CN113016821B - Antibacterial nano liquid drop and application thereof - Google Patents
Antibacterial nano liquid drop and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides an antibacterial nano liquid drop and application thereof in inhibiting bacterial biofilm. The preparation method of the antibacterial nano liquid drop comprises the following steps: placing the mixed solution of the oleic acid amide propyl dimethyl tertiary amine and the sulfur-doped carbon quantum dots at room temperature, centrifuging, removing supernatant to obtain a condensed phase of liquid-liquid phase separation, and dispersing in an aqueous solution. The nano liquid drop provided by the invention has a good bacteriostatic effect on staphylococcus aureus and escherichia coli. The destruction rate of 100 mu g/mL nano liquid drops to a staphylococcus aureus biomembrane can reach 82.68%, and the nano liquid drops have strong destruction effect on the biomembrane formed by the staphylococcus aureus.
Description
Technical Field
The invention belongs to the field of nanotechnology, and particularly relates to an antibacterial nano liquid drop based on a surfactant and a carbon quantum dot and application thereof in the aspect of inhibiting bacterial biofilms.
Background
Pathogenic bacteria (such as escherichia coli, staphylococcus epidermidis and staphylococcus aureus) are the main causes of death of patients with low immunity, and for the diseases, antibacterial drugs are mostly used for preventing and treating clinically. On one hand, the excessive use of antibacterial drugs easily causes the strains to generate drug resistance; on the other hand, pathogenic bacteria are easy to form biofilm, which not only leads to the enhancement of drug resistance of the bacteria, but also interferes with the recognition and killing mechanisms of the immune system of a patient. Research and development of novel antibacterial biofilm materials, capable of disrupting biofilm growth, are important in today's antibacterial therapy.
The carbon fluorescent quantum dots have excellent anti-biofilm activity, have no toxicity to normal cells, and provide an effective way for effectively destroying bacterial biofilms in food, medical supplies and food and medicine processing environments.
Chinese patent CN 108635371A discloses the application of carbon fluorescent quantum dots (CDs-LP) in inhibiting the formation of Escherichia coli biofilm. But the action time is longer and needs further improvement.
Chinese patent CN 111841599 a discloses a carbon quantum dot composite material (nitrogen-doped carbon quantum dot N-CQDs) with photocatalytic antibacterial properties, which is simple and convenient to operate, easy to control and low in preparation cost, and although the prepared nitrogen-doped carbon quantum has high photocatalytic antibacterial activity to escherichia coli and staphylococcus aureus, no report about the effect of the carbon quantum on biofilm is available at present.
Chinese patent CN 111214489A discloses a complex formed by combining graphene oxide and copper ions, the destruction rate of the prepared complex to the biofilm of staphylococcus aureus reaches 80% under the conditions of graphene (125 mu g/mL) and copper ions (1000 mu M), and the destruction rate of the nano liquid drop prepared by the invention to the biofilm of staphylococcus aureus can reach more than 80% when the concentration is 100 mu g/mL, so that the dosage is lower.
The nano-droplet has a nano-scale and a structure, and has properties of being used as liquid and mesoscopic substances, so that the nano-droplet has a wide prospect in the aspect of application. At present, no report about the fluorescent nano liquid drop with antibacterial performance is available.
Disclosure of Invention
In order to solve the problems, the invention provides an antibacterial nano liquid drop based on a surfactant and sulfur-doped carbon quantum dots and application thereof in inhibiting bacterial biofilms. The invention prepares the fluorescent nano liquid drop with antibacterial property for the first time.
The terms:
PKOO: oleic acid amidopropyl dimethyl tertiary amine.
S-CQDs: sulfur doped carbon quantum dots.
In one aspect, the invention provides a preparation method of liquid-liquid phase separation nano droplets based on a surfactant and sulfur-doped carbon quantum dots.
The preparation method comprises the following steps: placing the mixed solution of the oleic acid amide propyl dimethyl tertiary amine and the sulfur-doped carbon quantum dots at room temperature, centrifuging, removing supernatant to obtain a condensed phase of liquid-liquid phase separation, and dispersing the condensed phase in an aqueous solution.
Furthermore, the concentration of the amidopropyl dimethyl tertiary amine oleate in the mixed solution is 30-40mM, and the concentration of the sulfur-doped carbon quantum dots is 1-4 mg/mL.
Preferably, the concentration of the amidopropyl dimethyl tertiary amine oleate in the mixed solution is 34mM, and the concentration of the sulfur-doped carbon quantum dots is 2 mg/mL.
