Silver-carbon quantum dot composite nanomaterial loaded with antibacterial peptide and preparation method thereof

Abstract

This invention discloses a silver-carbon quantum dot composite nanomaterial loaded with an antimicrobial peptide, comprising silver-carbon quantum dots (AgCQDs) and an antimicrobial peptide CKKII14. The antimicrobial peptide CKKII14 is loaded onto the silver-carbon quantum dots (AgCQDs) via Ag-S bonds, and its serial number is CIKKIIKKIIKKII-NH2. This silver-carbon quantum dot composite nanomaterial loaded with the antimicrobial peptide enhances the antimicrobial properties of the peptide, exhibiting significant antimicrobial effects against *Escherichia coli* and *Staphylococcus aureus*. The optimal inhibitory concentration (MIC) against *Escherichia coli* reaches 3 µg / mL, and against *Staphylococcus aureus* reaches 5 µg / mL.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials technology, specifically to a silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides and its preparation method. Background Technology

[0002] Escherichia coli (E. coli) is a Gram-negative short rod-shaped bacterium that is widely found in the natural environment, water sources, soil, and the intestines of humans and warm-blooded animals. This bacterium is highly adaptable and proliferates rapidly in moist, nutrient-rich environments, making it one of the key pathogens monitored in environmental hygiene, food hygiene, and medical fields.

[0003] Staphylococcus aureus is a Gram-positive coccus. It can produce a variety of toxic factors such as enterotoxins, hemolysins, and coagulases, which can not only cause purulent skin infections and respiratory infections, but also easily contaminate food and produce heat-resistant enterotoxins, leading to food poisoning and causing acute poisoning symptoms such as nausea, vomiting, and abdominal pain.

[0004] In food processing environments, skin wounds, medical device surfaces, and public health settings, *Escherichia coli* and *Staphylococcus aureus* readily form mixed contamination and co-infections, and biofilms easily form when they coexist. Currently, most traditional antimicrobial materials suffer from narrow antimicrobial spectra, uneven inhibitory effects against Gram-negative and Gram-positive bacteria, poor long-term efficacy, insufficient stability, or inadequate biosafety, making it difficult to simultaneously achieve highly efficient and stable inhibition of both *Escherichia coli* and *Staphylococcus aureus*. Against this backdrop, developing novel antimicrobial materials that combine broad-spectrum antimicrobial activity, good stability, and biosafety is of significant research value.

[0005] Antimicrobial peptides, as a novel class of antimicrobial active substances, possess broad antimicrobial spectrum and rapid killing effect, exhibiting good inhibitory or bactericidal effects against various microorganisms, including Gram-positive bacteria, Gram-negative bacteria, and fungi. However, antimicrobial peptides, when used alone, still suffer from several drawbacks, such as poor stability, susceptibility to temperature and pH changes, and easy degradation by proteases. To overcome the shortcomings of single antimicrobial peptide applications, combining them with nanomaterials for antimicrobial activity has become a major research direction. Based on the combination of silver-carbon quantum dots, which possess the excellent biocompatibility and water solubility of carbon quantum dots, and the superior broad-spectrum antimicrobial properties of silver nanomaterials, this application develops a silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides. By combining antimicrobial peptides with silver-carbon quantum dots to construct multifunctional antimicrobial materials, on the one hand, the stability and local enrichment capacity of antimicrobial peptides can be improved by utilizing silver-carbon quantum dots, reducing enzymatic degradation and inactivation; on the other hand, the synergistic effect of antimicrobial peptides and silver-based antimicrobial mechanisms can be achieved, significantly enhancing overall antimicrobial activity and bactericidal efficiency. Summary of the Invention

[0006] To address the problem that Escherichia coli and Staphylococcus aureus cannot be simultaneously and efficiently inhibited, this invention provides a silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides and discloses a method for preparing the material.

[0007] The technical solution adopted in this invention is: a silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptide, comprising silver-carbon quantum dots AgCQDs and antimicrobial peptide CKKII14, wherein the antimicrobial peptide CKKII14 is loaded on AgCQDs through Ag-S bonds, and the amino acid sequence number of the antimicrobial peptide CKKII14 is CIKKIIKKIIKKII-NH2.

