Surface potential adjustable nanometer magnetic beads, preparation method and application thereof

Abstract

The application discloses a kind of nano magnetic beads with adjustable surface potential and its preparation method and application, the nano magnetic bead includes four iron oxide magnetic bead core, silica layer being coated in the four iron oxide magnetic bead core surface and ligand unit;The ligand unit is loaded on the surface of the silica layer by amide bond, and the ligand unit can be coordinated with metal ions connection.The nano magnetic bead in the application is modified with ligand molecule, and the ligand molecule can form coordination bond with metal ions, so that the nano magnetic bead surface has a large number of positive charges, ligand molecule itself is neutral or has negative electricity, and after losing coordinated metal ions, the surface of nano magnetic bead can be neutral or have a large number of negative charges, so the nano magnetic bead in the application can be adsorbed and eluted by changing ligand molecule to metal ions, so as to realize the purpose of adjusting its surface potential.

Description

Technical Field

[0001] The present invention relates to the field of magnetic beads, and in particular to nano magnetic beads with adjustable surface potential, a preparation method and application thereof. Background Art

[0002] Environmental nucleic acids (eNA) generally refer to the sum of nucleic acid fragments directly extracted from environmental samples (such as water, soil, and air). They are widely used in biodiversity research, tracking invasive and endangered species, detecting drug-resistance genes, and pathogen control. Compared to traditional methods (such as field surveys and clinical testing), eNA offers advantages such as non-invasiveness, low bias, and high efficiency and cost-effectiveness. However, environmental samples are often complex in composition and low in nucleic acid content, often requiring large sample volumes (100mL-10L) for subsequent analysis.

[0003] Currently, the commonly used method for extracting eNA from surface water samples (such as rivers, lakes, and oceans) is filtration. After filtering a 500-10,000 mL surface water sample through a 0.22 μm filter membrane, the filter membrane is collected in a centrifuge tube. Lysis buffer and proteinase K are added to fully release the nucleic acids on the filter membrane into the lysis buffer, and then the nucleic acids are purified and enriched. However, filtration is a complex and time-consuming method, with long sample processing and extraction times. It is not suitable for large-scale, high-frequency sampling applications, such as monitoring for drug-resistant genes and pathogenic microorganisms in aquatic environments. In addition, using the membrane filter method to extract nucleic acids can lead to the loss of some biological information. Studies have found that even after filtration with a 0.22 μm filter membrane, a considerable amount of eNA is still present in the filtrate. The nucleic acids in the filtrate are defined as "dissolved nucleic acids" and are primarily present in the form of membrane-encapsulated nucleic acids (such as bacteria smaller than 0.22 μm, cell debris, and extracellular vesicles) as well as small viral particles. Since "soluble nucleic acids" have a low concentration and are difficult to be retained by conventional filter membranes, and the sample volume is large, complex and time-consuming extraction techniques are required, such as precipitation, ultra-high-speed centrifugation, and ultrafiltration, which limits their application in research and practice. Commonly used methods for enriching nucleic acids in liquid samples in laboratories include precipitation, column extraction, and traditional magnetic bead methods. The extraction processes of these methods are often complex and time-consuming. At the same time, since a large amount of solvent needs to be added to the water sample during the nucleic acid extraction process (often 1-2 times the volume of the water sample), it is not suitable for the enrichment of nucleic acids in large-volume liquid samples (greater than 10 mL), such as urine samples and environmental water samples.

[0004] Current nucleic acid extraction technologies for large-volume liquid samples (greater than 10 mL) include PEG precipitation, filtration, and centrifugation. However, these technologies have the following technical limitations, limiting their large-scale use:

[0005] (1) The operation is complex, time-consuming and costly

[0006] Currently, commonly used techniques are complex and time-consuming. From sample pretreatment to nucleic acid elution and enrichment, it often takes 12-24 hours. They also require expensive equipment such as high-speed / ultra-high-speed centrifuges and ultrafiltration vacuum pumps, increasing costs and limiting their large-scale application.

[0007] (2) Low nucleic acid recovery rate, low enrichment, and bias

[0008] Currently, commonly used technologies have low recovery rates for free nucleic acids in samples (for example, membrane filtration and ultra-high-speed centrifugation methods have difficulty retaining free nucleic acids). Furthermore, due to equipment limitations (for example, the centrifuge's low capacity, the filter membrane can become clogged by impurities in the water, and large water sample volumes require frequent membrane replacement, which is cumbersome), it is difficult to increase the volume of water samples processed. Furthermore, to meet elution efficiency requirements, a large amount of eluent must be used (for example, the membrane filtration method often requires 400-1000 μL of eluent to improve nucleic acid elution efficiency). This makes it difficult to increase the degree of nucleic acid enrichment (generally, it can only enrich tens to hundreds of times), making low-concentration target nucleic acids difficult to detect. Furthermore, different technologies have certain biases in nucleic acid extraction. For example, membrane filtration and centrifugation methods have high extraction rates for intracellular nucleic acids, but low extraction rates for viral nucleic acids and free nucleic acids, affecting their applicability in different research fields. Summary of the Invention

[0009] In order to overcome at least one technical problem existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a nanomagnetic bead.

[0010] A second object of the present invention is to provide a method for preparing nanomagnetic beads.

[0011] A third object of the present invention is to provide a method for extracting negatively charged biomacromolecules.

[0012] A fourth object of the present invention is to provide an application of the above-mentioned method for extracting nanomagnetic beads and / or negatively charged biomacromolecules in the field of biological detection.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides a nanomagnetic bead, comprising a silica layer and a ligand unit coated on the surface of the magnetic bead core; the ligand unit is loaded on the surface of the silica layer via an amide bond; and the ligand unit can coordinate with a metal ion.

[0015] Preferably, the ligand unit is selected from At least one of .

[0016] Preferably, the ligand unit is selected from

[0017] A polymer material having at least one functional group.

[0018] The “*” in the above structural formula refers to the position where the group is connected.

[0019] The above-mentioned ligand units can all be modified on the silica layer of the nanomagnetic beads through an amidation reaction between amino and carboxyl groups, and can also form coordination bonds with different metal ions, thereby increasing the positive potential on the surface of the magnetic beads.

[0020] In some embodiments of the present invention, the ligand unit itself is neutral or negatively charged, and the surface of the ligand unit can be loaded with cations.

[0021] Preferably, the nanomagnetic beads further contain metal ions, and coordination bonds are formed between the metal ions and the ligand units.

[0022] Preferably, the metal ion is selected from Al 3+ 、Fe 3+ Sc 3+ 、Co 2+ 、Co 3+ 、Ni 2+ 、Cu 2+ Mg 2+ , Pt 2+ At least one of .

