A method for specifically targeting and destroying a kind of miscellaneous bacteria by surface functionalized magnetic nanoparticles

Abstract

The application discloses a method for specifically targeting and destroying miscellaneous bacteria by surface functionalized magnetic nanoparticles, and belongs to the technical field of microbial reactors. The method comprises the following steps: sequentially performing stabilization treatment and surface functionalization treatment on a magnetic base material or directly performing functionalization treatment, and placing the obtained surface functionalized magnetic nanoparticles in a biological reactor containing various bacteria to perform incubation. After the functionalized magnetic nanoparticles are specifically targeted and combined with the bacteria, the bacteria are caused to selectively die by adjusting a low-frequency magnetic induction intensity and a frequency. The low-frequency magnetic induction intensity is 40-75 mT, and the frequency is 10-50 Hz. The method has little influence on the life cycle activities of uncombined living bacteria, and under the induction of a low-frequency magnetic field, the magnetic mechanical force of the nanoparticles is used to destroy the structure of dead bacteria and release the contents.

Description

Technical Field

[0001] This invention belongs to the field of microbial reactor technology, and particularly relates to a method for the specific targeting / destruction of a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles. Background Technology

[0002] Based on different types of bioreactors, many stages of industrial biosynthesis / conversion are semi-open processes. Microbial contamination has become a common and critical scientific problem that needs to be rigorously addressed. The main causes of contamination include human factors such as poor management (e.g., incomplete sterilization, which is generally not continuous and leads to the failure of individual / batch reactors), seed-borne microorganisms (which will cause the contamination area to expand continuously, resulting in significant production losses), unreasonable equipment structure, improper process pipeline installation (requiring replacement / modification of process equipment, introducing cost issues), equipment leaks (high probability of contamination), and dead zones or airborne contamination (causing large-scale contamination of the reactor). Currently, in industrial fermentation, contamination problems caused by poor management are particularly prominent. Therefore, once a reactor is contaminated (or contains unfavorable strains), the introduction of byproducts leads to product impurities, reduced yields, or even reaction termination, resulting in huge economic losses.

[0003] In recent years, with the rapid development of nanoscience and technology, magnetic nanoparticles, due to their unique magnetic properties and good biocompatibility, have been widely used in biomedicine and environmental fields. Based on their magnetic responsiveness, the incorporation of specific molecules onto their surfaces will broaden their research scope and is highly favored by researchers. Magnetic nanoparticles are prone to aggregation due to their nanoscale characteristics (small size effect, surface effect), requiring surface stabilization treatment. However, there is a certain "trade-off" between stability and magnetic properties, leading to a decrease in magnetic performance. Further modification can enhance their nucleophilicity to bacterial nucleolar regions and reduce biotoxicity. Therefore, by fully balancing the relationship between the stabilizing layer, the functional layer, and the magnetic properties of the magnetic nanoparticles, and through synergy with low-frequency magnetic fields, it is expected to solve the problem of low efficiency in traditional bioreactors, which is of great significance for achieving zero-carbon sustainable development. Summary of the Invention

[0004] This invention addresses the problem of contamination by other microorganisms in existing bioreactors by providing a method for specifically targeting and destroying a class of microorganisms using surface-functionalized magnetic nanoparticles. This method has minimal impact on the life cycle activities of live bacteria. Under the induction of a low-frequency magnetic field, the magnetic mechanical force of the nanoparticles is used to destroy the structure of dead bacteria and release their internal solutions.

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

[0006] A method for specifically targeting / destroying a type of bacteria using surface-functionalized magnetic nanoparticles includes the following steps: sequentially stabilizing and surface-functionalizing a magnetic matrix material, or directly functionalizing it (the functional layer has a stabilizing effect); placing the obtained surface-functionalized magnetic nanoparticles in a bioreactor containing various bacteria for incubation; after the functionalized magnetic nanoparticles specifically target and bind to the bacteria, adjusting the intensity and frequency of a low-frequency magnetic field causes the bacteria to die under mechanical force; the low-frequency magnetic field intensity is 40-75 mT, and the frequency is 10-50 Hz. The type of low-frequency magnetic field is rotational or dynamic, etc.

[0007] Furthermore, the magnetization of the magnetic matrix material is >50 emu. -1 The magnetic mechanical force is >30 pN. The synthesis methods for this magnetic volumetric material include co-precipitation, high-temperature decomposition, hydrothermal synthesis, sol-gel synthesis, microemulsion synthesis, and ultrasonic chemical synthesis.

[0008] Furthermore, the stabilization treatment refers to forming a stabilizing layer on the surface of the magnetic substrate material; the stabilizing layer includes a polymer coating, a surfactant stabilizing layer, a noble metal coating, or a metal / non-metal oxide coating.