[0008] This application also provides a method for preparing the above-mentioned silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides, comprising the following steps: S1: Preparation of carbon quantum dots (CQDs): Citric acid and urea are mixed with water and then reacted at 145℃~155℃ for 7 h~9 h. After cooling to room temperature, the mixture is filtered and dialyzed to obtain carbon quantum dots (CQDs). Water is used as a solvent, and there are no requirements on the amount added; it is sufficient to dissolve citric acid and urea.

[0009] S2: Preparation of silver-carbon quantum dots AgCQDs: Sodium borohydride, silver nitrate and carbon quantum dots CQDs from step S1 are mixed evenly, stirred in the dark, centrifuged and washed to obtain silver-carbon quantum dots AgCQDs. S3: Preparation of silver-carbon quantum dot-loaded antimicrobial peptide AgCQDs@CKKII14: AgCQDs and the antimicrobial peptide CKKII14 were incubated on a shaker to obtain the silver-carbon quantum dot composite nanomaterial AgCQDs@CKKII14 loaded with the antimicrobial peptide. The antimicrobial peptide CKKII14 is derived from the paper DOI:10.1021 / acs.langmuir.5c03844.

[0010] Through long-term research, the applicant discovered that combining AgCQDs with CKKII14 can further enhance the antibacterial properties of CKKII14. The reasons are as follows: 1. Microbial cell membranes are typically negatively charged, while AgCQDs@CKKII14 are positively charged. They are strongly attracted by electrostatic attraction. CKKII14 binds to the phospholipid bilayer of the bacterial cell membrane through hydrophobic and electrostatic interactions. As the peptide accumulates on the phospholipid membrane, when the concentration reaches a certain threshold, CKKII14 forms micelles and decomposes the lipid membrane, causing leakage of core substances within the bacterial cell (such as proteins, nucleic acids, and potassium ions), leading to cell death; 2. AgCQDs accumulate around the bacterial membrane, forming indentations on the cell wall and causing some damage to the bacteria. The combined effect of AgCQDs and CKKII14 further enhances the antibacterial properties of the antimicrobial peptide.

[0011] Preferably, the mass ratio of citric acid to urea in S1 is 1:1.1~1.3.

[0012] Preferably, in S1, a 0.22 µM aqueous filter membrane is used for filtration, and dialysis is performed using an Mw1000 dialysis bag for 22 h to 26 h.

[0013] Preferably, the ratio of sodium borohydride, silver nitrate, and carbon quantum dots (CQDs) in S2 is 9 mM ~ 11 mM: 9 mM ~ 11 mM: 72 µg / mL ~ 76 µg / mL.

[0014] Preferably, the stirring time in S2 is 5 minutes, the centrifugal washing is performed 2-3 times, the rotation speed is 8000rpm~10000rpm, and the centrifugal washing time is 10min~15min each time.

[0015] Preferably, the mass ratio of silver-carbon quantum dots AgCQDs to antimicrobial peptide CKKII14 in S3 is 16:0.8~1.2.

[0016] Preferably, AgCQDs and antimicrobial peptide CKKII14 in S3 are incubated on a shaker at room temperature for 12 h to 15 h.

[0017] The beneficial effects of this invention are: This application enhances the antimicrobial properties of antimicrobial peptides by loading them onto silver-carbon quantum dots. The preparation method and operation process are simple (see details). Figure 1 The synthesized AgCQDs@CKKII1 showed significant antibacterial effects against Escherichia coli and Staphylococcus aureus, with the optimal inhibitory concentration against Escherichia coli reaching 3 µg / mL and against Staphylococcus aureus reaching 5 µg / mL. Attached Figure Description

