Immunomagnetic beads for bacteria and preparation method and application thereof

Abstract

The present invention discloses a kind of immunomagnetic beads for bacteria and its preparation method and application, which belong to the field of biomedical technology. The immunomagnetic beads include Fe3O4 magnetic beads with photocatalysis, and the Fe3O4 magnetic beads with photocatalysis are loaded with antimicrobial peptides; the Fe3O4 magnetic beads with photocatalysis are bismuth-metal bismuth salt @Fe3O4 heterojunctions. The antimicrobial peptide is vancomycin; the bismuth-metal bismuth salt @Fe3O4 heterojunctions are Bi-BFO@Fe3O4 magnetic beads. The present invention prepares Bi-BFO@Fe3O4 magnetic beads by laser bombardment, then loads vancomycin, uses vancomycin to capture bacteria, uses Fe3O4 magnetic beads to separate and enrich target bacteria from samples, and then performs photocatalytic sterilization through the action of Bi-BFO. The immunomagnetic beads for bacteria realize the four-in-one effects of bacterial capture, separation, enrichment and photocatalytic sterilization, and have broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to immunomagnetic beads for bacteria, and a preparation method and application thereof. Background Art

[0002] In recent years, food bacterial contamination, postoperative bacterial infection, and contamination of medical supplies such as stem cell preparations have become important issues threatening human life and health. Gram-positive bacteria are a large type of bacteria. Staphylococcus aureus, also known as "flesh-eating bacteria", is a representative of Gram-positive bacteria and can cause many serious infections, leading to pneumonia, pseudomembranous enterocolitis, pericarditis, and even systemic infections such as sepsis and septicemia. In addition, Gram-positive bacteria are represented by Listeria monocytogenes and Streptococcus pyogenes. Listeria monocytogenes is a zoonotic pathogen that can cause listeriosis in humans and animals, leading to sepsis and meningitis. Streptococcus pyogenes is widely present in nature, human and animal feces, and the nasopharynx of healthy people, causing various serious diseases such as purulent inflammation, scarlet fever, erysipelas, and neonatal sepsis. The identification and sterilization of bacteria have become key points in medical diagnosis, food safety, environmental hygiene, and drug preparation. In the existing technology, the efficiency of capturing bacteria is low, especially in low concentration or complex samples, it is difficult to effectively capture the target bacteria. Many capture methods have low sensitivity and specificity for target bacteria, which may lead to missed detection or false detection, and are prone to capture non-target microorganisms or impurities, resulting in inaccurate results. After capturing the bacteria, how to efficiently separate the target bacteria remains a challenge; traditional centrifugation and filtration methods may not be able to completely separate pure target bacteria. Antibiotic sterilization or chemical disinfectants may remain in the environment after use, causing harm to the human body and the ecological environment. Over the past few decades, researchers have been committed to developing rapid and efficient bacterial detection and sterilization methods in complex samples with unknown colonies and samples with low bacterial concentrations.

[0003] Immunomagnetic beads (IMB, short for magnetic beads) are a new immunological technology developed in recent years. It combines the unique advantages of solidification reagents with the high specificity of immunological reactions. Based on immunology, it penetrates into various fields such as pathology, physiology, pharmacology, microbiology, biochemistry, and molecular genetics. Through the directional modification of immunomagnetic beads to form a core-shell structure, it can move in a directional manner under the attraction of an external magnetic field, thereby achieving the purpose of separation, detection, and purification of genes, proteins, cells, microorganisms, etc. Therefore, immunomagnetic beads are mainly used for gene purification, protein purification, cell purification, and microbial enrichment, but there are currently no immunomagnetic beads with bactericidal effects. Since the magnetism of immunomagnetic beads gives them the ability to easily separate (capture) and enrich, if immunomagnetic beads are used to capture bacteria, the target bacteria can be easily separated, and then the immunomagnetic beads are given a bactericidal function, so that the separated target bacteria can be effectively killed, thereby achieving the separation of target bacteria and sterilization in complex samples, which has a good application prospect. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a bacterial immunomagnetic bead and a preparation method and application thereof. The present invention prepares Bi-BFO@Fe3O4 magnetic beads by laser bombardment, then loads vancomycin, and uses vancomycin to capture bacteria. The bacterial immunomagnetic beads prepared by the present invention can separate and enrich the target bacteria from the sample, and then perform photocatalytic sterilization through the action of Bi-BFO. The magnetic beads can achieve the four-in-one effects of bacterial capture, separation, enrichment and photocatalytic sterilization, and have broad application prospects.

