Carbon nanodots against multi-drug resistant bacterial biofilm and preparation method and application thereof

Abstract

The application discloses carbon nanodots for resisting multi-drug resistant bacterial biofilm and a preparation method and application thereof. The carbon nanodots are prepared as follows: taking dopamine hydrochloride and diethylene triamine as raw materials, dissolving in deionized water, transferring the reaction solution to a reaction kettle, reacting at 80-200 DEG C for 1-24h, and subjecting the reaction product to dialysis and freeze-drying to obtain the carbon nanodots. The prepared carbon nanodots are used for resisting multi-drug resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) biofilm. The results show that the carbon nanodots have excellent performance of inhibiting the formation of MRSA biofilm and eliminating mature MRSA biofilm. In addition, the carbon nanodots can be applied to the preservation of perishable foods such as strawberries. The preparation method is simple and easy to operate, has low cost and good reproducibility, provides entities and technical guidance for the prevention and treatment of biofilm related infections and food safety protection, and provides a basis for the preparation and application of multifunctional antimicrobial materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antimicrobial biofilm functional material preparation and application, and particularly relates to a carbon nanodot for resisting multi-drug resistant bacterial biofilm and a preparation method and application thereof. BACKGROUND

[0002] In recent years, with the abuse and misuse of antibiotics, the problem of bacterial drug resistance is escalating, and currently there are many super drug-resistant bacteria, which have become a difficult point in clinical treatment. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is the most common multi-drug resistant pathogen in the world today, which is resistant to all β-lactam antibacterial drugs including cephalosporins and carbapenems, and can cause fatal skin, pneumonia and implantable device infections in the human body. More than that, methicillin-resistant Staphylococcus aureus usually forms a dense three-dimensional structure of biofilm on the infected site of the human body, on the surface of food and on the surface of medical devices, to escape the damage of drugs such as antibiotics and external force, and is considered to be another key factor for drug resistance. The drug resistance produced is 10-1000 times that of planktonic bacteria, which has brought great pressure to infection treatment and has become one of the three most difficult diseases to treat. Therefore, it has great application value to develop a multifunctional antibacterial agent with high efficiency against methicillin-resistant Staphylococcus aureus biofilm.

[0003] With the rapid development of nanotechnology, various nanomaterials have been developed for the killing and anti-infection treatment of bacteria, fungi and their biofilms. Carbon nanodots have attracted high attention in the field of antimicrobials due to their unique physicochemical properties, nanoscale size, excellent biological safety, easy preparation and rich surface functional groups. The currently developed carbon nanodots are mainly applied to the killing research of Staphylococcus aureus and Escherichia coli in the planktonic state, while the killing effect in the biofilm is less studied, and the related antibacterial effect research is still in the embryonic stage, and the potential antimicrobial biofilm effect needs to be further studied. Therefore, it is of great significance to develop carbon nanodots with high efficiency against methicillin-resistant Staphylococcus aureus biofilm to promote the development of carbon nanodots in the field of microbial prevention and control. SUMMARY

[0004] The present application aims at the current multi-drug resistant pathogen methicillin-resistant Staphylococcus aureus biofilm caused related infection and the lack of carbon nanodots that can be applied to high efficiency against methicillin-resistant Staphylococcus aureus biofilm, and provides a carbon nanodot for resisting multi-drug resistant bacterial biofilm and a preparation method and application thereof. The present application realizes the killing of multi-drug resistant pathogen methicillin-resistant Staphylococcus aureus biofilm by using carbon nanodots, which has the properties of simple preparation process, high efficiency against biofilm and can be used for food protection of perishable fruits and the like.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing carbon nanodots for biofilms against multidrug-resistant bacteria includes the following steps: using dopamine hydrochloride and diethylenetriamine as raw materials, dissolving them in deionized water, then transferring the resulting reaction solution to a reaction vessel, reacting in an oven for 1-24 hours, and then dialyzing and freeze-drying the reaction product to obtain carbon nanodots for biofilms against multidrug-resistant bacteria.

[0007] Preferably, the molar ratio of dopamine hydrochloride to diethylenetriamine is 1:50-50:1.

[0008] Preferably, the mass-to-volume ratio of dopamine hydrochloride to deionized water is 1g:10-100mL, and the reaction temperature is 80-200℃.

[0009] The present invention also provides carbon nanodots prepared according to the above preparation method.

[0010] Furthermore, the present invention also provides the application of the above-mentioned carbon nanodots in biofilms against multidrug-resistant bacteria.