Further preferably, the preparation method comprises the following steps: standing the mixed solution containing 34mM oleic acid amide propyl dimethyl tertiary amine and 2mg/mL sulfur-doped carbon quantum dots at room temperature for 24h, centrifuging at 10000rpm for 30min, discarding the supernatant to obtain a condensed phase of liquid-liquid phase separation, and dispersing in an aqueous solution.
In another aspect, the invention provides a liquid-liquid phase separation nano-droplet based on a surfactant and sulfur-doped carbon quantum dots.
The surfactant is oleic acid amide propyl dimethyl tertiary amine.
The liquid-liquid phase separation nano liquid drop is prepared by the preparation method.
In still another aspect, the invention provides an application of the aforementioned liquid-liquid phase separation nano droplet based on the surfactant and the sulfur-doped carbon quantum dot in inhibiting the growth of bacterial biofilm.
The bacteria include gram positive and/or gram negative bacteria.
Preferably, the bacteria are staphylococcus aureus and/or escherichia coli.
The action concentration of the nano liquid drop is 0-150 mug/mL.
In yet another aspect, the present invention provides an antimicrobial agent.
The antibacterial agent comprises the liquid-liquid phase separation nano-droplet based on the surfactant and the sulfur-doped carbon quantum dot.
The types of antimicrobial agents include, but are not limited to, sprays, drops, gels, suspensions, emulsions.
The bacteria comprise gram-positive bacteria and/or gram-negative bacteria.
Preferably, the bacteria are staphylococcus aureus and/or escherichia coli.
The concentration of the nano liquid drops in the antibacterial agent is 0-150 mug/mL.
In a further aspect, the present invention provides the use of the aforementioned antibacterial agent for inhibiting the growth of a bacterial biofilm.
The bacteria comprise gram-positive bacteria and/or gram-negative bacteria.
Preferably, the bacteria are staphylococcus aureus and/or escherichia coli.
The invention has the beneficial effects that:
(1) the MIC value of the nano liquid drop to the minimum inhibitory concentration of staphylococcus aureus is 32.86 mu g/mL, the MIC value to the minimum inhibitory concentration of escherichia coli is 21.38 mu g/mL, and the nano liquid drop has a good antibacterial effect.
(2) The destruction rate of 100 mu g/mL nano liquid drops to a staphylococcus aureus biofilm can reach 82.68%, and the nano liquid drops have a good destruction effect on the staphylococcus aureus biofilm.
Drawings
FIG. 1 is the inhibitory curve of the antibacterial nano-droplet provided by the invention against Staphylococcus aureus.
FIG. 2 is a curve showing the inhibition of the antibacterial nanodroplets of the present invention against E.coli.
FIG. 3 shows the respective destruction rates of different concentrations of PKOO, S-CQDs and nanodroplets on Staphylococcus aureus biofilms.
FIG. 4 shows the respective destruction rates of PKOO and nanodroplets on Staphylococcus aureus biofilms (spread plate method).
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
In the following examples, the Staphylococcus aureus was deposited as ATCC 6538 and the Escherichia coli was deposited as ATCC 8739 from the China general culture Collection.
Example 1 preparation method of liquid-liquid phase separation nano-droplet based on surfactant and sulfur-doped carbon quantum dot
1. Taking the PKOO and the S-CQDs to prepare a mixed solution, wherein the concentration of the PKOO and the concentration of the S-CQDs in the mixed solution are 34mM and 2 mg/mL. Wherein PKOO is available from Shanghai Yincong New materials, Inc.; S-CQDs are synthesized by the following method: weighing 5g of citric acid and 0.8g of mercaptoethylamine, adding 40mL of pure water, stirring for dissolving, placing in 50mL of polytetrafluoroethylene lining, heating at 150 ℃ for 150min, dialyzing for 48h after the reaction is finished, and freeze-drying to obtain S-CQDs.
2. And (3) after the mixed solution in the step 1 is placed at room temperature for 24 hours, the solution becomes clear from turbidity, and is centrifuged at 10000rpm for 30min to remove supernatant, so that colloidal solid is obtained.
3. And (3) dissolving the colloidal solid obtained in the step (2) by using deionized water, preparing a solution with a certain concentration, and storing at room temperature for later use.
