CN113845907A - Quaternized carbon dots and preparation method and application thereof - Google Patents

Quaternized carbon dots and preparation method and application thereof Download PDF

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CN113845907A
CN113845907A CN202111266783.4A CN202111266783A CN113845907A CN 113845907 A CN113845907 A CN 113845907A CN 202111266783 A CN202111266783 A CN 202111266783A CN 113845907 A CN113845907 A CN 113845907A
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quaternized
polyethyleneimine
carbon dots
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CN113845907B (en
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赵丹
张蕊
郝健
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South Central Minzu University
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Abstract

The invention belongs to the technical field of preparation and application of carbon quantum dots, and particularly discloses a quaternized carbon dot and a preparation method and application thereof, wherein the preparation method comprises the following steps: s1, subjecting the polyethyleneimine to tertiary amination reaction and quaternization reaction in sequence to obtain quaternized polyethyleneimine; s2, uniformly mixing the quaternized polyethyleneimine obtained in the step S1, citric acid and water, reacting at 120-220 ℃ for 1-6 h, cooling the reaction liquid, dialyzing, and drying to obtain the solid quaternized carbon dots. The method takes quaternized polyethyleneimine and citric acid as raw materials, the quaternized carbon dots are synthesized by a one-step hydrothermal method, the preparation process is simple and easy to implement, the prepared carbon dots have good water solubility and light stability, and excellent broad-spectrum antimicrobial activity, can effectively inhibit and kill various microorganisms including gram-positive bacteria, gram-negative bacteria and fungi, and has the activity of inhibiting and eliminating biofilms, and the application prospect is wide.

Description

Quaternized carbon dots and preparation method and application thereof
Technical Field
The invention belongs to the technical field of preparation and application of carbon quantum dots, and particularly relates to a quaternized carbon dot and a preparation method and application thereof.
Background
The fluorescent nano material has the nanoscale effect and the fluorescence property of the fluorescent material, and is widely applied to the fields of optics, electronics, biology, medicine and the like. The fluorescent carbon nano material becomes a research hotspot of the biological material due to the unique optical performance, low cytotoxicity and excellent biocompatibility. Carbon quantum dots (CDs) are a novel zero-dimensional fluorescent carbon nanomaterial discovered after fullerenes, carbon nanotubes and graphene, and have the characteristics of good stability and water solubility, high quantum yield, easy surface modification and the like. Researches show that the carbon dots have antibacterial activity and provide feasibility for applying the carbon dots to the sterilization materials.
However, the carbon dots prepared by the existing reports have the defects of low antibacterial activity and no broad-spectrum antibacterial property. Chinese patent CN 113181212A discloses a ZIF-8/carbon dot anti-biofilm composite nano material, which is characterized in that lysine carbon dots are embedded into a ZIF-8 metal organic framework, and the minimum inhibitory concentration of the composite nano material to staphylococcus aureus is 62.5 mu g/mL. At present, 2, 3-epoxypropyltrimethylammonium chloride and allyldimethylammonium chloride are used as raw materials to synthesize surface quaternary ammonium modified carbon dots by a one-pot method, and the carbon dots are proved to have satisfactory antibacterial activity on gram-positive bacteria but have poor antibacterial effect on gram-negative bacteria.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a quaternized carbon dot and a preparation method and application thereof.
In order to achieve the above object, the present invention provides a method for preparing a quaternized carbon dot, comprising the steps of:
s1, subjecting the polyethyleneimine to tertiary amination reaction and quaternization reaction in sequence to obtain quaternized polyethyleneimine;
s2, uniformly mixing the quaternized polyethyleneimine obtained in the step S1, citric acid and water, reacting at 120-220 ℃ for 1-6 h, cooling the reaction liquid, dialyzing, and drying to obtain the solid quaternized carbon dots.
