Escherichia coli and Staphylococcus aureus.

[0025] Figure 7 This is the growth curve of TPN-CuAg synthesized in this example against Escherichia coli and Staphylococcus aureus. The OD value at 600 nm was measured with a UV spectrophotometer to evaluate and compare the antibacterial properties of TPN-CuAg within 8 h. The black lines in Figures (A) and (B) are the growth curves of Escherichia coli or Staphylococcus aureus within 8 h as the control group, and the green lines represent the growth curves of Escherichia coli or Staphylococcus aureus after TPN-CuAg within 8 h. The OD value after incubation with TPN-CuAg remains at the same level over time, indicating that at this concentration, TPN-CuAg has strong antibacterial ability and stability against Escherichia coli and Staphylococcus aureus.

[0026] Figure 8The antibacterial rate of the TPN-CuAg synthesized in this example against Escherichia coli and Staphylococcus aureus at the same concentration. Figure (A) shows the antibacterial rates calculated after incubating three materials in the experiment with Escherichia coli in a 37°C incubator for 16 h at concentrations of 32 μg / mL, 64 μg / mL, and 128 μg / mL. For Staphylococcus aureus, the antibacterial rates were calculated after incubating with three materials at concentrations of 128 μg / mL, 256 μg / mL, and 512 μg / mL for 16 h, as shown in Figure (B). It can be shown that the antibacterial rates at their respective MIC concentrations reached 90%.

[0027] Figure 9 It is a graph of the DPPH radical scavenging rate of the TPN-CuAg nanoparticles synthesized in this example. The antioxidant activity of TPN-CuAg increases with the increase in concentration. The scavenging rate of TPN-CuAg reaches about 90% at 0.13 mg / mL and reaches equilibrium after 0.2 mg / mL. Specific implementation method