Example 2 detection of inhibitory Capacity of Nanoparoplet to Staphylococcus aureus
The following experiment was carried out using the nanodroplets obtained in example 1:
1. preparation of lecithin-Tween 80 nutrient agar culture plate
The lecithin-Tween 80 nutrient agar culture medium (purchased from Guangdong Huanji Microbiol technology Co., Ltd., product number 1095521) is sterilized under high pressure for 30min, after being cooled to a certain temperature, the melted and cooled nutrient agar is poured into special disposable 9mm bacteria culture dishes for bacteria, about 15mL of nutrient agar is poured into each culture dish, solidification is carried out for standby application, the temperature of the poured culture dish is not too high, otherwise, water vapor exists around the culture dish, and experimental errors are easily caused by coating plates.
2. Adding 3mL of PBS buffer solution into a staphylococcus aureus culture inclined plane for flushing, taking out bacterial liquid from the staphylococcus aureus culture inclined plane, and diluting the bacterial liquid to OD by using PBS600Is 0.5-0.8.
3. And (3) adding 200 mu L of the bacterial liquid diluted in the step (2) into a nano liquid drop solution, setting the concentration of the nano liquid drop to be 10, 20, 30, 50 and 60 mu g/mL, incubating for 1h at 37 ℃ in a constant temperature incubator, carrying out gradient dilution on the reaction liquid, adding 100 mu L of the reaction liquid diluted to 10000 times and 100000 times respectively into the solidified culture medium, and coating. And (3) after the culture dish is reversely buckled, putting the culture dish into a constant-temperature incubator for incubation at 37 ℃ for 24 hours, and counting bacterial colonies to obtain the minimum inhibitory concentration MIC value of the nano liquid drop to the staphylococcus aureus, wherein FIG. 1 is an inhibition curve of the antibacterial nano liquid drop to the staphylococcus aureus. And finally obtaining the MIC value of the nano liquid drop to the minimum inhibitory concentration of staphylococcus aureus by curve fitting, wherein the MIC value is 32.86 mu g/mL.
Example 3 detection of inhibitory Capacity of Nanoparoplet to Escherichia coli
With reference to the experimental method of example 2, the inhibitory ability of the nano-droplets to escherichia coli was examined, and the concentration of the nano-droplets was set. 5. 10, 15, 20, 25, 30, 50 μ g/mL.
The inhibition curve of nanodroplets against E.coli is shown in FIG. 2.
And finally obtaining the minimum inhibitory concentration MIC value of the nano liquid drop to the escherichia coli by curve fitting, wherein the MIC value is 21.38 mu g/mL.
Example 4 application of Nanobroplets to inhibition and disruption of bacterial biofilms
The following experiment was carried out using the nanodroplets obtained in example 1:
1. the Staphylococcus aureus-containing LB broth (available from Kyork Biotech Co., Ltd., Cat. No. 3105726) was diluted to OD6000.8-1.5 in 96-well platesAdding 200 mu L of diluted bacteria liquid into each hole, incubating for 24h, discarding the culture medium after the biological membrane grows mature, and washing three times by using PBS solution to wash away free bacteria.
The following groupings are set:
(1)100 μ L LB broth +100 μ L PKOO at a concentration of 10 μ g/mL;
(2)100 μ L LB broth +100 μ L S-CQDs at a concentration of 10 μ g/mL;
(3)100 μ L LB broth +100 μ L nanodroplets at a concentration of 10 μ g/mL;
(4)100 μ L LB broth +100 μ L PKOO at a concentration of 20 μ g/mL;
(5)100 μ L LB broth +100 μ L S-CQDs at a concentration of 20 μ g/mL;
(6)100 μ L LB broth +100 μ L nanodroplets with a concentration of 20 μ g/mL.
(7)100 μ L LB broth +100 μ L PKOO at a concentration of 200 μ g/mL;
(8)100 μ L LB broth +100 μ L S-CQDs at a concentration of 200 μ g/mL;
(9)100 μ L LB broth +100 μ L nanodroplets at a concentration of 200 μ g/mL;
(10)100 μ L LB broth +100 μ L PKOO at a concentration of 300 μ g/mL;
(11)100 μ L LB broth +100 μ L S-CQDs at a concentration of 300 μ g/mL;
(12)100 μ L LB broth +100 μ L nanodroplets with a concentration of 300 μ g/mL.
Samples were loaded into 96-well plates in groups as described above, with 6 replicates per sample set as parallel controls.