Preferably, in step S1, the specific operation of the tertiary amination reaction is: dropwise adding excessive propylene oxide into polyethyleneimine, and stirring and reacting at 0-5 ℃ for at least 7 h; then raising the temperature of the reaction mixture to distill off unreacted propylene oxide to obtain a tertiary aminated polyethyleneimine solution; the specific operation of the quaternization reaction is as follows: adding excessive benzyl chloride into the tertiary amination polyethyleneimine solution, and stirring and reacting at the constant temperature of 48-52 ℃ for at least 30 hours; and removing unreacted benzyl chloride by extraction, and drying to obtain the solid quaternized polyethyleneimine.
Preferably, in step S1, the molecular weight of the polyethyleneimine is 600-10000.
Preferably, in step S2, the mass ratio of the quaternized polyethyleneimine to the citric acid is 1 (0.1-5).
More preferably, the concentration of the citric acid in the mixed solution of the quaternized polyethyleneimine, the citric acid and water is 1mg/mL to 100 mg/mL.
According to another aspect of the present invention there is provided a quaternized carbon dot prepared by the above-described method of preparation.
According to a further aspect of the invention there is provided the use of the quaternized carbon dots prepared according to the invention in the preparation of an antibacterial agent capable of inhibiting the growth of gram positive bacteria, gram negative bacteria or fungi.
Preferably, the gram-positive bacteria comprise at least one of staphylococcus aureus, methicillin-resistant staphylococcus aureus and bacillus subtilis, the gram-negative bacteria comprise at least one of pseudomonas aeruginosa and escherichia coli, and the fungi comprise saccharomyces cerevisiae.
According to another aspect of the present invention there is provided the use of quaternised carbon dots in the inhibition of bacterial biofilm and/or in the elimination of mature bacterial biofilm.
According to another aspect of the invention there is provided the use of the quaternised carbon dots prepared according to the invention in bacterial or fungal imaging.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) the invention provides a preparation method of novel quaternized fluorescent carbon dots, which is characterized in that quaternized polyethyleneimine and citric acid are used as raw materials and synthesized by a one-step hydrothermal method, the process is simple and easy to implement, and the prepared quaternized carbon dots have a broad-spectrum antimicrobial function.
(2) The quaternized carbon dots prepared by the invention have good water solubility and light stability, and can effectively inhibit and kill various microorganisms including gram-positive bacteria, gram-negative bacteria and fungi due to the abundant positive charges on the surface, so that the quaternized carbon dots have excellent broad-spectrum antimicrobial activity, and the minimum inhibitory concentration to bacteria can reach below 25 mu g/mL.
(3) The quaternized carbon dots prepared by the method have good inhibition effect on the growth of various microorganisms, can be used for preparing broad-spectrum antibacterial agents, have strong bactericidal activity, and avoid drug resistance of bacteria caused by overuse of antibiotics.
(4) The quaternized carbon dot prepared by the method has strong inhibitory activity to bacterial biofilm formation, and the minimum membrane inhibitory concentration is only 12.5 mug/mL; meanwhile, the bacterial biofilm elimination agent also shows excellent effect on the elimination of mature bacterial biofilms, and the minimum elimination membrane concentration is 75 mug/mL. The quaternized carbon dots are applied to an anti-biofilm, so that drug resistance of bacteria and resistance to host immune defense mechanisms are effectively prevented, and the quaternized carbon dots have important significance in the fields of biological medicines, foods and environmental protection industries.
(5) The quaternized carbon dots prepared by the method can keep the capability of emitting cell fluorescence after being taken up by microbial cells for 2 hours, show excellent multicolor (blue, green and red) fluorescence, and are very suitable for microbial cell fluorescence imaging.
Drawings
FIG. 1 is a transmission electron micrograph of quaternized carbon dots prepared in example 1 of the present invention.
FIG. 2 is a chart of the infrared spectra of the polyethyleneimine, quaternized polyethyleneimine, and quaternized carbon sites of example 1 of the present invention.
FIG. 3 is a graph showing the UV absorption spectrum and the emission spectrum under excitation light of 370nm wavelength of the quaternized carbon dots prepared in example 1 of the present invention.
FIG. 4 is a transmission electron micrograph of quaternized carbon dots prepared in example 2 of the present invention.
FIG. 5 is a graph showing the emission spectra of quaternized carbon dots prepared in example 2 of the present invention under excitation light with a wavelength of 370 nm.
