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

Abstract

The application discloses antibacterial carbon dots and a low-temperature and normal-pressure preparation method and application thereof. The method is characterized in that ascorbic acid and cetylpyridinium chloride are used as raw materials, and the antibacterial carbon dots are synthesized through a low-temperature and normal-pressure method. The preparation method has the advantages of simple process, low reaction temperature, short reaction time, no need for any functional modification and low cost. The prepared carbon dots have good water solubility and excellent antibacterial activity. In particular, the carbon dots exhibit significant antibacterial activity on drug-resistant bacteria, can inactivate the drug-resistant bacteria through multiple ways such as destroying cell membranes, inducing active oxygen and reducing the surface charge of bacteria, and have a good application prospect in antibacterial aspects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterials and biomedical applications thereof, and particularly relates to an antibacterial carbon dot and a low-temperature and normal-pressure preparation method and application thereof. BACKGROUND

[0002] Diseases caused by bacterial infections seriously threaten people's life safety. 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 leads to the evolution of bacterial drug resistance, and the emergence of drug-resistant bacteria promotes the surge of mortality and medical expenses. As a new type of functional nanomaterial, carbon dots 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., so that antibacterial materials based on carbon dots can not only effectively inactivate bacteria, but also avoid the generation of bacterial drug resistance.

[0003] Cetylpyridinium chloride (CPC) is an economical antibacterial carbon dot precursor, but 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. respectively prepared luminescent carbon dots using CPC as a precursor by a hydrothermal method, and the preparation temperature reached 150℃ and the reaction time needed 2h. In addition, they focused on the formation mechanism and fluorescence emission performance of carbon dots, and 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, and also did not evaluate the effect of the carbon dots on drug-resistant bacteria, and the published antibacterial performance was also not ideal. When the addition concentration of the carbon dots in LB solid medium was 75μg / mL and 150μg / mL respectively, the growth of Staphylococcus aureus and Escherichia coli could be completely inhibited (RSC Adv., 2022, 12, 22695).

[0004] In the current patents on antibacterial carbon dots, there are generally problems such as harsh preparation conditions, long reaction time and poor antibacterial performance. For example, the Chinese patent document with publication number CN 118546675 A discloses an iron-doped antibacterial carbon quantum dot material and its preparation method. The antibacterial carbon dots are obtained by hydrothermal method, with a reaction temperature as high as 200℃ and a reaction time as long as 12h. The antibacterial activity of the carbon dots is not evaluated in the patent document. The Chinese patent document with publication number CN 118545703 A discloses a preparation method and application of iron-doped biomass carbon dots. The preparation temperature of the carbon dots is in the range of 180-200℃, and the reaction time is in the range of 10-16h. The antibacterial working concentration of the carbon dots is 200μg / mL. The Chinese patent document with publication number CN 118419913 A discloses nitrogen-doped tannin carbon dots against drug-resistant bacteria, and its preparation method and application. The preparation process uses a high-pressure reaction kettle, with a reaction temperature in the range of 160-220℃ and a reaction time in the range of 5-24h. The carbon dots obtained by the method can only inhibit the growth of Escherichia coli and Staphylococcus aureus at a concentration as high as 1600μg / mL. SUMMARY

[0005] In view of the problems of harsh preparation conditions, long reaction time and insufficient antibacterial activity of the existing antibacterial carbon dots, the purpose of the present application is to provide a method for preparing antibacterial carbon dots at low temperature and normal pressure, using ascorbic acid (AA) and cetylpyridinium chloride (CPC) as precursors. The method can obtain carbon dots with high antibacterial activity in a short time by heating the aqueous solution of ascorbic acid and cetylpyridinium chloride at a relatively low temperature. In the preparation process, the reaction temperature is low (80℃), the reaction time is short (0.5h), the process is simple, and the cost is low. The use of high-boiling organic solvents and high-temperature high-pressure reaction kettles is avoided, which is more environmentally friendly and safer. At the same time, the carbon dots provided by the method have excellent antibacterial activity without any modification. They can not only effectively inhibit Gram-positive and Gram-negative bacteria, but also efficiently inactivate drug-resistant bacteria. The minimum inhibitory concentration of the carbon dots against methicillin-resistant Staphylococcus aureus is 1.25μg / mL, and the minimum inhibitory concentration against Escherichia coli is 5μg / mL. They have the potential to be prepared into bacteriostatic agents or bactericides.

[0006] The technical solutions adopted by the present application are as follows:

[0007] A low-temperature normal-pressure preparation method of antibacterial carbon dots is obtained by heating an alkaline aqueous solution of ascorbic acid and cetylpyridinium chloride. The method specifically includes the following steps: dissolving ascorbic acid and cetylpyridinium chloride solid powders in a sodium hydroxide (NaOH) solution with a certain concentration; then transferring the obtained mixture to an oil bath pot for heating treatment; naturally cooling to room temperature after the reaction is completed; and obtaining antibacterial carbon dots after the reaction solution is filtered, dialyzed and freeze-dried.

