Antibacterial carbon dots as well as low-temperature normal-pressure preparation method and application thereof

Abstract

The invention discloses an antibacterial carbon dot and a low-temperature normal-pressure preparation method and application thereof.According to the method, ascorbic acid and cetylpyridinium chloride serve as raw materials, the antibacterial carbon dot is synthesized through a low-temperature normal-pressure method, and the preparation method is simple in process, low in reaction temperature, short in reaction time, free of any functional modification and low in cost. The prepared carbon dots have good water solubility and excellent antibacterial activity. Particularly, the carbon dots show remarkable antibacterial activity on drug-resistant bacteria, can inactivate the drug-resistant bacteria through multiple ways of destroying cell membranes, inducing to generate active oxygen, reducing bacterial surface charges and the like, and have a good application prospect in the aspect of antibiosis.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial technology and its biomedical application, and specifically relates to an antibacterial carbon dot and a low-temperature and normal-pressure preparation method and application thereof. Background Art

[0002] Diseases caused by bacterial infections seriously threaten people's lives. The antibacterial mechanism of traditional antibiotics is mainly through inhibiting bacterial cell wall synthesis, enhancing bacterial cell membrane permeability, interfering with bacterial protein synthesis, inhibiting bacterial nucleic acid replication and transcription, etc. However, the abuse of antibiotics has led to the evolution of bacterial resistance, and the emergence of drug-resistant bacteria has led to a surge in mortality and medical costs. Carbon dots, as a new type of functional nanomaterial, have an antibacterial mechanism different from traditional antibiotics. In particular, carbon dots can inactivate drug-resistant bacteria by destroying cell membranes, inducing oxidative stress, etc. Therefore, antibacterial materials based on carbon dots can not only effectively inactivate bacteria, but also avoid the development of bacterial resistance.

[0003] Cetylpyridinium chloride (CPC) is an economical antibacterial carbon dot precursor. However, the reported carbon dot preparation technologies generally have technical defects such as harsh reaction conditions and insufficient antibacterial activity. For example, Zhu and Zheng et al. used CPC as a precursor and adopted a hydrothermal method to prepare luminescent carbon dots. The preparation temperature reached 150°C and the reaction time required 2 hours. In addition, they focused on the formation mechanism and fluorescence luminescence properties of carbon dots, but did not evaluate the antibacterial activity of carbon dots, especially the effect on drug-resistant bacteria (Nanoscale, 2020, 12, 12773; RSC Adv., 2015, 5, 11667); Yang et al. prepared antibacterial carbon dots by mixing CPC aqueous solution and sodium hydroxide (NaOH) aqueous solution. They also did not evaluate the effect of the carbon dots on drug-resistant bacteria, and the published antibacterial properties were not ideal. The growth of Staphylococcus aureus and Escherichia coli could only be completely inhibited when the added concentrations in LB solid culture medium reached 75 μg / mL and 150 μg / mL, respectively (RSC Adv., 2022, 12, 22695).

[0004] At present, patents on antibacterial carbon dots generally have problems such as harsh carbon dot preparation conditions, long reaction time and poor antibacterial performance. For example, the Chinese invention patent document with publication number CN 118546675 A discloses iron-doped antibacterial carbon quantum dot materials and their preparation methods. The antibacterial carbon dots are obtained by hydrothermal method. The reaction temperature of the preparation process is as high as 200°C and the reaction time is as long as 12h. The antibacterial activity is not evaluated in the patent document; the Chinese invention patent document with publication number CN 118545703 A discloses a preparation method and application of iron-doped biomass carbon dots. The carbon dot preparation temperature range is 180-200°C, the reaction time range is 10-16h, and the antibacterial working concentration of the carbon dots is 200μg / mL; the Chinese invention patent document with publication number CN118419913 A discloses a method for preparing iron-doped biomass carbon dots and its application. The carbon dot preparation temperature range is 180-200°C, the reaction time range is 10-16h, and the antibacterial working concentration of the carbon dots is 200μg / mL; A's Chinese invention patent document discloses a nitrogen-doped tannic acid carbon dot for resistance to drug-resistant bacteria, and its preparation method and application. The preparation process uses a high-pressure reactor with a reaction temperature range of 160-220°C and a reaction time range of 5-24h. The carbon dots obtained by this method can inhibit the growth of Escherichia coli and Staphylococcus aureus only when the concentration is as high as 1600μg / mL. Summary of the invention