FIG. 6 is a transmission electron micrograph of quaternized carbon dots prepared in example 3 of the present invention.
FIG. 7 is a graph showing the emission spectra of quaternized carbon dots prepared in example 3 of the present invention under excitation light with a wavelength of 370 nm.
FIG. 8 is a graph of the minimum inhibitory concentration of quaternized carbon dots of example 5 of the present invention against Staphylococcus aureus at various pH conditions.
FIG. 9 is OD showing the inhibition of Staphylococcus aureus biofilm growth in example 6 of the present invention600-a time profile.
FIG. 10 is a photograph of biofilm elimination after treatment of mature Staphylococcus aureus biofilms with quaternized carbon points in example 7 of the present invention and the corresponding OD600And detecting the value.
FIG. 11 is a fluorescence image of Staphylococcus aureus after incubation with quaternized carbon spots for 2h in example 8 of the present invention under excitation of light at 405nm (a), 488nm (b), and 556nm (c).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preparation method of a quaternized carbon dot, which comprises the following steps:
s1, subjecting Polyethyleneimine (PEI) to tertiary amination reaction and quaternization reaction in sequence to obtain quaternized polyethyleneimine (qPEI);
s2, uniformly mixing the quaternized polyethyleneimine obtained in the step S1, citric acid and water, reacting at 120-220 ℃ for 1-6 h, cooling the reaction liquid, dialyzing, and drying to obtain solid quaternized carbon dots (qCDs).
The method comprises the steps of preparing quaternized polyethyleneimine through two-step polymerization reaction, and then obtaining carbon dots with surface quaternary ammonium groups by using the quaternized polyethyleneimine and citric acid as substrates through a hydrothermal method. The carbon dots have stronger bacteriostatic effect on bacteria or fungi than the existing carbon dots and have broad spectrum, and the quaternized carbon dots can directly destroy microbial cell membranes to ensure that the microbes are difficult to generate drug resistance.
The method comprises the steps of alkylating primary amino and secondary amino on a polyethyleneimine macromolecular chain by using propylene oxide, and quaternizing the tertiary amino of alkylated polyethyleneimine by using benzyl chloride. Specifically, in step S1, the specific operation of the tertiary amination reaction is: dropwise adding excessive propylene oxide into polyethyleneimine, and stirring and reacting at 0-5 ℃ for at least 7 h; the temperature of the reaction mixture was then raised to distill off unreacted propylene oxide, resulting in a solution of tertiary aminated polyethyleneimine. Preferably, the temperature of the tertiary amination reaction is 3 ℃ and the propylene oxide distillation temperature is 35 ℃. The specific operation of the quaternization reaction is as follows: adding excessive benzyl chloride into the tertiary amination polyethyleneimine solution, and stirring and reacting at the constant temperature of 48-52 ℃ for at least 30 hours; and removing unreacted benzyl chloride by extraction, and drying to obtain the solid quaternized polyethyleneimine. Preferably, the temperature of the quaternization reaction is 50 ℃, after the reaction is finished, diethyl ether is used for extracting for a plurality of times, and then the transparent solid quaternization polyethyleneimine is obtained by vacuum drying.
In some embodiments, the polyethyleneimine has a molecular weight of 600-10000. If the molecular weight of polyethyleneimine is too large, the reaction effect is poor, and the particle size of the prepared carbon dots is too large.
In some embodiments, in step S2, the mass ratio of the quaternized polyethyleneimine to the citric acid is (1-5):1, preferably, the mass ratio is 1: (0.2-5), and more preferably, 2: 1. The concentration of the citric acid in the hydrothermal reaction mixture is 1mg/mL-100mg/mL, preferably, the citric acid concentration is 10mg/mL-60 mg/mL.
In some embodiments, in step S2, the solution containing blue fluorescent carbon spots is dialyzed against a 500Da cut-off dialysis bag to remove minute impurities, and then lyophilized to obtain solid quaternized carbon spots.
The invention also provides a quaternized carbon dot prepared by the preparation method of any one of the embodiments.