[0011] Preferably, the multidrug-resistant bacterial biofilm targets methicillin-resistant Staphylococcus aureus (MRSA) biofilms.

[0012] Preferably, the application includes the following steps:

[0013] Carbon nanodots were co-incubated with methicillin-resistant Staphylococcus aureus (MRSA) planktonic bacteria for a certain period of time. Subsequently, the ability of carbon nanodots to inhibit the formation of MRSA biofilms was investigated using crystal violet staining and confocal laser scanning microscopy. At the same time, carbon nanodots were applied to mature biofilms, and the ability of carbon nanodots to remove MRSA biofilms was investigated using crystal violet staining and confocal laser scanning microscopy.

[0014] Preferably, the working concentration of the carbon nanodots is 50-1000 μg / mL, and the co-incubation time is 24-72 h.

[0015] The present invention also provides the application of the above-mentioned carbon nanodots in the microbial protection of food.

[0016] Preferably, the food microbial protection targets perishable fruit such as strawberries.

[0017] The present invention has the following advantages and effects over the prior art:

[0018] (1) The carbon nanodots of the present invention have the characteristics of simple preparation operation, good reproducibility and high biosafety.

[0019] (2) It has the characteristics of safety and high efficiency in the application of biofilm against multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, and can be used for food preservation.

[0020] (3) This invention expands the range of multifunctional nanomaterials for biofilms against multidrug-resistant bacteria, providing technical support for the preparation of novel antimicrobial materials with excellent biosafety. Attached Figure Description

[0021] Figure 1 This is the X-ray photoelectron spectrum of carbon nanodots.

[0022] Figure 2 This is a transmission electron microscope (TEM) image of carbon nanodots.

[0023] Figure 3 The inhibition rate (left) and bactericidal rate (right) of carbon nanodots against methicillin-resistant Staphylococcus aureus.

[0024] Figure 4 This demonstrates the inhibitory effect of carbon nanodots on biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA). The left image shows the evaluation of the biofilm inhibition performance of carbon nanodots using crystal violet staining; the middle and right images are from a confocal laser scanning microscope.

[0025] Figure 5 This demonstrates the ability of carbon nanodots to scavenge mature biofilms of methicillin-resistant Staphylococcus aureus (MRSA). The left image shows the evaluation of carbon nanodots' ability to scavenge biofilm formation using crystal violet staining; the middle and right images are from a confocal laser scanning microscope.

[0026] Figure 6 The preservation effect of carbon nanodots on strawberries. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] Example 1

[0029] 189.64 mg of dopamine hydrochloride and 5.16 g of diethylenetriamine were weighed and dissolved in 18.96 mL of deionized water. After the reactants were completely dissolved and mixed, the resulting reaction solution was transferred to a reaction vessel and placed in an oven at 200 °C for 12 h. Subsequently, the reaction product was separated and purified by dialysis, and then freeze-dried to obtain carbon nanodots for biofilm formation against multidrug-resistant bacteria.

[0030] Example 2

[0031] 0.948 g of dopamine hydrochloride and 10.32 mg of diethylenetriamine were weighed and dissolved in 95 mL of deionized water. After the reactants were completely dissolved and mixed, the resulting reaction solution was transferred to a reaction vessel and placed in an oven at 80 °C for 24 h. Subsequently, the reaction product was separated and purified by dialysis, and then freeze-dried to obtain carbon nanodots for biofilm formation against multidrug-resistant bacteria.

[0032] Example 3

[0033] 1.896 g of dopamine hydrochloride and 1.032 g of diethylenetriamine were weighed and dissolved in 18.96 mL of deionized water. After the reactants were completely dissolved and mixed, the resulting reaction solution was transferred to a reaction vessel and placed in an oven at 150 °C for 12 h. Subsequently, the reaction product was separated and purified by dialysis, and then freeze-dried to obtain carbon nanodots for biofilm formation against multidrug-resistant bacteria.

[0034] Example 4

[0035] The carbon nanodots prepared in Example 1 of this invention were characterized in terms of chemical composition and morphology, such as... Figure 1 and 2 As shown. Figure 1 X-ray photoelectron spectroscopy shows that carbon nanodots are mainly composed of four elements: C, N, O and Cl, with N doping content as high as 28.36%. Figure 2 Transmission electron microscopy images show that the carbon nanodots have a size of less than 10 nm.

