Nanometer magnetic bead with adjustable surface potential as well as preparation method and application of nanometer magnetic bead

Abstract

The invention discloses a nano magnetic bead with adjustable surface potential and a preparation method and application thereof. The nano magnetic bead comprises a ferroferric oxide magnetic bead core, a silicon dioxide layer coating the surface of the ferroferric oxide magnetic bead core and a ligand unit, the ligand unit is loaded on the surface of the silicon dioxide layer through an amido bond, and the ligand unit can be in coordination connection with metal ions. The surface of the nano magnetic bead is modified with ligand molecules, the ligand molecules and metal ions can form coordinate bonds, so that the surface of the nano magnetic bead is provided with a large number of positive charges, the ligand molecules are neutral or are negatively charged, and after the metal ions losing coordination, the surface of the nano magnetic bead is neutral or is provided with a large number of negative charges, and therefore, the surface of the nano magnetic bead is neutral or is negatively charged. According to the nano magnetic bead, the purpose of adjusting the surface potential of the nano magnetic bead can be achieved by changing adsorption and elution of ligand molecules to metal ions.

Description

Technical Field

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

[0002] Environmental nucleic acid (eNA) generally refers to the sum of nucleic acid fragments directly extracted from environmental samples (such as water, soil, and air). It is widely used in biodiversity research, invasive or endangered species tracking, drug resistance gene detection, pathogenic microbial control, etc. Compared with traditional methods (such as field surveys, clinical testing, etc.), it has the advantages of non-invasiveness, low bias, high efficiency and economy. However, environmental samples are usually complex in composition and low in nucleic acid content, and large volumes (100mL-10L) of samples are often required for subsequent analysis.

[0003] At present, the method commonly used to extract eNA from surface water (rivers, lakes, oceans, etc.) samples is the filtration method. After 500-10000mL of surface water samples are filtered through a 0.22μm filter membrane, the filter membrane is collected in a centrifuge tube, lysis solution and proteinase K are added, and the nucleic acid on the filter membrane is fully released in the lysis solution, and then the nucleic acid is purified and enriched. However, the filtration method is a complex and time-consuming method. The sample processing and extraction time is long, and it is not suitable for large-scale high-frequency sampling application scenarios, such as monitoring of drug-resistant genes and pathogenic microorganisms in water environments. In addition, the use of the filter membrane method to extract nucleic acids will lead to the loss of some biological information. Studies have found that after filtration with a 0.22μm filter membrane, a considerable amount of eNA is still present in the filtrate. The nucleic acid in the filtrate is defined as "dissolved nucleic acid", which mainly exists in the form of membrane-wrapped nucleic acids (such as bacteria, cell fragments and extracellular vesicles smaller than 0.22μm) and small virus particles. Since "soluble nucleic acids" have a low concentration and are difficult to be retained by conventional filter membranes, and the sample size 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. Common methods for enriching nucleic acids in liquid samples in laboratories include precipitation, column extraction, and traditional magnetic beads. The extraction process of these methods is 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] At present, the nucleic acid extraction technologies for large volume liquid samples (greater than 10 mL) include PEG precipitation, filtration, centrifugation, etc., which have the following technical problems, limiting their large-scale use:

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

[0006] The commonly used techniques are complex and time-consuming. It often takes 12-24 hours from sample pretreatment to nucleic acid elution and enrichment. It also requires the use of expensive equipment such as high-speed / ultra-high-speed centrifuges and ultrafiltration vacuum pumps, which increases the cost of the technology and limits its large-scale application.

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

[0008] The commonly used technologies currently have a low recovery rate for free nucleic acids in samples (for example, membrane filter methods and ultra-high-speed centrifugation methods are difficult to retain free nucleic acids). At the same time, due to equipment limitations (for example, the capacity of the centrifuge is low, the filter membrane will be blocked by impurities in the water, and the large volume of water samples requires frequent replacement of the filter membrane, which is cumbersome to operate), it is difficult to increase the volume of the processed water sample, and the elution efficiency requires the use of a large amount of eluent (for example, the membrane filter method often requires the use of 400-1000μL eluent to improve the efficiency of nucleic acid elution), resulting in difficulty in improving the degree of nucleic acid enrichment (generally only tens to hundreds of times enrichment), making low-concentration target nucleic acids difficult to detect. In addition, different technologies have certain biases in nucleic acid extraction. For example, the membrane filter method and centrifugation method 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] The 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 through an amide bond; and the ligand unit can coordinately connect with metal ions.

[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 group connection position.