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

[0006] The first aspect of the present invention provides immunomagnetic beads for bacteria, the immunomagnetic beads comprising Fe3O4 magnetic beads with photocatalytic properties, on which antimicrobial peptides are loaded; the Fe3O4 magnetic beads with photocatalytic properties are bismuth-metal bismuth salt@Fe3O4 heterojunction magnetic beads; the bismuth-metal bismuth salt@Fe3O4 heterojunction magnetic beads are Bi-BFO@Fe3O4 magnetic beads.

[0007] Preferably, the antimicrobial peptide is vancomycin; and the particle size of the Fe3O4 magnetic beads with photocatalytic properties is 60-100 nm.

[0008] The second aspect of the present invention provides a method for preparing immunomagnetic beads for bacteria, comprising the following steps:

[0009] (1) Dispersing bismuth salt and Fe3O4 magnetic beads in ethylene glycol, stirring at room temperature, turning on the laser to bombard Fe3O4, centrifuging, washing and drying after the bombardment, and obtaining Bi-BFO@Fe3O4 magnetic beads;

[0010] (2) Carboxylating the Bi-BFO@Fe3O4 magnetic beads to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads; adding a coupling agent to couple the antimicrobial peptides with the carboxylated Bi-BFO@Fe3O4 magnetic beads to obtain bacterial immunomagnetic beads.

[0011] Preferably, in step (1), the bismuth salt is bismuth nitrate; the bismuth salt is bismuth nitrate; the ratio of the added amounts of bismuth nitrate, ethylene glycol and Fe3O4 magnetic beads is 1.2~1.7 g: 20~40 mL: 1.0~1.5 g.

[0012] Preferably, in step (1), the stirring time is 80-120 min; the parameters of the laser bombardment are: wavelength 1064 nm, energy 10-20 W, scanning speed 10-500 mm s –1 .

[0013] Preferably, in step (2), the carboxylation treatment is:

[0014] Bi-BFO@Fe3O4 magnetic beads, 2,3-dimercaptosuccinic acid and Na2CO3 were dispersed in a mixed solution of tetrahydrofuran and water, and after ultrasonic treatment, centrifugation, washing and freeze-drying were performed in sequence to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads.

[0015] Preferably, the mass ratio of the Bi-BFO@Fe3O4 magnetic beads, 2,3-dimercaptosuccinic acid and Na2CO3 is 1.0~1.5:1.5~2.0:0.75~1.5; and the ultrasonic treatment time is 30~50 min.

[0016] Preferably, in step (2), the coupling agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; and the antimicrobial peptide is vancomycin.

[0017] Preferably, the mass ratio of the carboxylated Bi-BFO@Fe3O4 magnetic beads, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, polyvinyl pyrrolidone and vancomycin is 1.0~1.5: 0.2~0.4: 0.5~1.0: 0.1~0.3: 0.3~0.5.

[0018] The third aspect of the present invention provides the use of immunomagnetic beads for bacteria in capturing, separating, enriching and photocatalytically killing bacteria.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention prepares Bi-BFO@Fe3O4 magnetic beads by laser bombardment, then loads vancomycin, uses vancomycin to capture bacteria, uses bacterial immunomagnetic beads to separate and enrich target bacteria from samples, and then uses Bi-BFO to perform photocatalytic sterilization. The magnetic beads can achieve the four-in-one functions of bacterial capture, separation, enrichment and photocatalytic sterilization, and have broad application prospects. In addition to vancomycin, other antimicrobial peptides can also be loaded to capture specific bacteria.

[0021] (2) The present invention prepares Bi-BFO@Fe3O4 heterojunction (magnetic beads) by laser bombardment of Fe3O4 magnetic beads, thereby increasing the bismuth content in the heterojunction, using the heterojunction to regulate the energy level band gap, improving the separation efficiency of photogenerated electrons and holes, improving its photocatalytic performance, and generating active oxygen. Therefore, the use of laser treatment further improves the bactericidal ability of the photocatalytic material, so that the immunomagnetic beads have efficient photocatalytic bactericidal ability under visible light conditions.

