Boric acid functionalized magnetic bead for enrichment and detection of viable bacteria and preparation method and application of boric acid functionalized magnetic bead

Abstract

The invention discloses a boric acid functionalized magnetic bead for enrichment and detection of viable bacteria, which is prepared by the following steps: preparing an iron-based nano magnetic bead by using ferric salt as a raw material, modifying the iron-based nano magnetic bead by using tetraethoxysilane as a silicon source in the presence of ammonia water by using an alkylating reagent containing double bonds to obtain an alkylated modified magnetic bead, and then preparing the boric acid functionalized magnetic bead for enrichment and detection of viable bacteria. A phenylboronic acid functional monomer containing double bonds, polyethylene glycol and polyethylene glycol diacrylate are adopted, azodiisobutyronitrile is used as an initiator, an alkylation modified magnetic bead is coated with a polymer layer containing the phenylboronic acid functional monomer, and the boric acid functionalized magnetic bead is prepared. The boric acid functionalized magnetic bead can efficiently capture and enrich low-abundance pathogenic bacteria from a biological sample, has no toxicity to the pathogenic bacteria, and can identify the pathogenic bacteria through plate culture. The invention also discloses application of the boric acid functionalized magnetic bead in preparation of a kit or a reagent for enriching and detecting pathogenic bacteria.

Description

Technical Field

[0001] The invention relates to boric acid functionalized magnetic beads, and in particular to boric acid functionalized magnetic beads for enriching and detecting low-abundance live bacteria, and a preparation method and application thereof. Background Art

[0002] Bacterial infections have always been a major killer of human health and are still the leading cause of death in many underdeveloped countries. More than 10 million people die each year from bacterial infectious diseases, some of which can cause serious illness and eventually death even at extremely low concentrations. [1] The high morbidity and mortality caused by bacterial infections have received widespread attention worldwide. In some serious cases, clinicians need to make a diagnosis based on accurate information on bacterial identification, which plays an important role in saving the lives of infected people. It can be seen that rapid and accurate identification of bacteria is the key to effectively controlling bacterial infections.

[0003] Clinical detection methods for pathogens mainly include: plate culture method, DNA polymerase chain reaction (PCR) and immunoassay method. Plate culture method is the gold standard for bacterial detection, with the characteristics of high accuracy and good repeatability. However, this method generally requires a bacterial solution with a concentration of more than 30 CFU / mL for culture before identification. However, in the early stage of pathogen infection, the bacterial concentration in biological samples (blood, urine, sweat and other body fluids) is low, and the plate culture method is difficult to achieve the culture and identification of pathogens. When the pathogens multiply in large numbers in the body, the plate culture method takes 24-72 hours to complete the identification, which is difficult to meet the needs of bacterial prevention and control. PCR is the most sensitive and reliable method for pathogen detection to date. However, the persistence of DNA after cell death makes PCR unable to distinguish between live and dead bacteria. It can be seen that the concentration of pathogens in the PCR test report only represents that the body has been infected with pathogens, and cannot provide guidance for further medication for doctors. At the same time, the complex pretreatment and decoding analysis procedures are cumbersome and time-consuming, and the expensive detection equipment and professional operators limit the application of PCR in the field of pathogen identification. Immunoassay is a method of bacterial identification using antigen-antibody specificity. Immunoassays such as enzyme-linked immunosorbent assay and flow cytometry are time-consuming and have good specificity and stability for the detection of pathogens. However, in the early stages of infection, due to low antibody levels, immunoassays are difficult to achieve rapid and accurate detection, and there are also problems such as high reagent costs, unstable antibodies, and high false positive rates. It can be seen that clinical detection methods for pathogens have various drawbacks. A method for accurately identifying low-abundance live bacteria from complex biological samples has extremely important scientific significance and market value.

[0004] At present, the methods for accurately identifying live bacteria mainly rely on identification with highly sensitive instruments or rapid enrichment of bacteria before identification. Highly sensitive instruments are expensive and require professionally trained personnel, which leads to high costs for bacterial identification. Bacterial enrichment mainly relies on new functional materials and is a hot topic and focus of current research.

[0005] At present, the main methods for enriching bacteria are: centrifugation [2] , filtration method [3] , immunomagnetic bead method, etc. The centrifugation method is to separate the target bacteria by centrifugal force according to the different specific gravity of bacteria and impurities in the liquid medium; its advantages are fast separation speed, high efficiency and good liquid clarity; its disadvantage is that other components with similar density to the target bacteria will also settle, and the centrifugal speed is difficult to control. The filtration method is based on the pore size and uses the different particle sizes of different components in the solution to achieve separation. After the liquid sample is filtered through the membrane, the filtrate is discarded, and the bacteria are trapped by the membrane and enriched; its advantages are low cost, easy operation, large processing volume and wide range of use; but it also has the problems of low sensitivity and specificity and easy clogging of the filter pores. Magnetic separation technology is currently one of the most effective ways to separate and enrich microorganisms. This technology uses magnetic beads to adsorb and apply an external magnetic field to drive the separation of targets (such as bacteria, viruses, parasites and cancer cells, etc.) from complex original samples. However, the existing magnetic separation technology mainly focuses on immunomagnetic beads. Immunomagnetic separation technology is to couple antibodies of specific pathogens to magnetic beads and capture pathogens through antigen-antibody reaction, but antibodies have problems such as poor stability, low resistance, difficulty in preservation and high cost. Therefore, it is of scientific and market value to develop a highly sensitive, easy to operate, reusable, safe and non-toxic bacterial capture and enrichment technology.

[0006] References:

[0007] [1] Hu, WC; Pang, J.; Biswas, S.; Wang, K.; Wang, C.;

[0008] [2]Hevia, A.; Delgado, S.; Margolles, A.; Sánchez, B., Application of density gradient for the isolation of the fecal microbial stool component and the potential use thereof. Sci Rep 2015,5,16807.