The quaternized carbon dots prepared by the method can be used for preparing broad-spectrum antibacterial agents, can inhibit the growth of bacteria including gram-positive bacteria and gram-negative bacteria and fungi, and has a better effect of inhibiting the bacteria. Specifically, the gram-positive bacteria include at least one of Staphylococcus aureus (Staphylococcus aureus), Methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis (Bacillus subtilis), the gram-negative bacteria include at least one of Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia coli (Escherichia coli), and the fungi include Saccharomyces cerevisiae (Saccharomyces cerevisiae).
The quaternized carbon dots prepared by the invention can be used for inhibiting the formation of bacterial biofilms or eliminating mature bacterial biofilms. The specific steps for inhibiting the formation of the bacterial biofilm are as follows: and mixing phosphate buffer salt solution containing the quaternized carbon points with the bacterial suspension, wherein the concentration of the quaternized carbon points in the mixed solution is at least 12.5 mu g/mL, and culturing in an environment suitable for the growth of bacteria. The effect of inhibiting biofilm formation can be verified by measuring the optical density value of the mixed liquor at a wavelength of 600nm over 48 h.
The specific steps for eliminating the mature bacterial biofilm are as follows: phosphate buffered saline containing at least 75 μ g/mL of quaternized carbon sites is added to the mature bacterial biofilm and incubated in an environment suitable for bacterial growth. After a period of incubation, the biofilm can be stained by crystal violet staining followed by OD measurement600A value; biofilm elimination can also be observed using a scanning electron microscope.
In some embodiments, the bacterial biofilm is a biofilm of staphylococcus aureus, methicillin-resistant staphylococcus aureus, bacillus subtilis, escherichia coli, or pseudomonas aeruginosa.
The quaternized carbon dots prepared by the method can also be used for imaging bacteria or fungi, and particularly have a good imaging effect on staphylococcus aureus. The specific operations of imaging bacteria or fungi are as follows: collecting bacterial or fungal cells, adding a quaternized carbon dot solution, incubating for 2h under the optimal growth condition of bacteria or fungi, and taking a fluorescence picture by using a confocal fluorescence microscope. Preferably, the co-incubation is at a bacterial or fungal concentration of 109CFU/mL, and the concentration of the quaternized carbon dots is 500 mug/mL, so that a better imaging effect can be achieved.
The above technical solution is described in detail below with reference to specific examples.
In the following examples, the methods used are those commonly used in the art unless otherwise specified. The strain, medium, biochemical reagent and the like used are commercially available unless otherwise specified.
EXAMPLE 1 preparation of quaternized carbon dots
1. Preparation of quaternized polyethyleneimine
An excess of propylene oxide, used as an alkylating agent, was added dropwise to a commercially available polyethyleneimine and the alkylation reaction was stirred at 3 ℃ for 7 h. Subsequently, the temperature of the mixture was raised to 35 ℃ and unreacted propylene oxide was distilled off to obtain a solution of tertiary amination reaction, i.e., a tertiary aminated polyethyleneimine solution. In the quaternization reaction, excess benzyl chloride was added to the tertiary aminated polyethyleneimine solution and the reaction was stirred at a constant temperature of 50 ℃ for 30 h. And extracting unreacted benzyl chloride with diethyl ether for several times to obtain a quaternized product solution. And finally obtaining transparent solid quaternized polyethyleneimine after vacuum drying.
2. Preparation of quaternised carbon dots
200mg of quaternized polyethyleneimine, 300mg of citric acid and 5mL of water are uniformly mixed, and the mixture is transferred to a hydrothermal reaction kettle, wherein the reaction temperature is 160 ℃, and the reaction time is 2 hours, so that a quaternized carbon dot (qCDs) solution is obtained. And cooling the obtained product, dialyzing by using a dialysis bag with the molecular weight cut-off of 500Da to remove micro impurities to obtain a solution containing blue fluorescent carbon dots, and freeze-drying to obtain solid qCDs.
Transmission electron microscope observation of the quaternized carbon dots obtained by the preparation method comprises the following steps:
dissolving the purified sample in deionized water, dropping 10 μ L onto 300 mesh copper net, and observing with transmission electron microscope (JEM-2100, JEOL Ltd., Japan); the image results and the quantitative results are shown in FIG. 1.