[0022] (3) The preparation method of the present invention is efficient, highly selective, easy to operate and moderately cost-effective. The prepared immunomagnetic beads can achieve efficient capture and separation in complex samples, thereby improving the sensitivity of bacterial detection. Photocatalytic sterilization technology is low-cost, environmentally friendly, and efficient. It can cope with different application scenarios and has strong practicality. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : XRD pattern of Bi-BFO@Fe3O4 prepared in Example;

[0024] Figure 2 : XRD pattern of BFO@Fe3O4 prepared in comparative example;

[0025] Figure 3 : X-ray energy spectrum analysis diagram of Bi-BFO@Fe3O4 prepared in Example;

[0026] Figure 4 : X-ray energy spectrum analysis diagram of BFO@Fe3O4 prepared in comparative example;

[0027] Figure 5 :External magnetic field controls the enrichment of immunomagnetic beads loaded with bacteria;

[0028] Figure 6 : Colony growth plate images of the immunomagnetic beads prepared in the examples and comparative examples before (control group) and after (experimental group) killing of Staphylococcus aureus, (a) colony growth plate image of the control group of the example, (b) colony growth plate image of the Bi-BFO@Fe3O4 experimental group prepared in the example 1 hour later, (c) colony growth plate image of the control group of the comparative example, (d) colony growth plate image of the BFO@Fe3O4 experimental group prepared in the comparative example 1 hour later. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] As introduced in the background technology section, immunomagnetic beads are mainly used for gene purification, protein purification, cell purification, and microbial enrichment, but there are currently no immunomagnetic beads with bactericidal effects. Bismuth and metal bismuth salts have photocatalytic effects. If metal bismuth salts are compounded with Fe3O4 magnetic beads, the magnetic beads can be endowed with photocatalytic bactericidal effects. Therefore, it is necessary to prepare a metal bismuth salt @Fe3O4 heterojunction, and the prior art generally adopts sol-gel-high temperature calcination method, hydrothermal method and other methods to prepare the heterojunction of metal bismuth salt, but the bismuth content is not high, so the bactericidal performance needs to be improved.

[0031] Based on this, the purpose of the present invention is to provide an immunomagnetic bead for bacteria and a preparation method and application thereof. The present invention prepares Bi-BFO@Fe3O4 magnetic beads by laser bombardment of bismuth nitrate and Fe3O4, mixes Bi-BFO@Fe3O4 magnetic beads with 2,3-dimercaptosuccinic acid to prepare carboxylated Bi-BFO@Fe3O4 magnetic beads, and couples carboxylated Bi-BFO@Fe3O4 magnetic beads and broad-spectrum antimicrobial peptide vancomycin through a coupling agent to prepare an immunomagnetic bead solution. Polyvinyl pyrrolidone is added when loading vancomycin to improve the stability of magnetic nanoparticles in the solution, avoid precipitation, and increase the water solubility and biocompatibility of the complex. Vancomycin is a broad-spectrum antimicrobial peptide that can capture Gram-positive bacteria. Bacterial capture is that vancomycin molecules are connected to D-Ala-D-Ala molecules on the surface of Gram-positive bacteria, which improves the capture efficiency. Vancomycin has a high binding rate with Gram-positive bacteria, so that Gram-positive bacteria and Gram-negative bacteria are separated. Figure 6 The enrichment of immunomagnetic beads loaded with bacteria is controlled by external magnetic field. It can be seen that the immunomagnetic beads loaded with bacteria can be enriched by external magnetic field control.

[0032] The immunomagnetic bead solution was mixed with cultured Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes and Streptococcus pyogenes, and the vancomycin molecules were connected to the D-Ala-D-Ala molecules on the surface of Gram-positive bacteria, which improved the efficiency of bacterial capture; the enrichment of immunomagnetic beads loaded with bacteria was manipulated by an external magnetic field, achieving bacterial capture, separation and enrichment. The increased bismuth content in the heterojunction enhanced the photocatalytic bactericidal effect, allowing the magnetic beads to achieve the four-in-one effect of bacterial capture, separation, enrichment and photocatalytic bactericidal.

[0033] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0034] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0035] Example: Preparation of bacterial immunomagnetic beads

[0036] (1) Disperse 1.2 g of bismuth nitrate and 1.0 g of Fe3O4 magnetic beads in 20 mL of ethylene glycol and stir at room temperature for 80 min to prepare a mixed solution. Adjust the laser parameters: wavelength 1064 nm, energy 15 W, scan rate 100 mm s –1 , turn on the laser to bombard the Fe3O4 magnetic beads in the mixed solution. After the bombardment, centrifuge, wash with water and ethanol alternately three times, and dry to obtain Bi-BFO@Fe3O4 magnetic beads.

[0037] (2) 1.0 g Bi-BFO@Fe3O4 magnetic beads, 1.5 g 2,3-dimercaptosuccinic acid, and 0.75 g Na2CO3 were mixed and dispersed in a mixed solution of tetrahydrofuran and water (V 四氢呋喃 :V 水 = 1:9), ultrasonically treated for 30 min, centrifuged, washed three times with water and ethanol alternately, and freeze-dried to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads. The carboxylated Bi-BFO@Fe3O4 magnetic beads were resuspended in sterile PBS solution with a pH of 7.4 to form a magnetic bead resuspension for later use (0.05 g mL –1 ).