[0009] [3] Tilmanne, A.; Kandet Yattara, HM; Herpol, M.; Vlaes, L.; Retore, P.; Quach, C.; Vandenberg, O.; Hallin, M.; Martiny, D., Multi-step optimization of the filtration method for the isolation of Campylobacter species from stoolsamples. European journal of clinical microbiology&infectious diseases: official publication of the European Society of Clinical Microbiology 2019,38(5),859-864. Summary of the invention

[0010] The purpose of the present invention is to provide a boric acid functionalized magnetic bead to address the problem of difficulty in detecting low-abundance live bacteria in complex samples, and to use the boric acid functionalized magnetic bead in a live bacteria enrichment technology for microbial culture methods, so as to enrich, culture and identify bacteria in a highly sensitive, rapid, economical and effective manner.

[0011] The objective of the present invention is achieved through the following technical solutions:

[0012] The invention discloses boric acid functionalized magnetic beads for enriching and detecting live bacteria. The boric acid functionalized magnetic beads (referred to as enrichment magnetic beads) are prepared by using iron salt as raw material to prepare iron-based nano magnetic beads, and then using tetraethyl orthosilicate as silicon source to modify the iron-based nano magnetic beads with an alkylating agent containing double bonds in the presence of ammonia water to obtain alkylated modified magnetic beads (referred to as modified magnetic beads), and using phenylboronic acid functional monomers containing double bonds and polyethylene glycol and polyethylene glycol diacrylate and azobisisobutyronitrile as initiator to coat the alkylated modified magnetic beads with a polymer layer containing phenylboronic acid functional monomers to obtain boric acid functionalized magnetic beads (referred to as enrichment magnetic beads).

[0013] Another object of the present invention is to provide a method for preparing boric acid functionalized magnetic beads for enrichment and detection of live bacteria, comprising:

[0014] Step (1), synthesis of iron-based nano-magnetic beads: dispersing iron salt, stabilizer and sodium acetate in a reaction solvent, reacting at a temperature of 100 to 300° C. for 6 to 24 hours; magnetically separating the reaction product, washing with water and anhydrous ethanol to remove unreacted reagents, and drying to obtain iron-based nano-magnetic beads;

[0015] Step (2), modification of nanomagnetic beads: disperse iron-based nanomagnetic beads in a reaction solvent, add ammonia water, stir and mix, then add tetraethyl orthosilicate, and stir at a temperature of 30 to 80° C. for 0.5 to 5 hours; add a double bond-containing silanization reagent at a temperature of 30 to 90° C. under light-proof conditions, maintain the temperature at 30 to 90° C., and stir for 3 to 12 hours; magnetically separate the reaction product, wash it with anhydrous ethanol, and dry it to obtain alkylated modified magnetic beads;

[0016] Step (3), coating with a polymer layer containing phenylboronic acid functional monomers: dispersing the alkylated magnetic beads in a reaction solvent, adding phenylboronic acid functional monomers containing double bonds, polyethylene glycol (porogen and solvent), polyethylene glycol diacrylate (PEGDA, crosslinking agent), and azobisisobutyronitrile (AIBN), dispersing them evenly, stirring and reacting at a temperature of 40 to 90° C. for 5 to 48 hours, magnetically separating the reaction products, washing with water and ethanol, and drying to obtain boronic acid functionalized magnetic beads.

[0017] In step (1), the iron salt is ferric chloride (FeCl3), ferric sulfate (Fe2(SO4)3), ferric acetate (Fe(OH)(CH3COO)2), or ferric nitrate (FeNO3)3).

[0018] The stabilizer is trisodium citrate dihydrate and polyacrylic acid. The stabilizer ensures electrostatic repulsion between the nano-magnetic beads and plays a role in dispersion and uniformity. For example, trisodium citrate has three carboxylic acid groups and has a strong coordination affinity for trivalent iron ions, which is conducive to the attachment of citric acid on the surface of magnetite nanocrystals and prevents aggregation into large single crystals.

[0019] The mass ratio of the iron salt to the stabilizer is (0.5-10):(1-10), preferably 1:0.5-1:2.

[0020] The mass ratio of the iron salt to sodium acetate is (0.5-10):(1-10), preferably 1:0.25-1:2.

[0021] The mass volume ratio of the iron salt to the reaction solvent is (0.5-10):(50-100) g / mL, preferably 1:10-1:100 g / mL.

[0022] The reaction solvent is ethylene glycol, polyethylene glycol, glycerol, an aqueous solution of polyvinyl alcohol with a concentration of 1-10% m / v, or an aqueous solution of cross-linked polyvinyl pyrrolidone with a concentration of 1-10% m / v. The reaction solvent also has a certain dispersibility and is both a reaction solvent and a reducing agent.

[0023] The synthesis of the iron-based nano-magnetic beads is carried out in a high-pressure reactor.

[0024] The water and anhydrous ethanol cleaning method is to use ultrapure water and anhydrous ethanol to alternately clean the reaction product 3 times, specifically: the reaction solution is cooled to room temperature, the reaction product is fixed by an external magnetic field, the solvent is discarded, ultrapure water is added for ultrasonic cleaning, the reaction product is fixed by an external magnetic field, the solution is discarded, anhydrous ethanol is added for ultrasonic cleaning, the reaction product is fixed by an external magnetic field, and the solution is discarded; the reaction product is alternately cleaned 3 times with ultrapure water and anhydrous ethanol.

[0025] In step (2), the mass volume ratio of the iron-based nanomagnetic beads to the reaction solvent is (100-1000):(100-1000) mg / mL, preferably 1:0.5-1:1.

[0026] The reaction solvent is an organic solvent such as anhydrous ethanol, anhydrous toluene, and n-octane.

[0027] The mass volume ratio of the iron-based nano-magnetic beads and ammonia water is (200-800):(2-20) mg / mL, preferably 25:1-50:1 mg / mL.

[0028] The mass fraction of the ammonia water is 10% to 40%. After adding the ammonia water, stir at a temperature of 30 to 80° C. for 10 minutes.

[0029] The mass volume ratio of the iron-based nano-magnetic beads and tetraethyl orthosilicate is (100-500):(0.5-10) mg / mL, preferably 50:1-200:1 mg / mL.