[0009] Furthermore, the polymer coating is polyethylene glycol, polydopamine, propyl 3-(trimethoxysilyl)methacrylate, or ammoniated starch; the surfactant stabilizing layer is oleic acid, stearic acid, lauric acid, or dodecylphosphonic acid; the noble metal coating is gold; and the metal / non-metal oxide coating is SiO2 or Al2O3.

[0010] Furthermore, the surface functionalization treatment refers to placing a magnetic substrate with a stable layer in specific molecules for reaction to generate magnetically responsive functionalized nanoparticles with a size of 160-480 nm.

[0011] Furthermore, the specific molecules include biomolecules, aptamers / oligonucleotides, sugars / peptides, and small molecule / polymer ligands.

[0012] Furthermore, the biomolecules are antibodies, enzymes, or amino acids; the aptamers / oligonucleotides are DNA / RNA type aptamers; the sugars / peptides are glucosamine or antimicrobial peptides; and the small molecule / polymer ligands are folic acid, gentamicin, zinc(II)-dimethylpyridinium chloride copolymer, or 3-aminopropyltriethoxysilane.

[0013] Further, the reactions include direct crosslinking, click chemistry, linking chemistry, physical interaction, or hybridization methods. Direct crosslinking is based on the reaction of functional groups such as -NH2, R-NH-R, and HC=O on the surface of magnetic nanoparticles with succinimide esters, isothiocyanates, and -SH. Click chemistry is based on the reaction of terminal alkyne groups, cyclic alkyne groups, and non-alcohol carbonyl groups with azidobutyrate NHS esters and azides. Linking chemistry is based on the reaction of -COOH and -NH2 on the surface of magnetic nanoparticles before intermediate molecules containing N-hydroxysuccinimide groups, followed by the reaction of maleimide groups and heterocyclic compounds in the intermediate molecules with -SH and -NH2. Physical interaction is based on the electrostatic interactions, hydrophobic interactions, host-guest interactions, and non-covalent interactions generated by the surface charge properties, hydrophilicity / hydrophobicity, shape and structure, physicochemical properties of magnetic nanoparticles, and specific molecules. Hybridization methods are based on the hybridization reaction of oligonucleotides and aptamers on the surface of magnetic nanoparticles.

[0014] Furthermore, the incubation time is 12-24 hours to ensure that the surface ligands of the surface-functionalized magnetic nanoparticles fully bind to the receptors on the surface of bacterial cells.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] (1) This invention develops and designs the specificity of the surface of magnetic nanoparticles through different methods and different types of specific molecules, so that they have specific targeting for different types of bacteria. While expanding the universality of application, it also has super selectivity. Compared with the traditional method of dealing with bacteria, this method has higher efficiency in removing bacteria and is more operable.

[0017] (2) The magnetic nanoparticles stabilized by the present invention have significantly improved dispersion and stability, enhanced reaction sites on the surface of nanoparticles, and the surface functionalized magnetic nanoparticles can achieve a high recovery rate (~90%) based on their magnetic responsiveness, thus having certain environmental and economic benefits.

[0018] (3) This invention introduces a low-frequency magnetic field composed of neodymium iron boron (NdFeB) strong permanent magnets, causing functionalized magnetic nanoparticles to move in the culture medium according to a specific trajectory and amplitude. By changing the number and placement of the permanent magnets, as well as the rotation frequency of the device, the movement of the surface-functionalized magnetic nanoparticles can be remotely controlled. Under certain magnetic generation conditions (10-50Hz, 40-75mT), they can destroy harmful bacteria. Research on the remote induction and control of surface-functionalized magnetic nanoparticles to specifically destroy dead bacterial communities based on low-frequency magnetic fields contributes to the construction of a novel, efficient, low-cost, and sustainable bioreactor operation process. Surface-functionalized magnetic nanoparticles have magnetic recyclability, easy remote induction, and biocompatibility, which greatly promotes the self-sustainability of bioreactors and has promising prospects for industrial application. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0020] Figure 1 This is a flowchart of the technology of the present invention;

[0021] Figure 2 This is a technical concept diagram of the present invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, "room temperature" in this invention refers to 25±2℃.