2. And (2) incubating the sample solution in the step (1) with the bacterial solution for 24h, sucking the bacterial solution in a 96-well plate, washing with ultrapure water for three times to remove free bacteria, adding 50 mu L of methanol for fixing for 15min, sucking out and naturally drying, adding 200 mu L of 0.1% crystal violet for dyeing for 15min, sucking out the crystal violet dye solution after dyeing is finished, washing with ultrapure water for three times, naturally drying, adding 200 mu L of 33% acetic acid into each well, incubating for 30min at 37 ℃ to dissolve the crystal violet, and measuring the OD value at 590nm by using a multifunctional microplate reader after dissolution is finished. The destruction rate of the sample to the biofilm formed by staphylococcus aureus was calculated by the following calculation formula (see fig. 3):
Destruction Ratio(％)＝(OD(PBS)-OD(sample))/OD(PBS)×100。
example 5 application of the plate coating method to the measurement of the nano-droplets for the inhibition and disruption of bacterial biofilms the following experiment was carried out using the nano-droplets obtained in example 1:
1. staphylococcus aureus LB broth diluted to OD600After 0.8-1.5, 200. mu.L of diluted bacteria solution was added to each well of a 96-well plate, incubated for 24h, and after the biofilm was grown to maturity, the medium was discarded and washed three times with PBS to wash away free bacteria.
The following groups were set to load the samples into the 96-well plate:
(1)100 μ L LB broth and 100 μ L PKOO at a concentration of 100 μ g/mL;
(2)100 μ L LB broth and 100 μ L nanodroplets at a concentration of 100 μ g/mL.
After incubation of the solution for 2h, the 96-well plate was sonicated for 10min to redisperse the adherent growth of Staphylococcus aureus into a suspension.
2. The suspension obtained in step 1 was serially diluted 100000 times, and 100. mu.L of the diluted solution was applied to nutrient agar plates. The agar plates were incubated at 37 ℃ for 18h before colony counting. The destruction rate of the pre-fabricated biofilm was calculated according to the following formula (see fig. 4):
Destruction Ratio(％)＝(C(PBS)-C(sample))/CPBS)×100。
Claims (5)
1. the antibacterial agent is characterized by comprising liquid-liquid phase separation nano liquid drops based on a surfactant and sulfur-doped carbon quantum dots, wherein the type of the antibacterial agent is one of spray, drops, a sol, a suspension and an emulsion, and the upper limit of the concentration of the nano liquid drops in the antibacterial agent is 150 mu g/mL;
the preparation method of the liquid-liquid phase separation nano liquid drop based on the surfactant and the sulfur-doped carbon quantum dot comprises the following steps: placing the mixed solution of the oleic acid amide propyl dimethyl tertiary amine and the sulfur-doped carbon quantum dots at room temperature, centrifuging, removing supernatant to obtain a condensed phase with liquid-liquid phase separation, and dispersing in an aqueous solution;
the concentration of the amidopropyl dimethyl tertiary amine oleate in the mixed solution is 30-40mM, and the concentration of the sulfur-doped carbon quantum dots is 1-4 mg/mL.
2. The antibacterial agent according to claim 1, wherein the mixed solution has a concentration of amidopropyl dimethyl tertiary amine oleate of 34mM and a concentration of sulfur-doped carbon quantum dots of 2 mg/mL.
3. The antimicrobial agent of claim 2, wherein the preparation method comprises the steps of: standing the mixed solution containing 34mM oleic acid amide propyl dimethyl tertiary amine and 2mg/mL sulfur-doped carbon quantum dots at room temperature for 24h, centrifuging at 10000rpm for 30min, discarding the supernatant to obtain a condensed phase with liquid-liquid phase separation, and dispersing in an aqueous solution.
4. Use of an antibacterial agent as claimed in any one of claims 1 to 3 in the manufacture of a medicament for inhibiting the growth of a bacterial biofilm, wherein the bacteria are gram positive and/or gram negative bacteria.
5. Use according to claim 4, wherein the bacteria are Staphylococcus aureus and/or Escherichia coli.
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| CN110982518A (en) * | 2019-11-19 | 2020-04-10 | 上海应用技术大学 | Nitrogen-sulfur co-doped carbon quantum dot fluorescent probe for cysteine detection and preparation and application thereof |
| CN111849475A (en) * | 2020-08-25 | 2020-10-30 | 中南民族大学 | Nitrogen and sulfur co-doped carbon dot and preparation method and application thereof |
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| CN105567228A (en) * | 2016-01-27 | 2016-05-11 | 山西大学 | N, P and S-codoped fluorescent carbon quantum dot and preparation method and application thereof |
| CN110982518A (en) * | 2019-11-19 | 2020-04-10 | 上海应用技术大学 | Nitrogen-sulfur co-doped carbon quantum dot fluorescent probe for cysteine detection and preparation and application thereof |
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