Application of carbon dots as antibacterial material

Abstract

The application relates to application of carbon dots as antibacterial materials and belongs to the technical field of antibacterial agents. The carbon dots are synthesized by solvent thermal treatment of a p-phenylenediamine solution. The carbon dots as antibacterial materials are synthesized by simple solvent thermal treatment of p-phenylenediamine, and the p-phenylenediamine molecules serving as the only precursor for the synthesis of the carbon dots have two -NH2, which have positive charges (-NH3 + ) at physiological pH, so that the p-phenylenediamine can make the surface of the synthesized carbon dots covered by quaternary ammonium groups as the source of the quaternary ammonium groups. Since the quaternary ammonium groups are an effective antibacterial group, the carbon dots have a large number of positively charged quaternary ammonium groups on the surface, so that the carbon dots can be adsorbed on the negatively charged bacterial cell membrane through electrostatic action, the cell membrane is destroyed, and the bacteria are killed, so that the carbon dots have strong spectral antibacterial activity and cannot cause bacterial drug resistance.

Description

Technical Field

[0001] This invention relates to the application of carbon dots as an antibacterial material, belonging to the field of antibacterial agent technology. Background Technology

[0002] With the emergence of multidrug-resistant bacteria, commonly used antibiotics are no longer effective, and bacterial infections are considered one of the world's largest public health threats. Therefore, the discovery and design of novel, highly effective antibacterial drugs, distinct from traditional small-molecule antibiotics, is an urgent task. Recently, the rapid development of nanoscience and nanotechnology has provided promising alternatives for antibacterial therapy. Nanomaterials, with their large surface area, can effectively contact bacteria through electrostatic interactions, thereby disrupting bacterial cell membrane permeability and respiratory function, ultimately leading to bacterial death. Furthermore, nanoparticles endocytosed by bacteria can induce programmed apoptosis by disrupting amino acids and affecting DNA synthesis. Some noble metal nanoparticles (e.g., Ag, Au nanoparticles) and metal oxide nanoparticles (e.g., TiO2, ZnO, Fe2O3, CuO) exhibit good antibacterial activity. Compared to traditional antibiotics (small organic molecules), nanoparticles, because they cause bacterial death by disrupting cell membranes and affecting the exchange of substances between cells and the external environment, are considered to have the potential for multiple antibacterial effects and have been shown not to induce bacterial resistance. Although these metal and metal oxide nanoparticles have excellent antibacterial activity, in practical applications, these nanoparticles will continuously release metal particles, which have potential cytotoxicity to the human body. Therefore, the biosafety of these nanoparticles for long-term use needs further research.

[0003] As a novel type of metal-free nanoparticle, carbon dots have attracted widespread attention due to their ease of synthesis and modification, extremely small size, excellent fluorescence properties, low cytotoxicity, and superior water solubility. These fascinating properties have led to the widespread application of carbon dots 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. In most studies on antibacterial carbon dots, the antibacterial mechanism is achieved by inducing oxidative stress in bacteria using reactive oxygen species (ROS) generated by the carbon dots. However, the generation of ROS often requires the addition of light or hydrogen peroxide. Unfortunately, the addition of hydrogen peroxide or light stimulation can complicate the treatment process and may also cause unintended damage to sensitive tissues in the human body. Therefore, there is a strong desire to explore carbon dots with antibacterial activity without exogenous stimulation. Huang et al., in their paper "Synthesis of Self-Assembled Spermidine-Carbon Quantum Dots Effective Against Multidrug-Resistant Bacteria" (Adv. Healthcare Mater. 2016, 5:2545-2554), disclosed a method for obtaining carbon dots through solid-state pyrolysis of citric acid at 180℃, followed by modification of these carbon dots with spermidine at a high temperature of 260℃. This two-step synthetic method overcomes the common problem of requiring photostimulation or hydrogen peroxide addition for antibacterial carbon dots, and the resulting spermidine-modified carbon dots exhibit good antibacterial activity. While this method eliminates the stress stimulation issue, the synthetic steps are relatively complex. Summary of the Invention

[0004] The purpose of this invention is to provide an application of carbon dots synthesized using a simple method as an antibacterial material.