[0018] Figure 1 A schematic diagram illustrating the preparation and antibacterial principle of a silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides; Figure 2 A is the UV-Vis spectrum of the CQDs from Example 1. Figure 2 B is the UV-Vis spectrum of AgCQDs from Example 1; Figure 2 C is the excitation spectrum of the CQDs in Example 1; Figure 2 D is the fluorescence spectrum of CQDs and AgCQDs in Example 1; Figure 2 E is a histogram of Zeta potentials for CQDs, AgCQDs, and AgCQDs@CKKII14 in Example 1; Figure 2 F is a TEM image of AgCQDs from Example 1; Figure 3A is the full spectrum analysis diagram of AgCQDs in Example 1; Figure 3 B is the high-resolution spectrum of element C in AgCQDs of Example 1; Figure 3 C is the high-resolution spectrum of N element in AgCQDs of Example 1; Figure 3 D is the high-resolution spectrum of the O element in the AgCQDs of Example 1; Figure 3 E is a high-resolution spectrum of Ag elements in AgCQDs from Example 1; Figure 4 Figure A shows the minimum inhibitory concentration (MIC) of AgCQDs against Escherichia coli in Example 1. Figure 4 B is the minimum inhibitory concentration (MIC) result of CKKII14 against Escherichia coli in Example 1. Figure 4 C represents the minimum inhibitory concentration (MIC) of AgCQDs@CKKII14 against Escherichia coli. Figure 5 A is a graph showing the minimum inhibitory concentration of AgCQDs against Staphylococcus aureus in Example 1; Figure 5 B is a graph showing the minimum inhibitory concentration (MIC) of CKKII14 against Staphylococcus aureus in Example 1. Figure 5 C represents the minimum inhibitory concentration (MIC) of AgCQDs@CKKII14 against Staphylococcus aureus. Figure 6 The graph shows the bactericidal concentration results of the composite material AgCQDs@CKKII14 in Example 1 against Escherichia coli. Figure 7 The graph shows the bactericidal concentration results of the composite material AgCQDs@CKKII14 in Example 1 against Staphylococcus aureus. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and a preferred embodiment.

[0020] Unless otherwise specified, all raw materials used in the following examples are commercially available.

[0021] The reagents and instruments used in this experiment are as follows: LB broth, item number HB0128, was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd. Agar powder, item number A8190, was purchased from Beijing Solarbio Technology Co., Ltd. Citric acid, urea, and silver nitrate were all purchased from Sinopharm Chemical Reagent Co., Ltd. Sodium borohydride was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. The antimicrobial peptide CKKII14 sequence number is CIKKIIKKIIKKII-NH2. Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were both provided by Changzhou Second People's Hospital.

[0022] The culture media used in this invention are: LB liquid culture medium, which is composed of 5g LB broth and 200 mL water; and LB solid culture medium, which is composed of 5g LB broth, 3.5g agar powder and 200 mL water.

[0023] UV-Vis spectra were measured using a UV-Vis spectrophotometer (Shimadzu UV-1900i, Japan). Potentiometric analysis of each sample was performed at 25°C using a Zetasizer Nano ZS nanoparticle size analyzer (Malvern, UK). Fluorescence spectra were determined using an integrated fluorescence spectrometer (Fs5, Edinburgh, UK). Absorbance values ​​of the bacterial suspensions at 600 nm were measured using a Varioskan LUX multi-functional microplate reader (Thermo Fisher Scientific, USA). Centrifugation of samples was performed using a Centrifuge 5418 R refrigerated benchtop microcentrifuge (Eppendorf, Germany). Example 1

[0024] ①The synthesis steps of carbon quantum dots (CQDs) are as follows: Weigh 20 g of citric acid and 20 g of urea, add 60 mL of water to dissolve and mix evenly, put into a high temperature and high pressure reactor, react at 150℃ for 8 h, cool to room temperature, filter with a 0.22 µM aqueous filter membrane, dialyze with an Mw1000 dialysis bag for 24 h to obtain carbon quantum dots (CQDs), freeze-dry for later use.

[0025] ②The synthesis steps of silver-carbon quantum dots (AgCQDs) are as follows: Weigh 0.3026 g sodium borohydride (10 mM), 1.3589 g silver nitrate (10 mM) and 0.06 g carbon quantum dots (CQDs) (75 µg / mL), add 800 mL of water, mix well, protect from light, stir for 5 min, and when the reaction is complete, centrifuge and wash 3 times, 10 min each time, at 8000 rpm, and freeze dry for later use.

[0026] ③The synthesis steps of AgCQDs@CKKII14 are as follows: AgCQDs (160 µg) and antimicrobial peptide CKKII14 (10 µg) were incubated on a shaker for 12 h to obtain 1 mL of silver-carbon quantum dot composite nanomaterial AgCQDs@CKKII14 loaded with antimicrobial peptide.