[0023] Preferably, the magnetic bead core is selected from at least one of ferroferric oxide magnetic bead cores, nickel oxide magnetic bead cores, and cobalt oxide magnetic bead cores.

[0024] When metal ions are present, the surface of the nanomagnetic beads of the present invention has a high positive charge and a high adsorption capacity for nucleic acids. The nucleic acid extraction rate is close to 100%, and the degree of nucleic acid enrichment can reach 100-1000 times. After the nucleic acids are adsorbed on the surface of the nanomagnetic beads, they can be eluted by an eluent. The elution efficiency is high, the elution effect is good, the elution speed is fast, the eluent has no destructive effect on the nucleic acids, and the extracted nucleic acids can be directly used for detection and analysis.

[0025] The present invention modifies nanomagnetic beads with ligand molecules. These molecules form coordination bonds with metal ions, stably binding the metal ions to the bead surface. This imparts a positive charge to the bead surface in an aqueous environment, allowing it to adsorb negatively charged biomacromolecules such as nucleic acids, proteins, phospholipid membranes, cells, bacteria, viruses, and vesicles. Adding an eluent containing a high concentration of chelating agents and anions to the system can compete with the metal ions on the bead surface, restoring the nanomagnetic bead surface to its previous neutral or negative charge, thereby achieving efficient elution of the biomacromolecules.

[0026] The second aspect of the present invention provides a method for preparing the nanomagnetic beads according to the first aspect of the present invention, comprising the following steps:

[0027] S1: Mixing amino-containing magnetic silica beads, ligand molecules, 1,3-diisopropylcarbodiimide, and N,N-diisopropylethylamine to obtain coordinated nanomagnetic beads;

[0028] S2: mixing the coordinated nanomagnetic beads with a solution containing metal ions to react to obtain the nanomagnetic beads.

[0029] In the present invention, 1,3-diisopropylcarbodiimide and N,N-diisopropylethylamine both participate in the amidation reaction of amino groups and carboxyl groups. The role of 1,3-diisopropylcarbodiimide is to activate the amino groups on the amino-containing magnetic beads and silica balls, making it easier for them to react with the carboxyl groups in the ligand molecules to form amide bonds. N,N-diisopropylethylamine, as an acid-binding agent, can improve the efficiency of the reaction between the amino-containing magnetic beads and silica balls and the ligand molecules.

[0030] Preferably, the ligand molecule is selected from

[0031] or at least one of the polymers having the above ligand molecules as side chain functional groups.

[0032] Preferably, the pH of the solution containing metal ions is 2-7.

[0033] Preferably, the method for preparing the amino-containing magnetic bead silicon spheres comprises the following steps:

[0034] A1: Mix magnetic metal salt, acetate, and ethylene glycol, heat the mixture, and separate the magnetic bead cores by magnetic attraction.

[0035] A2: mixing the magnetic bead core with ammonia water and tetraethoxysilane to react so that the surface of the magnetic bead core is coated with a silicon dioxide layer;

[0036] A3: Then, the mixture is mixed with 3-aminopropyltriethoxysilane to react to obtain the amino-containing magnetic bead silica spheres.

[0037] Preferably, the magnetic metal salt is selected from at least one of iron salts, nickel salts and cobalt salts.

[0038] Preferably, the temperature rising reaction in step A1 is specifically as follows: firstly, the temperature is raised to 190-210°C at a heating rate of 80-90°C / h, kept at that temperature for 2-12h, and then the temperature is lowered to 20-40°C at a cooling rate of 80-90°C / h.

[0039] A third aspect of the present invention provides a method for extracting negatively charged biomacromolecules, comprising the following steps:

[0040] (1) mixing the nanomagnetic beads with a test sample, allowing negatively charged biomacromolecules in the test sample to be adsorbed on the surface of the nanomagnetic beads, and separating the adsorbed nanomagnetic beads;

[0041] (2) mixing the adsorbed nanomagnetic beads with an eluent and eluting the mixture to desorb the negatively charged biomacromolecules from the surface of the nanomagnetic beads;

[0042] The eluent contains anions.

[0043] The nanomagnetic beads described in the first aspect of the present invention contain metal ions, which make the surface charge of the nanomagnetic beads positive. Nanomagnetic beads with positive surface charge and metal coordination are used to adsorb negatively charged biomacromolecules such as nucleic acids, proteins, phospholipid membranes, cells, viruses, bacteria, and vesicles in water samples through electrostatic adsorption interaction. The present invention modifies the surface of the nanomagnetic beads with ligand molecules, and through the coordination of the ligand molecules and the metal ions, the surface of the magnetic beads is positively charged in a liquid environment, thereby achieving adsorption of biomacromolecules such as nucleic acids. After the nucleic acid is adsorbed, an eluent is added. The chelating agent and anion in the eluent compete with the ligands on the magnetic beads for the metal ions, thereby reducing the surface potential of the magnetic beads and improving the elution ability of the nucleic acid.

[0044] Preferably, the negatively charged biomacromolecules include at least one of nucleic acids, proteins, phospholipid bilayers, cells, bacteria, viruses, and vesicles.

[0045] Preferably, the nucleic acid comprises DNA, RNA or PNA.

[0046] Preferably, the eluent further contains a chelating agent and a cell lysis agent, wherein the concentration of the chelating agent is 0.02 to 0.2 mol / L, and the concentration of the anion is 0.05 to 0.2 mol / L; based on the total mass percentage of the eluent as 100%, the mass percentage of the cell lysis agent in the eluent is 0.1 to 2%, and the pH of the eluent is 6.5 to 10.

[0047] Preferably, the eluent further contains a buffer. The buffer in the present invention is used to stabilize the pH of the eluent.

[0048] Preferably, the buffer comprises TE buffer.

[0049] Preferably, the concentration of Tris(hydroxymethyl)aminomethane in the TE buffer is 8 to 12 mmol / L.

[0050] Preferably, the chelating agent contains at least one of ethylenediaminetetraacetic acid (EDTA), salicylate, and oxalate.

[0051] Preferably, the cell lysis agent is selected from at least one of sodium dodecyl sulfate (SDS), protease, and cell wall-breaking enzyme.

[0052] Preferably, the anion is selected from PO4 3- 、SO4 2- 、F - 、Cl - At least one of .

[0053] Preferably, the mixed elution temperature is 60-100°C; further preferably, the mixed elution temperature is 70-90°C.

[0054] Preferably, the mixed elution time is 20 to 60 minutes; further preferably, the mixed elution time is 20 to 40 minutes.

[0055] The fourth aspect of the present invention provides the application of the nanomagnetic beads described in the first aspect of the present invention and / or the method for extracting negatively charged biomacromolecules described in the third aspect of the present invention in the field of biological detection.