[0028] This invention synthesizes different magnetic matrix materials using various methods, employing two processing modes for different magnetic matrices: 1) After surface stabilization, specific molecules react with the magnetic matrix stabilization layer to form magnetically responsive functionalized nanoparticles of a certain size; 2) Directly reacting with specific molecules to bind to the magnetic matrix surface, thus forming surface-functionalized magnetic nanoparticles that integrate surface stabilization and functionalization. These surface-functionalized magnetic nanoparticles are then thoroughly mixed in a bioreactor containing various bacteria. The specific molecules on the surface of the functionalized magnetic nanoparticles can specifically bind to the surface of one type of bacteria without affecting the metabolic activities of other bacteria. After co-incubation for a period of time, a low-frequency magnetic field with a certain magnetic field strength and frequency is used to remotely control the rotation or linear movement of the functionalized magnetic nanoparticles. The bacteria, under the influence of external mechanical force, undergo a series of enzyme cascade reactions, inducing programmed cell death or directly destroying the cell membrane / cell wall, leading to bacterial death. This technology can effectively eliminate problems such as bioreactor failure due to contamination by other microorganisms, and is beneficial for removing unfavorable bacterial species, improving the stability of the reaction system, and ensuring the purity and singleness of the product.

[0029] The specific technical solution is as follows: A method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles includes the following steps: sequentially stabilizing and surface-functionalizing a magnetic matrix material; incubating the resulting surface-functionalized magnetic nanoparticles in a bioreactor containing multiple bacteria, or directly performing functionalization treatment (the functional layer has a stabilizing effect); and selectively killing bacteria by adjusting the low-frequency magnetic induction intensity and frequency; wherein the low-frequency magnetic induction intensity is 40-75 mT and the frequency is 10-50 Hz. The low-frequency magnetic field type is rotational or dynamic, etc.

[0030] In some preferred embodiments, the magnetization of the magnetic matrix material is >50 emu. -1 The magnetomechanical force generated in the magnetic field is >30pN. The synthesis methods for this magnetic matrix material include co-precipitation, high-temperature decomposition, hydrothermal synthesis, sol-gel synthesis, microemulsion synthesis, and ultrasonic chemical synthesis.

[0031] In some preferred embodiments, the magnetic matrix material can be prepared by dissolving 0.1-0.2M anhydrous FeCl3 and 40-80mM trisodium citrate dihydrate in 20-30mL of diethylene glycol, and then vigorously stirring the resulting mixture in a water bath at 50-80℃ until fully dissolved. Then, 0.3-0.6M NaAc is added and stirred until homogeneous, and then transferred to a stainless steel autoclave lined with polytetrafluoroethylene. After treatment at 180-200℃ for 5-10h, a Fe3O4 magnetic matrix with a particle size of 2-10nm is obtained. Alternatively, the magnetic matrix material can be prepared by dissolving 1.45g FeCl3·6H2O and 50mg polyvinylpyrrolidone (PVP) in 60mL of 70% (v / v) ethanol solution in a round-bottom three-necked flask, and then ultrasonically dispersing and mixing for 10min. Under nitrogen protection and vigorous mechanical stirring, 200 μL of tetraethyl silicate was first added, followed by 20 mL of 1 mol / L sodium borohydride solution at a rate of 5 mL / min. The reaction system temperature was maintained at 15℃ for 2 h. The black magnetic precipitate was then collected, washed several times with ethanol and distilled water, and vacuum dried at 50-60℃ for 12-24 h. Alternatively, the magnetic matrix material can be prepared as follows: 2-6 g of citric acid and 0.12-0.36 g of urea were used as a carbon source, added to 50-80 mL of deionized water, and dissolved by ultrasonication. Then, 50-100 μL each of 0.1-0.2 mol / L FeCl2 and FeCl3 were added as magnetic sources. After thorough mixing in an anaerobic environment, a precursor solution was formed. The precursor solution was placed in the center of a microwave oven and reacted at 700W high power for 4-5 min to obtain crude magnetic nanoparticles. The obtained brownish-black substance was dissolved in 40-50 mL of deionized water, then centrifuged for 10-15 min (10000 r / min). The supernatant was filtered through a 0.22 μm or 0.45 μm filter membrane, and then dialyzed for 24 h using a dialysis bag with a cutoff of 500 D or 1000 D (water changed every 3 h or 6 h) to obtain a pure functionalized magnetic nanoparticle solution. The purified solution was frozen in liquid nitrogen and freeze-dried in a vacuum dryer at a vacuum degree of 0.055-0.060 mBar, a temperature of -70 to -80 °C, and a compressor temperature of 15-17 °C for 12-24 h to obtain the magnetic matrix. The magnetic matrix materials prepared by the above method in the following examples all meet the requirement of magnetization > 50 emu. -1 , Magnetomechanical force > 30pN.