TEM was used to characterize the size of the quaternized carbon dots. TEM results show that the quaternized carbon dots are uniform spheres and have good dispersibility; the particle size is normally distributed, and the average particle size is about 2.46 nm. (based on statistical analysis of 100 points).
Infrared spectrum scanning of the quaternized carbon dots obtained by the preparation method:
the freeze-dried samples were examined for infrared absorption spectra of carbon spots by FTIR (Nicolet 6700, Thermo Scientific, USA) and the results are shown in FIG. 2.
In the infrared spectrum of Polyethyleneimine (PEI), 1572cm-1The band at (A) is the flexural vibration absorption of the N-H bond of the primary amine group, 1106cm-1The band at (B) is the flexural vibration absorption of the N-H bond of the secondary amine group. In the spectrum of quaternized polyethyleneimine (qPEI), 1578cm-1The band is weakened, indicating that the primary amine group has changed to a secondary amineOr a tertiary amine group. At the same time, 2974cm appeared-1-2834cm-1Band of (b) when-CH3And>-CH2the tensile vibration of the C-H bond is absorbed when the groups coexist. Since there is no-CH on PEI3Radical, the appearance of these peaks being indicative of the-CH of propylene oxide3Groups have been introduced into qPEI macromolecules. In addition, at 1371cm-1The characteristic absorption band of the hydroxyl O-H bond appears, indicating that the ring-opening reaction of propylene oxide occurs. In the qPEI spectrum, the characteristic vibration absorption band of the benzene ring appears at 702cm-1、746cm-1、1643cm-1、1456cm-1And 3029cm-1Therein, 702cm-1And 746cm-1Is a characteristic peak of mono-substituted benzene ring. The appearance of these bands indicated that the alkylated PEI had reacted with benzyl chloride and produced quaternary ammonium groups. In addition, 1058cm appeared-1And 1134cm-1New bands of (b), which are characteristic absorptions of quaternary ammonium groups. This result further confirms that a reaction has occurred between the tertiary amine group and benzyl chloride and that a quaternized product has been formed. On the basis of qPEI, the quaternized carbon points (qCDs) are originally 3300cm, except for the characteristic absorption of ammonium groups and aromatic rings-1The broadening of the characteristic peak of the absorbed amino groups is probably due to the addition of-OH. Two new absorption peaks of 1716cm appear-1And 1585cm-1Stretching vibration and aromatic ring stretching vibration belonging to C ═ O and C ═ C respectively. Comparison of the infrared absorption spectra of both qPEI and qCDs shows that the addition of Citric Acid (CA) and the hydrothermal reaction lead to a change in microstructure.
Zeta potential detection of the quaternized carbon dots obtained by the preparation method:
the samples obtained were dissolved in deionized water dispersion and evaluated using a Zetasizer (Nano ZS, Malvern Instruments, Worcestershire, UK).
The surface potential of qCDs with quaternary ammonium groups is measured by Zeta potential, and the measured result is +5.03mV, which indicates that the surface of qCDs has abundant positive charges and is beneficial to destroying the bacterial cell membrane when carbon points are close to bacteria.
Fluorescence and ultraviolet spectrum scanning of the quaternized carbon dots obtained by the preparation method:
the obtained sample was dispersed and dissolved in deionized water, and the resulting solution was placed in a fluorescence spectrophotometer (LS55, PerkinElmer Company) detection cell for measuring the emission Wavelength (wavelet) and the fluorescence intensity (PL intensity), and an ultraviolet-visible spectrophotometer (Lambda-35, PerkinElmer Company) detection cell for measuring the Absorbance (Absorbance) at different wavelengths (wavelet), and the measurement results are shown in FIG. 3.
FIG. 3 shows fluorescence and UV-visible absorption spectra of the qCDs prepared. In the UV-visible absorption spectrum, a peak appears at 220nm, which is characteristic absorption of the π - π transition of conjugated benzene rings. In addition, the peak at 290nm is due to an n-pi electron transition from a C-O bond, and the slight absorption at 350nm is due to the n-pi transition corresponding to the surface carbonyl/amine functional group. The fluorescence emission spectrum shows that the emission wavelength of the quaternized carbon dots reaches about 468nm at the excitation wavelength of 370 nm. The quantum yield of the carbon dots was found to be 11.18%.