[0056] The beneficial effects of the present invention are as follows: the surface of the nanomagnetic beads in the present invention is modified with ligand molecules, which can form coordination bonds with metal ions, so that the surface of the nanomagnetic beads has a large amount of positive charge. The ligand molecules themselves are neutral or negatively charged. After losing the coordinated metal ions, the surface of the nanomagnetic beads can be made neutral or have a large amount of negative charge. Therefore, the nanomagnetic beads in the present invention can achieve the purpose of adjusting their surface potential by changing the adsorption and elution of metal ions by the ligand molecules.

[0057] After the nanomagnetic beads in the present invention adsorb metal ions, their surface carries a large amount of positive charge, making them suitable for the extraction of biological macromolecules such as nucleic acids or substances with negatively charged surfaces from different types of liquid samples (including blood, urine, wastewater, seawater, etc.) of different volumes (10μL to 10L). No additional solvent is required during the nucleic acid extraction process. Simply add a few milligrams of magnetic beads to the original sample (10mL-1L) and incubate for 5-30 minutes to achieve nearly 100% nucleic acid adsorption.

[0058] When using the nanomagnetic beads of the present invention to extract negatively charged biomacromolecules, the entire extraction process can be completed in 1-2 hours, significantly less than currently used nucleic acid extraction methods such as membrane filtration, ultracentrifugation, and PEG precipitation. Furthermore, the nucleic acid elution conditions employed in the present invention are mild, without affecting nucleic acid properties. Furthermore, the eluate composition has been systematically optimized, allowing it to be directly used in subsequent conventional nucleic acid analyses such as Qubit, PCR, and sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 FTIR images of the nanomagnetic beads with adjustable surface potential in Examples 1 to 4.

[0060] Figure 2 This is a surface potential test diagram of the nanomagnetic beads used for nucleic acid extraction in Example 5.

[0061] Figure 3 Schematic diagram of the extraction mechanism of nucleic acids, microorganisms, etc. by nanomagnetic beads in Example 5.

[0062] Figure 4 This is a schematic diagram of the process of extracting nucleic acids using nanomagnetic beads in Example 5.

[0063] Figure 5 These are SEM images of the QL magnetic beads, Al(III)-coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution in the examples of the present invention.

[0064] Figure 6 These are EDS test images of the QL magnetic beads, Al(III) coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution in the embodiments of the present invention.

[0065] Figure 7 These are surface potential test diagrams of the QL magnetic beads, Al(III)-coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution in the embodiments of the present invention.

[0066] Figure 8 Schematic diagram of the charge change of the QL magnetic beads coordinated with Al(III) and after Al(III) elution in an embodiment of the present invention.

[0067] Figure 9 This is a test diagram of nucleic acid enrichment and recovery using Fe(III) coordinated LF magnetic beads in an embodiment of the present invention.

[0068] Figure 10 This is a test diagram of nucleic acid enrichment and recovery after the nanomagnetic beads in Examples 1 to 4 of the present invention are coordinated with Al ions.

[0069] Figure 11 This is a test diagram during the optimization process of the eluent composition and elution conditions of the eluent in the embodiment of the present invention.

[0070] Figure 12 This is a test diagram of the extraction bias of Al(III)-coordinated QL magnetic beads for different nucleic acid fragments in the example and an SEM image after adsorption of viruses and bacteria.

[0071] Figure 13 This is a test chart of daily monitoring of antibiotic resistance genes in different environmental water samples using Al(III) coordinated QL magnetic beads in the example.

[0072] Figure 14 This is a test diagram for screening antibiotic resistance genes in the environment using Al(III)-coordinated QL magnetic beads and metagenomic technology in the example.

[0073] Figure 15 This is a test diagram of the nanomagnetic beads in an embodiment of the present invention when used for biodiversity surveys and tracking endangered / harmful species.

[0074] Figure 16 This is a test diagram of the nanomagnetic beads in an embodiment of the present invention being used for identifying and screening drug-resistant genes in surface water and effluent from sewage treatment plants.

[0075] Figure 17 This is a diagram of the use of nanomagnetic beads in an embodiment of the present invention for daily monitoring of antibiotic resistance genes in surface water. DETAILED DESCRIPTION

[0076] The specific implementation of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0077] Example 1

[0078] This example provides a method for preparing nanomagnetic beads with adjustable surface potential. The specific steps are as follows:

[0079] (1) Synthesis of amino magnetic beads

[0080] First, ferroferric oxide magnetic beads were synthesized by a hydrothermal method, and the specific steps were as follows: a. Fix a 500 mL three-necked flask on a magnetic stirring stand, quickly weigh 5.4 g of ferric chloride hexahydrate (FeCl3.6H2O) and pour it into the three-necked flask; b. Measure 200 mL of ethylene glycol solution and pour it into the three-necked flask in step a, adjust the stirrer speed to 300 rpm, and stir for 30 minutes; c. Weigh 14.4 g of sodium acetate and pour it into the three-necked flask in step b, continue stirring for 30 minutes, and small bubbles will appear on the upper layer of the solution in the flask after stirring; d. Add the solution in the three-necked flask to the flask. Transfer to the reactor, tighten, and place in the oven; e. Set the oven temperature as follows: 0-2h: increase from 30℃ to 200℃; 2h-12h: 200℃; 12-14h: decrease from 200℃ to 30℃; f. After the reaction is completed, take out the reactor, cool to room temperature, transfer the solution in the reactor to a 50mL centrifuge tube, place it on a 50mL magnetic stand for magnetic adsorption, and discard the clarified liquid after clarification; g. Use ultrapure water for three times of shaking and ultrasonic cleaning, and anhydrous ethanol for three times of shaking and ultrasonic cleaning; h. After cleaning, place the magnetic beads in a clean room temperature to dry to obtain ferroferric oxide magnetic beads.

[0081] The magnetic beads synthesized at this time are easily oxidized and need to be coated with silicon, that is, a layer of silicon dioxide is synthesized on its surface. The specific steps are as follows: a. Weigh 7.5g of the ferroferric oxide magnetic beads prepared by the above method into a 250mL round-bottom flask, add 44.5mL of prepared 0.1mol / L hydrochloric acid, and ultrasonicate for 30min. During the hydrothermal period, change the water once; b. Transfer the magnetic bead solution in the flask to a 50mL centrifuge tube, dissolve it with ultrapure water, and shake and ultrasonicate for 3 times; c. Transfer the magnetic beads to a 1000mL round-bottom flask with 160mL of ultrapure water, add 640mL of anhydrous ethanol, and ultrasonicate the mixed magnetic bead solution in the flask for 10min ; d. Fix the flask on the stirrer and turn on the switch to stir. Adjust the stirrer speed to 300 rpm, add 8 mL of ammonium hydroxide solution to the flask in turn, and then slowly add 6 mL of tetraethoxysilane (TEOS) solution dropwise and react for 1 hour; e. After the reaction is completed, transfer the magnetic bead solution in the round-bottom flask to a 50 mL centrifuge tube, place it on a 50 mL magnetic stand, and after magnetic absorption, clarify and discard the clarified liquid; f. Use anhydrous ethanol to shake and ultrasonically clean it 3 times, ultrapure water to shake and ultrasonically clean it 3 times, and then use anhydrous ethanol to shake and ultrasonically clean it 2 times; g. After cleaning, place it in a clean place at room temperature to dry to obtain magnetic beads coated with a silica layer on the surface, which are recorded as silicon-coated magnetic beads.