[0032] In some preferred embodiments, the stabilization treatment of the magnetic substrate surface can be a polymer coating stabilizing layer, specifically polyethylene glycol, polydopamine, propyl 3-(trimethoxysilyl)methacrylate, ammoniated starch, etc. The stabilization treatment of the magnetic substrate surface can also be a surfactant stabilizing layer, specifically oleic acid, stearic acid, lauric acid, dodecylphosphonic acid, etc. The stabilization treatment of the magnetic substrate surface can also be a noble metal coating, specifically gold, etc. The stabilization treatment of the magnetic substrate surface can also be a metal / non-metal oxide coating, specifically SiO2, Al2O3, etc. In some preferred embodiments, the method for stabilizing the magnetic substrate material can be: adding 0.1-0.2g of magnetic nanoparticle matrix solution to 50-100mL of tetramethoxysilane / ethanol solution (volume ratio 30%), and stirring for 12-24h.

[0033] In some preferred embodiments, the surface functionalization treatment refers to reacting a magnetic substrate with a stable layer in specific molecules to generate magnetically responsive functionalized nanoparticles with a size of 160-480 nm. The specific molecules include biomolecules, aptamers / oligonucleotides, sugars / peptides, and small molecule / polymer ligands. The biomolecules are antibodies, enzymes, or amino acids; the aptamers / oligonucleotides are DNA / RNA type aptamers; the sugars / peptides are glucosamides or antimicrobial peptides; and the small molecule / polymer ligands are folic acid, gentamicin, zinc(II)-dimethylpyridinium chloride copolymers, or 3-aminopropyltriethoxysilane. The reaction includes direct crosslinking, click chemistry, linker chemistry, physical action, or hybridization methods. The direct crosslinking is based on the reaction of functional groups such as -NH2, R-NH-R, and HC=O on the surface of the magnetic nanoparticles with succinimide esters, isothiocyanates, and -SH. The click chemistry is based on the reaction of terminal alkyne groups, cyclic alkyne groups, and non-alcoholic carbonyl groups with NHS azidobutyrate esters and azides. The linking chemistry is based on the reaction of the -COOH and -NH2 groups on the surface of magnetic nanoparticles before the reaction of intermediate molecules containing N-hydroxysuccinimide groups, followed by the reaction of the maleimide groups and heterocyclic compounds in the intermediate molecules with -SH, -NH2, etc. The physical interactions are based on the surface charge properties, hydrophilicity / hydrophobicity, shape and structure, physicochemical properties of the magnetic nanoparticles, and the electrostatic interactions, hydrophobic interactions, host-guest interactions, and non-covalent interactions generated by specific molecules. The hybridization method is based on the hybridization reaction of oligonucleotides and aptamers on the surface of magnetic nanoparticles.

[0034] In some preferred embodiments, the preparation method for functionalizing the magnetic matrix material can be as follows: 0.1-0.2 g of Fe3O4 magnetic matrix is ​​dispersed in 100 mL of arginine aqueous solution (Fe3O4 to arginine mass ratio is 1:1), and ultrasonically reacted for 20-30 min. After ultrasonic treatment, the black precipitate obtained is collected magnetically, washed 3-5 times with deionized water, and dried under vacuum at 50-60℃ for 12-24 h. Alternatively, 0.01-0.02 g of N-(3-triethoxysilylpropyl)glucamide can be added to the stabilized magnetic nanoparticle solution, stirred for 12-24 h, washed 2-3 times alternately with ethanol and deionized water, and vacuum dried overnight at 50-60℃. Another method is to dissolve 0.03-0.06 g of magnetic matrix, 0.05-0.1 g of glycine, and 0.05-0.1 g of L-tryptophan in 40-50 mL of ultrapure water. The mixture is sonicated for 10-15 minutes, reacted in a reactor at 200℃ for 6-8 hours, then centrifuged at 10000-12000 rpm for 10-15 minutes, repeated 3-4 times, and dried in a vacuum environment at 50-60℃ for 12-24 hours.

[0035] In some preferred embodiments, the process of functionalized magnetic nanoparticles targeting / destroying dead bacteria includes the following steps: First, the operating environment area of ​​the magnetic field generator is sterilized with ultraviolet light. After sterilization, a culture dish containing 10-20 mL of bacterial culture medium solution is placed in the center of the reactor, and functionalized magnetic nanoparticles are added. After thorough mixing and co-incubation for a period of time, a low-frequency magnetic field is introduced into the system. The dead bacterial structure is destroyed by the rapid magnetophoresis or magnetomechanical force generated by the magnetic nanoparticles. The incubation time is 12-24 hours to ensure that the surface ligands of the functionalized magnetic nanoparticles fully bind to the receptors on the bacterial cell surface. The low-frequency magnetic field has a frequency of 10-50 Hz and a magnetic induction intensity of 40-75 mT. The low-frequency magnetic field type can be rotating or dynamic. In the embodiments of this invention, the low-frequency magnetic field is composed of neodymium iron boron strong permanent magnets.