[0030] The mass volume ratio of the iron-based nano-magnetic beads and the double-bond-containing silanization agent is (100-500):(1-15) mg / mL, preferably 6:1-100:1 mg / mL.

[0031] The double bond-containing silanization agent includes, but is not limited to, methacryloxypropyltriethoxysilane, allyltriethoxysilane, vinyltriethoxysilane, and 3-[tri(1-methylethoxy)silyl]propyl methacrylate.

[0032] The anhydrous ethanol cleaning method is as follows: the reaction solution is cooled to room temperature while stirring, the reaction product is fixed by an external magnetic field, the solvent is discarded, anhydrous ethanol is added for ultrasonic cleaning, and the cleaning is performed 3 times, each cleaning lasting 5 minutes.

[0033] In step (3), the mass ratio of the alkylated modified magnetic beads to the double-bond-containing phenylboronic acid functional monomer is (0.1-5):(0.1-5), preferably 1:1-1.5:1.

[0034] The double-bond-containing phenylboronic acid functional monomers include, but are not limited to, 4-vinylphenylboronic acid, 3-vinylphenylboronic acid, 3-methacrylamidephenylboronic acid, and 3-allylformamidophenylboronic acid.

[0035] The mass volume ratio of the alkylated modified magnetic beads and polyethylene glycol is (0.1-5):(0.2-10) g / mL, preferably 1:1-1:4.

[0036] The mass volume ratio of the alkylated modified magnetic beads and polyethylene glycol diacrylate is (0.1-5):(0.1-10) g / mL, preferably 1:0.3-1:2 g / mL.

[0037] The mass ratio of the alkylated modified magnetic beads to azobisisobutyronitrile is (100-5000):1.

[0038] The mass volume ratio of the alkylated modified magnetic beads and the reaction solvent is (0.1-5):(10-100) g / mL, preferably 1:20-1:400 g / mL.

[0039] The reaction solvent is a polyvinyl alcohol aqueous solution with a concentration of 1 to 10% m / v or a cross-linked polyvinyl pyrrolidone aqueous solution with a concentration of 1 to 10% m / v.

[0040] The polyethylene glycol described in the present invention is selected from PEG200, PEG400 or PEG800.

[0041] The boric acid functionalized magnetic beads of the present invention can efficiently capture and enrich pathogenic bacteria from low-abundance biological samples, and are non-toxic to pathogenic bacteria. The pathogenic bacteria in the boric acid functionalized magnetic beads / pathogenic bacteria complex are live bacteria, and can be identified by plate culture, which can be used as a good pre-diagnosis treatment. The boric acid functionalized magnetic beads can identify Gram-positive pathogenic bacteria and Gram-negative pathogenic bacteria, and have higher selectivity for Gram-positive bacteria. The boric acid functionalized magnetic beads are easy to carry and are suitable for making portable kits or reagents for enriching and detecting pathogenic bacteria.

[0042] Another object of the present invention is to provide the use of the boric acid functionalized magnetic beads in the preparation of a kit for enriching and detecting pathogenic bacteria.

[0043] Another object of the present invention is to use the boric acid functionalized magnetic beads in the preparation of reagents for enriching and detecting pathogenic bacteria.

[0044] Preferably, the application is to add the boric acid functionalized magnetic beads to a liquid sample containing pathogenic bacteria, adjust the pH value to 8.5-9, shake at room temperature, magnetically separate the boric acid functionalized magnetic beads enriched with pathogenic bacteria, and perform plate culture and identification.

[0045] The liquid sample containing pathogenic bacteria is a human or animal liquid sample containing pathogenic bacteria, specifically urine, small intestinal fluid, etc. containing pathogenic bacteria.

[0046] The pathogenic bacteria are Gram-positive pathogenic bacteria and Gram-negative pathogenic bacteria, preferably Gram-positive pathogenic cocci, and can be specifically selected from Staphylococcus aureus, hemolytic Staphylococcus, and Streptococcus.

[0047] Beneficial effects of the present invention:

[0048] 1. The boric acid functionalized magnetic beads of the present invention have a large specific surface area: the particle size of the magnetic beads is nanometer-scale, providing abundant adsorption sites (boric acid ligands).

[0049] 2. The boric acid functionalized magnetic beads of the present invention have the ability to enrich pathogenic bacteria from low-abundance (≤1 CFU / mL) biological samples, and the captured bacteria are live bacteria.

[0050] 3. The present invention uses an alkylating agent containing double bonds to modify the double bonds on the surface of the magnetic beads. The double bonds participate in the copolymerization of the boric acid monomer, making the link between the magnetic beads and the functional monomer tighter and not easy to peel off. The boric acid functionalized magnetic beads of the present invention can be reused. After three cycles, the enrichment efficiency can still be maintained at more than 80% of the first use. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Scanning electron micrographs of the alkylated modified magnetic beads (A) and the boric acid functionalized magnetic beads (B) prepared in Example 1.

[0052] Figure 2 The results of culturing the supernatant on plates after the alkylated magnetic beads (ie, the modified magnetic beads in the figure) and the boric acid functionalized magnetic beads (ie, the enriched magnetic beads in the figure) prepared in Example 1 recognized Staphylococcus aureus.

[0053] Figure 3 The particle size distribution diagram of the alkylated modified magnetic beads (A) and the boronic acid functionalized magnetic beads (B) prepared in Example 2, and the SEM image of the boronic acid functionalized magnetic beads (C).

[0054] Figure 4This is the result of culturing the supernatant on plates after the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in Example 2 recognized hemolytic Staphylococcus.

[0055] Figure 5 The particle size distribution diagram of the alkylated modified magnetic beads (A) and the boric acid functionalized magnetic beads (B) prepared in Example 3.

[0056] Figure 6 This is the result of culturing the supernatant on plates after the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in Example 3 recognized Streptococcus.

[0057] Figure 7 The results of recognition and cultivation of Streptococcus by the boric acid functionalized magnetic beads prepared in Example 4.

[0058] Figure 8 The results of recognition and culture of hemolytic Staphylococcus by the boric acid functionalized magnetic beads prepared in Example 5.