[0082] Then, amino groups were modified on the silica-coated magnetic beads to obtain amino magnetic beads. The specific steps are as follows: a. 400 mg of silica-coated magnetic beads were placed in a 50 mL centrifuge tube; b. Ultrasonic cleaning was performed three times with anhydrous ethanol; c. The magnetic beads were transferred to a 250 mL round-bottom three-necked flask with 120 mL of anhydrous ethanol, with a total volume of 120 mL, and ultrasonicated again for 10 minutes; d. The round-bottom three-necked flask was fixed to an overhead stirrer for stirring, and 1.2 mL of 3-aminopropyltriethoxysilane was slowly added and stirred overnight; e. The amino magnetic bead reaction solution reacted overnight was collected, ultrasonically cleaned three times with anhydrous ethanol, and then ultrasonically cleaned three times with ultrapure water, rinsed once with anhydrous ethanol, and dried to obtain amino magnetic beads, which were recorded as AJ magnetic beads.

[0083] (2) Surface modification of amino magnetic beads

[0084] The ligand molecule 3,4-dihydroxyphenylpropionic acid was modified on the surface of amino magnetic beads. The specific modification method was as follows: a. 3,4-dihydroxyphenylpropionic acid was dissolved in N,N-dimethylformamide (DMF) with a concentration of 5 mmol / L, 1,3-diisopropylcarbodiimide (DIC, final concentration of 5 mmol / L) and N,N-diisopropylethylamine (DIPEA, final concentration of 10 mmol / L) were added, and stirred at room temperature for 4 hours; b. 100 mg of amino magnetic beads were added, stirred at room temperature for 8 hours, washed twice with DMF and water, washed once with ethanol, and dried at room temperature to obtain nanomagnetic beads with adjustable surface potential in this example, which were recorded as LF magnetic beads.

[0085] Example 2

[0086] The preparation method of the surface potential adjustable nanomagnetic beads in this example is different from that in Example 1 only in that the ligand molecule used in the surface modification of the amino magnetic beads in this example is terpyridine-4-carboxylic acid, and the surface potential adjustable nanomagnetic beads in this example are recorded as SD magnetic beads.

[0087] Example 3

[0088] The preparation method of the surface potential adjustable nanomagnetic beads in this example is different from that in Example 1 only in that the ligand molecule used in the surface modification of the amino magnetic beads in this example is 8-hydroxyquinoline-5-carboxylic acid, and the surface potential adjustable nanomagnetic beads in this example are recorded as QL magnetic beads.

[0089] Example 4

[0090] The preparation method of the surface potential-adjustable nanomagnetic beads in this example is different from that in Example 1 only in that the ligand molecule used in the surface modification of the amino magnetic beads in this example is 3,4-(benzo-12-crown-4)benzoic acid, and the surface potential-adjustable nanomagnetic beads in this example are recorded as GM magnetic beads.

[0091] The structural formulas of the ligand molecules used in Examples 1 to 4 are as follows:

[0092]

[0093] The FTIR images of the nanomagnetic beads with adjustable surface potential in Examples 1 to 4 were tested respectively. The specific test results are as follows: Figure 1 As shown by Figure 1 It can be seen that the surface potential of the nanomagnetic beads with adjustable surface potential in Examples 1 to 4 is 1035 cm -1 and 588cm -1 There are strong characteristic absorption peaks of Si-O-Si bond and Fe-O bond at 1650cm -1 The characteristic absorption peaks of aromatic rings are generated on the left and right, proving that the ligand molecules are successfully modified on the surface of the magnetic beads.

[0094] Example 5

[0095] This example provides a nanomagnetic bead for nucleic acid extraction, and the specific preparation steps are as follows:

[0096] The surface potential adjustable nanometer magnetic beads in examples 1-4 are respectively incubated with metal ions to make the surface of the nanometer magnetic beads positive, and the specific process is as follows: a. metal chloride compounds (FeCl3, AlCl3, ScCl3, CoCl2) are dissolved in water (concentration is 25 mmol / L), and sodium hydroxide solution is used to adjust to appropriate pH to obtain different metal ion solutions with different pH, and the specific pH is as follows: FeCl3 solution with pH 2, FeCl3 solution with pH 3, FeCl3 solution with pH 4, AlCl3 solution with pH 5, AlCl3 solution with pH 6, AlCl3 solution with pH 7, ScCl3 solution with pH 4, ScCl3 solution with pH 5, ScCl3 solution with pH 6, CoCl3 solution with pH 4, and CoCl3 solution with pH 5; b. 10 mL of the above metal ion solution with adjusted pH is taken, 100 mg of ligand magnetic beads is added, and incubation is carried out at room temperature for 24 hours to obtain a series of magnetic beads with different surface potentials, which are the nanometer magnetic beads for nucleic acid extraction in the example, and then the surface potential of the nanometer magnetic beads for nucleic acid extraction in the example is tested, and in order to facilitate comparison, the surface potential of the nanometer magnetic beads in examples 1-4 is tested as a control, and the specific test results are shown in Figure 2 , and it can be known from Figure 2 that the surface potential adjustable nanometer magnetic beads in the application can obtain nanometer magnetic beads with positive surface charges by incubation with different metal ion solutions, and the surface potential of the nanometer magnetic beads can be adjusted by adjusting the type of metal ions and the pH of the solution during incubation.

[0097] In the process of co-incubation of the surface potential adjustable nanometer magnetic beads in the application with metal ions, the metal ions are loaded on the surface of the surface potential adjustable nanometer magnetic beads to make the surface of the surface potential adjustable nanometer magnetic beads positive, and then the nucleic acid, microorganism and the like with negative surface charges in the environmental water sample are adsorbed on the surface of the nanometer magnetic beads through the electrostatic adsorption of positive and negative charges, and under the elution of the eluent, the metal ions adsorbed on the surface of the nanometer magnetic beads are desorbed, the surface of the nanometer magnetic beads is negative, and due to the repulsion between negative charges, the nucleic acid, microorganism and the like are detached from the surface of the nanometer magnetic beads, so that the extraction of the nucleic acid and microorganism is realized, and the specific principle diagram is shown in Figure 3 .