[0027] The obtained AgCQDs, CQDs, and AgCQDs@CKKII14 were diluted with water at a volume ratio of 9:1. Potential analysis was performed on each diluted sample group. The results are detailed in [link to results]. Figure 2 E.

[0028] Figure 2 A is the UV-Vis spectrum of CQDs, which shows that carbon quantum dot CQDs have an absorption peak near 650 nm, indicating that CQDs were successfully prepared. Figure 2 B is the UV-Vis spectrum of AgCQDs, which shows that AgCQDs have an absorption peak near 400 nm, indicating that AgCQDs were successfully prepared. Figure 2 C is the excitation spectrum of carbon quantum dots (CQDs), which shows that the optimal excitation wavelength of CQDs is around 350 nm, indicating that CQDs were successfully prepared. Figure 2 D shows the fluorescence spectra of AgCQDs (yellow curve) and CQDs (green curve), indicating that CQDs are fluorescent. After silver doping, AgCQDs exhibit fluorescence quenching, indicating that AgCQDs were successfully synthesized. Figure 2 E is a histogram of Zeta potentials for AgCQDs, CQDs, and AgCQDs@CKKII14, indicating that AgCQDs are negatively charged, CQDs are negatively charged, and AgCQDs@CKKII14 are positively charged, indicating that antimicrobial peptides were successfully loaded onto AgCQDs. Figure 2 F is a TEM image of AgCQDs, indicating that AgCQDs were successfully prepared.

[0029] Figure 3 A indicates that AgCQDs contain four elements: C, N, O, and Ag; the corresponding element peak values ​​are 284.08 eV, 407.08 eV, 532.08 eV, and 368.08 eV, respectively. Figure 3 B indicates that AgCQDs contain C=C, CN, and C=O groups in the high-resolution C 1s XPS spectrum, with corresponding group peaks of 285.18 eV, 284.78 eV, and 283.28 eV, respectively. Figure 3 C is the high-resolution N 1s spectrum of AgCQDs. AgCQDs contain groups such as CNC, NH, and C=N, with corresponding peak values ​​of 406.98 eV, 406.18 eV, and 405.68 eV, respectively. Figure 3 D is the high-resolution O 1s spectrum of AgCQDs. AgCQDs contain groups such as C=O, -COOH, and C=O / CO, with corresponding peak values ​​of 530.48 eV, 531.48 eV, and 534.18 eV, respectively. Figure 3 E shows that the two peaks of Ag 3d originate from Ag 3d5 / 2 and Ag 3d3 / 2, with peak values ​​of 373.98 eV and 367.98 eV, respectively. These results indicate that AgCQDs were successfully synthesized. Example 2

[0030] ①The synthesis steps of carbon quantum dots (CQDs) are as follows: Weigh 20 g of citric acid and 22 g of urea, add 60 mL of water to dissolve and mix evenly, put into a high temperature and high pressure reactor, react at 145℃ for 7 h, cool to room temperature, filter with a 0.22 µM aqueous filter membrane, dialyze with an Mw1000 dialysis bag for 22 h to obtain carbon quantum dots (CQDs), freeze-dry for later use.

[0031] ②The synthesis steps of silver-carbon quantum dots (AgCQDs) are as follows: Weigh 0.2724 g sodium borohydride (9 mM), 1.2231 g silver nitrate (9 mM) and 0.0576 g carbon quantum dots (CQDs) (72 µg / mL), add 800 mL of water and mix well. Protect from light and stir for 5 min. After the reaction is complete, centrifuge and wash twice, 12 min each time, at 9000 rpm. Freeze-dry for later use.

[0032] ③The synthesis steps of AgCQDs@CKKII14 are as follows: AgCQDs (160 µg) and antimicrobial peptide CKKII14 (8 µg) were incubated on a shaker for 13 h to obtain 1 mL of silver-carbon quantum dot composite nanomaterial AgCQDs@CKKII14 loaded with antimicrobial peptide.

[0033] Example 3 ①The synthesis steps of carbon quantum dots (CQDs) are as follows: Weigh 20 g of citric acid and 26 g of urea, add 60 mL of water to dissolve and mix evenly, put into a high temperature and high pressure reactor, react at 155℃ for 9 h, cool to room temperature, filter with a 0.22 µM aqueous filter membrane, dialyze with an Mw1000 dialysis bag for 26 h to obtain carbon quantum dots (CQDs), freeze-dry for later use.