[0005] In view of the problems of harsh preparation conditions, long reaction time and insufficient antibacterial activity of existing antibacterial carbon dots, the purpose of the present invention is to provide a method for preparing antibacterial carbon dots under low temperature and normal pressure conditions using ascorbic acid (AA) and cetylpyridinium chloride (CPC) as precursors. In this method, an aqueous solution of ascorbic acid and cetylpyridinium chloride is heated and refluxed at a relatively low temperature to obtain carbon dots with high antibacterial activity in a short time. In the preparation process, the reaction temperature is low (80°C), the reaction time is short (0.5h), the process is simple, the cost is low, and the use of high-boiling organic solvents and high-temperature and high-pressure reactors is avoided, which is more environmentally friendly and safer. At the same time, the carbon dots provided by this method do not require any modification, that is, they have excellent antibacterial activity, can effectively inhibit Gram-positive and Gram-negative bacteria, and can efficiently inactivate drug-resistant bacteria. The minimum inhibitory concentration for methicillin-resistant Staphylococcus aureus is 1.25μg / mL and the minimum inhibitory concentration for Escherichia coli is 5μg / mL, and they have the potential to be prepared into antibacterial agents or bactericides.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A low-temperature and normal-pressure preparation method for antibacterial carbon dots is obtained by heating an alkaline aqueous solution of ascorbic acid and cetylpyridinium chloride. Specifically, the following steps are included: dissolving ascorbic acid and cetylpyridinium chloride solid powders in a sodium hydroxide (NaOH) solution of a certain concentration; then transferring the obtained mixed solution to an oil bath for heating treatment, cooling naturally to room temperature after the reaction is completed, filtering, dialyzing, and freeze-drying the reaction solution to obtain antibacterial carbon dots.

[0008] Preferably, the molar ratio of ascorbic acid to cetylpyridinium chloride is 1:1-10.

[0009] Preferably, the concentration of the NaOH solution of a certain concentration is 0.1-0.5 mol / L.

[0010] Preferably, the heating treatment is carried out at a temperature of 60 to 80° C. and for a time of 15 to 60 minutes.

[0011] Preferably, the molecular weight cut-off of the dialysis is 1000Da, and the dialysis time is 12 to 96 hours.

[0012] Preferably, the pore size of the filtering membrane is 0.22-0.45 μm.

[0013] The present invention also claims protection for the antibacterial carbon dots prepared by the above preparation method. The antibacterial carbon dots prepared by the present invention can inactivate Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), methicillin-resistant Staphylococcus aureus (MRSA), etc., and have excellent antibacterial activity.

[0014] Therefore, the present invention also claims to protect the use of the antibacterial carbon dots in the preparation of bacterial imaging agents or antibacterial agents.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The preparation method provided by the present invention can quickly (0.5 h) obtain antibacterial carbon dots under low temperature (80°C) and normal pressure conditions, avoiding the use of organic solvents and high temperature and high pressure conditions, and is safer and more environmentally friendly. In addition, the preparation process of the method is simple and has strong operability.

[0017] (2) The antibacterial carbon dots prepared in the present invention have good water solubility. They have excellent solubility in aqueous solution, and the maximum concentration of the aqueous solution can reach 4 mg / mL. They can be stably stored at room temperature for at least 6 months.

[0018] (3) The carbon dots prepared by the present invention have excellent antibacterial activity and have the potential to be used as bacterial imaging agents and antibacterial agents. Compared with other reported antibacterial carbon dots, the carbon dots prepared by this method can significantly inhibit the growth of drug-resistant bacteria in a low concentration range (1.25 μg / mL).

[0019] (4) The surface of the carbon dots prepared by the present invention is rich in functional groups such as hydroxyl groups, quaternary amine groups, and amine groups, which facilitates the introduction of other groups or the formation of composite materials to obtain more functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a transmission electron microscopy image of the antibacterial carbon dots prepared in Example 1, with a scale of 20 nm.

[0021] Figure 2 This is the X-ray photoelectron spectrum of the antibacterial carbon dots prepared in Example 1.

[0022] Figure 3 The ultraviolet absorption spectrum and fluorescence emission spectrum of the antibacterial carbon dots prepared in Example 2.

[0023] Figure 4 The growth curves of MRSA and E. coli in the presence of the antibacterial carbon dots prepared in Example 2.

[0024] Figure 5 Scanning electron microscope images of MRSA and E. coli bacterial cells in the presence of the antibacterial carbon dots prepared in Example 2. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the reagents and materials used are all commercially available unless otherwise specified.

[0027] Example 1

[0028] 1 mmol (0.1761 g) of ascorbic acid and 1 mmol (0.340 g) of hexadecylpyridinium chloride solid were dissolved in 20 mL of NaOH solution (0.1 mol / L), and the mixture was placed in a water bath at 80°C for constant temperature reaction for 1 hour. After the reaction was completed, it was cooled to room temperature. The brown reaction solution was filtered through a 0.22 μm filter head and dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 1000 Da. During the dialysis process, the water in the beaker was replaced every 4 hours. After 24 hours of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 1. The transmission electron microscope image and X-ray photoelectron spectrum of the antibacterial carbon dots 1 prepared in this example are shown as follows: Figure 1 and Figure 2 As shown, the antibacterial carbon dots 1 are spherical particles with a size of about 5 nm, and the surface of the carbon dots is rich in functional groups such as hydroxyl groups, quaternary amine groups, and amine groups.