[0098] The LF magnetic beads in example 1 are incubated with FeCl3 solution to make the surface of the LF magnetic beads positive, and the specific process is as follows: a. FeCl3 is dissolved in water (concentration is 25 mmol / L), and sodium hydroxide solution is used to adjust pH to 4, 5 and 6 respectively to obtain FeCl3 solutions with different pH, b. 10 mL of the above FeCl3 solution with adjusted pH is taken, 100 mg of the LF magnetic beads in example 1 is added, and incubation is carried out at room temperature for 24 hours to obtain Fe(III) coordination LF magnetic beads.

[0099] The nanomagnetic beads in Examples 1 to 3 were respectively incubated with AlCl3 solution to make their surfaces positively charged. The specific process is as follows: a. AlCl3 was dissolved in water (concentration of 25 mmol / L), and the pH was adjusted to 6 with sodium hydroxide solution to obtain AlCl3 solution. b. 10 mL of the above-mentioned pH-adjusted AlCl3 solution was taken, and 100 mg of the nanomagnetic beads in Examples 1 to 3 were added respectively. The mixture was incubated at room temperature for 24 hours to obtain Al(III) coordinated LF magnetic beads, Al(III) coordinated SD magnetic beads, Al(III) coordinated QL magnetic beads, and Al(III) coordinated GM magnetic beads, respectively.

[0100] The nanomagnetic beads in Example 1 were incubated with a scandium trichloride solution to make their surface positively charged. The specific process was as follows: a. ScCl3 was dissolved in water (concentration was 25 mmol / L), and the pH was adjusted to 6 with sodium hydroxide solution to obtain a ScCl3 solution. b. 10 mL of the pH-adjusted ScCl3 solution was taken, and 100 mg of the nanomagnetic beads in Example 1 were added respectively. The mixture was incubated at room temperature for 24 hours to obtain Sc(III)-coordinated LF magnetic beads.

[0101] Example 6

[0102] This example provides a method for extracting nucleic acids. The specific steps are as follows:

[0103] Reference Figure 4 Schematic diagram of the nucleic acid extraction process in the example of surface water sample (lake water). Take 100mL of lake water in a glass bottle, add 5mg of Al(III) coordinated QL magnetic beads (separate the Al(III) coordinated QL magnetic beads from the aluminum chloride storage solution before use and wash twice with ultrapure water), shake and incubate on a shaker at room temperature for 30 minutes, use a magnetic stand to collect the magnetic beads and transfer them to a 1.5mL centrifuge tube, and discard all the supernatant. Add 100μL of eluent (the concentration of ethylenediaminetetraacetic acid in this eluent is 0.1mol / L, PO4 3- The concentration of 0.1 mol / L and the mass percentage of sodium dodecyl sulfate are 2%. It is prepared by dissolving EDTA, disodium hydrogen phosphate, and sodium dodecyl sulfate in 1× TE buffer (the eluent has a pH of 7.5). The solution is then incubated in a water bath at 80°C for 30 minutes to elute the nucleic acids from the magnetic beads. The magnetic beads obtained after elution are designated Al(III)-eluted QL magnetic beads. After incubation, the magnetic beads are separated from the eluent by magnetic attraction, and the eluent is collected in a new centrifuge tube. The nucleic acids are now present in the eluent and can be used for subsequent analysis.

[0104] Scanning electron microscopy was used to test the surface morphology of QL magnetic beads, Al(III) coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution. The specific test results are as follows: Figure 5As shown by Figure 5 It can be seen that the surface morphology of the Al(III) coordinated QL magnetic beads is quite different from that of the original QL magnetic beads, while the surface morphology of the QL magnetic beads after Al(III) elution is consistent with that of the original QL magnetic beads, indicating that the elution effect of the eluent is obvious.

[0105] The EDS data of AJ magnetic beads, QL magnetic beads, Al(III) coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution were tested respectively. The specific test results are as follows Figure 6 As shown by Figure 6 It can be seen that after the QL magnetic beads were incubated with AlCl3 solution, the Al content in the QL magnetic beads was significantly increased, while after Al(III) elution, the Al content in the QL magnetic beads was significantly reduced after elution with the eluent, which was equivalent to the Al content in the QL magnetic beads.

[0106] The surface potentials of AJ magnetic beads, QL magnetic beads, Al(III) coordinated QL magnetic beads, and QL magnetic beads after Al(III) elution were tested respectively. The specific test results are as follows: Figure 7 As shown by Figure 7 It can be seen that after the QL magnetic beads are incubated with AlCl3 solution, the surface of the QL magnetic beads carries a large amount of positive charge, which can be used to adsorb nucleic acids. After Al(III) is eluted, the potential on the surface of the QL magnetic beads turns to a negative value after being eluted with the eluent, and the QL magnetic beads carry a negative charge. This further shows that the nanomagnetic beads in the present invention have the characteristic of adjustable surface potential and can be used for the adsorption and desorption of nucleic acids. After incubation with AlCl3, the surface of the Al(III)-coordinated QL magnetic beads carries a positive charge. When eluted with the eluent, the surface of the QL magnetic beads carries a negative charge after Al(III) is eluted. Specifically, Figure 8 shown.

[0107] Use a commercial kit to extract genomic DNA from cells, and use Qubit to detect the DNA concentration in the extract. The DNA concentration in the initial solution is 40ng / μL. Take 100μL of the initial solution in three 1.5mL centrifuge tubes, and then add 0.5mg of Fe(III) coordinated LF magnetic beads, Al(III) coordinated LF magnetic beads, and Sc(III) coordinated LF magnetic beads respectively. Incubate on a shaker at room temperature for 30 minutes, collect the magnetic beads using a magnetic stand, and discard all the supernatant. Add 100μL of elution solution (the concentration of ethylenediaminetetraacetic acid in the elution solution is 25mmol / L, PO4 3- The concentration of 0.1 mol / L and the mass percentage of sodium dodecyl sulfate are 0.1%, which is prepared by dissolving ethylenediaminetetraacetic acid, phosphate, and sodium dodecyl sulfate in 1×TE buffer (pH of the eluent = 7.5), incubating in a water bath at 80°C for 30 minutes, and then testing the DNA concentration in the eluate and supernatant respectively. The specific test results are as follows: Figure 9As shown. Figure 9 It can be seen that compared with LF nanomagnetic beads coordinated by other metal ions, Al(III) coordinated LF magnetic beads have better nucleic acid adsorption and elution effects.