[0034] ②The synthesis steps of silver-carbon quantum dots (AgCQDs) are as follows: Weigh 0.3329 g sodium borohydride (11 mM), 1.4949 g silver nitrate (11 mM) and 0.0608 g carbon quantum dots (CQDs) (76 µg / mL), add 800 mL of water and mix well. Protect from light and stir for 5 min. After the reaction is complete, centrifuge and wash 3 times, 15 min each time, at 10000 rpm. Freeze dry for later use.

[0035] ③The synthesis steps of AgCQDs@CKKII14 are as follows: AgCQDs (160 µg) and antimicrobial peptide CKKII14 (12 µg) were incubated on a shaker for 15 h to obtain 1 mL of silver-carbon quantum dot composite nanomaterial AgCQDs@CKKII14 loaded with antimicrobial peptide.

[0036] Example 4 The minimum inhibitory concentration (MIC) was determined by the microdilution method: The following materials are required: Escherichia coli culture medium: composed of 0.1% Escherichia coli and 99.9% LB liquid medium; Staphylococcus aureus culture medium: composed of 0.1% Staphylococcus aureus and 99.9% LB liquid medium; E. coli experimental group: The AgCQDs prepared in Example 1 were diluted with water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL, 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL, 80 µg / mL and 160 µg / mL, respectively. CKKII14 was prepared into solutions with concentrations of 1 µg / mL, 2 µg / mL, 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL and 80 µg / mL using water. The AgCQDs@CKKII14 prepared in Example 1 was dissolved in water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL and 5 µg / mL, respectively.

[0037] Staphylococcus aureus experimental group The AgCQDs prepared in Example 1 were diluted with water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL, 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL, 80 µg / mL and 160 µg / mL, respectively. CKKII14 was prepared into solutions with concentrations of 1 µg / mL, 2 µg / mL, 5 µg / mL, 10 µg / mL, 20 µg / mL, 40 µg / mL, 80 µg / mL and 160 µg / mL using water. The AgCQDs@CKKII14 prepared in Example 1 was dissolved in water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL, 5 µg / mL and 8 µg / mL, respectively.

[0038] Escherichia coli control group: Six groups of 100 µL E. coli bacterial suspensions were mixed with six groups of 100 µL sterile water to obtain six mixtures. These mixtures were then transferred to 96-well plates and incubated on a shaker (37℃, 160 rpm) for 18 h. The absorbance (OD) of each of the six samples was measured at 600 nm using a microplate reader. 600 nm The average value of the obtained data is the concentration of E. coli in the control group sample.

[0039] Escherichia coli experimental group: Eight groups of 100 µL E. coli bacterial suspensions were mixed with 100 µL of AgCQDs solution of the above concentration to obtain eight mixed solutions; seven groups of 100 µL E. coli bacterial suspensions were mixed with 100 µL of CKKII14 solution of the above concentration to obtain seven mixed solutions; three groups of 100 µL E. coli bacterial suspensions were mixed with 100 µL of AgCQDs@CKKII14 solution of the above concentration to obtain three mixed solutions. Each of the above mixed solutions was transferred to a 96-well plate and incubated on a shaker (37℃, 160 rpm) for 18 h to obtain each sample. The absorbance (OD) of each sample at 600 nm was measured using a microplate reader. 600 nm To ensure experimental accuracy, the above experimental conditions and procedures were kept unchanged and repeated 5 times. The average value of each group of samples was then taken to obtain the concentration of *E. coli* in each group of experimental samples. The survival rate was determined by the ratio of the *E. coli* concentration in the experimental group to that in the control group. Detailed data are shown in the appendix. Figure 4 .

[0040] Staphylococcus aureus control group: Six groups of 100 µL Staphylococcus aureus bacterial suspensions were mixed with six groups of 100 µL sterile water to obtain six mixtures. These mixtures were then transferred to 96-well plates and incubated on a shaker (37℃, 160 rpm) for 18 h. The absorbance (OD) of each of the six samples was measured at 600 nm using a microplate reader. 600 nm The average value of the obtained data is the concentration of Staphylococcus aureus in the control group sample.