[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0006] An application of carbon dots as an antibacterial material, wherein the carbon dots are synthesized by solvothermal treatment of a p-phenylenediamine solution.

[0007] The carbon dots of this invention, serving as antibacterial materials, are synthesized in one step via simple solvothermal treatment of p-phenylenediamine. The p-phenylenediamine molecule, the sole precursor for carbon dot synthesis, has two -NH2 groups, which carry a positive charge (-NH3) at physiological pH. +Therefore, p-phenylenediamine can serve as a source of quaternary ammonium groups, allowing the surface of the synthesized carbon dots to be covered with quaternary ammonium groups. Since quaternary ammonium groups are effective antibacterial groups, the abundant positively charged quaternary ammonium groups on the carbon dot surface allow the carbon dots to adsorb onto the negatively charged bacterial cell membrane through electrostatic interactions, leading to bacterial death by disrupting the cell membrane. Therefore, this carbon dot exhibits strong broad-spectrum antibacterial activity and does not induce bacterial resistance. Furthermore, because positive charges reside on the outer side of the membrane (positive membrane potential) and negative charges reside on the inner side (negative membrane potential) when the cell is at rest, this carbon dot is almost non-cytotoxic under normal conditions.

[0008] This invention investigates the antibacterial activity of carbon dots synthesized by solvothermal treatment of p-phenylenediamine against typical Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus). The results show that the minimum bactericidal concentration (MBC) of the synthesized carbon dots against both bacteria is lower than previously reported, indicating that these carbon dots can be used as broad-spectrum antibacterial agents.

[0009] The carbon dots synthesized using p-phenylenediamine as the sole carbon source in this invention have a large amount of -NH4+ coating on their surface. 3+ Positively charged groups can significantly enhance the antibacterial activity of carbon dots, and the sterilization process is very simple, requiring no light stimulation or the addition of H2O2.

[0010] Furthermore, the antibacterial material is a Gram-positive antibacterial material. The Gram-positive bacteria can be Gram-negative or Gram-positive. More specifically, the Gram-positive bacteria are Staphylococcus aureus or Escherichia coli. Staphylococcus aureus is a typical Gram-positive bacterium, and Escherichia coli is a typical Gram-negative bacterium. The carbon dots exhibit significant bactericidal effects against both Staphylococcus aureus and Escherichia coli.

[0011] Furthermore, the temperature of the solvent heat treatment is 140–200°C, for example 180°C, and the time is 6–20 h, for example 12 h.

[0012] Furthermore, the carbon dots are prepared using a method comprising the following steps: a p-phenylenediamine solution is subjected to solvothermal treatment followed by solid-liquid separation, and the solvent is removed from the resulting liquid phase to obtain the carbon dots. Solvothermal treatment not only greatly simplifies the synthesis and modification steps of antibacterial carbon dots but also allows for better control of the functional groups and charge on the carbon dot surface, resulting in excellent antibacterial activity. The solid-liquid separation involves centrifugation followed by filtration of the supernatant from the centrifugation process using a microporous membrane.

[0013] Furthermore, the solid-liquid separation includes first centrifuging the system obtained by solvothermal treatment, and then filtering the supernatant from the centrifugation using a microporous membrane. The centrifugation speed is 8000–12000 rpm, for example, 10000 rpm; the centrifugation time is 8–12 min, for example, 10 min. The pore size of the microporous membrane is preferably 0.22 μm.

[0014] Furthermore, the concentration of p-phenylenediamine in the p-phenylenediamine solution is 5 × 10⁻⁶. -3 ~9×10 -3 mol / L. It is understood that the solvent for the p-phenylenediamine solution is an organic solvent. Further, the solvent for the p-phenylenediamine solution is an alcohol solvent, such as ethanol. Attached Figure Description

[0015] Figure 1 The images show the UV-Vis absorption spectra of the carbon dots (curve b) and p-phenylenediamine (curve a) prepared in Example 1, and the fluorescence emission spectrum of the carbon dots (curve c). The inset shows photographs of the filtrate taken under sunlight (left) and a 365nm UV lamp (right).