[0108] Use cell genomic DNA extract to prepare a test sample with a DNA concentration of 10 ng / μL as the initial solution. Add 100 μL of the initial solution to 5 1.5 mL centrifuge tubes, and then add 0.5 mg of nanomagnetic beads (AJ magnetic beads, Al (III) coordinated LF magnetic beads, Al (III) coordinated SD magnetic beads, Al (III) coordinated QL magnetic beads, Al (III) coordinated GM magnetic beads). Incubate on a shaker at room temperature for 30 minutes, collect the magnetic beads using a magnetic stand, and discard all the supernatant. Add 100 μL of elution solution (the concentration of ethylenediaminetetraacetic acid in the elution solution is 25 mmol / L, PO4 3- The concentration of 0.1 mol / L and the mass percentage of sodium dodecyl sulfate are 0.1%, which is prepared by dissolving ethylenediaminetetraacetic acid, phosphate, and sodium dodecyl sulfate in 1×TE buffer (pH of the eluent = 7.5), incubating in a water bath at 80°C for 30 minutes, and then testing the DNA concentration in the eluate and supernatant respectively. The specific test results are as follows: Figure 10 As shown. Figure 10 It can be seen that compared with other nanomagnetic beads, Al(III) coordinated QL magnetic beads have better adsorption and elution effects on nucleic acids, further proving that better nucleic acid extraction effects can be achieved by modifying nanomagnetic beads with 8-hydroxyquinoline-5-carboxylic acid ligand molecules and Al(III) ions.

[0109] In order to further optimize the elution conditions, the present invention optimized the temperature, pH, chelating agent and anion type during elution. The specific test method is as follows: according to the nucleic acid extraction method described above, Al(III) coordinated QL magnetic beads are used for extraction, and then different eluents are used for elution under different conditions. First, the present invention attempts to elute nucleic acids using different concentrations of anion and chelating agent solutions alone, including 10-200mmol / L salicylic acid ( Figure 11 a) 10-200mmol / L oxalate ( Figure 11 b) 10-200mmol / L fluoride ion ( Figure 11 c) 1-200mmol / L EDTA ( Figure 11 d) and 50-500mmol / L phosphate ( Figure 11 e), the specific test results are as follows Figure 11As shown in (ae), the eluent containing 200mmol / L phosphate ions has a better elution effect on nucleic acids. Different concentrations of EDTA and 0.1mol / L phosphate were used as eluent components to elute nucleic acids adsorbed on nanomagnetic beads. The elution rate test results are shown in Figure 2. Figure 11 As shown in (f), Figure 11 (f) It can be seen that when 200mmol / L EDTA is used in combination with phosphate, the elution effect is better than when EDTA or phosphate is used alone. The elution rate test results are shown in Figure 2. Figure 11 (g) shows that Figure 11 (g) It can be seen that the elution efficiency of adsorbed nucleic acid is the highest under the elution condition of 80℃. Elution was performed using elution buffers (containing 200mmol / L EDTA and 100mmol / L phosphate) with different pH values ​​(6.5, 7, 8, 9). The elution rate test results are shown in Figure 2. Figure 11 As shown in (h), Figure 11 (h) It can be seen that under the elution condition of pH = 7, the elution efficiency of adsorbed nucleic acids is the highest.

[0110] Al(III)-coordinated QL magnetic beads were used to adsorb and elute different forms of nucleic acids (including free nucleic acids of different fragment sizes, nucleic acids wrapped in virus shells, and nucleic acids inside bacteria). The specific test results are as follows: Figure 12 As shown in Table 3. Al(III)-coordinated QL magnetic beads were used to extract DNA of different fragment sizes, and the DNA concentrations of different fragments in the original solution, the supernatant after adsorption, and the eluate were detected by gel electrophoresis. The results are shown in Table 3. Figure 12 (A) As shown. Then the SEM images of the Al(III)-coordinated QL magnetic beads after adsorption of viruses and bacteria were tested respectively. Figure 12 (B) and Figure 12 (C), and qPCR was used to detect the concentrations of bacteria and viruses in the original solution and the eluate, see Table 3. Figure 12 As shown in Table 3, the nanomagnetic beads of the present invention have a good recovery effect on DNA of different fragment sizes (45-2000 bp), and have a strong adsorption capacity for viruses and bacteria, with a recovery rate close to 100% (Table 3).

[0111] It can be seen that the nanomagnetic beads for nucleic acid extraction in the present invention can enrich nucleic acids from large volume liquid samples without adding any additional solvent, and are easy to elute, and can achieve 100% adsorption efficiency of nucleic acids ( Figure 10 ) and 90% elution efficiency ( Figure 11(h)), can achieve 100-1000 times nucleic acid enrichment (the Ct value of free nucleic acid in the eluate decreased by about 9.5 compared with that in the initial solution in Table 1, which means that the nucleic acid in the eluate was enriched by about 700 times), and is applicable to different fragment lengths ( Figure 12 ), different forms of DNA extraction (Table 1, Figure 12 ), the operation process and elution buffer composition are gentle and do not affect the properties of nucleic acids. The extracted nucleic acids can be directly used for subsequent routine analyses, such as Qubit, PCR, sequencing, etc.

[0112] Environmental water is an important transmission medium for drug-resistant pathogens and antibiotic resistance genes (ARGs). Daily monitoring of ARGs and drug-resistant bacteria (ARB) in water bodies is of great value for assessing regional ARGs risks and ARGs prevention and control. The surface potential-adjustable nanomagnetic beads of the present invention are suitable for the rapid extraction of bacteria and their nucleic acids in environmental water samples. The surface potential-adjustable nanomagnetic beads are incubated with metal ions to make the surface of the nanomagnetic beads positively charged, and can efficiently adsorb free nucleic acids, viruses, and bacteria in water bodies through electrostatic interactions. In the verification test, we added known concentrations of free nucleic acids, virus particles and bacteria to different types of environmental water samples (surface water, laboratory building wastewater, teaching building wastewater, etc.), enriched and extracted them using the Al(III)-coordinated QL magnetic beads of the present invention, and used the corresponding qPCR primers to detect the concentrations of free nucleic acids, bacteria and viruses in the initial solution and the extract. The specific test results are shown in Table 1 below.

[0113] Table 1 qPCR detection results of free nucleic acids, virus particles, and bacteria-related nucleic acids in different environmental water samples

[0114]

[0115] The Ct values ​​in Table 1 are the results of qPCR testing. A decrease of 1 in the Ct value indicates a doubling of the nucleic acid concentration. A decrease of x in the Ct value indicates a x-fold increase in concentration. Sd refers to the standard deviation of multiple replicate test results.