[0059] Fig. 9 This is a diagram of the reuse efficiency of the boric acid functionalized magnetic beads prepared in Example 6.

[0060] Fig.10 The results of the boric acid functionalized magnetic beads prepared in Example 6 identifying bacteria in real samples. DETAILED DESCRIPTION

[0061] Example 1

[0062] Take 0.5g FeCl3, 1g trisodium citrate dihydrate, and 1g sodium acetate, disperse them in 50mL ethylene glycol, react in a high-pressure reactor at 100°C for 6h, and cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, and the magnetic beads are fixed by an external magnet. The solvent is discarded, 50mL ultrapure water is added, and ultrasonic cleaning is performed for 5min. The magnetic beads are fixed by an external magnet, and the solution is discarded. 30mL anhydrous ethanol is added, and ultrasonic cleaning is performed for 5min. The magnetic beads are fixed by an external magnet, and the solution is discarded. In this way, the magnetic beads are washed alternately with ultrapure water and anhydrous ethanol for 3 times; dried to constant weight, iron-based nanomagnetic beads are obtained.

[0063] 100 mg of iron-based nano-magnetic beads were evenly dispersed in 100 mL of anhydrous ethanol, 2 mL of ammonia water (analytical grade, 25-28%, the same below) was added, and the mixture was stirred at 30°C for 10 min. 0.5 mL of tetraethyl orthosilicate was added, and the mixture was stirred at 30°C for 0.5 h. Under light-proof conditions, 1 mL of methacryloxypropyltriethoxysilane was added at 30°C, and the mixture was stirred at 30°C for 3 h after the addition was completed. The mixture was cooled to room temperature while stirring, and the magnetic beads were fixed with an external magnet. The solvent was discarded, and anhydrous ethanol was added for ultrasonic cleaning for 3 times, with each cleaning time lasting 5 min. The mixture was dried to constant weight to obtain alkylated magnetic beads ( Figure 1 A).

[0064] 0.1 g of alkylated magnetic beads were dispersed in 10 mL of 1% m / v polyvinyl alcohol (CAS: 9002-89-5; Mw average molecular weight 13000-23000; the same below) aqueous solution, 0.1 g of 4-vinylbenzeneboronic acid, 0.2 mL of polyethylene glycol 200 (PEG200), 0.1 mL of PEGDA (polyethylene glycol diacrylate CAS: 26570-48-9; molecular weight 10000; the same below), and 1 mg of AIBN were added, and the mixture was uniformly dispersed, stirred and reacted at 40° C. for 5 h, and cooled to room temperature; the magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, and ultrasonic cleaning was performed for 5 min, the magnetic beads were fixed with a magnet, the solution was discarded, and 30 mL of anhydrous ethanol was added, and ultrasonic cleaning was performed for 5 min, the magnetic beads were fixed with a magnet, and the solution was discarded; in this way, the magnetic beads were alternately washed with ultrapure water and anhydrous ethanol, and the alternating cycle was repeated 3 times; the magnetic beads were collected and dried to constant weight to obtain boric acid functionalized magnetic beads ( Figure 1 B).

[0065] contrast Figure 1 A and Figure 1 B It can be found that the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in this example are both spherical, and their particle size distribution is uniform, with an average particle size of about 200 nm; the average particle size of the boric acid functionalized magnetic beads is slightly larger than that of the silanized magnetic beads.

[0066] Take 1 mg of the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in this example and add them to 10 3 CFU / mL Staphylococcus aureus suspension, 0.1M NaOH aqueous solution was adjusted to pH 9, and the Staphylococcus aureus was enriched by shaking at room temperature for 30 minutes; the magnetic beads were separated by magnetic separation to obtain the supernatant. The supernatant was spread on a plate to show ( Figure 2 ), the boric acid functionalized magnetic beads can effectively identify and enrich Staphylococcus aureus, and the enrichment rate of Staphylococcus aureus at this concentration can reach 98.43%, that is, 98.43% of Staphylococcus aureus is recognized on the surface of the boric acid functionalized magnetic beads; while the enrichment rate of Staphylococcus aureus by the alkylated modified magnetic beads is only 17.29%.

[0067] Example 2

[0068] Take 10g Fe2(SO4)3, 10g polyacrylic acid (CAS: 9003-01-4; Mw average molecular weight 450 000; the same below), and 10g sodium acetate, disperse them in 100mL polyethylene glycol 600, react in a high-pressure reactor at 300℃ for 24h, and cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, the magnetic beads are fixed by an external magnet, the solvent is discarded, 50mL ultrapure water is added for ultrasonic cleaning for 5min, the magnetic beads are fixed by an external magnet, the solution is discarded, and 30mL anhydrous ethanol is added for ultrasonic cleaning for 5min, the magnetic beads are fixed by an external magnet, and the solution is discarded; in this way, the magnetic beads are washed alternately with ultrapure water and anhydrous ethanol for 3 times; dried to constant weight, iron-based nanomagnetic beads are obtained.

[0069] 500 mg of iron-based nano-magnetic beads were evenly dispersed in 250 mL of anhydrous ethanol, and 20 mL of ammonia water was added, and the mixture was stirred at 80 ° C for 10 min. 10 mL of tetraethyl orthosilicate was added, and the mixture was stirred at 80 ° C for 5 h. Under light-proof conditions, 15 mL of allyltriethoxysilane was added at 90 ° C, and the mixture was stirred at 90 ° C for 12 h after the addition was completed. The mixture was cooled to room temperature while stirring, and the magnetic beads were fixed with an external magnet. The solvent was discarded, and anhydrous ethanol was added for ultrasonic cleaning for 3 times, with each cleaning time of 5 min. The mixture was dried to constant weight to obtain alkylated magnetic beads ( Figure 3 A).