[0016] Figure 2 Transmission electron microscope image (A) and particle size distribution map (B) of the carbon dots prepared in Example 1;

[0017] Figure 3 Agar plate images (A) showing the bacterial survival rates of Escherichia coli and Staphylococcus aureus after 3 hours of treatment with different concentrations of carbon dots and 16 hours of incubation at 37°C, and (B) showing the bacterial survival rates of Escherichia coli and Staphylococcus aureus after treatment with different concentrations of carbon dots. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0019] Example 1

[0020] The application of carbon dots as an antibacterial material in this embodiment is the application of carbon dots as an antibacterial material for Staphylococcus aureus.

[0021] The carbon dots used are prepared by a method including the following steps:

[0022] 1) After adding p-phenylenediamine to ethanol, sonicate for 30 minutes to ensure complete dissolution, yielding a concentration of 9 × 10⁻⁶. -3 A mol / L solution of p-phenylenediamine.

[0023] 2) The obtained p-phenylenediamine solution was then transferred to a high-pressure autoclave lined with polytetrafluoroethylene and heated in an oven at 180°C for 12 hours. After natural cooling to room temperature (about 20°C), a dark red suspension was obtained.

[0024] 3) Centrifuge the obtained dark red suspension at 10,000 rpm for 10 minutes in a high-speed centrifuge, then pass the supernatant through a 0.22 μm microporous membrane filter to remove larger particles, and vacuum dry the filtrate to obtain purified carbon dots.

[0025] To verify the successful synthesis of carbon dots in Example 1, 1 mg of the purified carbon dots from Example 1 was accurately weighed and dispersed in 1 mL of pure water to obtain a carbon dot dispersion. The UV-Vis absorption spectrum of the carbon dot dispersion was then measured and compared with the absorption spectrum of the starting material, p-phenylenediamine. Figure 1 As shown. By Figure 1 It can be seen that the carbon dot dispersion (curve a) exhibits two distinct UV absorptions at 245 nm and 285 nm, originating from the electronic transitions of the C-C and C=C bonds (π-π*) and the CO and C=O bonds (n-π*), respectively. However, the reactant p-phenylenediamine (curve b) only has a sharp peak at 240 nm and no UV absorption peak at 510 nm, while the carbon dot dispersion has a distinct absorption peak at 510 nm. Furthermore, under excitation at 510 nm, the carbon dot dispersion produces strong fluorescence emission at 580 nm (curve c). Figure 1 The illustration shows that the carbon dot dispersion appears light pink under sunlight, while it produces bright red fluorescence under 365nm ultraviolet light.

[0026] Accurately weigh 1 mg of the purified carbon dots prepared in Example 1 and disperse them in 1 mL of pure water to obtain a carbon dot dispersion; perform transmission electron microscopy on the obtained carbon dot dispersion, and the results are as follows. Figure 2 As shown in Figure A. Figure 2 As shown in A, the prepared carbon dots are spherical and well dispersed in water. Under a high-resolution transmission electron microscope, we can see uniform lattice fringes with a fringe spacing of 0.21 nm, which corresponds to the 100-plane lattice of graphitized carbon.

[0027] The carbon dots obtained in Example 1 were subjected to particle size analysis using dynamic light scattering, and the results are as follows: Figure 2 As shown in Figure B, the diameter of the synthesized carbon dots is mainly distributed between 2.5 and 4.0 nm, with the largest content (over 35%) being carbon dots with a diameter of 3.2 nm. These results demonstrate that Example 1 successfully synthesized carbon dots using p-phenylenediamine, and the carbon dots have uniform particle size and good dispersibility in water.

[0028] Example 2

[0029] The application of carbon dots as an antibacterial material in this embodiment is the application of carbon dots as an antibacterial material for Escherichia coli. The carbon dots used are the same as in Example 1.