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

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

[0010] Preferably, the heating treatment temperature is 60-80℃, and the time is 15-60 min.

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

[0012] Preferably, the filter membrane has a pore size of 0.22-0.45 μm.

[0013] The application also claims the antibacterial carbon dots prepared by the above preparation method. The antibacterial carbon dots prepared by the application can inactivate S. aureus, E. coli, MRSA, etc., and have excellent antibacterial activity.

[0014] Therefore, the application also claims the use of the antibacterial carbon dots in preparing bacterial imaging reagents or antibacterial agents.

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

[0016] (1) The preparation method provided by the application can obtain antibacterial carbon dots under low temperature (80℃) and normal pressure conditions, and the method can be fast (0.5 h), avoids the use of organic solvents and high temperature and high pressure conditions, and is more safe and environmentally friendly. In addition, the method has simple preparation process and strong operability.

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

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

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

[0020] Figure 1Transmission electron microscope picture of the antibacterial carbon dots prepared in Example 1, scale 20 nm.

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

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

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

[0024] Figure 5 Scanning electron microscope picture 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 further illustrations of the application and are not intended to limit the same.

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

[0027] Example 1

[0028] 1 mmol (0.1761 g) of ascorbic acid and 1 mmol (0.340 g) of cetylpyridinium chloride solid were dissolved in 20 mL of NaOH solution (0.1 mol / L), and the mixture was placed in a 80°C water bath for constant temperature reaction for 1 h. After the reaction was completed, the brown reaction solution was cooled to room temperature, filtered through a 0.22 μm filter head, and dialyzed in a dialysis bag with a molecular weight cut-off of 1000 Da for 24 h. The water in the beaker was replaced every 4 h during the dialysis, and after dialysis for 24 h, the solution in the dialysis bag was removed and freeze-dried to obtain antibacterial carbon dots 1. The transmission electron microscope picture and the X-ray photoelectron spectrogram of the antibacterial carbon dots 1 prepared in this example are shown in Figure 1 and Figure 2 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 antibacterial activity of the antibacterial carbon dots 1 on methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by the broth dilution method. The slant-preserved strain was inoculated in LB medium and incubated in a constant temperature 37°C shaker overnight. 50 μL of the bacterial solution with a density of 10 6The bacterial suspension with CFU / mL was mixed with 50 μL of different concentrations of antibacterial carbon dots 1 (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 change of optical density value of the bacterial suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. The results showed that 2.5 μg / mL of antibacterial carbon dots 1 could significantly inhibit the growth of MRSA, and 10 μg / mL of antibacterial carbon dots 1 could significantly inhibit the growth of E. coli.

[0030] Example 2

[0031] 1 mmol (0.1761 g) of ascorbic acid and 1 mmol (0.340 g) of cetylpyridinium chloride solid were dissolved in 20 mL of NaOH solution (0.1 mol / L), and the mixture was placed in a 80°C water bath for constant temperature reaction for 0.5 h. After the reaction was completed, it was cooled to room temperature, and the obtained brown reaction solution was filtered through a 0.22 μm filter head and dialyzed in a dialysis bag with a molecular weight cut-off of 1000 Da for 24 h. The water in the beaker was replaced every 4 h during dialysis, and after 24 h 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 example are shown in Figure 3 , which has bright fluorescence emission near 560 nm wavelength, and therefore may have potential application in bacterial cell imaging.

[0032] The growth inhibition activity of antibacterial carbon dots 2 on methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli) was determined by broth dilution method. The slant-preserved bacterial strain was inoculated in LB medium and incubated in a constant temperature 37°C shaker overnight. 50 μL of bacterial suspension with a density of 10 6 CFU / mL was mixed with 50 μL of different concentrations of antibacterial 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 change of optical density value of the bacterial suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. As shown in Figure 4 and Figure 5 , 1.25 μg / mL of antibacterial carbon dots 2 could significantly inhibit the growth of MRSA, and 5 μg / mL of antibacterial carbon dots 2 could significantly inhibit the growth of E. coli, and compared with the control group of bacterial cells (without antibacterial carbon dots 2), the MRSA and E. coli bacterial cell integrity treated with antibacterial carbon dots 2 was obviously damaged.