[0036] In some preferred embodiments, the process of functionalized magnetic nanoparticles targeting / destroying dead bacteria includes the following steps: The operating environment area of ​​the magnetic field generator is sterilized with ultraviolet light. After sterilization, 1-5 mg of the prepared functionalized magnetic nanoparticles are added to a petri dish containing 10-20 mL of Bacillus subtilis / S. aromatic amino acid culture medium solution (the concentration ratio of Bacillus subtilis to S. aromatic amino acid is 1:1). After uniform mixing for 12-24 h, the system is placed in a low-frequency magnetic field. The magnetic field frequency is set to 10-50 Hz and the magnetic induction intensity to 40-75 mT by adjusting the number of magnets and instrument parameters. After treatment for 5-8 h, the bacterial structure is destroyed based on the magnetomechanical force generated by carbon dots.

[0037] All raw materials used in the following embodiments of the present invention are commercially available.

[0038] In this invention, Bacillus subtilis (G+) and aromatic amino acid strains (G-) were used to simulate the microbial community structure in a microbial reactor, and all reagents used were of analytical grade.

[0039] The following embodiments are further illustrations of the technical solution of the present invention.

[0040] Example 1

[0041] (1) Synthesis steps of magnetic nanoparticle matrix:

[0042] 0.1 M anhydrous FeCl3 and 40 mM trisodium citrate dihydrate were dissolved in 30 mL of diethylene glycol. The resulting mixture was vigorously stirred in a 50 °C water bath until fully dissolved. Then, 0.3 M NaAc was added and stirred until homogeneous. The mixture was then transferred to a stainless steel autoclave lined with polytetrafluoroethylene. After treatment at 200 °C for 10 h, a Fe3O4 magnetic matrix with a particle size of approximately 8.8 nm was obtained.

[0043] (2) Surface stabilization treatment

[0044] 0.025 g of dopamine and 0.25 g of Fe3O4 magnetic matrix were dispersed in 50 mL of 0.05 mol / L Tris alkaline buffer solution with pH = 8.50. After stirring for 3 h, the matrix was washed three times each with deionized water and ethanol, and dried under vacuum at 60 °C for 12 h to obtain dopamine-stabilized Fe3O4 magnetic matrix.

[0045] (3) Functionalization steps of surface-functionalized magnetic nanoparticles:

[0046] 0.1 g of dopamine-stabilized Fe3O4 magnetic matrix was dispersed in 100 mL of 1 g / L arginine solution and sonicated for 30 min. After sonication, the resulting black precipitate was collected magnetically, washed five times with deionized water, and dried under vacuum at 60 °C for 12 h.

[0047] (4) The targeting / destructive efficacy of surface-functionalized magnetic nanoparticles against Bacillus subtilis and aromatic amino acid-containing Solomon's seal:

[0048] The operating environment of the magnetic field generator was sterilized with ultraviolet light. After sterilization, 5 mg of the above-mentioned functionalized magnetic nanoparticles were added to a petri dish containing 20 mL of Bacillus subtilis / S. aromatic amino acid culture medium solution (the concentration ratio of Bacillus subtilis to S. aromatic amino acid was 1:1). After uniform mixing for 24 h, the system was placed in a low-frequency magnetic field. The magnetic field frequency was set to 50 Hz and the magnetic induction intensity to 45 mT by adjusting the number of magnets and instrument parameters. After 8 h of treatment, the bacterial structure was destroyed by the magnetomechanical force generated by the magnetic nanoparticles.

[0049] (5) Performance evaluation of surface-functionalized magnetic nanoparticles targeting / destroying Bacillus subtilis and aromatic amino acid strains:

[0050] 1) After the low-frequency magnetic field treatment, the bacterial dead / live ratio was analyzed by bacterial staining and flow cytometry. The results showed that the number of intact cells decreased from 100% to 0%, indicating that the surface-functionalized magnetic nanoparticles have specific targeting properties for Bacillus subtilis and Solomon's seal.

[0051] 2) Using a 5mL sterile syringe, 4mL of solution was drawn and immediately passed through a 0.22μm aqueous filter membrane. The polysaccharide, protein, and COD levels were measured using a UV-Vis spectrophotometer to evaluate the bacterial lysis effect. The results showed that polysaccharide, protein, and COD levels were increased by 47%, 53%, and 51%, respectively, compared to the control group (i.e., without the addition of magnetic nanoparticles), indicating that the bacteria in the culture medium were effectively destroyed, releasing intracellular lysates.