EXAMPLE 2 preparation of quaternized carbon dots
1. Preparation of quaternized polyethyleneimine
The procedure for the preparation of the quaternized polyethyleneimine is as in step 1 of example 1.
2. Preparation of quaternised carbon dots
200mg of quaternized polyethyleneimine, 100mg of citric acid and 5mL of water are uniformly mixed, and the mixture is transferred to a hydrothermal reaction kettle, wherein the reaction temperature is 220 ℃, and the reaction time is 4 hours, so that a quaternized carbon dot (qCDs) solution is obtained. And cooling the obtained product, dialyzing by using a dialysis bag with the molecular weight cut-off of 500Da to remove micro impurities to obtain a solution containing blue fluorescent carbon dots, and freeze-drying to obtain solid qCDs.
By observing the prepared solid quaternized carbon dots by using a transmission electron microscope, as shown in fig. 4, the carbon dots are uniform spheres, the dispersibility is good, the average particle size is about 3.02nm, and the particle size of the quaternized carbon dots prepared in the embodiment is slightly increased. The emission wavelength and fluorescence intensity of qCDs were measured by a spectrofluorometer, and as shown in FIG. 5, the fluorescence intensity of the carbon dots was slightly lower than that of example 1. The reason may be that the temperature is increased, the carbonization degree is higher, the fluorescence is gradually reduced due to the quantum size effect or the change of surface defects, and the particle size is increased.
EXAMPLE 3 preparation of quaternized carbon dots
1. Preparation of quaternized polyethyleneimine
The procedure for the preparation of the quaternized polyethyleneimine is as in step 1 of example 1.
2. Preparation of quaternised carbon dots
And uniformly mixing 50mg of quaternized polyethyleneimine, 50mg of citric acid and 5mL of water, transferring to a hydrothermal reaction kettle, and reacting at 120 ℃ for 6 hours to obtain a quaternized carbon dot (qCDs) solution. And cooling the obtained product, dialyzing by using a dialysis bag with the molecular weight cut-off of 500Da to remove micro impurities to obtain a solution containing blue fluorescent carbon dots, and freeze-drying to obtain solid qCDs.
The obtained solid quaternized carbon dots are observed by a transmission electron microscope, and as shown in fig. 6, the carbon dots are uniform spheres, have good dispersibility and an average particle size of about 2.71 nm. The emission wavelength and fluorescence intensity of qCDs were measured by a spectrofluorometer, and as shown in FIG. 7, the emission wavelength of the carbon dots was 465nm, and the quantum yield of the carbon dots was measured to be 8.56%.
Example 4 detection of inhibitory Effect of quaternized carbon points on different types of microorganisms
50 μ L of PBS solutions (pH 7.4) containing different concentrations of the quaternized carbon dots prepared in example 1 were prepared and added to 96-well plates, respectively. Then 50. mu.L of the solution was added to a concentration of 2X 107CFU/mL of different microbial species suspensions were added to each PBS solution. The 96-well plate was incubated at 37 ℃ in a constant temperature incubator. At the end of the 12h incubation period, 5 μ L MTT per well was added and further incubated for 20 min. Thereafter, the formed precipitate was dissolved in dimethyl sulfoxide (DMSO) for visual observation, and the experiment was repeated 3 times. The results obtained are shown in table 1.
TABLE 1 Minimum Inhibitory Concentration (MIC) of quaternized carbon points for different species of microorganisms
Figure BDA0003327059720000101
As is apparent from table 1, the quaternized carbon dots prepared according to the present invention can effectively inhibit the growth of gram-positive bacteria (e.g., s.aureus, MRSA, and b.subtilis), gram-negative bacteria (p.aeruginosa and e.coli), and fungi (s.cerevisiae). Wherein the minimum inhibitory concentration to bacillus subtilis is as low as 7.8 mug/mL, and the minimum inhibitory concentration to gram-negative bacteria escherichia coli is only 12.5 mug/mL.