[0070] 5 g of alkylated modified magnetic beads were dispersed in 100 mL of 1% m / v cross-linked polyvinyl pyrrolidone (CAS: 9003-39-8; Mw average molecular weight 40000; the same below) aqueous solution, 5 g of 3-vinylbenzeneboronic acid, 10 mL of polyethylene glycol 400 (PEG400), 10 mL of PEGDA, and 1 mg of AIBN were added, and the mixture was uniformly dispersed. The mixture was stirred at 90° C. for 48 h and then cooled to room temperature. The magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, and ultrasonic cleaning was performed for 5 min. The magnetic beads were fixed with a magnet, the solution was discarded, and 30 mL of anhydrous ethanol was added, and ultrasonic cleaning was performed for 5 min. The magnetic beads were fixed with a magnet, and the solution was discarded. In this way, the magnetic beads were alternately washed with ultrapure water and anhydrous ethanol for 3 times. The magnetic beads were collected and dried to constant weight to obtain boric acid functionalized magnetic beads. Figure 3 C It can be observed that the surface of the boric acid functionalized magnetic beads presents a porous structure.

[0071] contrast Figure 3 A and Figure 3 B It can be found that the alkylated magnetic beads and the boric acid functionalized magnetic beads prepared in this example are both spherical, and the particle size distribution of both is uniform, with an average particle size of about 1000 nm; the average particle size of the boric acid functionalized magnetic beads is slightly larger than that of the silanized magnetic beads. Figure 1 and Figure 3It can be found that the higher the polymer (PEGDA) content used in preparing the boronic acid functionalized magnetic beads, the larger the average particle size of the magnetic beads.

[0072] Take 1 mg of the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in this example and add them to 10 3 CFU / mL Staphylococcus haemolyticus suspension, 0.1M NaOH aqueous solution was adjusted to pH 8.5, and the mixture was shaken at room temperature for 30 minutes; the magnetic beads were separated by magnetic separation to obtain the supernatant. The supernatant was spread on a plate and displayed ( Figure 4 ), the enrichment rate of hemolytic Staphylococcus aureus by boric acid functionalized magnetic beads at this concentration can reach 92.86%, that is, 92.86% of hemolytic Staphylococcus aureus are recognized on the surface of boric acid functionalized magnetic beads; while the enrichment rate of hemolytic Staphylococcus aureus by alkylated modified magnetic beads is only 13.38%.

[0073] Example 3

[0074] Take 3gFe(OH)(CH3COO)2, 2g trisodium citrate dihydrate, and 3g sodium acetate, disperse them in 80mL propylene glycol, react at 200℃ in a high-pressure reactor for 12h, and cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, and the magnetic beads are fixed by an external magnet. The solvent is discarded, 50mL ultrapure water is added, and ultrasonic cleaning is performed for 5min. The magnetic beads are fixed by an external magnet, and the solution is discarded. 30mL anhydrous ethanol is added, and ultrasonic cleaning is performed for 5min. The magnetic beads are fixed by an external magnet, and the solution is discarded. In this way, the magnetic beads are washed alternately with ultrapure water and anhydrous ethanol for 3 times; dried to constant weight, and iron-based nanomagnetic beads are obtained.

[0075] 200 mg of iron-based nano-magnetic beads were added to 200 mL of anhydrous ethanol and dispersed evenly. 6 mL of ammonia water was added and stirred at 50°C for 10 min. 2 mL of tetraethyl orthosilicate was added and stirred at 50°C for 2 h. 9 mL of vinyl triethoxysilane was added at 50°C under light-proof conditions. After the addition was completed, stirring was continued at 50°C for 4 h. The beads were cooled to room temperature while stirring. The magnetic beads were fixed with an external magnet. The solvent was discarded. Anhydrous ethanol was added for ultrasonic cleaning for 3 times, each time for 5 min. The beads were dried to constant weight to obtain alkylated magnetic beads ( Figure 5 A).

[0076] 1 g of alkylated modified magnetic beads were dispersed in 50 mL of 10% m / v polyvinyl alcohol aqueous solution, and 1 g of 3-methylacrylamidophenylboronic acid, 1 mL of polyethylene glycol 600 (PEG600), 2 mL of PEGDA, and 1 mg of AIBN were added, and the mixture was uniformly dispersed and stirred at 60°C for 24 h; the mixture was cooled to room temperature; the magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, and ultrasonic cleaning was performed for 5 min, the magnetic beads were fixed with a magnet, the solution was discarded, and 30 mL of anhydrous ethanol was added, and ultrasonic cleaning was performed for 5 min, the magnetic beads were fixed with a magnet, and the solution was discarded; the magnetic beads were washed alternately with ultrapure water and anhydrous ethanol, and the alternating cycle was repeated 3 times; the magnetic beads were collected and dried to constant weight to obtain boric acid functionalized magnetic beads ( Figure 5 B).

[0077] contrast Figure 5 A and Figure 5 B It can be found that the alkylated magnetic beads and boric acid functionalized magnetic beads prepared in this example have uniform particle size distribution, and the average particle size is about 200 nm; the average particle size of the boric acid functionalized magnetic beads is slightly larger than the particle size of the silanated magnetic beads.

[0078] Take 1 mg of boric acid functionalized magnetic beads and add to 10 3 CFU / mL Streptococcus uberis suspension, 0.1M NaOH aqueous solution was adjusted to pH 8.8, shaken at room temperature for 30 minutes, and magnetic beads were separated by magnetic separation to obtain the supernatant. The supernatant was spread on a plate to show ( Figure 6 ), the enrichment rate of streptococci by boric acid functionalized magnetic beads at this concentration can reach 96.55%, that is, 96.55% of streptococci are recognized on the surface of boric acid functionalized magnetic beads; while the enrichment rate of streptococci by alkylated modified magnetic beads is only 9.56%.

[0079] Example 4

[0080] Take 5g Fe(NO3)3, 6g polyacrylic acid, and 2g sodium acetate, disperse them in 60mL 1% m / v polyvinyl alcohol aqueous solution, react at 180°C in a high-pressure reactor for 10 hours, and cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, and the magnetic beads are fixed by an external magnet, the solvent is discarded, 50mL ultrapure water is added, ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, the solution is discarded, and 30mL anhydrous ethanol is added, ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, and the solution is discarded; in this way, the magnetic beads are alternately cleaned 3 times with ultrapure water and anhydrous ethanol; dry to constant weight to obtain iron-based nanomagnetic beads.