[0052] Example 2

[0053] (1) Synthesis steps of magnetic nanoparticle matrix:

[0054] In a round-bottom three-necked flask, 1.45 g of FeCl3·6H2O and 50 mg of polyvinylpyrrolidone were dissolved in 60 mL of 70% (v / v) ethanol solution and ultrasonically dispersed for 10 min. Under nitrogen protection and vigorous mechanical stirring, 20 mL of 1 mol / L sodium borohydride solution was added dropwise at a rate of 5 mL / min, and the reaction system temperature was maintained at 15 °C for 2 h. The black magnetic precipitate was then collected, washed several times with ethanol and distilled water, and dried under vacuum at 60 °C for 12 h.

[0055] (2) Synthesis steps of stabilized magnetic nanoparticles:

[0056] Add 0.1 g of magnetic nanoparticle matrix solution to 50 mL of tetramethoxysilane / ethanol solution (volume ratio 30%) and stir for 12 h.

[0057] (3) Synthesis steps of functionalized magnetic nanoparticles:

[0058] Add 0.01 g N-(3-triethoxysilylpropyl)glucamide to the above reaction solution, stir for 12 h, wash three times each with alternating ethanol and deionized water, and dry under vacuum at 60 °C for 12 h.

[0059] (4) Surface-functionalized magnetic nanoparticles target / destroy aromatic amino acid bacteria without targeting / destroying Bacillus subtilis efficacy:

[0060] The operating environment of the magnetic field generator was sterilized with ultraviolet light. After sterilization, 5 mg of the above-mentioned functionalized magnetic nanoparticles were added to a petri dish containing 20 mL of Bacillus subtilis / S. aromatic amino acid culture medium solution (the concentration ratio of Bacillus subtilis to S. aromatic amino acid was 1:1). After uniform mixing for 24 h, the system was placed in a low-frequency magnetic field. The magnetic field frequency was set to 50 Hz and the magnetic induction intensity to 45 mT by adjusting the number of magnets and instrument parameters. After 8 h of treatment, the bacterial structure was destroyed by the magnetomechanical force generated by the magnetic nanoparticles.

[0061] (5) Performance evaluation of surface-functionalized magnetic nanoparticles targeting / destroying aromatic amino acid bacteria without targeting / destroying Bacillus subtilis:

[0062] 1) After the low-frequency magnetic field treatment, the bacterial dead / live ratio was analyzed by bacterial staining and flow cytometry. The results showed that the number of intact cells decreased from 100% to 47%, and all of them were Bacillus subtilis (showing blue-purple fluorescence), indicating that the surface-functionalized magnetic nanoparticles have specific targeting to aromatic amino acid strains.

[0063] 2) Using a 5mL sterile syringe, 4mL of solution was drawn and immediately passed through a 0.22μm aqueous filter membrane. The polysaccharide, protein, and COD indices were measured using a UV-Vis spectrophotometer to evaluate the bacterial cytolysis effect. The results showed that the polysaccharide, protein, and COD were increased by 18%, 30%, and 27%, respectively, compared with the blank group (i.e., without the addition of magnetic nanoparticles), indicating that the aromatic amino acid strain of Solomon's seal was effectively destroyed, releasing the intracellular lysate of the bacteria.

[0064] Example 3

[0065] (1) Steps for synthesizing magnetic nanoparticles:

[0066] A carbon source consisting of 6g citric acid and 0.36g urea was added to 50mL of deionized water and sonicated to dissolve. Then, 100μL each of 0.2mol / L FeCl2 and FeCl3 were added as magnetic sources. The mixture was thoroughly stirred in an anaerobic environment to form a precursor solution. This precursor solution was placed in the center of a microwave oven and reacted at 700W for 4 minutes to obtain crude magnetic nanoparticles. The resulting brownish-black substance was dissolved in 40mL of deionized water and centrifuged for 15 minutes (10000r / min). The supernatant was filtered through a 0.22μm filter membrane and then dialyzed for 24 hours using a dialysis bag with a 1000D cutoff (water changed every 3 hours) to obtain a purified functionalized magnetic nanoparticle solution. The purified solution was frozen in liquid nitrogen and freeze-dried for 24 hours in a vacuum dryer at a vacuum level of 0.055mBar, a temperature of -70℃, and a compressor temperature of -17℃ to obtain the magnetic matrix.

[0067] (2) Synthesis steps of surface-functionalized magnetic nanoparticles:

[0068] 0.03 g of magnetic matrix, 0.05 g of glycine, and 0.05 g of L-tryptophan were dissolved in 50 mL of ultrapure water. The mixture was sonicated for 10 min, reacted in a reactor at 200 °C for 6 h, centrifuged at 10,000 rpm for 10 min, and repeated 3 times. The mixture was then dried in a vacuum environment at 60 °C for 12 h.