[0029] The growth inhibition activity of antimicrobial carbon dots 1 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6CFU / mL of bacterial suspension was mixed with 50μL of different concentrations of antibacterial carbon dots 1 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40μg / mL). The mixed bacterial suspension was incubated at 37°C for 24 hours, and the optical density of the bacterial suspension at 600nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that 2.5μg / mL of antibacterial carbon dots 1 can significantly inhibit the growth of MRSA, and 10μg / mL of antibacterial carbon dots 1 can significantly inhibit the growth of E.coli.

[0030] Example 2

[0031] 1mmol (0.1761g) of ascorbic acid and 1mmol (0.340g) of hexadecylpyridinium chloride solid were dissolved in 20mL of NaOH solution (0.1mol / L), and the mixture was placed in a water bath at 80°C for constant temperature reaction for 0.5h. After the reaction was completed, it was cooled to room temperature. The brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 2. The ultraviolet absorption spectrum and fluorescence emission spectrum of the antibacterial carbon dots 2 prepared in this embodiment are shown in Figure 2. Figure 3 As shown, the carbon dots have bright fluorescence emission near 560 nm and thus may have application potential in bacterial cell imaging.

[0032] The growth inhibition activity of antimicrobial carbon dots 2 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6 The bacterial suspension with CFU / mL was mixed with 50 μL of different concentrations of antimicrobial carbon dots 2 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40 μg / mL). The mixed bacterial suspension was incubated at 37°C for 24 h, and the optical density of the bacterial suspension at 600 nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. Figure 4 and Figure 5 As shown, 1.25 μg / mL of antibacterial carbon dots 2 can significantly inhibit the growth of MRSA, and 5 μg / mL of antibacterial carbon dots 2 can significantly inhibit the growth of E. coli. Compared with the bacterial cells in the control group (without antibacterial carbon dots 2), the integrity of MRSA and E. coli bacterial cells treated with antibacterial carbon dots 2 was significantly damaged.

[0033] Example 3

[0034] 1mmol (0.1761g) of ascorbic acid and 1mmol (0.340g) of hexadecylpyridinium chloride solid were dissolved in 20mL of NaOH solution (0.1mol / L), and the mixture was placed in a 60℃ water bath for constant temperature reaction for 0.5h. After the reaction was completed, it was cooled to room temperature, and the obtained brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 3.

[0035] The growth inhibition activity of antimicrobial carbon dots 3 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6 CFU / mL bacterial suspension was mixed with 50μL of different concentrations of antibacterial carbon dots 3 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40μg / mL). The mixed bacterial suspension was incubated at 37℃ for 24h, and the optical density of the bacterial suspension at 600nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that antibacterial carbon dots 3 showed significant inhibitory activity against both MRSA and E.coli.

[0036] Example 4

[0037] 10mmol (1.761g) of ascorbic acid and 1mmol (0.340g) of hexadecylpyridinium chloride solid were dissolved in 20mL of NaOH solution (0.1mol / L), and the mixture was placed in a water bath at 80℃ for constant temperature reaction for 0.5h. After the reaction was completed, it was cooled to room temperature, and the brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 4.

[0038] The growth inhibition activity of antimicrobial carbon dots 4 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6CFU / mL bacterial suspension was mixed with 50μL of different concentrations of antibacterial carbon dots 4 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40μg / mL). The mixed bacterial suspension was incubated at 37℃ for 24h, and the optical density of the bacterial suspension at 600nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that antibacterial carbon dots 4 showed significant inhibitory activity against both MRSA and E.coli.

[0039] Example 5

[0040] 10mmol (1.761g) of ascorbic acid and 1mmol (0.340g) of hexadecylpyridinium chloride solid were dissolved in 20mL of NaOH solution (0.1mol / L), and the mixture was placed in a water bath at 80℃ for constant temperature reaction for 15min. After the reaction was completed, it was cooled to room temperature, and the obtained brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 5.

[0041] The growth inhibition activity of antimicrobial carbon dots 5 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6 CFU / mL bacterial suspension was mixed with 50 μL of different concentrations of antibacterial carbon dots 5 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40 μg / mL). The mixed bacterial suspension was incubated at 37°C for 24 hours, and the optical density of the bacterial suspension at 600 nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that antibacterial carbon dots 5 showed significant inhibitory activity against both MRSA and E. coli.

