Metal-doped antibacterial carbon dots based on tannic acid as well as preparation method and application of metal-doped antibacterial carbon dots

Abstract

The invention discloses metal-doped antibacterial carbon dots based on tannic acid and a preparation method and application of the metal-doped antibacterial carbon dots based on tannic acid, and the method comprises the following steps: S1, respectively adding tannic acid and soluble metal salt into ultrapure water to form a tannic acid solution and a soluble metal salt solution; s2, the tannic acid solution and the soluble metal salt solution are mixed according to a certain proportion to form metal chelate dispersion liquid, and the metal chelate dispersion liquid is evenly mixed through ultrasonic treatment; s3, transferring the metal chelate dispersion liquid into a reaction kettle, reacting at 160-220 DEG C for 4-12 hours, and cooling to room temperature; s4, carrying out centrifugal treatment on the solution cooled to the room temperature, and carrying out suction filtration treatment on supernate obtained by centrifugation to obtain a clear solution A; and S5, dialyzing and purifying the clear solution A, and freeze-drying the dialysate to obtain the metal-doped antibacterial carbon dots based on the tannic acid. The preparation method is simple in process, low in cost and high in benefit, and the prepared metal-doped carbon dots have excellent antibacterial performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon quantum dot antibacterial, and in particular relates to a tannic acid-based metal-doped antibacterial carbon dot and a preparation method and application thereof. Background Art

[0002] With the emergence of multidrug-resistant bacteria, commonly used antibiotics are no longer effective, and bacterial infections are being recognized as one of the world's greatest public health threats. Therefore, it is an urgent task to discover and design new and highly effective antibacterial drugs that are different from traditional small molecule antibiotics. Recently, the rapid development of nanoscience and nanotechnology has provided promising alternatives for antibacterial therapy. Nanomaterials have the advantage of large specific surface area and can fully contact bacteria through electrostatic effects, thereby destroying the permeability and respiratory function of bacterial cell membranes, ultimately leading to bacterial death. In addition, nanoparticles endocytosed by bacteria can cause programmed apoptosis of bacteria by destroying amino acids and affecting DNA synthesis. Some precious metal nanoparticles (such as Ag and Au nanoparticles) and metal oxide nanoparticles (such as TiO2, ZnO, Fe2O3, CuO) have good antibacterial activity. Compared with traditional antibiotics (organic small molecules), nanoparticles are considered to have the potential for multiple antibacterial effects because they destroy cell membranes and affect the exchange of substances between cells and the outside world, thereby causing bacterial death, and have been shown not to induce bacterial resistance. Although these metal and metal oxide nanoparticles have excellent antibacterial activity, in actual applications these nanoparticles will continuously release metal particles and have potential cytotoxicity to the human body. Therefore, the biosafety of long-term use of these nanoparticles needs further study.

[0003] As an emerging nanoparticle, carbon dots have attracted widespread attention due to their easy synthesis and modification, extremely small size, excellent fluorescence properties, low cytotoxicity and excellent water solubility. These fascinating properties have made carbon dots widely used in photocatalysis, energy conversion, bioimaging, biosensors, photothermal therapy, drug delivery and cell-based tissue engineering. Recently, researchers have also turned their attention to the antibacterial activity of carbon dots. However, most of the current technical solutions are to couple carbon dots, which do not have antibacterial properties, with certain materials with antibacterial functions such as antibiotics, quaternary ammonium salts, etc. to give them antibacterial properties. These methods have the disadvantages of cumbersome preparation process, low stability, high cost and poor antibacterial performance. Summary of the invention

[0004] In order to solve the above problems, the present invention proposes a tannic acid-based metal-doped antibacterial carbon dot and a preparation method and application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing metal-doped antibacterial carbon dots based on tannic acid comprises the following steps:

[0007] S1. Adding tannic acid and a soluble metal salt into ultrapure water respectively to form a tannic acid solution and a soluble metal salt solution;

[0008] S2, mixing the tannic acid solution and the soluble metal salt solution in a certain proportion to form a metal chelate dispersion, and subjecting the metal chelate dispersion to ultrasonic treatment to mix the metal chelate dispersion uniformly;

[0009] S3, transferring the metal chelate dispersion to a reaction kettle, reacting at 160-220° C. for 4-12 hours, and then cooling to room temperature;

[0010] S4, centrifuging the solution after cooling to room temperature, and filtering the supernatant obtained by centrifugation to obtain a clear solution A;

[0011] S5. The clarified solution A is dialyzed for purification, and the dialyzate is freeze-dried to obtain tannic acid-based metal-doped antibacterial carbon dots.

