Preparation method and application of blue light-induced antibacterial nitrogen-containing carbon quantum dots

Abstract

The present invention belongs to the technical field of antibacterial materials, and specifically relates to a method for preparing nitrogen-containing carbon quantum dots with blue light-induced antibacterial properties. The present invention studies a method for preparing nitrogen-doped carbon quantum dots based on a water-soluble nitrogen source doped with a mulberry dry water extract, and the determination of the antibacterial properties of nitrogen-doped carbon quantum dots under blue light irradiation. Purple-red anthocyanin is extracted from mulberry dry water, a water-soluble nitrogen source is dissolved in the anthocyanin extract, and nitrogen-doped carbon quantum dots are prepared by a hydrothermal method. The nitrogen-doped quantum dot aqueous solution appears transparent and light orange, the preparation process is green and pollution-free, and the product has high biosafety. Under blue light irradiation, the quantum dot solution has an antibacterial rate of 90% against Escherichia coli and Staphylococcus aureus at a concentration of 50 μg / mL, and an antibacterial rate of 99% against Candida albicans at a concentration of 400 μg / mL, showing significant antibacterial properties.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibacterial nanomaterials, and specifically relates to a preparation method and application of blue light-induced antibacterial nitrogen-containing carbon quantum dots. Background Art

[0002] Currently, carbon quantum dots (CQDs) have become a clinical candidate for PDT because they typically produce or stimulate bacteria to produce highly toxic reactive oxygen species under visible light, causing bacterial lysis and death. However, the low quantum yield of carbon quantum dots and insufficient utilization of visible light limit their application as photosensitizers. In addition, the depth of light penetration into tissue is closely related to the wavelength of the incident light. Ultraviolet light can only reach a depth of 50-150nm, while blue light (425nm-475nm) can reach 1mm. Pure blue light can be used in medical fields such as skin diseases and cancer, and is less harmful to the human body than blue-violet light. Therefore, modifying its fluorescence properties and expanding its range are the current research focuses. The surface state of carbon quantum dots is the cause of their photoluminescence. Studies have shown that S or N doping of CQDs affects their maximum excitation and maximum emission. S-doped CQDs, due to the presence of negatively charged groups such as sulfonic acid groups on their surface, are not conducive to binding to bacterial cell walls. In N-doped CQDs, the functionalization of carbon quantum dots with surface amino groups (-NH2) induces a red shift in photoluminescence. In addition, the -NH2 groups enhance the affinity of carbon quantum dots to biological structures.

[0003] Patent CN202210984320.X discloses a photodynamic antibacterial agent, a quaternary ammonium salt formed from a photosensitizer containing tertiary amine nitrogen and a halogenated long-chain aliphatic hydrocarbon. Different types of photosensitizers are reacted with the halogenated long-chain aliphatic hydrocarbon. The antibacterial agent has an inhibition rate exceeding 90% against Staphylococcus aureus and Pseudomonas aeruginosa. However, the photosensitizer is expensive and unstable, making it unsuitable for practical production applications.

[0004] Patent CN202111623476.7 discloses a vacancy-rich silver-loaded tungsten oxide nanowire for enhancing visible light and near-infrared photodynamic antibacterial properties. On the one hand, the one-dimensional structure facilitates the directional transport of charge carriers, thereby promoting the production of more toxic reactive oxygen species in the material. On the other hand, the LSPR effect is used to enhance the material's photoresponsiveness, induce the rapid separation of photogenerated charge carriers, increase the generation of photoinduced reactive oxygen species, and improve the photodynamic antibacterial efficiency. However, the doping of metal particles reduces the biocompatibility of the material and increases production costs.

[0005] According to literature reports, researchers have mostly chosen 405nm blue-violet light as the light source for photodynamic antibacterial of carbon quantum dots. However, the depth that ultraviolet light can reach is only 50-150nm, and blue light (425nm-475nm) can reach 1mm ( S, Z, Budimir M, et al. Pharmaceutics, 2023. A study (Nour El Din S, El-Tayeb TA, Abou-Aisha K, et al. International Journal of Nanomedicine, 2016) reported that nanoparticles combined with 460nm blue light had a significant inhibitory effect on multidrug-resistant Pseudomonas aeruginosa. Therefore, materials capable of photodynamic antibacterial activity at 450nm-460nm have great development prospects.