[0042] Example 6

[0043] 1mmol (0.1761g) of ascorbic acid and 10mmol (3.40g) of hexadecylpyridinium chloride solid were dissolved in 20mL of NaOH solution (0.1mol / L), and the mixture was placed in a water bath at 80℃ for constant temperature reaction for 0.5h. After the reaction was completed, it was cooled to room temperature, and the brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain antibacterial carbon dots 6.

[0044] The growth inhibition activity of antibacterial carbon dots 6 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6 CFU / mL bacterial suspension was mixed with 50μL of different concentrations of antibacterial carbon dots 6 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40μg / mL). The mixed bacterial suspension was incubated at 37℃ for 24h, and the optical density of the bacterial suspension at 600nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that antibacterial carbon dots 6 showed significant inhibitory activity against both MRSA and E.coli.

[0045] Comparative Example 7

[0046] 1mmol (0.340g) of hexadecylpyridinium chloride solid was dissolved in 20mL NaOH solution (0.1mol / L), and the mixture was placed in an 80℃ water bath for constant temperature reaction for 0.5h. After the reaction was completed, it was cooled to room temperature, and the obtained brown reaction solution was filtered through a 0.22μm filter head and dialyzed for 24h with a dialysis bag with a molecular weight cutoff of 1000Da. During the dialysis process, the water in the beaker was replaced every 4h. After 24h of dialysis, the solution in the dialysis bag was taken out and freeze-dried to obtain comparative carbon point 7.

[0047] The growth inhibition activity of Cdot 7 against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The bacteria stored on the slant were inoculated into LB medium and cultured overnight in a constant temperature shaker at 37°C. 50 μL of the 10 6 CFU / mL of bacterial suspension was mixed with 50μL of different concentrations of comparative carbon dots 7 (final concentration: 0, 0.625, 1.25, 2.5, 5.10, 20, 40μg / mL). The mixed bacterial suspension was incubated at 37°C for 24h, and the optical density of the bacterial suspension at 600nm was recorded as the incubation time changed. Each concentration was repeated at least 3 times. The results showed that the minimum inhibitory concentrations of comparative carbon dots 7 against MRSA and E.colli were 5μg / mL and 10μg / mL, respectively.

[0048] The minimum inhibitory concentrations of the antibacterial carbon dots obtained in the above-mentioned embodiments are shown in Table 1. As can be seen from Table 1, the antibacterial carbon dots prepared in Examples 1-6 of the present invention have good inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), and the antibacterial effect is related to the reactant ratio, reaction temperature and reaction time for preparing the carbon dots. The best minimum inhibitory concentrations for methicillin-resistant Staphylococcus aureus are 1.25 μg / mL and for Escherichia coli are 5 μg / mL (Examples 2 and 6).

[0049] Table 1 Inhibitory activity of antibacterial carbon dots prepared in different examples against methicillin-resistant Staphylococcus aureus and Escherichia coli

[0050]

[0051] In summary, the present invention provides a method for preparing antibacterial carbon dots under low temperature and normal pressure conditions, which avoids the use of high temperature and high pressure conditions and is safer and more environmentally friendly. In addition, the carbon dots prepared by this method do not require any modification, and exhibit excellent antibacterial activity against both Gram-positive and Gram-negative bacteria, and can efficiently inactivate drug-resistant bacteria, and have the potential to be prepared into antibacterial agents or bactericides.

[0052] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing antibacterial carbon dots at low temperature and normal pressure, characterized in that: The following steps are involved: (1) firstly dissolving ascorbic acid and cetylpyridinium chloride solid powder in a sodium hydroxide solution; then transferring the mixed solution to an oil bath pot for heating, and after the reaction is completed, naturally cooling to room temperature to obtain a brown reaction solution; (2) The brown reaction solution obtained in step (1) is filtered and dialyzed to obtain antibacterial carbon dots.

2. The preparation method according to claim 1, characterized in that: In the step (1), the molar ratio of ascorbic acid to cetylpyridinium chloride is 1:1-10.

3. The preparation method according to claim 1, characterized in that: In the step (1), the concentration of the sodium hydroxide solution is 0.1-0.5 mol / L.

4. The preparation method according to claim 1, characterized in that: In the step (1), the reaction temperature is 60-80°C and the reaction time is 15-60 minutes.

5. The preparation method according to claim 1, characterized in that: In the step (2), the molecular weight cut-off of dialysis is 1000Da, and the dialysis time is 12 to 96 hours.

6. The preparation method according to claim 1, characterized in that: In the step (2), the pore size of the filter membrane is 0.22 to 0.45 μm.

7. The antibacterial carbon dots prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the antibacterial carbon dots according to claim 7 in the preparation of bacterial imaging agents.

9. Use of the antibacterial carbon dots according to claim 7 in the preparation of antibacterial agents.

10. The use according to claim 9, characterized in that: The antibacterial agent includes preparations against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Escherichia coli.

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