[0012] Preferably, the soluble metal salt in step S1 is one of acetate, zinc nitrate or chloride of zinc, manganese, iron, copper or rare earth elements.

[0013] Preferably, the mass ratio of tannic acid to soluble metal salt in step S1 is 100:(1-20).

[0014] Preferably, the mass concentrations of the tannic acid solution and the soluble metal salt solution in step S1 are both 2-20 mg / mL.

[0015] Preferably, in step S2, the liquid volume addition ratio of the tannic acid solution and the soluble metal salt solution is 100:(1-5).

[0016] Preferably, the inner liner of the reactor in step S3 is made of polytetrafluoroethylene, and the outer shell is made of 304 stainless steel.

[0017] Preferably, the centrifuge speed of the centrifuge in step S4 is 6000-9000 rpm, the centrifugation time is 10-30 minutes, and the filter membrane used for filtration is a water filter membrane with a pore size of 0.22 μm.

[0018] Preferably, in step S5, the clarified solution A is dialyzed for 12 to 72 hours using a 500 to 3500 Da dialysis cut-off bag.

[0019] Disclosed are tannic acid-based metal-doped antibacterial carbon dots, which are prepared by using the tannic acid-based metal-doped antibacterial carbon dots preparation method.

[0020] The invention discloses an application of metal-doped antibacterial carbon dots based on tannic acid. The metal-doped antibacterial carbon dots based on tannic acid are used as antibacterial and antibacterial materials in the medical field.

[0021] After adopting the above technical scheme, the present invention has the following beneficial effects: The present invention discloses a method for preparing metal-doped antibacterial carbon dots based on tannic acid, and uses tannic acid carbon source with a wide source and low price to prepare carbon dots. Tannic acid is a high molecular weight water-soluble polyphenol, which is widely present in various types of plants. Due to its unique structural characteristics, tannic acid is endowed with outstanding chemical properties and various biological activities, especially strong antibacterial activity against various pathogens. The carbon dots prepared with tannic acid as the carbon source have good antibacterial properties against typical Gram-negative bacteria and Gram-positive bacteria such as Escherichia coli and Staphylococcus aureus. After the carbon dots are subjected to metal doping modification treatment, they have more excellent antibacterial properties and stable properties. The aqueous dispersion of the carbon dots still has excellent antibacterial properties against Escherichia coli and Staphylococcus aureus after long-term static treatment. The invention has simple process, low manufacturing cost and good stability. The obtained material has both low toxicity and high antibacterial property and is pollution-free to the environment. It is a green antibacterial functional material, which effectively solves the technical problems of complicated preparation process, low stability, high cost, high toxicity and poor antibacterial property of antibacterial materials in the prior art. It is also expected to replace the technical problems of traditional antibiotics and drugs and has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a transmission electron microscopy image of tannic acid carbon dots in Example 1 of the present invention;

[0023] Figure 2 The Fourier infrared spectrum of tannic acid carbon dots in Comparative Example 1 and Examples 1 to 5 of the present invention;

[0024] Figure 3 The UV-visible absorption spectra of tannic acid carbon dots in Comparative Example 1 and Examples 1 to 5 of the present invention are shown;

[0025] Figure 4 This is a graph exploring the antibacterial performance of tannic acid carbon dots against Escherichia coli and Staphylococcus aureus in Comparative Example 1 and Examples 1 to 5 of the present invention;

[0026] Figure 5 The in vitro antibacterial test diagram of tannic acid carbon dots on Escherichia coli under different conditions in Example 1 of the present invention and Comparative Example 1 is shown;

[0027] Figure 6 The in vitro antibacterial test diagram of tannic acid carbon dots on Staphylococcus aureus under different conditions in Example 1 of the present invention and Comparative Example 1 is shown;

[0028] Figure 7These are experimental diagrams of the antibacterial zones of tannic acid carbon dots doped with different metals in Examples 9 to 13 of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] like Figures 1 to 7 shown.