[0028] A preparation method of an antibacterial TPN-CuAg nanocluster, characterized in that the antibacterial material is formed by the complexation of copper ions and silver ions with the thiol groups in tiopronin to form extremely small nanoclusters, which form a similar network structure through self-assembly. The preparation process of the antibacterial material TPN-CuAg includes the following two steps: The first step is the preparation of tiopronin, copper nitrate, and silver nitrate standard solutions: First, weigh 1.0000 - 2.0000 g of tiopronin with an electronic balance with a precision of 0.0001 g, place it in a 150 mL beaker, add 40 - 60 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask, and make up to the scale line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 Tiopronin, add KIO 3 Dry it in an oven at 105°C for 2 hours, cool it to room temperature, accurately weigh about 0.0178 g of KIO 3 , place it in a 250 mL iodine flask, add 50 mL of distilled water, shake until completely dissolved, add 5 mL of 6M HCl to make the solution strongly acidic, add 2 g of KI solid, shake well and let it stand in the dark for 10 min. The reaction generates I 2, titrate with tiopronin solution until the solution turns light yellow, add 2 mL of starch indicator, the solution turns blue, continue titrating until the blue color disappears, record the volume of tiopronin consumed, and calculate the concentration of the tiopronin standard solution; then, weigh 2.0000 - 3.0000 g of copper nitrate trihydrate and place it in a 150 mL beaker, add 30 - 40 mL of deionized water to the beaker and stir to dissolve, transfer the dissolved solution to a 100 mL volumetric flask, and make up to the mark with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 copper nitrate, accurately weigh 3.7224 g of disodium EDTA, dissolve it in deionized water, transfer it to a 1 L volumetric flask and make up to the mark, shake well for standby. Use a pipette to accurately measure 25.00 mL of a 0.01 M Cu(NO 3 ) 2 solution into a conical flask, add 10 mL of HAc-NaAc buffer solution with a pH of 5.0, add 2 - 3 drops of PAN indicator (ethanol solution of 0.1% 1-(2-pyridylazo)-2-naphthol), the solution turns blue, titrate with the EDTA standard solution until the solution changes from blue to bright yellow suddenly, record the volume of EDTA consumed, repeat the titration 2 - 3 times, take the average value of the two results with a volume difference ≤ 0.02 mL, and accurately calculate the concentration of the copper nitrate solution; finally, weigh 1.0000 - 2.0000 g of silver nitrate and place it in a 150 mL beaker, add 20 - 30 mL of deionized water to the beaker and stir to dissolve, transfer the dissolved solution to a 100 mL volumetric flask, and make up to the mark with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 silver nitrate, weigh 0.1461 g of dried NaCl, dissolve the NaCl in deionized water, transfer it to a 100 mL volumetric flask, make up to the mark and shake well to obtain a 0.025 mol•L -1 NaCl standard solution. Use a pipette to accurately transfer 25.00 mL of the NaCl standard solution to a conical flask, add 0.1 g of calcium carbonate to make the solution pH 6.5 - 10.5 (neutral to weakly alkaline), add 1 mL of 5% K 2 CrO 4 solution, shake well, titrate with the prepared 0.01 mol•L -1 to determine AgNO 3 solution, shake while titrating until the solution shows a brick-red Ag 2 CrO 4 precipitate as the end point, record the volume consumed, perform parallel determinations 3 times, take the average value, and the relative deviation should be ≤ 0.2%. Store in the dark for standby; 1.2 The second step is the preparation of antibacterial TPN-CuAg nanoclusters: Measure 20 - 40 mL of the above-prepared tiopronin standard solution and add it to a 250 mL three-necked flask. Place the three-necked flask in a magnetic stirring water bath. Then measure 5 - 15 mL of the above-prepared copper nitrate standard solution and add it dropwise to the three-necked flask. React at a speed of 500 - 800 rpm at 20 - 30 °C for 30 - 50 min to obtain a reaction mixture solution. Finally, measure 5 - 15 mL of the above-prepared silver nitrate standard solution and add it to the above reaction mixture solution. React at a speed of 600 - 800 rpm at 20 - 30 °C for 16 - 18 h. The color of the reaction mixture solution gradually changes from green to orange-yellow. Divide the reaction solution equally into two 50 mL centrifuge tubes, centrifuge at a speed of 8000 - 1000 rpm for 10 min to collect the lower-layer precipitate, and wash and centrifuge it 3 times repeatedly with deionized water to obtain antibacterial TPN-CuAg nanoclusters. Freeze-dry them. Finally, put the obtained orange-yellow powder into a refrigerator at 4 °C for storage and standby. Specific embodiments