[0116] As shown in Table 1, the Al(III)-coordinated QL magnetic beads of the present invention have a significant enrichment capacity for nucleic acids, viruses, and bacteria in different types of environmental water samples, with enrichment levels reaching 100-1000 times. Compared with traditional methods for enriching nucleic acids in environmental water (such as membrane filtration, which has a low retention rate for free nucleic acids and is complex and time-consuming, requiring 12 to 24 hours), the entire process is simpler and faster (1-2 hours), without the need for special equipment such as centrifuges and vacuum pumps. It can be used for nucleic acid extraction in the environment and is therefore useful in applications such as environmental biodiversity surveys, research on endangered invasive species, and routine monitoring of drug-resistant bacteria and ARGs.

[0117] In order to verify the feasibility of the nanometer magnetic beads in the ARGs background investigation and daily monitoring in the application, we regularly collected environmental water samples for different application scenarios (surface rivers, sewage plant effluent, seawater, and mariculture wastewater), extracted the ARGs therein using the Al(III) coordination QL magnetic beads in the application according to the method described above, and detected using qPCR. The specific test results are shown in FIGS. 1-3. Figure 13 As shown in FIGS. 1-3, Figure 13 (A) is a daily monitoring diagram of antibiotic resistance genes in a community lake; Figure 13 (B) is a daily monitoring diagram of antibiotic resistance genes in a river section of a domestic sewage treatment plant effluent; Figure 13 (C) is a monitoring diagram of antibiotic resistance genes in seawater and mariculture wastewater. Figure 13 As can be seen from FIGS. 1-3, the nanometer magnetic beads in the application are convenient and fast in the whole process of extraction and detection of antibiotic resistance genes, and meet the large-scale and high-frequency daily monitoring work of large-volume environmental water samples.

[0118] In addition, we analyzed the environmental nucleic acids enriched by the Al(III) coordination QL magnetic beads in the application using metagenomic technology, and compared with the integrated ARGs database to screen out the ARGs with the highest relative abundance and the most serious risk. The specific test results are shown in FIG. 4. Figure 14 As shown in FIG. 4, Figure 14 As can be seen from FIG. 4, the nanometer magnetic beads in the application have feasibility in ARGs background investigation and screening, and discovery of potential ARGs.

[0119] The commonly used nucleic acid extraction techniques (precipitation method, column extraction method, traditional magnetic bead method, etc.) need to add a large amount of high-salt solvent, which is not suitable for the enrichment and extraction of nucleic acids in large-volume samples. The filter membrane method and PEG precipitation method have the limitations of complex operation and time-consuming, and dependence on special equipment. Based on the metal ion exchange strategy, the nanometer magnetic beads with adjustable surface potential in the application are prepared by co-incubation with metal ions to obtain nanometer magnetic beads with positive surface charge. Through electrostatic interaction, the efficient adsorption of biological macromolecules and cells in large-volume water samples is realized. By changing the surface potential of the nanometer magnetic beads, efficient elution of the adsorbed molecules is realized, and the nucleic acid recovery rate reaches 90%. The nanometer magnetic beads in the application can also realize the efficient enrichment of nucleic acids in environmental water samples and urine samples. Combined with PCR or sequencing technology, it is successfully applied to different scenarios, for example:

[0120] (1) Liquid biopsy

[0121] Urine is an easily accessible biological sample, and the nucleic acids therein show important potential in disease diagnosis, monitoring and research. The known sequence DNA in environmental water samples and urine was extracted using the Al(III) coordination QL magnetic beads in the application according to the nucleic acid extraction and enrichment method described above. The specific test results are shown in Table 2.

[0122] Table 2 extraction results of DNA in environmental water samples and urine samples

[0123]

[0124] As can be seen from Table 2, the nano magnetic beads in the application can be used for enrichment extraction of free nucleic acid in large volume biological samples such as environmental water samples, urine and the like, and the enrichment multiple can reach more than 100 times; combined with PCR, sequencing and the like, it can be applied to non-invasive disease diagnosis, gene mutation detection and the like.

[0125] (2) Monitoring of pathogenic microorganisms in the environment

[0126] Research shows that monitoring pathogenic microorganisms in the water environment can accurately reflect the prevalence rate of viral lineage, and can discover new viral variants earlier than clinical samples. The pathogenic microorganisms and viruses in the environmental water samples are extracted by using the Al(III) coordination QL magnetic beads in the application according to the above nucleic acid extraction and enrichment method, and the specific extraction results are as shown in Table 3.

[0127] Table 3 extraction effect of pathogenic bacteria and viruses

[0128]

[0129] The nano magnetic beads in the application can be suitable for rapid extraction of pathogenic microorganism related nucleic acid in environmental water samples, and can be used for epidemiological daily investigation.

[0130] (3) Ecological investigation

[0131] Environmental DNA refers to the sum of nucleic acid fragments directly extracted from environmental samples (such as water, soil, air), and has wide application in the fields of biodiversity research, monitoring of invasive species, monitoring of endangered species and the like. The nano magnetic beads in the application can realize rapid enrichment extraction of water environmental DNA, help relevant personnel to carry out ecological investigation and research, including biodiversity investigation, monitoring of endangered / invasive species and the like, and the specific process is as shown in Figure 15 .

[0132] (4) Screening and daily monitoring of antibiotic resistance genes

[0133] The environment is an important medium for the spread of ARGs, and the wastewater discharged from hospitals, breeding farms, sewage treatment plants and the like is an important source of ARGs entering the environment. Comprehensive investigation and daily monitoring of ARGs in related wastewater and surface water are the premise for formulating management policies and controlling the spread of ARGs. The nano magnetic beads in the application can be used for identification and screening of drug resistance genes in surface water and effluent from sewage treatment plants, and the specific process is as shown in Figure 16 . The nano magnetic beads in the application can be used for screening and daily monitoring of antibiotic resistance genes in surface water, and the specific process is as shown in Figure 17As shown, the nanomagnetic beads of the present invention are easy to use for drug resistance gene identification and screening, enabling rapid in situ extraction of nucleic acids from large volumes of environmental water, meeting the needs of large-scale, high-frequency sample processing in routine ARGs monitoring. In previous studies, we enriched and extracted ARGs from various water bodies and, through sequencing and qPCR, enabled the screening and routine monitoring of major ARGs pollutants.

[0134] In summary, the nanomagnetic beads of the present invention have the following advantages:

[0135] (1) Wide range of applications

[0136] It can be applied to the extraction of biological macromolecules such as nucleic acids or substances with negatively charged surfaces from different types of liquid samples (including blood, urine, wastewater, seawater, etc.) of different volumes (10μL to 10L).

[0137] (2) No need to add additional solvent

[0138] Traditional magnetic bead-based nucleic acid extraction methods often require the addition of large amounts of highly concentrated salt solutions to help nucleic acids in liquid samples aggregate and adsorb onto the magnetic beads. This makes them difficult to apply to large-volume, low-concentration samples, such as urine or environmental water. The nanomagnetic beads of the present invention are capable of enriching and extracting nucleic acid molecules from liquid samples without the addition of any solvent. Experiments have shown that simply adding a few milligrams of magnetic beads to the original sample (10mL-1L) and incubating for 5-30 minutes can achieve nearly 100% nucleic acid adsorption.