[0031] Example 1

[0032] Using tannic acid as a carbon source, carbon dots were prepared by hydrothermal method. Prepare a 100mL polytetrafluoroethylene reactor and add 60mL ultrapure water. Take 240mg tannic acid and add it to ultrapure water, then add 6.73mg zinc acetate, and ultrasonically vibrate for 10 minutes. The hydrothermal precursor appears milky white. Then put the reactor into a constant temperature blast drying oven and react at 200℃ for 4 hours. During this period, the tannic acid metal chelate decomposes, polymerizes, and carbonizes. After the reaction, wait for the hydrothermal reactor to cool naturally to room temperature. After cooling to room temperature, centrifuge the hydrothermal product at a speed of 9000rpm for 15 minutes. The supernatant obtained by centrifugation is filtered with a water filter membrane with a pore size of 0.22μm. The filtrate is dialyzed for 48 hours using a 500Da dialysis cutoff bag to remove small organic molecules and residual small molecular ions. Finally, the carbon dot dispersion after dialysis is freeze-dried for 3 days and sealed for storage after freeze-drying. Then, a zinc-doped carbon dot powder solid was obtained.

[0033] In Examples 2 to 13, referring to the specific preparation process of Example 1, and using the process parameters in Table 1 respectively, tannic acid-based metal-doped antibacterial carbon dots were finally prepared.

[0034] Comparative Example 1

[0035] In Comparative Example 1, tannic acid was used to prepare biomass carbon quantum dots without metal ion doping. Specifically, 240 mg of tannic acid was accurately weighed in a beaker, 60 mL of ultrapure water was added, and ultrasonic vibration was performed for 10 minutes to make it evenly dispersed. The solution was transferred to a reactor lined with 100 mL of polytetrafluoroethylene, and the reactor was placed in a constant temperature blast drying oven. The reaction was carried out at 200 ° C for 4 hours. After cooling to room temperature, the centrifuge was used to centrifuge at a speed of 9000 rpm for 15 minutes. The supernatant obtained by centrifugation was filtered with a water filter membrane with a pore size of 0.22 μm, and the filtrate was dialyzed for 48 hours using a 500 Da dialysis cutoff bag to remove organic small molecules and residual small molecule ions. Finally, the carbon point dispersion after dialysis was freeze-dried for 3 days, and sealed and stored after freeze-drying. Undoped tannic acid carbon point powder solid was obtained.

[0036] The process parameters of the antibacterial carbon dots of Comparative Example 1 and Examples 1-13 are shown in Table 1.

[0037] Table 1: Process parameters of antibacterial carbon dots of Comparative Example 1 and Examples 1-13

[0038]

[0039] In order to further verify and illustrate the superiority of the technology of the present invention, the inventors have carried out structural characterization and performance measurement on the prepared carbon dots, the specific contents are as follows:

[0040] Figure 1 (a) is the structural formula of tannic acid. Figure 1 (b) is an aqueous solution of tannic acid carbon dots. Figure 1 (c) is a transmission electron microscope (TEM) image of tannic acid carbon dots, where the particle size of the carbon dots is distributed in the range of 3 to 8 nm. The particle size distribution is uniform.

[0041] Figure 2 The Fourier transform infrared spectra of carbon dots prepared under different conditions in Examples 1 to 5 and Comparative Example 1 are shown. It has been verified that the tannic acid carbon dots prepared by changing the doping ratio of the Zn element without changing the total substance concentration of the hydrothermal reaction precursor have the same functional group structure.