[0038] Dissolve 0.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide in 5 mL of sterile PBS solution at pH 7.4 to prepare 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution; dissolve 0.2 g of N-hydroxysuccinimide in 5 mL of sterile PBS solution at pH 7.4 to prepare N-hydroxysuccinimide solution; dissolve 0.3 g of vancomycin in 5 mL of sterile PBS solution at pH 7.4 to prepare vancomycin solution.

[0039] Mix 20 mL of magnetic bead resuspension with 5 mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and 5 mL of N-hydroxysuccinimide solution, shake at 37°C for 0.8 h to obtain activated magnetic beads, wash the activated magnetic beads three times in 20 mL of sterile PBS solution with a pH of 7.4, and resuspend in 20 mL of sterile PBS solution with a pH of 7.4 to form an activated magnetic bead solution.

[0040] The activated magnetic bead solution was mixed with 5 mL of vancomycin solution and 0.1 g of polyvinyl pyrrolidone, shaken in a dark box at 37 °C for 8 min, washed three times in 20 mL of sterile PBS solution with a pH of 7.4, and resuspended in 20 mL of sterile PBS solution with a pH of 7.4 to form an immunomagnetic bead solution.

[0041] Comparative Example

[0042] The difference from Example 1 is that step (1) adopts the sol-gel-high temperature calcination method to prepare the heterojunction. The specific method is:

[0043] 1.2 g bismuth nitrate, 1.0 g Fe3O4, and 0.1 g polyvinyl pyrrolidone were blended and dispersed in 20 mL ethylene glycol, and stirred at room temperature for 80 min to prepare a mixed solution. The mixed solution was dried at 80 °C overnight, ground into powder, transferred to a tube furnace and heated to 400 °C, retained for 30 min, and then heated to 500 °C and retained for 30 min to obtain BFO@Fe3O4 magnetic beads.

[0044] Finally, an immunomagnetic bead solution is obtained.

[0045] Characterization:

[0046] Figure 1 This is the XRD pattern of Bi-BFO@Fe3O4 prepared in step (1) of the example. Figure 2 This is the XRD pattern of BFO@Fe3O4 prepared in comparative example step (1). It can be seen that Figure 1 In addition to BFO and Fe3O4, the peak of Bi was also detected. Figure 2 Only the peaks of BFO and Fe3O4 were detected. This shows that the laser bombardment method is not only simple and has a short synthesis time, but also contains Bi in the prepared heterojunction. Figure 1 It can be seen that both Bi and BFO are crystalline, and both have photocatalytic effects, which can further improve the photocatalytic sterilization effect.

[0047] Figure 3 This is the X-ray energy spectrum analysis diagram of Bi-BFO@Fe3O4 prepared in step (1) of the embodiment. Figure 4 This is an X-ray energy spectrum analysis diagram of BFO@Fe3O4 prepared in step (1) of the comparative example. It can be seen that the bismuth content in the Bi-BFO@Fe3O4 prepared in the example is higher than that in the BFO@Fe3O4 prepared in step (1) of the comparative example, which further illustrates that the Bi-BFO@Fe3O4 prepared in the example has better photocatalytic performance.

[0048] Experimental example: Immunomagnetic capture, enrichment, separation and sterilization

[0049] The experiment was divided into a control group and an experimental group. The control group: the original bacterial solution before capture in the embodiment or comparative example, the specific preparation method is: 992 μL of sterile PBS solution with a pH of 7.4 and 8 μL of a bacterial concentration of 10 7 CFU mL –1 The Staphylococcus aureus was added to the centrifuge tube and mixed evenly. 100 μL of the supernatant was spread on a nutrient agar plate (Qingdao Haibo Biological) to obtain a control group. Experimental group: The bacterial solution captured in the embodiment or comparative example was specifically prepared as follows: 30 μL of 150 μg mL –1 The immunomagnetic bead solution prepared in the embodiment or comparative example, 962 μL of sterilized PBS solution with a pH of 7.4 and 8 μL of a bacterial concentration of 10 7 CFU mL –1 aureus and mixed them evenly, and then irradiated with a xenon lamp to simulate sunlight. –2 Irradiate with visible light (λ>420 nm) for 1 h. After irradiation, take 100 μL of supernatant from the centrifuge tube and spread it on the nutrient agar plate (Qingdao Haibo Biotechnology) to obtain the experimental group. If the experimental group spreads the nutrient agar plate with immunomagnetic beads, there will be magnetic beads in the colony growth plate set, which will affect the judgment of the colony number. Therefore, when taking the supernatant of the experimental group, you can apply the following Figure 5 The external magnetic field shown enriches the sterilized immunomagnetic beads together, and then the supernatant is taken for coating. Figure 6 The figures are colony growth plates of the immunomagnetic beads before (control group) and after (experimental group) killing of Staphylococcus aureus under different illumination times. It can be seen that the sterilization rate of the immunomagnetic beads prepared in the embodiment against Staphylococcus aureus is greater than 99%, and the sterilization rate of the immunomagnetic beads prepared in the comparative example against Staphylococcus aureus is about 90%.