[0006] Patent CN202310666863.1 applies for a kind of carbon quantum dots (AMQDs) prepared based on an anthocyanin-rich water extract to inhibit Escherichia coli, Staphylococcus aureus and Candida albicans under visible light (405nm) irradiation. On this basis, the present invention improves the quantum yield and red-shifts the maximum emission by doping a certain proportion of nitrogen, thereby achieving an inhibitory effect on Escherichia coli, Staphylococcus aureus and Candida albicans at lower concentrations under visible light (450nm-460nm). However, at the same concentration, carbon quantum dots (AMQDs) have no significant antibacterial properties against these three bacteria under visible light (450nm-460nm), and the optical properties and antibacterial effects of quantum dots cannot be expected based on existing research mechanisms. Summary of the Invention

[0007] To address the above issues, the present invention provides a method for preparing nitrogen-containing carbon quantum dots with blue light-induced antibacterial properties. Nitrogen-doped carbon quantum dots are prepared by dissolving a water-soluble nitrogen source in a mulberry dry water extract, resulting in a material with antibacterial activity and excellent fluorescence properties.

[0008] The technical solution adopted by the present invention to solve the technical problem is to prepare nitrogen-containing carbon quantum dots with blue light-induced antibacterial properties. The preparation method comprises the following steps:

[0009] (1) Weigh dried mulberries, add deionized water, and place in a 50°C oven for 1 hour. Stir with a glass rod 2-3 times, each time for 3-4 minutes. After stirring for 1 hour, filter and remove the dried mulberry residue to obtain an aqueous mulberry anthocyanin extract (AM).

[0010] (2) Take the water extract of dried mulberry anthocyanins in a beaker, place it in an oven and dry it to constant weight, and calculate the concentration.

[0011] (3) A water-soluble nitrogen source was dissolved in the mulberry dry water extract, placed in a high-pressure reactor, and reacted at 180°C for 12 hours. After the reaction was completed and cooled to room temperature, it was filtered with a 0.22 μm water filter membrane to obtain a light orange nitrogen-doped carbon quantum dot solution (N-CQDs), which was stored in a refrigerator for later use.

[0012] (4) Mix the light orange nitrogen-doped carbon quantum dot solution (N-CQDs) and PBS buffer solution, and prepare a carbon quantum dot antibacterial solution with a concentration of ≥50 μg / mL according to the proportion.

[0013] Furthermore, the water-soluble nitrogen source is urea. And the mass concentration ratio of anthocyanin water extract to urea is c 桑葚干水提取液 :c 尿素 =5:1~13:1. Among them, when the mass concentration ratio is 9:1, the N-CQDs obtained have the best antibacterial effect.

[0014] Furthermore, when the nitrogen-doped carbon quantum dot antibacterial solution is used, the wavelength is 20mW / cm2 at 405nm-460nm. 2 Irradiation with blue light enhances antibacterial properties. Furthermore, irradiation with 450nm-460nm blue light demonstrates excellent antibacterial properties against Escherichia coli and Staphylococcus aureus at a concentration of 50μg / mL. At a concentration of 400μg / mL, the N-CQDs solution demonstrated >99% antibacterial activity against Escherichia coli, >99% against Staphylococcus aureus, and 97% against Candida albicans.

[0015] The prepared nitrogen-doped carbon quantum dot (N-CQDs) antibacterial solution exhibited significant inhibitory effects against Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and Candida albicans under 450nm-460nm illumination. Compared with AMQDs, N-CQDs exhibited lower antibacterial concentrations, longer maximum excitation and emission wavelengths, and stronger fluorescence intensity at the same concentration.

[0016] The beneficial effects of the present invention are:

[0017] (1) The nitrogen-doped carbon quantum dots prepared by the present invention significantly reduced the antibacterial concentrations against Escherichia coli, Staphylococcus aureus and Candida albicans.

[0018] (2) The fluorescent nitrogen-doped carbon quantum dots N-CQDs prepared by the present invention have significant antibacterial properties under 450nm-460nm light, and can reach a deeper level in treatments involving tissue penetration.