[0042] Figure 3 The UV-visible absorption spectra of the carbon dots prepared under different conditions in Examples 1 to 5 and Comparative Example 1 are shown. After diluting the carbon dot solution, it was found that the carbon dots strongly absorbed light in the ultraviolet band below 400nm. The hydroxyl group has an absorption peak near 260nm-270nm, proving that the TA-CDs surface has -OH.

[0043] In order to verify the antibacterial effect of the tannic acid carbon dots and the zinc-doped tannic acid carbon dots of the present invention, the following experiments were conducted:

[0044] (1) Preparation of bacterial culture medium

[0045] Weigh 2.1 g of LB broth and dissolve it in a conical flask filled with 100 mL of deionized water, weigh 3.6 g of LB agar and dissolve it in a conical flask filled with 100 mL of deionized water, and sterilize them in an autoclave at 120° C. for 60 minutes to obtain liquid culture medium and solid culture medium, respectively.

[0046] (2) Preparation of bacterial suspension

[0047] Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were added to the liquid culture medium, cultured in a constant temperature incubator at 37°C for 12 hours, and then centrifuged at 7000rpm for 2 minutes to obtain bacterial precipitates. The bacterial precipitates of Escherichia coli and Staphylococcus aureus were then dispersed in PBS and the absorbance OD was adjusted. 600 At around 0.35 to 0.4, a bacterial suspension is obtained.

[0048] (3) Inhibition zone test

[0049] Take 3mL of the bacterial suspension prepared in the above steps and mix it evenly with 100mL of LB agar medium. Take 20mL of the mixed LB agar medium and pour it into a standard culture dish. After standing for 30 minutes and cooling completely, use a 6.0mm diameter puncher to punch holes in the agar culture dish. Add 50μL of tannic acid carbon dot samples with the same concentration and different concentrations to the experimental holes. The blank group is treated with deionized water. Three groups are tested in parallel. Incubate in a constant temperature incubator at 37℃ for 12h, and record the diameter of the antibacterial zone of each group of samples at the end. The size of the antibacterial zone diameter reflects the difference in antibacterial performance between different samples.

[0050] Figure 4 (a) is a comparison chart of the antibacterial performance of carbon dots prepared under different conditions in Examples 1 to 5 and Comparative Example 1 against Staphylococcus aureus. It can be seen from the figure that under the same carbon dot concentration in each example, 1% Zn-doped tannic acid carbon dots have the best antibacterial performance against Staphylococcus aureus. Similarly, Figure 4 (b) Comparison of the antibacterial performance of carbon dots prepared under different conditions against Escherichia coli in Examples 1 to 5 and Comparative Example 1. The tannic acid carbon dots doped with 1% Zn have the best antibacterial performance against golden Staphylococcus. When the Zn doping content increases, the diameter of the antibacterial zone decreases and the antibacterial performance decreases.

[0051] (4) Antibacterial test

[0052] The carbon dots prepared in Example 1 and Comparative Example 1 were configured into a 5.0 mg / mL aqueous solution, and diluted to different concentrations by the binary method. The bacterial suspensions of Escherichia coli and Staphylococcus aureus prepared above were diluted 1000 times with LB broth. Take 50 μL of each of the carbon dot solutions of different concentrations and the diluted bacterial suspension, mix them evenly in a 96-well plate, and then place the mixed solutions in the dark and under LED white light for co-culture for 12 hours. Then take 50 μL of the acted bacterial suspension and spread it on the agar culture plate, invert it in a constant temperature incubator at 37°C and culture it for 12 hours. Three groups of parallel experiments were conducted to observe the growth of the colonies and count them using the plate count method. The results are as follows: Figure 5 and Figure 6 shown.

[0053] Depend on Figure 5 and Figure 6 It can be seen that under the same carbon dot concentration condition, the antibacterial performance of the tannic acid carbon dots in Example 1 is significantly improved after zinc doping. When the tannic acid carbon dots have a lower concentration, they still have an antibacterial effect on Escherichia coli and Staphylococcus aureus. The above results show that the carbon dots have a broad-spectrum antibacterial performance against Escherichia coli and Staphylococcus aureus.