[0041] Staphylococcus aureus experimental group: Eight groups of 100 µL Staphylococcus aureus bacterial suspensions were mixed with 100 µL of AgCQDs solution of the above concentration to obtain eight mixed solutions; eight groups of 100 µL Staphylococcus aureus bacterial suspensions were mixed with 100 µL of CKKII14 solution of the above concentration to obtain eight mixed solutions; four groups of 100 µL Staphylococcus aureus bacterial suspensions were mixed with 100 µL of AgCQDs@CKKII14 solution of the above concentration to obtain four mixed solutions. Each of the above mixed solutions was transferred to a 96-well plate and incubated on a shaker (37℃, 160 rpm) for 24 h to obtain each sample. The absorbance (OD) of each sample at 600 nm was measured using a microplate reader. 600 nm To ensure experimental accuracy, the above experimental conditions and procedures were kept unchanged and repeated 5 times. The average value of each group of samples was then taken to obtain the concentration of Staphylococcus aureus in each group of samples in experimental group 1. The survival rate was determined by the ratio of the Staphylococcus aureus concentration in the experimental group to that in the control group. Detailed data are shown in the appendix. Figure 5 .

[0042] Figure 4 The graph shows the minimum inhibitory concentrations (MICs) of AgCQDs, CKKII14, and AgCQDs@CKKII14 against Escherichia coli. Figure 4 A indicates that the optimal inhibitory concentration of AgCQDs against Escherichia coli is 80 µg / mL; Figure 4 B indicates that the optimal inhibitory concentration of CKKII14 against Escherichia coli is 5 µg / mL; Figure 4 C indicates that the optimal inhibitory concentration of AgCQDs@CKKII14 against Escherichia coli is 3 µg / mL. The results show that, compared to AgCQDs and CKKII14 alone, AgCQDs@CKKII14 exhibits the best inhibitory effect against Escherichia coli at a lower concentration.

[0043] Figure 5 The graph shows the minimum inhibitory concentrations (MICs) of AgCQDs, CKKII14, and AgCQDs@CKKII14 against Staphylococcus aureus. Figure 5 A indicates that the optimal inhibitory concentration of AgCQDs against Staphylococcus aureus is 80 µg / mL; Figure 5 B indicates that the optimal inhibitory concentration of CKKII14 against Staphylococcus aureus is 160 µg / mL; Figure 5 C indicates that the optimal inhibitory concentration of AgCQDs@CKKII14 against Staphylococcus aureus is 5 µg / mL. The results show that compared to AgCQDs and CKKII14 alone, AgCQDs@CKKII14 exhibits the best antibacterial effect at a significantly lower inhibitory concentration against Staphylococcus aureus. Example 5

[0044] The minimum bactericidal concentration (MBC) was determined by the spread inoculation method: The following materials are required: Escherichia coli culture medium: composed of 0.1% Escherichia coli and 99.9% LB liquid medium; Staphylococcus aureus culture medium: composed of 0.1% Staphylococcus aureus and 99.9% LB liquid medium; Escherichia coli experimental group: AgCQDs@CKKII14 prepared in Example 1 was diluted with water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL and 5 µg / mL respectively.

[0045] Staphylococcus aureus experimental group: AgCQDs@CKKII14 prepared in Example 1 was diluted with water to prepare solutions with concentrations of 1 µg / mL, 2 µg / mL, 3 µg / mL, 4 µg / mL, 5 µg / mL and 6 µg / mL respectively.

[0046] Escherichia coli experimental group: Five mixtures of 100 µL E. coli bacterial suspension were mixed with 100 µL of AgCQDs@CKKII14 solution of the above concentration to obtain five mixed solutions. These mixtures were transferred to 96-well plates and incubated at 37°C and 160 rpm for 18 h. Then, 30 µL of each mixed solution was inoculated onto LB agar and incubated at 37°C for 12 h. The colony count on each LB agar plate was observed the following day. To ensure experimental accuracy, the above experimental conditions and procedures were repeated twice, and the colony count was observed.