[0036] Example 5

[0037] (1) In this embodiment, the carbon nanodots prepared in Example 1 of the present invention were used to investigate their growth inhibition ability against methicillin-resistant Staphylococcus aureus (MRSA), a multidrug-resistant bacterium: freshly grown bacterial solution (10 μL / 3000 μL) was subjected to logarithmic growth. 6 CFU / mL was co-incubated with different concentrations of carbon nanodots (0-800 μg / mL) for 24 h. The absorbance of the corresponding bacterial solutions at 600 nm was then measured using a multi-functional microplate reader to calculate and evaluate the inhibitory effect of carbon nanodots on the growth of methicillin-resistant Staphylococcus aureus (Bacterial survival rate = OD). 600-样品组 / OD 600-对照组 (×100%), the result is as follows Figure 3 As shown in the left figure, all samples are in triplicate. Figure 3 As shown in the left figure, carbon nanodots have a good inhibitory effect on the growth of methicillin-resistant Staphylococcus aureus, with a minimum inhibitory concentration of 400 μg / mL.

[0038] (2) The bactericidal ability of the carbon nanodots prepared in Example 1 of this invention against methicillin-resistant Staphylococcus aureus was investigated: freshly grown bacterial solution (10 6CFU / mL was incubated with different concentrations of carbon nanodots (0-800 μg / mL) at 37℃ (180 rpm) for 24 h. Subsequently, the bacterial suspension was serially diluted using the plate count method, and 100 μL of the suspension was spread onto solid agar plates. Growth was continued for 24-48 h, and colonies were counted. Finally, the bactericidal activity of carbon nanodots against methicillin-resistant Staphylococcus aureus (CRSA) was analyzed and evaluated (Log). 10 (colony count × dilution factor)), results are as follows Figure 3 As shown in the right figure, all samples are in triplicate. Figure 3 As shown in the right figure, carbon nanodots have the same minimum inhibitory concentration and minimum bactericidal concentration, both of which are 400 μg / mL, indicating that carbon nanodots have excellent killing ability against methicillin-resistant Staphylococcus aureus.

[0039] Example 6

[0040] This embodiment uses the carbon nanodots prepared in Example 1 of the present invention to evaluate their inhibitory ability on the formation of methicillin-resistant Staphylococcus aureus biofilm: fresh bacterial culture in the logarithmic growth phase (10 7 CFU / mL was mixed with carbon nanodots of different concentrations and incubated at 37°C for 48 h. The culture medium was then removed, airborne bacteria were washed away, and the samples were stained with crystal violet. After washing and dissolving in ethanol, the absorbance (OD) values ​​of different sample groups were measured at 595 nm using a multi-functional microplate reader. 595 All samples were tested in triplicate, and the results are as follows: Figure 4 The left figure shows (biofilm mass = OD). 595-样品组 / OD 595-对照组 ×100%). Simultaneously, the bacterial solution (10 7 Biofilms co-incubated with 400 μg / mL carbon nanodots for 48 h were stained with SYTO9 / PI live / dead dye. The ability of carbon nanodots to inhibit biofilm formation was observed using confocal laser scanning microscopy. The results are as follows: Figure 4 The middle image (control group) and the right image (sample group) are shown. Figure 4 It is known that carbon nanodots have an excellent ability to inhibit the formation of biofilms.

[0041] Example 7

[0042] This embodiment uses the carbon nanodots prepared in Example 1 of the present invention to evaluate their ability to remove mature biofilms of methicillin-resistant Staphylococcus aureus: fresh bacterial culture in the logarithmic growth phase (10 7The samples were incubated at 37℃ for 72 h with CFU / mL. The culture medium was then removed, and the airborne bacteria were washed away. The samples were then incubated with different concentrations of carbon nanodots (in the culture medium) for another 24 h. The absorbance (OD) values ​​of different sample groups were measured at 595 nm using a multi-functional microplate reader with crystal violet staining. 595 All samples were tested in triplicate, and the results are as follows: Figure 5 The left figure shows (biofilm clearance rate = (OD) 595-对照组 -OD 595-样品组 ) / OD 595-对照组 (×100%). Simultaneously, the cultured mature biofilm was co-incubated with 400 μg / mL carbon nanodots for 24 h, stained with SYTO9 / PI live / dead dye, and the ability of carbon nanodots to remove biofilm was observed using confocal laser scanning microscopy. The results are as follows: Figure 5 The middle image (control group) and the right image (sample group) are shown. Figure 5 It is known that carbon nanodots have excellent biofilm removal capabilities.