[0029] The preparation of antibacterial TPN-CuAg nanoclusters is achieved by the complexation of copper ions and silver ions with the sulfhydryl groups in tiopronin, forming a network structure through self-assembly. The preparation process of TPN-CuAg nanoclusters includes the following two steps: The first step is the preparation of tiopronin, copper nitrate, and silver nitrate standard solutions: First, weigh 1.6319 g of tiopronin with an electronic balance with an accuracy of 0.0001 g, place it in a 150 mL beaker, add 50 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask and make up the volume to the calibration line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 tiopronin, and dry KIO 3 in an oven at 105 °C for 2 hours, cool to room temperature, accurately weigh about 0.0178 g of KIO 3 , place it in a 250 mL iodine flask, add 50 mL of distilled water, shake until completely dissolved, add 5 mL of 6M HCl to make the solution strongly acidic, add 2 g of KI solid, shake well and let it stand in the dark for 10 min. React to generate I 2, titrate with tiopronin solution until the solution turns light yellow, add 2 mL of starch indicator, the solution turns blue, continue titrating until the blue color disappears, record the volume of tiopronin consumed, and calculate the concentration of the tiopronin standard solution; then, weigh 2.4160 g of copper nitrate trihydrate and place it in a 150 mL beaker, add 35 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask, and make up to the calibration line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 copper nitrate. Accurately weigh 3.7224 g of EDTA disodium salt, dissolve it in deionized water, transfer it to a 1 L volumetric flask and make up to volume, shake well for standby. Use a pipette to accurately measure 25.00 mL of a 0.01 M Cu(NO 3 ) 2 solution into a conical flask, add 10 mL of HAc-NaAc buffer solution with a pH of 5.0, add 2 - 3 drops of PAN indicator (ethanol solution of 0.1% 1-(2-pyridylazo)-2-naphthol), the solution turns blue, titrate with the EDTA standard solution until the solution changes from blue to bright yellow suddenly, record the volume of EDTA consumed. Repeat the titration 2 - 3 times, take the average value of the two results with a volume difference ≤0.02 mL, and accurately calculate the concentration of the copper nitrate solution; finally, weigh 1.6987 g of silver nitrate and place it in a 150 mL beaker, add 20 - 30 mL of deionized water to the beaker and stir to dissolve. Transfer the dissolved solution to a 100 mL volumetric flask, and make up to the calibration line of the volumetric flask with deionized water to prepare a solution with a concentration of 0.01 mol•L -1 silver nitrate. Weigh 0.1461 g of dried NaCl, dissolve the NaCl in deionized water, transfer it to a 100 mL volumetric flask, make up to volume and shake well to obtain a 0.025 mol•L -1 NaCl standard solution. Use a pipette to accurately transfer 25.00 mL of the NaCl standard solution to a conical flask, add 0.1 g of calcium carbonate to make the solution pH 6.5 - 10.5 (neutral to weakly alkaline), add 1 mL of 5% K 2 CrO 4 solution, shake well, and titrate with the prepared 0.01 mol•L -1 to determine AgNO 3 solution while shaking. When the solution shows a brick-red Ag 2 CrO 4 precipitate as the end point, record the consumed volume, perform parallel determinations 3 times, take the average value, and the relative deviation should be ≤0.2%. Store in the dark for standby; The second step is the preparation of antibacterial TPN-CuAg nanoclusters: Measure 30 mL of the above-prepared tiopronin standard solution and add it to a 250 mL three-necked flask. Place the three-necked flask in a magnetic stirring water bath. Then measure 10 mL of the above-prepared copper nitrate standard solution and add it dropwise to the three-necked flask. React at 25 °C for 40 min at a rotation speed of 600 rpm to obtain a reaction mixture solution. Finally, measure 10 mL of the above-prepared silver nitrate standard solution and add it to the above reaction mixture solution. React at 25 °C for 17 h at a rotation speed of 700 rpm. The color of the reaction mixture solution gradually changes from green to orange-yellow. Divide the reaction solution equally into two 50 mL centrifuge tubes, centrifuge at 9000 rpm for 10 min to collect the lower-layer precipitate, and wash and centrifuge it repeatedly 3 times with deionized water to obtain antibacterial TPN-CuAg nanoclusters. Freeze-dry them. Finally, put the obtained orange-yellow powder into a refrigerator at 4 °C for storage and standby.