[0019] (3) The fluorescent nitrogen-doped carbon quantum dots prepared by the present invention have a red-shifted maximum excitation and maximum emission compared with the carbon quantum dots prepared in patent CN202310666863.1 at the same concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Fluorescence emission spectra of N-CQDs and AMQDs at the same concentration.

[0021] Figure 2EDX spectrum of N-CQDs.

[0022] Figure 3 High-resolution transmission electron microscopy image of N-CQDs. DETAILED DESCRIPTION

[0023] Prepare the dried mulberry aqueous extract: Weigh 3.0 g of dried mulberries into a beaker, add 30 mL of deionized water, and heat in a 50°C oven for 1 hour. Stir with a glass rod 2-3 times (3-4 minutes each time). After 1 hour, filter to remove the dried mulberry residue to obtain the dried mulberry anthocyanin aqueous extract (AM).

[0024] Preparation of nitrogen-doped carbon quantum dots: Dissolve the water-soluble nitrogen source urea in the mulberry dry water extract, place the solution in a high-pressure reactor, react at 180°C for 12 hours, wait for the reaction to end and cool to room temperature, filter with a 0.22um water filter membrane to obtain a light orange nitrogen-doped carbon quantum dot solution (N-CQDs), the nitrogen-doped carbon quantum dot solution (N-CQDs), store in the refrigerator for use. Take 1mL of nitrogen-doped carbon quantum dot solution in a beaker, place it in an oven and dry it to constant weight, and calculate the concentration. The mass concentration ratios of anthocyanin water extract and urea are c 桑葚干水提取液 :c 尿素 =5:1, 9:1, 13:1, and the corresponding theoretical N doping amounts in nitrogen-doped carbon quantum dots are 7.66%, 4.6%, and 3.28%.

[0025] Preparation of cysteine-doped carbon quantum dots: Dissolve cysteine ​​in mulberry dry water extract. Place the solution in an autoclave and react at 180°C for 12 hours. After the reaction is complete and cooled to room temperature, filter through a 0.22 μm water filter to obtain a nitrogen-doped carbon quantum dot solution (N-CQDs-1). Store in a refrigerator until needed. Place 1 mL of the nitrogen-doped carbon quantum dot solution (N-CQDs-1) in a beaker and dry in an oven to constant weight. Calculate the concentration.

[0026] Preparation of glutathione-doped carbon quantum dots: Dissolve glutathione in mulberry dry water extract. Place the solution in an autoclave and react at 180°C for 12 hours. After the reaction is complete and cooled to room temperature, filter through a 0.22 μm water filter to obtain a nitrogen-doped carbon quantum dot solution (N-CQDs-2). Store in a refrigerator until needed. Place 1 mL of the nitrogen-doped carbon quantum dot solution (N-CQDs-2) in a beaker and dry in an oven to constant weight. Calculate the concentration.

[0027] Preparation of carbon quantum dots: Place the mulberry dry water extract in an autoclave and react at 180°C for 10 hours. After the reaction is complete and cooled to room temperature, filter through a 0.22 μm water filter to obtain a light orange carbon quantum dot solution, mulberry dry water extract carbon quantum dots (AMQDs), which is stored in a refrigerator until used. Place 1 mL of the carbon quantum dot solution in a beaker and dry in an oven to constant weight. Calculate the concentration.

[0028] PBS buffer: Dissolve 2.84 g of disodium hydrogen phosphate and 1.36 g of potassium dihydrogen phosphate in 1000 mL of water.

[0029] The nitrogen-doped carbon quantum dot solution (N-CQDs) and PBS buffer were mixed, and antibacterial solutions of different concentrations were prepared according to the proportion (for example, 50 μg / mL solution means that the mass of nitrogen-doped carbon quantum dots in 1 mL of antibacterial solution is 50 μg).

[0030] All examples and comparative examples were used to determine the antibacterial rate of the materials according to the suspension quantitative antibacterial test method in WS / T 650-2019 "Antibacterial and antibacterial effect evaluation method".

[0031] Figure 1 The fluorescence emission spectra of N-CQDs and AMQDs at the same concentration are shown in Figure 2. Figure 1 The conclusion obtained is that at the same concentration, different materials have different maximum excitation and maximum emission, and different fluorescence intensities.