[0069] (3) Surface-functionalized magnetic nanoparticles target / destroy Bacillus subtilis and do not target / destroy the efficacy of aromatic amino acid-containing Solomon's bacterium:

[0070] The operating environment of the magnetic field generator was sterilized with ultraviolet light. After sterilization, 5 mg of the above-mentioned functionalized magnetic nanoparticles were added to a petri dish containing 20 mL of Bacillus subtilis / S. aromatic amino acid culture medium solution (the concentration ratio of Bacillus subtilis to S. aromatic amino acid was 1:1). After uniform mixing for 24 h, the system was placed in a low-frequency magnetic field. The magnetic field frequency was set to 50 Hz and the magnetic induction intensity to 45 mT by adjusting the number of magnets and instrument parameters. After 8 h of treatment, the bacterial structure was destroyed by the magnetomechanical force generated by the magnetic nanoparticles.

[0071] (4) Performance evaluation of surface-functionalized magnetic nanoparticles targeting / destroying Bacillus subtilis without targeting / destroying aromatic amino acid-producing Solomon's seal:

[0072] 1) After the low-frequency magnetic field treatment, the bacterial dead / live ratio was analyzed by bacterial staining and flow cytometry. The results showed that the number of intact cells decreased from 100% to 48-49%, and all of them were aromatic amino acid strains (showing red fluorescence), indicating that the surface-functionalized magnetic nanoparticles have specific targeting properties for Bacillus subtilis.

[0073] 2) Using a 5mL sterile syringe, 4mL of solution was drawn and immediately passed through a 0.22μm aqueous filter membrane. The polysaccharide, protein, and COD indices were measured using a UV-Vis spectrophotometer to evaluate the bacterial cytolysis effect. The results showed that the polysaccharide, protein, and COD were increased by 27%, 33%, and 21%, respectively, compared with the blank group (i.e., without the addition of magnetic nanoparticles), indicating that Bacillus subtilis was effectively destroyed and intracellular lysates were released.

[0074] Comparative Example 1

[0075] Same as Example 1, except that the magnetic field frequency is set to 10Hz and the magnetic induction intensity is 30mT.

[0076] Using this comparative method to treat and destroy the bacterial efficacy of Bacillus subtilis and Solomon's seal, the results showed that the number of intact cells decreased from 100% to 82%, and the polysaccharide, protein and COD decreased by 87%, 79% and 85% respectively compared with the control group (i.e., magnetic field frequency of 50 Hz and magnetic induction intensity of 45 mT), indicating that the lower magnetic field frequency and magnetic induction intensity failed to effectively destroy the bacteria.

[0077] Comparative Example 2

[0078] Same as Example 2, except that step 2 is not performed.

[0079] Using this comparative method to treat and destroy the bacterial efficacy of Bacillus subtilis and Arnechoys aromatic amino acid strains, the results showed that the magnetic nanoparticles exhibited corona discharge (i.e., adsorption of proteins in the culture medium) and aggregation, indicating decreased stability. The number of intact cells decreased from 100% to 62%. Compared with the control group (i.e., stabilized magnetic nanoparticles), polysaccharides, proteins, and COD decreased by 57%, 49%, and 35%, respectively, indicating that the unstabilized magnetic nanoparticles reduced the targeting / destructive performance against Arnechoys aromatic amino acid strains.

[0080] Comparative Example 3

[0081] Same as Example 2, except that step 3 is omitted.

[0082] Using this comparative method to treat and destroy the bacterial efficacy of Bacillus subtilis and Arnechoys aromatica, the results showed that the number of intact cells decreased from 100% to 98%. Compared with the control group (i.e., functionalized magnetic nanoparticles), polysaccharides, proteins and COD decreased by 98%, 99% and 98%, respectively. This indicates that the unfunctionalized magnetic nanoparticles do not have specific targeting to Arnechoys aromatica and show good biocompatibility.

[0083] Comparative Example 4

[0084] Same as Example 1, except that in step 1), the synthesized magnetic matrix is ​​a core-shell magnetic MnFe2O4.

[0085] The comparative method was used to investigate the efficacy of MnFe2O4 in destroying Bacillus subtilis and Argus roxburghii (a type of bacteria with aromatic amino acids). The results showed that MnFe2O4 reduced the destruction time of Bacillus subtilis and Argus roxburghii by 48% compared to the control group (i.e., surface-functionalized nanoparticles with Fe3O4 as the magnetic matrix). This was attributed to the superior magnetic properties of MnFe2O4 compared to Fe3O4, but Mn... 2+ Its biotoxicity to live bacteria is greater than that to Fe. 2+ Therefore, sufficient stabilization treatment is required.