EXAMPLE 5 inhibitory Effect of quaternized carbon points on Staphylococcus aureus growth under different pH conditions
50 μ L of PBS solutions (20mM) containing different concentrations of the quaternized carbon dots prepared in example 1 and different pH (pH 6.2, 6.4, 6.8, 7.0, 7.4, 7.8) were prepared and added to 96-well plates, respectively. Then 50. mu.L of the solution was added to a concentration of 2X 107CFU/mL of Staphylococcus aureus suspension was added to each PBS solution. The 96-well plate was incubated at 37 ℃ in a constant temperature incubator. At the end of the 12h incubation period, 5 μ L MTT per well was added and further incubated for 20 min. Thereafter, the formed precipitate was dissolved in DMSO for visual observation, and the experiment was repeated 3 times. The resulting antimicrobial MIC is shown in fig. 8.
In practical antibacterial experiments, environmental pH is an important influencing condition that is not negligible. The influence of the environmental pH on the antibacterial performance of the quaternized carbon points is continuously researched, and the MICs of the quaternized carbon points in different pH environments to S. As shown in FIG. 8, at pH 6.2-7.0, MIC were all 12.5. mu.g/mL; however, when the pH was further increased to 7.4 and 7.8, the MIC increased to 15. mu.g/mL and 20. mu.g/mL, respectively. From the above results, it can be seen that such changes in the state of presence of the quaternized carbon sites caused by changes in the environmental pH indirectly result in a slight increase in their antibacterial MIC, but it cannot be denied that their antibacterial activity is still strong.
Example 6 detection of inhibitory Activity of quaternized carbon points on Staphylococcus aureus biofilm formation
50 μ L of PBS solutions containing different concentrations (0 μ g/mL (i.e., positive control), 1.25 μ g/mL, 2.5 μ g/mL, 5.0 μ g/mL, 12.5 μ g/mL, 15.0 μ g/mL, 20.0 μ g/mL, 25.0 μ g/mL, 30.0 μ g/mL) of the quaternized carbon dots prepared in example 1 were prepared and added to 96-well plates, respectively. Then will be50 μ L at a concentration of 2X 107CFU/mL of Staphylococcus aureus suspension was added to each well. Negative control wells were 50 μ L of PBS solution without quaternized carbon spots plus 50 μ L of bacteria-free medium. The 96-well plate was incubated in a biochemical incubator at 37 ℃. The optical density values of the solutions in the 96-well plate at 600nm were measured over 48h, and the optical density change curves for different times of incubation at different concentrations of the quaternized carbon dots were plotted, as shown in FIG. 9.
OD of S.aureus bacterial liquid treated by quaternized carbon points with different concentrations within 48h600Values showing inhibitory activity of the quaternized carbon points on bacterial growth and biofilm formation; and it is clear that the inhibition rate is positively correlated with the concentration of the antibacterial agent. The quaternised carbon dots of 12.5. mu.g/mL completely inhibited biofilm formation within 48h, i.e.a minimum membrane inhibitory concentration (MBIC) of 12.5. mu.g/mL. These results indicate that the quaternized carbon dots prepared have excellent inhibitory effect on biofilm formation.
Example 7 detection of the Effect of quaternized carbon points on the elimination of mature Staphylococcus aureus biofilms
The concentration of 100 mu L is 2X 107And respectively adding the CFU/mL staphylococcus aureus suspension into each hole of a 96-well plate, placing the 96-well plate in an incubator for culturing for 48 hours at a constant temperature, slightly pouring off redundant culture medium and planktonic bacteria, and washing the cells for 1-2 times by using 200 mu L PBS. 50 μ L of PBS solutions containing the quaternized carbon dots obtained in example 1 at different concentrations (0. mu.g/mL, 25.0. mu.g/mL, 50.0. mu.g/mL, 75.0. mu.g/mL) were prepared, added to each well of a 96-well plate to which the bacterial suspension was added, and incubated at constant temperature for 12 hours. Excess medium and planktonic bacteria were decanted and washed 3 times with 200. mu.L PBS. Then fixed with glutaraldehyde or methanol for 30min, followed by staining with 50 μ L of 0.1% crystal violet solution for 15 min. Pouring out crystal violet solution, washing off residual color with 200 μ L of LPBS, dissolving the stained biological membrane with 200 μ L of 95% ethanol, shaking, mixing, placing 100 μ L in a new 96-well plate, and determining OD600The value is obtained.