[0032] Figure 2 It is doped with N 4.6% (c 桑葚干水提取液 :c 尿素 =9:1) prepared by EDX spectrum of N-CQDs, the utilization rate of doped N can be calculated.

[0033] Example 1:

[0034] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 7.66%) were mixed with PBS buffer and a 50 μg / mL solution was prepared according to the ratio.

[0035] Example 2:

[0036] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer and a 50 μg / mL solution was prepared according to the ratio.

[0037] Example 3:

[0038] Nitrogen-doped carbon quantum dots (N-CQDs) (3.28% N doping) and PBS buffer were mixed to prepare a 50 μg / mL solution according to the ratio.

[0039] Example 4:

[0040] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed and a 50 μg / mL solution was prepared according to the proportion.

[0041] Table 1 Antibacterial rate of N-CQDs and AMQDs at 50 μg / mL under 450 nm-460 nm illumination for 1 h

[0042]

[0043] The data in Table 1 show that under 450nm-460nm light irradiation for one hour, the nitrogen doping ratio in N-CQDs affects the antibacterial effect. At a concentration of 50μg / mL, N-CQDs doped with 4.6% nitrogen achieved an inhibition rate of around 90% against Escherichia coli and Staphylococcus aureus. AMQDs showed no antibacterial activity against any of the three bacteria at 50μg / mL.

[0044] Example 5:

[0045] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 7.66%) were mixed with PBS buffer and a 400 μg / mL solution was prepared according to the ratio.

[0046] Example 6:

[0047] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer to prepare a 400 μg / mL solution according to the ratio.

[0048] Example 7:

[0049] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 3.28%) were mixed with PBS buffer to prepare a 400 μg / mL solution according to the ratio.

[0050] Example 8:

[0051] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed and a 400 μg / mL solution was prepared according to the proportion.

[0052] Example 9: Nitrogen-doped carbon quantum dots (N-CQDs-1) (doped with 4.6% N) and PBS buffer were mixed to prepare a 400 μg / mL solution according to the ratio.

[0053] Example 10: Nitrogen-doped carbon quantum dots (N-CQDs-2) (doped with 4.1% N) and PBS buffer were mixed to prepare a 400 μg / mL solution according to the ratio.

[0054] Table 2 Antibacterial rate of N-CQDs and AMQDs at 400 μg / mL under 450 nm-460 nm illumination for 1 h

[0055]

[0056] The data in Table 2 show that under 450nm-460nm illumination for 1 hour, N-CQDs at a concentration of 400μg / mL exhibited an inhibition rate exceeding 99% against both Escherichia coli and Staphylococcus aureus. N-CQDs doped with 4.6% nitrogen also exhibited an inhibition rate of 97% against Candida albicans. At the same concentration, AMQDs exhibited an inhibition rate of 77% and 86% against E. coli and S. aureus, respectively, but showed no antibacterial activity against C. albicans. Doping with cysteine ​​and glutathione also showed no antibacterial activity, indicating that the choice of nitrogen source influences the antibacterial properties of quantum dots.

[0057] Comparative Example 1:

[0058] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer to prepare a 400 μg / mL solution according to the ratio.

[0059] Comparative Example 2:

[0060] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer to prepare a 700 μg / mL solution according to the ratio.

[0061] Comparative Example 3:

[0062] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed and a 500 μg / mL solution was prepared according to the proportion.

[0063] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed and a 400 μg / mL solution was prepared according to the proportion.

[0064] Comparative Example 4:

[0065] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed to prepare a 6000 μg / mL solution according to the proportion.

[0066] Table 3 Antibacterial rate of N-CQDs and AMQDs under 405nm light irradiation for 1h

[0067]

[0068]

[0069] The data in Table 3 show that the antibacterial activity of N-CQDs doped with 4.6% N against Escherichia coli, Staphylococcus aureus, and Candida albicans under 405 nm illumination for 1 hour was slightly lower than that observed under 450-460 nm illumination for 1 hour. N-CQDs exhibited significant antibacterial activity against Candida albicans at 700 μg / mL, a significant improvement over AMQDs.

[0070] Comparative Example 5:

[0071] PBS buffer solution.