[0081] 300 mg of iron-based nano-magnetic beads were added to 150 mL of anhydrous ethanol and dispersed evenly. 8 mL of ammonia water was added and stirred at 60°C for 10 min. 3 mL of tetraethyl orthosilicate was added and stirred at 60°C for 3 h. Under light-proof conditions, 3 mL of 3-[tri(1-methylethoxy)silyl]propyl methacrylate was added at 80°C. After the addition was completed, stirring was continued at 80°C for 6 h. The beads were cooled to room temperature while stirring. The magnetic beads were fixed with an external magnet, the solvent was discarded, and anhydrous ethanol was added for ultrasonic cleaning for 3 times, each time for 5 min. The beads were dried to constant weight to obtain alkylated magnetic beads.

[0082] 3 g of alkylated modified magnetic beads were dispersed in 90 mL of 10% m / v cross-linked polyvinyl pyrrolidone aqueous solution, and 3 g of 3-allylcarboxamidophenylboronic acid, 8 mL of polyethylene glycol 800 (PEG800), 4 mL of PEGDA, and 1 mg of AIBN were added, dispersed evenly, and stirred at 70°C for 12 hours; cooled to room temperature; the magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, the solution was discarded, and 30 mL of anhydrous ethanol was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, and the solution was discarded; in this way, the magnetic beads were alternately washed with ultrapure water and anhydrous ethanol, and the alternating cycle was repeated 3 times; the magnetic beads were collected and dried to constant weight to obtain boronic acid functionalized magnetic beads.

[0083] Take 1 mg of boric acid functionalized magnetic beads and add to 10 3 CFU / mL Streptococcus uberis suspension, 0.1M NaOH aqueous solution was adjusted to pH 8.8, and the suspension was shaken at room temperature for 30 minutes. The magnetic beads were separated by magnetic separation to obtain the supernatant and the boric acid functionalized magnetic beads / streptococcus complex. The supernatant was plated and the boric acid functionalized magnetic beads / streptococcus complex was plated. Figure 7 The supernatant plate coating showed that the supernatant basically did not contain streptococci, indicating that the boric acid functionalized magnetic beads could efficiently identify streptococci; after the boric acid functionalized magnetic beads / streptococcus complex was cultured on the plate, a large number of streptococci appeared on the plate, indicating that the boric acid functionalized magnetic beads were not toxic to streptococci, and the streptococci in the boric acid functionalized magnetic beads / streptococcus complex were live bacteria and could be cultured and identified on the plate.

[0084] Example 5

[0085] Take 7g FeCl3, 8g trisodium citrate dihydrate, and 2g sodium acetate, disperse them in 100mL 1% m / v cross-linked polyvinyl pyrrolidone aqueous solution, react at 200°C in a high-pressure reactor for 18h, and cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, the magnetic beads are fixed by an external magnet, the solvent is discarded, 50mL ultrapure water is added, ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, the solution is discarded, and 30mL anhydrous ethanol is added, ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, and the solution is discarded; in this way, the magnetic beads are alternately washed 3 times with ultrapure water and anhydrous ethanol; dried to constant weight to obtain iron-based nanomagnetic beads.

[0086] Add 300 mg of iron-based nano-magnetic beads to 200 mL of anhydrous ethanol and disperse them evenly. Add 5 mL of ammonia water and stir at 40°C for 10 min. Add 6 mL of tetraethyl orthosilicate and stir at 40°C for 4 h. Add 5 mL of methacryloxypropyltriethoxysilane at 60°C under light-proof conditions and continue stirring at 60°C for 4 h. Cool to room temperature while stirring, fix the magnetic beads with an external magnet, discard the solvent, add anhydrous ethanol for ultrasonic cleaning, and wash 3 times, each time for 5 min. Dry to constant weight to obtain alkylated magnetic beads.

[0087] 3 g of alkylated modified magnetic beads were dispersed in 100 mL of 6% m / v cross-linked polyvinyl pyrrolidone aqueous solution, and 2 g of 4-vinylbenzeneboronic acid, 6 mL of PEG800, 1 mL of PEGDA, and 1 mg of AIBN were added, dispersed evenly, and stirred at 80°C for 10 hours; cooled to room temperature; the magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, the solution was discarded, and 30 mL of anhydrous ethanol was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, and the solution was discarded; in this way, the magnetic beads were alternately washed with ultrapure water and anhydrous ethanol, and the alternating cycle was repeated 3 times; the magnetic beads were collected and dried to constant weight to obtain boronic acid functionalized magnetic beads.

[0088] Take 1 mg of boric acid functionalized magnetic beads and add to 10 2 CFU / mL hemolytic Staphylococcus aureus suspension was adjusted to pH 9 with 0.1M NaOH aqueous solution and shaken at room temperature for 30 min. The magnetic beads were separated by magnetic separation to obtain the supernatant and the boric acid functionalized magnetic beads / hemolytic Staphylococcus aureus complex. The supernatant was plated and the boric acid functionalized magnetic beads / hemolytic Staphylococcus aureus complex was plated. Figure 8The results of supernatant plate culture showed that the supernatant basically did not contain hemolytic Staphylococcus, indicating that boric acid functionalized magnetic beads can efficiently identify hemolytic Staphylococcus, and obtain boric acid functionalized magnetic beads / hemolytic Staphylococcus complex; after the boric acid functionalized magnetic beads / hemolytic Staphylococcus complex was plate cultured, a large number of hemolytic Staphylococcus appeared in the plate, indicating that the boric acid functionalized magnetic beads were not toxic to hemolytic Staphylococcus, and the hemolytic Staphylococcus in the boric acid functionalized magnetic beads / streptococcus complex was a live bacterium and could be used for plate culture and identification; the number of hemolytic Staphylococcus in the enriched and re-cultured plate increased significantly, indicating that the boric acid functionalized magnetic beads have the ability to enrich hemolytic Staphylococcus.