[0043] Example 8

[0044] This embodiment uses the carbon nanodots prepared in Example 1 of the present invention to evaluate their preservative ability on perishable foods, taking strawberries as an example. The carbon nanodots were dissolved in an aqueous solution containing 2.5% chitosan and 0.625% glycerol to a final concentration of 50 μg / mL. Fresh strawberries were then immersed in the solution for 1 minute. The resulting strawberries were placed on a table near a window, and the changes in the strawberries were observed. A solution without carbon nanodots was used as a control. The results are as follows: Figure 6 As shown. By Figure 6 It can be seen that the prepared carbon nanodots have excellent preservation and anti-corrosion capabilities.

[0045] Comparative Example 1

[0046] Similar to Example 1, 189.64 mg of dopamine hydrochloride and 5.16 g of ethylenediamine were weighed and dissolved in 18.96 mL of deionized water. After the reactants were completely dissolved and mixed, the resulting reaction solution was transferred to a reaction vessel and placed in an oven at 200 °C for 12 h. Subsequently, the product was separated and purified by dialysis and then freeze-dried to obtain a solid powder. Surface charge characterization and anti-biofilm testing of the product were performed. The results showed that although the prepared carbon nanodots had similar charges, their ability to inhibit the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms was significantly weakened, and they did not have the ability to remove MRSA biofilms. The results are shown in Table 1.

[0047] Comparative Example 2

[0048] Similar to Example 1, 189.64 mg of p-phenylenediamine and 5.16 g of diethylenetriamine were weighed and dissolved in 18.96 mL of deionized water. After the reactants were completely dissolved and mixed, the resulting reaction solution was transferred to a reaction vessel and placed in an oven at 200 °C for 12 h. Subsequently, the product was separated and purified by dialysis and then freeze-dried to obtain a solid powder. Surface charge characterization and anti-biofilm testing of the product were performed. The results showed that the charge of the prepared carbon nanodots increased, but their ability to inhibit the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms and to remove mature biofilms was significantly weakened. The results are shown in Table 1.

[0049] Table 1. Surface charge and anti-biofilm properties of carbon nanodots in each embodiment and comparative example.

[0050]

[0051] In summary, the carbon nanodots of this invention exhibit excellent resistance to multidrug-resistant bacterial biofilm formation and food preservation capabilities, demonstrating that the carbon nanodots prepared by the technical solution of this invention possess optimal resistance to multidrug-resistant bacterial biofilm formation.

[0052] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, 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 considered within the scope of protection of the present invention.

Claims

1. A method for preparing carbon nanodots for biofilms resistant to multidrug-resistant bacteria, characterized in that, The process includes the following steps: using dopamine hydrochloride and diethylenetriamine as raw materials, dissolving them in deionized water, then transferring the resulting reaction solution to a reaction vessel and reacting in an oven for 1-24 hours; the product after reaction is dialyzed and freeze-dried to obtain carbon nanodots for biofilm formation against multidrug-resistant bacteria; the molar ratio of dopamine hydrochloride to diethylenetriamine is 1:50-50:1; the mass-to-volume ratio of dopamine hydrochloride to deionized water is 1g:10-100mL; and the reaction temperature is 80-200℃.

2. Carbon nanodots prepared by the method according to claim 1.

3. The application of the carbon nanodots according to claim 2 in biofilms against multidrug-resistant bacteria, characterized in that, The multidrug-resistant bacterial biofilm targets methicillin-resistant Staphylococcus aureus (MRSA) biofilms for elimination.

4. The application according to claim 3, characterized in that, Includes the following steps: Carbon nanodots were co-incubated with methicillin-resistant Staphylococcus aureus (MRSA) planktonic bacteria for a certain period of time. Subsequently, the ability of carbon nanodots to inhibit the formation of MRSA biofilms was investigated using crystal violet staining and confocal laser scanning microscopy. At the same time, carbon nanodots were applied to mature biofilms, and the ability of carbon nanodots to remove MRSA biofilms was investigated using crystal violet staining and confocal laser scanning microscopy.

5. The application according to claim 4, characterized in that, The working concentration of the carbon nanodots is 50-1000 μg / mL, and the co-incubation time is 24-72 h.

6. The application of the carbon nanodots according to claim 2 in the microbial protection of food.

7. The application according to claim 6, characterized in that, The aforementioned food microbial protection targets strawberries, a perishable fruit.

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