Claims

1. A method for preparing antibacterial TPN-CuAg nanoclusters, characterized in that The nano clusters are formed by self-assembly of the thiol complex copper ions and silver ions in tiopronin to form a network structure. The preparation process of the TPN-CuAg nano clusters includes the following two steps: 1.1 The first step is to prepare the standard solutions of tiopronin, copper nitrate and silver nitrate: First, weigh 1.0000-2.0000 g of tiopronin using an electronic balance with an accuracy of 0.0001 g, place it in a 150 mL beaker, add 40-60 mL of deionized water and stir to dissolve it in the beaker, transfer the dissolved solution to a 100 mL volumetric flask, and dilute to the mark on the volumetric flask with deionized water to prepare a concentration of 0.01 mol•L -1 Tiopronin: Dry KIO3 in an oven at 105℃ for 2 hours, cool to room temperature, accurately weigh about 0.0178g KIO3, place in a 250mL iodine volumetric flask, add 50mL distilled water, shake until completely dissolved, add 5mL 6M HCl to make the solution strongly acidic, add 2g KI solid, shake well and stand in the dark for 10min, the reaction generates I2, titrate with tiopronin solution until the solution turns light yellow, add 2mL starch indicator, the solution turns blue, continue titrating until the blue disappears, record the volume of tiopronin consumed, and calculate the concentration of tiopronin standard solution; then, weigh 2.0000~3.0000g copper nitrate trihydrate and place in a 150mL beaker, add 30~40mL deionized water in the beaker and stir to dissolve, transfer the dissolved solution to a 100mL volumetric flask, dilute to the scale line of the volumetric flask with deionized water, and prepare a concentration of 0.01mol•L -1 Copper nitrate: accurately weigh 3.7224 g EDTA disodium salt, dissolve it in deionized water, transfer it to a 1 L volumetric flask, shake it for later use, use a pipette to accurately measure 25.00 mL of 0.01 M Cu(NO3)2 solution in a conical flask, add 10 mL of HAc-NaAc buffer solution with a pH of 5.0, add 2-3 drops of PAN indicator (0.1% 1-(2-pyridyl azo)-2-naphthol in ethanol), the solution is blue, titrate with EDTA standard solution until the solution suddenly changes from blue to bright yellow, record the volume of EDTA consumed, repeat the titration 2-3 times, take the average of the two results with a volume difference of ≤0.02 mL, and accurately calculate the concentration of the copper nitrate solution; finally, weigh 2.0000 ~3.0000 g of silver nitrate and place it in a 150 mL beaker, add 20 ~ 30 mL of deionized water in the beaker and stir to dissolve, and transfer the dissolved solution into a 100 mL volumetric flask, dilute to the mark with deionized water to obtain a concentration of 0.01 mol•L -1 Silver nitrate, weigh 0.1461g of dried NaCl, dissolve the NaCl in deionized water, transfer to a 100mL volumetric flask, dilute to volume and shake well to obtain 0.025 mol•L -1 NaCl standard solution: Use a pipette to accurately transfer 25.00 mL of NaCl standard solution to a conical flask, add 0.1 g of calcium carbonate to adjust the solution pH to 6.5-10.5 (neutral to weakly alkaline), add 1 mL of 5% K2CrO4 solution, shake well, and use the prepared 0.01 mol•L -1 Titrate with a fixed AgNO3 solution, shaking while dropping, until the solution shows brick-red Ag2CrO4 precipitation, which is the end point. Record the consumed volume, measure three times in parallel, and take the average value. The relative deviation should be ≤0.2%. Store away from light for later use. 1.2 The second step is the preparation of antibacterial TPN-CuAg nanoclusters: 20 ~ 40 mL of the prepared thiopronin standard solution was measured and added to a 250 mL three-necked flask, and the three-necked flask was placed in a water bath with magnetic stirring, and then 5 ~ 15 mL of the prepared copper nitrate standard solution was measured and added dropwise to the three-necked flask, and the mixture was reacted at 500 ~ 800 rpm and 20 ~ 30 ° C for 30 ~ 50 min to obtain a reaction mixed solution. Finally, 5 ~ 15 mL of the prepared silver nitrate standard solution was measured and added to the reaction mixed solution, and the mixture was reacted at 600 ~ 800 rpm and 20 ~ 30 ° C for 16 ~ 18 h. The color of the reaction mixed solution gradually changed from green to orange-yellow. The reaction solution was evenly divided into two 50 mL centrifuge tubes and centrifuged at 8000 ~ 1000 rpm for 10 The lower precipitate was collected and washed repeatedly with deionized water and centrifuged three times to obtain antibacterial TPN-CuAg nanoclusters, which were freeze-dried and finally the orange-yellow powder was stored in a 4°C refrigerator for later use.

2. The method for preparing an antibacterial TPN-CuAg nanocluster according to claim 1, characterized in that The antibacterial TPN-CuAg nanoclusters have an inhibitory effect on Escherichia coli and Staphylococcus aureus.

3. The method for preparing an antibacterial TPN-CuAg nanocluster according to claim 1, characterized in that The antibacterial TPN-CuAg nanoclusters can effectively scavenge DPPH free radicals.

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