As shown in FIG. 10, the map and OD can be observed from ethanol dissolution600As a result, it was found that the mature biofilm was significantly eliminated by 75. mu.g/mL of the quaternized carbon dots, i.e., minimal eliminationThe concentration of the membrane removed was 75. mu.g/mL.
Example 8 fluorescence confocal imaging of Staphylococcus aureus Using quaternized carbon dots
1mL of Staphylococcus aureus suspension (. apprxeq.10)9CFU/mL) was centrifuged at 8000rpm for 5min, the supernatant was removed, the bacterial cells were collected, and 1mL of a solution containing the quaternized carbon dots prepared in example 1 (500. mu.g/mL) was added and incubated at 37 ℃ for 2 h. To take the fluorescence image, a confocal fluorescence microscope 60X objective was used to collect the pictures.
As shown in FIG. 11, labeling Staphylococcus aureus with light excitation at 405nm, 488nm, and 556nm wavelengths, respectively, resulted in blue, green, and red fluorescence. The quaternized carbon dots can retain their ability to fluoresce from cells after 2h of incubation uptake by the cells and exhibit excellent polychromatic fluorescence. These results indicate that polyquaternised carbon dots are good candidates for fluorescence imaging of bacterial cells.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A method for preparing quaternized carbon dots, characterized by comprising the steps of:
s1, subjecting the polyethyleneimine to tertiary amination reaction and quaternization reaction in sequence to obtain quaternized polyethyleneimine;
s2, uniformly mixing the quaternized polyethyleneimine obtained in the step S1, citric acid and water, reacting at 120-220 ℃ for 1-6 h, cooling the reaction liquid, dialyzing, and drying to obtain the solid quaternized carbon dots.
2. The method for preparing quaternized carbon dots according to claim 1, wherein in step S1, the tertiary amination reaction is specifically performed by: dropwise adding excessive propylene oxide into polyethyleneimine, and stirring and reacting at 0-5 ℃ for at least 7 h; then raising the temperature of the reaction mixture to distill off unreacted propylene oxide to obtain a tertiary aminated polyethyleneimine solution; the specific operation of the quaternization reaction is as follows: adding excessive benzyl chloride into the tertiary amination polyethyleneimine solution, and stirring and reacting at the constant temperature of 48-52 ℃ for at least 30 h; and removing unreacted benzyl chloride by extraction, and drying to obtain the solid quaternized polyethyleneimine.
3. The method of making quaternized carbon dots of claim 1, wherein: in step S1, the molecular weight of the polyethyleneimine is 600-10000.
4. The method of making quaternized carbon dots of claim 1, wherein: in step S2, the mass ratio of the quaternized polyethyleneimine to the citric acid is 1 (0.1-5).
5. The method of making quaternized carbon dots of claim 4, wherein: the concentration of the citric acid in the mixed liquid after the quaternized polyethyleneimine, the citric acid and the water are mixed is 1mg/mL-100 mg/mL.
6. A quaternized carbon dot produced by the production method according to any one of claims 1 to 5.
7. Use of the quaternized carbon dots of claim 6 in the preparation of an antimicrobial agent, characterized in that: the antimicrobial agent is capable of inhibiting the growth of gram-positive bacteria, gram-negative bacteria or fungi.
8. Use according to claim 7, characterized in that: the gram-positive bacteria comprise at least one of staphylococcus aureus, methicillin-resistant staphylococcus aureus and bacillus subtilis, the gram-negative bacteria comprise at least one of pseudomonas aeruginosa and escherichia coli, and the fungi comprise saccharomyces cerevisiae.
9. Use of the quaternized carbon dots of claim 6 for inhibiting bacterial biofilm and/or eliminating mature bacterial biofilm.
10. Use of the quaternized carbon dots of claim 6 in bacterial or fungal imaging.
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