[0072] Comparative Example 6:

[0073] PBS buffer solution was exposed to light for 1 h (illumination conditions: 405 nm 20 mW / cm 2 (Irradiation with blue light)

[0074] Comparative Example 7:

[0075] PBS buffer solution was exposed to light for 1 h (illumination conditions: 450 nm-460 nm 20 mW / cm 2 (Irradiation with blue light)

[0076] Comparative Example 8:

[0077] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 7.66%) were mixed with PBS buffer to prepare a 700 μg / mL solution according to the ratio.

[0078] Comparative Example 9:

[0079] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer to prepare a 700 μg / mL solution according to the ratio.

[0080] Comparative Example 10:

[0081] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 3.28%) were mixed with PBS buffer to prepare a 700 μg / mL solution according to the ratio.

[0082] Comparative Example 11:

[0083] The mulberry dry water extract carbon quantum dots (AMQDs) solution and PBS buffer were mixed and a 500 μg / mL solution was prepared according to the proportion.

[0084] Comparative Example 12:

[0085] Nitrogen-doped carbon quantum dots (N-CQDs) (doped with 4.6%) were mixed with PBS buffer and a 4 mg / mL solution was prepared according to the ratio.

[0086] Table 4 Antibacterial rates of N-CQDs and AMQDs in the absence of light

[0087]

[0088] The data in Table 4 show that in the absence of light, N-CQDs and AMQDs showed no antibacterial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. PBS solution showed no antibacterial activity against the three bacteria, regardless of whether or not it was irradiated with visible light, eliminating the possibility that light irradiation could affect their growth.

[0089] In summary, nitrogen-doped carbon quantum dots (N-CQDs) can enhance the inhibitory effect on the three bacteria, and the doping ratio affects the antibacterial effect. 50 μg / mL of N-CQDs can achieve an antibacterial rate of 90% against Escherichia coli and Staphylococcus aureus under 450nm-460nm light for 1 hour, and 400 μg / mL of N-CQDs can achieve an antibacterial rate of 99% against Candida albicans. Compared with the AMQDs prepared in patent CN202310666863.1, the present invention reduces the antibacterial concentration of the three bacteria and also reduces the possibility of damage to normal tissues caused by the light source used.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing blue light-induced antibacterial nitrogen-doped carbon quantum dots, characterized by: The preparation method of nitrogen-doped carbon quantum dots is as follows: (1) Extracting anthocyanin aqueous extract from mulberry dry raw materials; (2) dissolving urea in anthocyanin aqueous extract to prepare a mixed solution; (3) The mixed solution was placed in a high-pressure reactor and reacted at 180°C for 12 hours. After the reaction was completed, it was cooled to room temperature and filtered to obtain a light orange nitrogen-doped carbon quantum dot solution; The prepared nitrogen-doped carbon quantum dots can exhibit antibacterial activity against Escherichia coli and / or Staphylococcus aureus under irradiation with blue light of 450nm-460nm wavelength.

2. The method for preparing blue light-induced antibacterial nitrogen-doped carbon quantum dots according to claim 1, characterized in that: The concentration ratio of anthocyanin water extract to urea in the solution is c 桑葚干水提取液 :c 尿素 =5:1~13:

1.

3. The method for preparing blue light-induced antibacterial nitrogen-doped carbon quantum dots according to claim 2, characterized in that: The concentration ratio of anthocyanin water extract to urea in the solution is c 桑葚干水提取液 :c 尿素 =9:

1.

4. An antibacterial solution, characterized in that: The blue light-induced antibacterial nitrogen-doped carbon quantum dots prepared by the method of any one of claims 1 to 3 are mixed with PBS buffer to obtain an antibacterial solution.

5. The antibacterial solution according to claim 4, wherein: The concentration of nitrogen-doped carbon quantum dots in the antibacterial solution is ≥50 μg / mL.

6. Use of the blue light-induced antibacterial nitrogen-doped carbon quantum dots prepared according to the method according to any one of claims 1 to 3 in the preparation of antibacterial products.

7. Use of the blue light-induced antibacterial nitrogen-doped carbon quantum dots prepared according to the method according to any one of claims 1 to 3 as a photosensitizer.

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