[0139] (3) Quick and easy operation

[0140] The entire nucleic acid extraction process of the present invention can be completed within 1-2 hours (the specific time varies depending on the large volume of liquid sample), which is much lower than the currently commonly used environmental nucleic acid extraction methods such as membrane filtration, ultracentrifugation, and PEG precipitation.

[0141] (4) Mild elution conditions

[0142] The nucleic acid elution conditions during the nucleic acid extraction process of the present invention are mild and do not affect the properties of the nucleic acids. At the same time, the eluate components have been systematically optimized so that they can be directly used for subsequent conventional nucleic acid analysis, such as Qubit, PCR, sequencing, etc.

[0143] (5) Low cost, nanomagnetic beads can be reused

[0144] The present invention does not rely on expensive equipment, such as high-speed centrifuges, high-power vacuum pumps, etc. The nanomagnetic beads can be reused multiple times while ensuring high nucleic acid recovery efficiency, greatly reducing costs.

[0145] (6) High nucleic acid adsorption and elution efficiency

[0146] The amino magnetic bead method commonly used at present also uses electrostatic interaction to adsorb nucleic acid molecules in a liquid sample, which is similar to the principle of the present application, however, the adsorption and elution efficiency thereof is not as good as the surface potential adjustable nanometer magnetic bead used in the present application. The present application uses 1.25 μg of Al(III) coordination QL magnetic beads per μL to adsorb nucleic acid in an initial solution with a nucleic acid concentration of 49.67 ng / μL, and then tests the nucleic acid concentration in the supernatant after the magnetic beads are adsorbed and in the eluent, and calculates the adsorption capacity of the magnetic beads to the nucleic acid according to the following formula: (nucleic acid concentration in the initial solution - nucleic acid concentration in the supernatant after the magnetic beads are adsorbed) / magnetic bead dosage. The specific test results are shown in Table 4 below.

[0147] Table 4 Test data of adsorption capacity of Al(III) coordination QL magnetic beads to nucleic acid in water

[0148]

[0149]

[0150] As can be seen from Table 4, the adsorption capacity of the Al(III) coordination QL magnetic beads used in the present application to nucleic acid (including DNA and RNA) in water is as high as 18 ng of nucleic acid per μg of magnetic beads, which is much higher than that of the commonly used amino magnetic beads (1-2 ng of nucleic acid per μg of magnetic beads). The nucleic acid elution efficiency after adsorption is more than 90% (known from Figure 11 ).

[0151] (7) Different forms of nucleic acid can be recovered

[0152] The forms of nucleic acid in a liquid sample are complex, including free nucleic acid, intracellular nucleic acid, and adsorbed nucleic acid. The nucleic acid extraction method in the present application realizes efficient extraction of DNA and RNA of different fragment sizes, intracellular nucleic acid, viral nucleic acid, and free nucleic acid. In addition, the extraction efficiency of the method for nucleic acid in free form is excellent, and the problem of difficulty in recovering free nucleic acid in a large volume of water sample is solved.

[0153] (8) The extraction process does not produce any chemical waste liquid

[0154] The methods for extracting "soluble nucleic acid" (nucleic acid capable of passing through a 0.22 μm filter) in a large volume sample at present are mainly PEG method and coagulation precipitation method, both of which will produce harmful waste liquid (PEG waste liquid or metal ion waste liquid) equivalent to the volume of the liquid sample, which needs to be treated additionally. The present application does not need to add any chemical reagent to the liquid sample, and is green and environmentally friendly.

[0155] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A nanomagnetic bead, characterized in that: It comprises a magnetic bead core, a silicon dioxide layer coated on the surface of the magnetic bead core, and a ligand unit; the ligand unit is loaded on the surface of the silicon dioxide layer through an amide bond; the ligand unit can coordinate with the metal ion; The ligand unit is selected from 、 、 、 At least one of; The nanomagnetic beads also contain metal ions, which form coordination bonds with the ligand units; The metal ion is selected from Al 3+ 、Fe 3+ Sc 3+ 、Co 2+ 、Co 3+ At least one of; When the metal ion is Co 2+ or Co 3+ When the ligand unit is or ; The nanomagnetic beads are prepared by a preparation method comprising the following steps: S1: Mixing amino-containing magnetic silica beads, ligand molecules, 1,3-diisopropylcarbodiimide, and N,N-diisopropylethylamine to obtain coordinated nanomagnetic beads; S2: mixing the coordinated nanomagnetic beads with a solution containing metal ions to react to obtain the nanomagnetic beads; The pH of the solution containing metal ions is 3 to 7; The ligand molecule is selected from 、 、 、 At least one of .

2. The method for preparing the nanomagnetic beads according to claim 1, wherein: The following steps are involved: S1: Mixing amino-containing magnetic silica beads, ligand molecules, 1,3-diisopropylcarbodiimide, and N,N-diisopropylethylamine to obtain coordinated nanomagnetic beads; S2: mixing the coordinated nanomagnetic beads with a solution containing metal ions to react to obtain the nanomagnetic beads; The pH of the solution containing metal ions is 3-7.

3. A method for extracting negatively charged biomacromolecules, characterized in that: The following steps are involved: (1) mixing the nanomagnetic beads described in claim 1 with a sample to be tested, so that negatively charged biomacromolecules in the sample to be tested are adsorbed on the surface of the nanomagnetic beads, and separating the adsorbed nanomagnetic beads; (2) mixing the adsorbed nanomagnetic beads with an elution solution and eluting the mixture to desorb the negatively charged biomacromolecules from the surface of the nanomagnetic beads; The eluent contains anions; The negatively charged biomacromolecule is at least one of nucleic acid, bacteria, and virus.

4. The method for extracting negatively charged biomacromolecules according to claim 3, wherein: The eluent also contains a chelating agent and a cell lysis agent, wherein the concentration of the chelating agent is 0.02-0.2 mol / L and the concentration of the anion is 0.05-0.2 mol / L; based on the total mass percentage of the eluent as 100%, the mass percentage of the cell lysis agent in the eluent is 0.1-2%, and the pH of the eluent is 6.5-10.

5. The method for extracting negatively charged biomacromolecules according to claim 3, wherein: The mixed elution temperature is 60-100°C; And / or, the mixed elution time is 20 to 60 minutes.

6. Use of the nanomagnetic beads according to claim 1 and / or the method for extracting negatively charged biomacromolecules according to any one of claims 3 to 5 in the field of biological detection for purposes other than disease diagnosis.

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