[0089] Example 6

[0090] Take 5g Fe(NO3)3, 3g trisodium citrate dihydrate, and 7g sodium acetate, disperse them in 80mL 10% m / v polyvinyl alcohol aqueous solution, and react at 160°C in a high-pressure reactor for 18h; cool to room temperature; the magnetic beads in the reaction solution are precipitated to the bottom of the reactor, and the magnetic beads are fixed by an external magnet, the solvent is discarded, 50mL ultrapure water is added, and ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, the solution is discarded, and 30mL anhydrous ethanol is added, and ultrasonic cleaning is performed for 5min, the magnetic beads are fixed by an external magnet, and the solution is discarded; in this way, the magnetic beads are washed alternately with ultrapure water and anhydrous ethanol for 3 times; dry to constant weight to obtain iron-based nanomagnetic beads.

[0091] Add 100 mg of iron-based nano-magnetic beads to 100 mL of anhydrous ethanol and disperse evenly, add 2 mL of ammonia water, stir at 70 ° C for 10 min, add 1 mL of tetraethyl orthosilicate, and stir at 70 ° C for 2 h; add 15 mL of methacryloxypropyltriethoxysilane at 60 ° C under light-proof conditions, and continue to stir at 60 ° C for 3 h after the addition is complete; cool to room temperature while stirring, fix the magnetic beads with an external magnet, discard the solvent, add anhydrous ethanol for ultrasonic cleaning, wash 3 times, each time for 5 minutes; dry to constant weight to obtain alkylated magnetic beads.

[0092] 0.2 g of alkylated modified magnetic beads were dispersed in 80 mL of 2% m / v polyvinyl alcohol aqueous solution, and 0.2 g of 3-methylacrylamidophenylboronic acid, 0.8 mL of PEG400, 0.3 mL of PEGDA, and 1 mg of AIBN were added, dispersed evenly, and stirred at 50°C for 28 hours; cooled to room temperature; the magnetic beads were fixed with a magnet, the solvent was discarded, 50 mL of ultrapure water was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, the solution was discarded, 30 mL of anhydrous ethanol was added, ultrasonic cleaning was performed for 5 minutes, the magnetic beads were fixed with a magnet, and the solution was discarded; in this way, the magnetic beads were washed alternately with ultrapure water and anhydrous ethanol, and the alternating cycle was repeated 3 times; the magnetic beads were collected and dried to constant weight to obtain boronic acid functionalized magnetic beads.

[0093] Take 1 mg of boric acid functionalized magnetic beads and add to 10 2 The suspension of CFU / mL Staphylococcus aureus was adjusted to pH 9 with 0.1M NaOH aqueous solution, and the suspension was shaken at room temperature for 30 min. The boric acid functionalized magnetic beads were magnetically separated. After the strain was cultured on a plate, the magnetic beads were washed with pH 4 acetic acid-PBS buffer (the pH of the PBS buffer was adjusted to 4 with acetic acid), washed with pure water until neutral, and then incubated again for 10 min. 2 CFU / mL Staphylococcus aureus suspension was shaken at room temperature for 30 minutes, and the boric acid functionalized magnetic beads were magnetically separated. After the strain was cultured on a plate, the boric acid functionalized magnetic beads were washed in the same steps, and this was repeated 5 times. Fig. 9 It can be seen that after the boric acid functionalized magnetic beads are reused for 3 times, the recognition efficiency of Staphylococcus aureus can still reach more than 80%. It can be seen that the boric acid functionalized magnetic beads have good reusability.

[0094] Staphylococcus aureus was added to the urine of healthy women (the final concentration of Staphylococcus aureus in urine was 10 2 CFU / mL), 1 mg of boric acid functionalized magnetic beads was added to 1 mL of urine supplemented with Staphylococcus aureus, the pH was adjusted to 9 with 0.1 M NaOH aqueous solution, and the mixture was shaken at room temperature for 30 min. The magnetic beads were separated by magnetic separation to obtain the supernatant and the magnetic bead / bacteria complex. The supernatant was plated and the magnetic bead / bacteria complex was plated. The results are shown in Fig.10 A. The results of supernatant plate culture showed that in addition to Staphylococcus aureus, there were other strains in the urine with added Staphylococcus aureus; after adding boric acid functionalized magnetic beads, no added Staphylococcus aureus strains appeared in the supernatant, and only a small number of strains were not captured by the boric acid functionalized magnetic beads, indicating that the boric acid functionalized magnetic beads still have good bacterial capture ability in real samples; after the magnetic bead / bacteria complex was plate cultured (i.e., enrichment magnetic beads were added to the sample in the figure and then cultured), a large number of Staphylococcus aureus and a small number of unknown strains appeared in the plate, indicating that the boric acid functionalized magnetic beads are not toxic to Staphylococcus aureus and unknown strains in urine, and the bacteria in the boric acid functionalized magnetic beads / bacteria complex are live bacteria and can be used for plate culture and identification; the number of bacteria in the enriched and cultured plate is significantly higher than that in the original bacterial solution, indicating that the boric acid functionalized magnetic beads have the ability to enrich bacteria from low-abundance samples.

[0095] Staphylococcus aureus was added to the urine of healthy men (final concentration of Staphylococcus aureus in urine was 0.5 CFU / mL), 1 mg of boric acid functionalized magnetic beads was taken and added to 100 mL of urine with Staphylococcus aureus added, pH was adjusted to 9 with 0.1 M NaOH aqueous solution, and the solution was shaken at room temperature for 30 min. The magnetic beads were separated by magnetic separation to obtain the supernatant and the boric acid functionalized magnetic beads / bacteria complex. The urine with low concentration of Staphylococcus aureus added and the supernatant were plated and the boric acid functionalized magnetic beads / bacteria complex was plated. The results are shown in Fig.10 B. No strains were seen in urine with low concentration of Staphylococcus aureus added; no strains were seen in the supernatant after adding boric acid functionalized magnetic beads; a large number of Staphylococcus aureus appeared in the plate after the boric acid functionalized magnetic beads / bacteria complex was cultured on the plate, indicating that the boric acid functionalized magnetic beads have the ability to enrich bacteria from low-abundance biological samples and are not toxic to Staphylococcus aureus. The bacteria in the boric acid functionalized magnetic beads / bacteria complex are live bacteria and can be used for plate culture and identification.