[0047] Escherichia coli control group: Three groups of 100 µL E. coli bacterial suspensions were mixed with three groups of 100 µL sterile water to obtain three mixtures. These mixtures were then transferred to 96-well plates and incubated at 37°C and 160 rpm for 18 h. 30 µL of each mixture was then inoculated onto LB agar and incubated at 37°C for 12 h. The colony counts on the LB agar plates were observed every other day. By comparing the colony counts of the experimental and control groups, the minimum concentration required to kill E. coli was determined.

[0048] Staphylococcus aureus experimental group: Six mixtures of 100 µL Staphylococcus aureus culture were prepared by mixing 100 µL of AgCQDs@CKKII14 solution of the above concentration. These mixtures were then transferred to 96-well plates and incubated at 37°C and 160 rpm for 24 h. 30 µL of each mixture was then inoculated onto LB agar and incubated at 37°C for 12 h. Colony counts on the LB agar were observed the following day. To ensure accuracy, the experimental conditions and procedures were repeated twice, and colony counts were observed.

[0049] Staphylococcus aureus control group: Three groups of 100 µL Staphylococcus aureus bacterial suspensions were mixed with three groups of 100 µL sterile water to obtain three mixtures. These mixtures were then transferred to 96-well plates and incubated at 37°C and 160 rpm for 18 h. After incubation, 30 µL of each mixture was inoculated onto LB agar and cultured at 37°C for 12 h. The colony counts on the LB agar plates were observed the following day. By comparing the colony counts of the experimental and control groups, the minimum concentration required to kill Staphylococcus aureus was determined.

[0050] Figure 6 This indicates that the lowest concentration of AgCQDs@CKKII14 that kills Escherichia coli is 3 µg / mL; Figure 7 This indicates that the minimum concentration of AgCQDs@CKKII14 to kill Staphylococcus aureus is 5 µg / mL.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also within the protection scope of the present invention.

Claims

1. A silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides, characterized in that, It includes silver-carbon quantum dots AgCQDs and antimicrobial peptide CKKII14, wherein the antimicrobial peptide CKKII14 is loaded on the silver-carbon quantum dots AgCQDs via Ag-S bonds, and the amino acid sequence number of the antimicrobial peptide CKKII14 is CIKKIIKKIIKKII-NH2.

2. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 1, characterized in that, The preparation steps of the silver-carbon quantum dot composite nanomaterial are as follows: S1: Preparation of carbon quantum dots (CQDs): Citric acid and urea are mixed with water and then reacted at 145℃~155℃ for 7 h~9 h. After cooling to room temperature, the mixture is filtered and dialyzed to obtain carbon quantum dots (CQDs). S2: Preparation of silver-carbon quantum dots AgCQDs: Sodium borohydride, silver nitrate and carbon quantum dots CQDs from step S1 are mixed evenly, stirred in the dark, centrifuged and washed to obtain silver-carbon quantum dots AgCQDs. S3: Preparation of silver-carbon quantum dot-loaded antimicrobial peptide AgCQDs@CKKII14: AgCQDs and antimicrobial peptide CKKII14 were incubated on a shaker to obtain silver-carbon quantum dot composite nanomaterials AgCQDs@CKKII14 loaded with antimicrobial peptide.

3. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, The mass ratio of citric acid to urea in S1 is 1:1.1~1.

3.

4. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, S1 uses a 0.22 µM aqueous filter membrane for filtration, and Mw1000 dialysis bags for dialysis for 22 h to 26 h.

5. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, The ratio of sodium borohydride, silver nitrate, and carbon quantum dots (CQDs) in S2 is 9 mM ~ 11 mM: 9 mM ~ 11 mM: 72 µg / mL ~ 76 µg / mL.

6. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, The stirring time in S2 is 5 minutes, the number of centrifugal washing cycles is 2-3, the rotation speed is 8000 rpm~10000 rpm, and the centrifugal washing time for each cycle is 10 min~15 min.

7. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, In S3, the mass ratio of silver-carbon quantum dots AgCQDs to antimicrobial peptide CKKII14 is 16:0.8~1.

2.

8. The silver-carbon quantum dot composite nanomaterial loaded with antimicrobial peptides according to claim 2, characterized in that, AgCQDs and antimicrobial peptide CKKII14 in S3 were incubated on a shaker at room temperature for 12-15 hours.

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