[0050] Sterilization rate (%) = (number of colonies in the control group - number of colonies in the experimental group) / number of colonies in the control group × 100%.

[0051] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An immunomagnetic bead for bacteria, characterized in that The immunomagnetic beads include Fe3O4 magnetic beads with photocatalytic properties, and the antimicrobial peptides are loaded on the Fe3O4 magnetic beads with photocatalytic properties; the Fe3O4 magnetic beads with photocatalytic properties are bismuth-metal bismuth salt@Fe3O4 heterojunction magnetic beads; the bismuth-metal bismuth salt@Fe3O4 heterojunction magnetic beads are Bi-BFO@Fe3O4 magnetic beads; The antimicrobial peptide is vancomycin; the particle size of the photocatalytic Fe3O4 magnetic beads is 60-100 nm; The bacterial immunomagnetic beads are prepared by the following method: (1) Dispersing bismuth salt and Fe3O4 magnetic beads in ethylene glycol, stirring at room temperature, turning on the laser to bombard Fe3O4, centrifuging, washing and drying after the bombardment, and obtaining Bi-BFO@Fe3O4 magnetic beads; (2) Carboxylating the Bi-BFO@Fe3O4 magnetic beads to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads; mixing the antimicrobial peptide with polyvinyl pyrrolidone, and then adding a coupling agent to couple the antimicrobial peptide with the carboxylated Bi-BFO@Fe3O4 magnetic beads to obtain bacterial immunomagnetic beads.

2. The method for preparing the immunomagnetic beads for bacteria according to claim 1, characterized in that: The following steps are involved: (1) Dispersing bismuth salt and Fe3O4 magnetic beads in ethylene glycol, stirring at room temperature, turning on the laser to bombard Fe3O4, centrifuging, washing and drying after the bombardment, and obtaining Bi-BFO@Fe3O4 magnetic beads; (2) Carboxylating the Bi-BFO@Fe3O4 magnetic beads to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads; mixing the antimicrobial peptide with polyvinyl pyrrolidone, and then adding a coupling agent to couple the antimicrobial peptide with the carboxylated Bi-BFO@Fe3O4 magnetic beads to obtain bacterial immunomagnetic beads.

3. The preparation method according to claim 2, characterized in that: In step (1), the bismuth salt is bismuth nitrate; the ratio of the added amounts of bismuth nitrate, ethylene glycol and Fe3O4 magnetic beads is 1.2~1.7 g: 20~40 mL: 1.0~1.5 g.

4. The preparation method according to claim 2, characterized in that: In step (1), the stirring time is 80-120 min; the parameters of the laser bombardment are: wavelength 1064 nm, energy 10-20 W, scanning speed 10-500 mm s –1 .

5. The preparation method according to claim 2, characterized in that: In step (2), the carboxylation treatment is: Bi-BFO@Fe3O4 magnetic beads, 2,3-dimercaptosuccinic acid and Na2CO3 were dispersed in a mixed solution of tetrahydrofuran and water, and after ultrasonic treatment, centrifugation, washing and freeze-drying were performed in sequence to obtain carboxylated Bi-BFO@Fe3O4 magnetic beads.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the Bi-BFO@Fe3O4 magnetic beads, 2,3-dimercaptosuccinic acid and Na2CO3 is 1.0-1.5:1.5-2.0:0.75-1.5; and the ultrasonic treatment time is 30-50 min.

7. The preparation method according to claim 2, characterized in that: In step (2), the coupling agents are N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; and the antimicrobial peptide is vancomycin.

8. The preparation method according to claim 7, characterized in that: The mass ratio of the carboxylated Bi-BFO@Fe3O4 magnetic beads, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, polyvinyl pyrrolidone and vancomycin is 1.0-1.5: 0.2-0.4: 0.5-1.0: 0.1-0.3: 0.3-0.

5.

9. Use of the bacterial immunomagnetic beads according to claim 1 in capturing, separating, enriching and photocatalytically killing bacteria.

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