[0086] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles, characterized in that, Includes the following steps: Surface functionalization treatment was performed on the magnetic matrix material, and the resulting surface functionalized magnetic nanoparticles were placed in a bioreactor containing various bacteria for incubation. After the functionalized magnetic nanoparticles specifically targeted and bound to the bacteria, the bacteria were killed under mechanical force by adjusting the low-frequency magnetic induction intensity and frequency. The low-frequency magnetic induction intensity is 40-75mT, and the frequency is 10-50Hz; The magnetization of the magnetic matrix material is >50 emu. -1 Magnetomechanical force > 30 pN; The surface functionalization treatment refers to placing a magnetic substrate with a stable layer into specific molecules for reaction, generating magnetically responsive functionalized nanoparticles with a size of 160-480 nm. The magnetic matrix material can be prepared by: Dissolve 0.1-0.2M anhydrous FeCl3 and 40-80mM trisodium citrate dihydrate in 20-30mL of diethylene glycol. Vigorously stir the resulting mixture in a water bath at 50-80℃ until fully dissolved. Then add 0.3-0.6M NaAc and stir until homogeneous. Transfer the mixture to a stainless steel autoclave lined with polytetrafluoroethylene. After treatment at 180-200℃ for 5-10h, obtain a Fe3O4 magnetic matrix with a particle size of 2-10nm. Alternatively, in a round-bottom three-necked flask, 1.45 g FeCl3·6H2O and 50 mg polyvinylpyrrolidone are dissolved in 60 mL of 70% (v / v) ethanol solution and ultrasonically dispersed for 10 min. Under nitrogen protection and mechanical stirring, 200 μL of tetraethyl silicate is added first, followed by 20 mL of 1 mol / L sodium borohydride solution at a rate of 5 mL / min. The reaction system temperature is maintained at 15 °C for 2 h. The black magnetic precipitate is then collected, washed with ethanol and distilled water, and vacuum dried at 50-60 °C for 12-24 h. Alternatively, use 2-6g citric acid and 0.12-0.36g urea as a carbon source, add it to 50-80mL of deionized water, sonicate to dissolve, and then add 50-100μL of 0.1-0.2mol / L urea. FeCl2 and FeCl3 were used as magnetic sources and thoroughly mixed in an anaerobic environment to form a precursor solution. The precursor solution was placed in the center of a microwave oven and reacted at 700W high power for 4-5 minutes to obtain crude magnetic nanoparticles. The resulting brownish-black substance was dissolved in 40-50 mL of deionized water and then centrifuged at 10000 r / min for 10-15 minutes. The supernatant was filtered through a 0.22 μm or 0.45 μm filter membrane and then dialyzed for 24 hours using a dialysis bag with a cutoff of 500D or 1000D, with the water changed every 3 hours or 6 hours, to obtain a pure functionalized magnetic nanoparticle solution. The purified solution was frozen in liquid nitrogen and freeze-dried in a vacuum dryer under vacuum conditions of 0.055-0.060 mBar, temperature of -70 to -80℃, and compressor temperature of 15-17℃ for 12-24 hours to obtain the magnetic matrix.

2. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 1, characterized in that, It also includes stabilization treatment of the magnetic matrix material.

3. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 1 or 2, characterized in that, The stabilization treatment refers to forming a stabilizing layer on the surface of the magnetic substrate material; the stabilizing layer includes a polymer coating, a surfactant stabilizing layer, a noble metal coating, a metal oxide coating, or a non-metal oxide coating.

4. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 3, characterized in that, The polymer coating is polyethylene glycol, polydopamine, propyl 3-(trimethoxysilyl)methacrylate, or ammoniated starch; the surfactant stabilizing layer is oleic acid, stearic acid, lauric acid, or dodecylphosphonic acid; the noble metal coating is gold; and the metal / non-metal oxide coating is SiO2 or Al2O3.

5. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 1, characterized in that, The specific molecules include biomolecules, aptamers / oligonucleotides, sugars / peptides, and small molecule / polymer ligands.

6. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 5, characterized in that, The biomolecules are antibodies or enzymes; the aptamers / oligonucleotides are DNA / RNA type aptamers; the sugars / peptides are glucosamides or antimicrobial peptides; and the small molecule / polymer ligands are folic acid, gentamicin, zinc(II)-dimethylpyridinium chloride copolymers, or 3-aminopropyltriethoxysilane.

7. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 1, characterized in that, The surface functionalization treatment refers to the reaction in which a magnetic matrix with a stable layer is placed in a specific molecule for reaction, including direct crosslinking, click chemistry, link chemistry, physical action, or hybridization methods.

8. The method for specifically targeting / destroying a class of miscellaneous bacteria using surface-functionalized magnetic nanoparticles according to claim 1 or 2, characterized in that, The incubation time is 12-24 hours.

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