Claims

1. A boric acid functionalized magnetic bead, characterized in that: The method uses iron salt as a raw material to prepare iron-based nano-magnetic beads, and then uses tetraethyl orthosilicate as a silicon source. In the presence of ammonia water, an alkylating agent containing a double bond is used to modify the iron-based nano-magnetic beads to obtain alkylated modified magnetic beads. Phenylboronic acid functional monomers containing double bonds, polyethylene glycol, and polyethylene glycol diacrylate are used, and azobisisobutyronitrile is used as an initiator to coat a polymer layer containing phenylboronic acid functional monomers on the alkylated modified magnetic beads to obtain boric acid functionalized magnetic beads.

2. The boric acid functionalized magnetic beads according to claim 1, characterized in that: The iron salt is ferric chloride, ferric sulfate, ferric acetate and ferric nitrate.

3. The boric acid functionalized magnetic beads according to claim 1, characterized in that: The double-bond-containing silanization agent is methacryloxypropyltriethoxysilane, allyltriethoxysilane, vinyltriethoxysilane or 3-[tri(1-methylethoxy)silyl]propyl methacrylate.

4. The boric acid functionalized magnetic beads according to claim 1, characterized in that: The double-bond-containing phenylboronic acid functional monomer is 4-vinylphenylboronic acid, 3-vinylphenylboronic acid, 3-methacrylamidephenylboronic acid or 3-allylformylaminophenylboronic acid.

5. A method for preparing the boric acid functionalized magnetic beads according to any one of claims 1 to 4, characterized in that: include: Step (1), synthesis of iron-based nano-magnetic beads: dispersing iron salt, stabilizer and sodium acetate in a reaction solvent, reacting at a temperature of 100 to 300° C. for 6 to 24 hours; magnetically separating the reaction product, washing with water and anhydrous ethanol, and drying to obtain iron-based nano-magnetic beads; Step (2), modification of nanomagnetic beads: disperse iron-based nanomagnetic beads in a reaction solvent, add ammonia water, mix well, then add tetraethyl orthosilicate, and stir at a temperature of 30 to 80° C. for 0.5 to 5 hours; add a double bond-containing silanization reagent at a temperature of 30 to 90° C. under light-proof conditions, maintain the temperature at 30 to 90° C., and stir for 3 to 12 hours; magnetically separate the reaction product, wash it with anhydrous ethanol, and dry it to obtain alkylated modified magnetic beads; Step (3), coating with a polymer layer containing phenylboronic acid functional monomers: dispersing the alkylated magnetic beads in a reaction solvent, adding phenylboronic acid functional monomers containing double bonds, polyethylene glycol, polyethylene glycol diacrylate, and azobisisobutyronitrile, stirring the reaction at a temperature of 40 to 90° C. for 5 to 48 hours, magnetically separating the reaction products, washing with water and ethanol, and drying to obtain boronic acid functionalized magnetic beads.

6. The method for preparing boric acid functionalized magnetic beads according to claim 5, characterized in that: In step (1), the stabilizer is trisodium citrate dihydrate and polyacrylic acid; the mass ratio of the iron salt to the stabilizer is (0.5-10):(1-10), preferably 1:0.5-1:2; the mass ratio of the iron salt to sodium acetate is (0.5-10):(1-10), preferably 1:0.25-1:2; the reaction solvent is ethylene glycol, polyethylene glycol, glycerol, an aqueous solution of polyvinyl alcohol with a concentration of 1-10% m / v, or an aqueous solution of cross-linked polyvinyl pyrrolidone with a concentration of 1-10% m / v.

7. The method for preparing boric acid functionalized magnetic beads according to claim 5, characterized in that: In step (2), the mass volume ratio of the iron-based nano-magnetic beads and the reaction solvent is (100-1000): (100-1000) mg / mL, preferably 1:0.5-1:1; the reaction solvent is ethanol, toluene or n-octane; the mass volume ratio of the iron-based nano-magnetic beads and ammonia water is (200-800): (2-20) mg / mL, preferably 25:1-50:1 mg / mL; the mass volume ratio of the iron-based nano-magnetic beads and tetraethyl orthosilicate is (100-500): (0.5-10) mg / mL, preferably 50:1-200:1 mg / mL; the mass volume ratio of the iron-based nano-magnetic beads and the silanization agent containing double bonds is (100-500): (1-15) mg / mL, preferably 6:1-100:1 mg / mL.

8. The method for preparing boric acid functionalized magnetic beads according to claim 5, characterized in that: In step (3), the mass ratio of the alkylated magnetic beads to the double-bond-containing phenylboronic acid functional monomer is (0.1-5):(0.1-5), preferably 1:1-1.5:1; the mass volume ratio of the alkylated magnetic beads to polyethylene glycol is (0.1-5):(0.2-10) g / mL, preferably 1:1-1:4; the mass volume ratio of the alkylated magnetic beads to polyethylene glycol diacrylate is (0.1-5):(0.1-10) g / mL, preferably 1:0.3-1:2 g / mL; the mass ratio of the alkylated magnetic beads to azobisisobutyronitrile is (100-5000):1; the reaction solvent is a polyvinyl alcohol aqueous solution with a concentration of 1-10% m / v or a cross-linked polyvinyl pyrrolidone aqueous solution with a concentration of 1-10% m / v.

9. Use of the boric acid functionalized magnetic beads according to any one of claims 1 to 4 in preparing a kit or reagent for enriching and detecting pathogenic bacteria.

10. The use according to claim 9, characterized in that: The application is to add the boric acid functionalized magnetic beads into a liquid sample containing pathogenic bacteria, adjust the pH value to 8.5-9, shake at room temperature, magnetically separate the boric acid functionalized magnetic beads enriched with pathogenic bacteria, and perform plate culture and identification.

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