[0030] Example 3

[0031] The antibacterial agent in this embodiment is an antibacterial agent for Staphylococcus aureus or Escherichia coli. Specifically, the antibacterial agent is a carbon dot dispersion with a concentration of 2 to 30 μg / L formed by dispersing the carbon dots prepared in Example 1 in water, for example, carbon dot concentrations of 2, 5, 10, 20, and 30 μg / L.

[0032] Experimental Example

[0033] In this experimental example, typical Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria Staphylococcus aureus (S. aureus) were selected as research subjects to investigate the antibacterial activity of the carbon dots prepared in Example 1.

[0034] Prepare multiple bacterial suspensions using the following method: Take 100 μL of 10 5 The bacterial suspension of CFU / mL was diluted in 800 μL of pH 7.2 PBS buffer to obtain the bacterial culture.

[0035] Different concentrations of carbon dot dispersions, diluted with PBS buffer, were added to different bacterial cultures. The mixtures were shaken thoroughly to achieve carbon dot concentrations of 1, 2, 5, 10, 20, and 30 μg / mL, respectively, and incubated at 37°C for 3 hours. Then, 100 μL of the incubated bacterial suspension was plated onto an agar plate and incubated upside down at 37°C for 17 hours. Three parallel experiments were conducted, and colony growth was observed using plate counting. The control group used PBS buffer instead of the carbon dot solution. Results are as follows: Figure 3 As shown.

[0036] Figure 3 A represents agar plate images showing the effects of reacting two types of bacteria with different concentrations of carbon dots for 3 hours, followed by incubation at 37°C for 16 hours. It is clearly visible that as the carbon dot concentration increases, the colony counts of both Gram-negative and Gram-positive bacteria decrease significantly. Figure 3 B represents the bacterial survival rate of *Escherichia coli* and *Staphylococcus aureus* after treatment with different concentrations of carbon dots; specific values ​​are shown in Table 1. The results indicate that the carbon dots synthesized in Example 1 possess broad-spectrum antibacterial properties.

[0037] Table 1. Survival rate of bacteria in antibacterial experiments

[0038]

Claims

1. An application of carbon dots as an antibacterial material, characterized in that: The carbon dots are synthesized by solvothermal treatment of a p-phenylenediamine solution; specifically, the p-phenylenediamine solution is solvothermal treated, followed by solid-liquid separation, and the resulting filtrate is vacuum dried to obtain purified carbon dots. The concentration of p-phenylenediamine in the p-phenylenediamine solution is 5 × 10⁻⁶. -3 ~9×10 -3 mol / L.

2. The application of carbon dots as an antibacterial material according to claim 1, characterized in that: The antibacterial material is a Gram-specific antibacterial material.

3. The application of carbon dots as an antibacterial material according to claim 2, characterized in that: The Gram bacteria mentioned are Staphylococcus aureus or Escherichia coli.

4. The application of carbon dots as an antibacterial material according to any one of claims 1 to 3, characterized in that: The solvent heat treatment is performed at a temperature of 140–200°C for a time of 6–20 hours.

5. The application of carbon dots as an antibacterial material according to any one of claims 1 to 3, characterized in that: The carbon dots are prepared by a method including the following steps: a p-phenylenediamine solution is subjected to solvothermal treatment followed by solid-liquid separation, and the solvent is removed from the resulting liquid phase to obtain the carbon dots.

6. The application of carbon dots as an antibacterial material according to claim 3, characterized in that: When the bacterial strain is Escherichia coli, the concentration of added carbon dots is 20~30 μg / mL; when the bacterial strain is Staphylococcus aureus, the concentration of added carbon dots is 2~30 μg / mL.

7. The application of carbon dots as an antibacterial material according to claim 5, characterized in that: The solid-liquid separation includes first centrifuging the system obtained by solvent heat treatment, and then filtering the supernatant of the centrifuged solution using a microporous membrane; the microporous membrane has a pore size of 0.22 μm.

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