[0054] The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the carbon dots prepared in Example 1 and Comparative Example 1 under light and dark conditions, respectively, are shown in Table 2.

[0055] Table 2: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration of the carbon dots prepared in Example 1 and Comparative Example 1 under light and dark conditions, respectively

[0056]

[0057]

[0058] The results in Table 1 and Figure 5 , Figure 6 It can be seen from the in vitro antibacterial test results that the zinc-doped carbon dots prepared in Example 1 and Comparative Example 1 of the present invention have excellent antibacterial effects on Escherichia coli and Staphylococcus aureus under both dark and light conditions, and the carbon dots prepared in Example 1 and Comparative Example 1 have better antibacterial effects on Escherichia coli and Staphylococcus aureus under light conditions. Under light-assisted conditions, carbon dots with lower concentrations can also have good antibacterial effects on Escherichia coli and Staphylococcus aureus.

[0059] Figure 7The experimental diagram of the inhibition zone of tannic acid carbon dots doped with different metal ions on Escherichia coli and Staphylococcus aureus. It can be seen from the experimental results that the tannic acid carbon dots were doped with five metal ions, Zn, Cu, Fe, Mn, and Eu, respectively, and the metal element doping amount was 10%. The five metal-doped carbon dots have good antibacterial effects on Escherichia coli and Staphylococcus aureus, and present different sizes of inhibition zones in the same experiment. Among them, the diameter of the inhibition zone of Zn-doped tannic acid carbon dots on Escherichia coli and Staphylococcus aureus is the largest. The antibacterial effect is the best. The above results show that the metal-doped carbon dots prepared with tannic acid as the biomass carbon source have good antibacterial effects.

[0060] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing metal-doped antibacterial carbon dots based on tannic acid, characterized in that: The following steps are involved: S1. Adding tannic acid and a soluble metal salt into ultrapure water respectively to form a tannic acid solution and a soluble metal salt solution; S2, mixing the tannic acid solution and the soluble metal salt solution in a certain proportion to form a metal chelate dispersion, and subjecting the metal chelate dispersion to ultrasonic treatment to mix the metal chelate dispersion uniformly; S3, transferring the metal chelate dispersion to a reaction kettle, reacting at 160-220° C. for 4-12 hours, and then cooling to room temperature; S4, centrifuging the solution after cooling to room temperature, and filtering the supernatant obtained by centrifugation to obtain a clear solution A; S5. The clarified solution A is dialyzed for purification, and the dialyzate is freeze-dried to obtain tannic acid-based metal-doped antibacterial carbon dots.

2. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The soluble metal salt in step S1 is one of acetate, zinc nitrate or chloride of zinc, manganese, iron, copper or rare earth elements.

3. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The mass ratio of tannic acid to soluble metal salt in step S1 is 100:(1-20).

4. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The mass concentrations of the tannic acid solution and the soluble metal salt solution in step S1 are both 2-20 mg / mL.

5. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The liquid volume addition ratio of the tannic acid solution and the soluble metal salt solution in step S2 is 100:(1-5).

6. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The inner liner of the reactor in step S3 is made of polytetrafluoroethylene, and the outer shell is made of 304 stainless steel.

7. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: The centrifuge speed of the centrifuge in step S4 is 6000-9000 rpm, the centrifugation time is 10-30 minutes, and the filter membrane used for filtration is a water filter membrane with a pore size of 0.22 μm.

8. The method for preparing metal-doped antibacterial carbon dots based on tannic acid according to claim 1, characterized in that: In step S5, the clarified solution A is dialyzed for 12 to 72 hours using a 500 to 3500 Da dialysis cut-off bag.

9. A metal-doped antibacterial carbon dot based on tannic acid, characterized in that: The tannic acid-based metal-doped antibacterial carbon dots are prepared by the method for preparing tannic acid-based metal-doped antibacterial carbon dots according to any one of claims 1 to 8.

10. An application of the metal-doped antibacterial carbon dots based on tannic acid as claimed in claim 9, characterized in that: The tannic acid-based metal-doped antibacterial carbon dots are used as antibacterial and antibacterial materials and are applied in the medical field.

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