[0033] Example 3

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

[0035] The broth dilution method was used to determine the growth inhibition activity of the antibacterial carbon dots 3 on methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). The slant-preserved strain was inoculated in LB medium and incubated in a constant temperature 37 °C shaker overnight. 50 μL of bacterial suspension with a density of 10 6 CFU / mL was mixed with 50 μL of antibacterial carbon dots 3 of different concentrations (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 change of optical density value of the bacterial suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. The results showed that the antibacterial carbon dots 3 exhibited significant inhibition activity on MRSA and E. coli.

[0036] Example 4

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

[0038] The broth dilution method was used to determine the growth inhibition activity of the antibacterial carbon dots 4 on methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). The slant-preserved strain was inoculated in LB medium and incubated in a constant temperature 37 °C shaker overnight. 50 μL of bacterial suspension with a density of 10 6The bacterial suspension of 10 CFU / mL was mixed with 50 μL of different concentrations of antibacterial carbon dots 4 (final concentrations: 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 change of optical density value of the bacterial suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. The research results show that antibacterial carbon dots 4 exhibit significant inhibitory activity on MRSA and E. coli.

[0039] Example 5

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

[0041] The broth dilution method was used to determine the growth inhibition activity of antibacterial carbon dots 5 on methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli). The slant-preserved strain was inoculated in LB medium and incubated in a constant temperature 37°C shaker overnight. 50 μL of bacterial suspension with a density of 10 6 The bacterial suspension of 10 CFU / mL was mixed with 50 μL of different concentrations of antibacterial carbon dots 5 (final concentrations: 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 change of optical density value of the bacterial suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. The research results show that antibacterial carbon dots 5 exhibit significant inhibitory activity on MRSA and E. coli.

[0042] Example 6

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

[0044] The broth dilution method was used to determine the growth inhibition activity of antibacterial carbon dots 6 against methicillin-resistant Staphylococcus aureus (MRSA) and E. coli. The bacteria preserved in the slant were inoculated in LB medium and incubated in a constant temperature 37 °C shaker overnight. 50 μL of bacteria suspension with a density of 10 6 CFU / mL was mixed with 50 μL of antibacterial carbon dots 6 of different concentrations (final concentration: 0, 0.625, 1.25, 2.5, 5, 10, 20, 40 μg / mL). The mixed bacteria suspension was incubated at 37 °C for 24 h, and the change of optical density value of the bacteria suspension at 600 nm with incubation time was recorded. Each concentration was repeated at least 3 times. The results of the study showed that antibacterial carbon dots 6 exhibited significant inhibitory activity against MRSA and E. coli.

[0045] Comparative Example 7

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

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

[0048] The minimum inhibitory concentration of the antibacterial carbon dots obtained by the above embodiments is shown in Table 1. As can be seen from Table 1, the antibacterial carbon dots prepared by Examples 1-6 of the present application have good inhibitory activity on methicillin-resistant Staphylococcus aureus (MRSA) and E. coli, and the inhibitory effect is related to the proportion of the reactants, the reaction temperature and the reaction time for preparing the carbon dots. The best minimum inhibitory concentration of methicillin-resistant Staphylococcus aureus can reach 1.25 μg / mL and the minimum inhibitory concentration of E. coli can reach 5 μg / mL (Examples 2, 6).

[0049] Table 1 Inhibitory activity of antibacterial carbon dots prepared by different embodiments on methicillin-resistant Staphylococcus aureus and E. coli

[0050]

[0051] In summary, the present application 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 more safe and environmentally friendly. In addition, the carbon dots prepared by the method do not need any modification and exhibit excellent antibacterial activity on gram-positive and gram-negative bacteria, and can efficiently inactivate drug-resistant bacteria, having the potential to be prepared into bacteriostatic or bactericidal agents.

[0052] The above is only the preferred embodiment of the present application, and it should be noted that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A method for low-temperature and normal-pressure preparation of antibacterial carbon dots, characterized in that, The method comprises the following steps: (1) first, dissolve ascorbic acid and cetylpyridinium chloride solid powder in a sodium hydroxide solution with a concentration of 0.1-0.5 mol / L according to a molar ratio of 1:1-10; then, transfer the mixed solution to an oil bath pot for heating, and react at 60-80 ℃ for 15-60 min; after the reaction, naturally cool to room temperature to obtain a brown reaction solution; (2) filter and dialyze the brown reaction solution obtained in step (1) to obtain antibacterial carbon dots.

2. The production method according to claim 1, characterized by, In step (2), the molecular weight cut-off of dialysis is 1000 Da, and the dialysis time is 12-96 h.

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

4. The antibacterial carbon dots prepared by the preparation method according to any one of claims 1-3.

5. The antibacterial carbon dots according to claim 4 are used for preparing a bacterial imaging reagent.

6. The antibacterial carbon dots according to claim 4 are used for preparing an antibacterial agent.

7. Use according to claim 6, characterized in that, The antibacterial agent includes preparations for resisting Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Escherichia coli.

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