[0019] The above ligand units can be modified on the silica layer of the nanomagnetic beads through an amidation reaction between the amino group and the carboxyl group, and can 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 contained, the surface of the nanomagnetic beads in the present invention has a high positive charge and a high adsorption capacity for nucleic acids. The extraction rate of nucleic acids is close to 100%, and the enrichment degree of nucleic acids can reach 100-1000 times. After the nucleic acids are adsorbed on the surface of the nanomagnetic beads, they can be eluted by an elution solution, and the elution efficiency and elution effect are high, the elution speed is fast, the elution solution 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 uses ligand molecules to modify nanomagnetic beads. The ligand molecules form coordination bonds with metal ions to stably bind the metal ions to the surface of the magnetic beads, so that the surface of the magnetic beads is positively charged in an aqueous environment, so that negatively charged biological macromolecules can be adsorbed, such as nucleic acid molecules, proteins, phospholipid membranes, cells, bacteria, viruses, vesicles, etc. Adding an eluent containing a high concentration of chelating agents and anions to the system can compete with the metal ions on the surface of the magnetic beads, so that the surface of the nanomagnetic beads can be restored to the previous neutral or negatively charged state, thereby achieving efficient elution of biological macromolecules.

[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: Mix and react the amino-containing magnetic silica beads, ligand molecules, 1,3-diisopropylcarbodiimide and N,N-diisopropylethylamine to obtain coordinated nano magnetic beads;

[0028] S2: mixing the coordinated nanomagnetic beads with a solution containing metal ions 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 bead silica balls, making it easier 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 bead 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-mentioned 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 the magnetic metal salt, acetate and ethylene glycol, heat the reaction, and separate the magnetic bead core 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, so as to obtain the amino-containing magnetic bead silica sphere.

[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] The 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 the 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 for elution, so that the negatively charged biomacromolecules are desorbed 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, so that the surface charge of the nanomagnetic beads is positive, and the surface charge is positive metal-coordinated nanomagnetic beads, and the surface negatively charged biomacromolecules such as nucleic acids, proteins, phospholipid membranes, cells, viruses, bacteria, and vesicles in the water sample are adsorbed by electrostatic adsorption interaction force. The present invention modifies the ligand molecules on the surface of the nanomagnetic beads, 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 the adsorption of biomacromolecules such as nucleic acids. After the nucleic acid is adsorbed, an eluent is added, and the chelating agent and anion in the eluent compete with the ligand on the magnetic beads for 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 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.

[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 lysing 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 min; further preferably, the mixed elution time is 20 to 40 min.

[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, and the ligand molecules can form coordination bonds with metal ions, so that the surface of the nanomagnetic beads carries a large amount of positive charge, and the ligand molecules themselves are neutral or negatively charged. After losing the coordinated metal ions, the surface of the nanomagnetic beads can be neutral or carry 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 the ligand molecules to the metal ions.

[0057] After the nanomagnetic beads in the present invention adsorb metal ions, their surfaces carry a large amount of positive charges, and can be used for the extraction of biological macromolecules such as nucleic acids or substances with negatively charged surfaces in different types of liquid samples (including blood, urine, wastewater, seawater, etc.) of different volumes (10 μL to 10L). During the extraction of nucleic acids, no additional solvent is required. Only a few milligrams of magnetic beads need to be added to the original sample (10mL-1L) and incubated for 5-30 minutes to achieve nearly 100% nucleic acid adsorption.

[0058] When the nanomagnetic beads of the present invention are used to extract biomacromolecules with negative surface charge, the entire extraction process can be completed within 1-2 hours, which is much lower than the currently commonly used nucleic acid extraction methods, such as membrane filtration, ultracentrifugation, PEG precipitation, etc. In addition, the nucleic acid elution conditions of the present invention are mild and do not affect the properties of nucleic acids. At the same time, the components of the eluent have been systematically optimized so that it can be directly used for subsequent conventional nucleic acid analysis, such as Qubit, PCR, sequencing, etc. 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 acid 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 the QL magnetic beads after Al(III) elution in an embodiment of the present invention.

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

[0068] Fig.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] Fig.11 It is a test diagram of the eluent composition and elution condition optimization process of the eluent in the embodiment of the present invention.

[0070] Fig.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 a SEM image after adsorption of viruses and bacteria.

[0071] Fig.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] Fig.14 This is a test diagram of the use of Al(III) coordinated QL magnetic beads and metagenomic technology to screen antibiotic resistance genes in the environment in the example.

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

[0074] Fig.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] Fig.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 is 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 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, and 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 500mL three-necked flask on a magnetic stirring rack, quickly weigh 5.4g of ferric chloride hexahydrate (FeCl3.6H2O) and pour it into the three-necked flask; b. Measure 200mL of ethylene glycol solution and pour it into the three-necked flask in step a, adjust the stirrer speed to 300rpm, and stir for 30min; c. Weigh 14.4g of sodium acetate into the three-necked flask in step b, continue stirring for 30min, and small bubbles will appear on the upper layer of the solution in the flask after stirring; d. Pour the solution in the three-necked flask into the three-necked flask; Transfer to the reactor, tighten, and put into 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 absorption, and discard the clarified liquid after clarification; g. Use ultrapure water for three times of oscillation and ultrasonic cleaning, and anhydrous ethanol for three times of oscillation and ultrasonic cleaning; h. After cleaning, place the magnetic beads at clean room temperature to dry to obtain ferroferric oxide magnetic beads.

[0081] The synthesized magnetic beads 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 the prepared 0.1mol / L hydrochloric acid, and ultrasonicate for 30min, changing the water once during hydrothermal treatment; b. Transfer the magnetic bead solution in the flask to a 50mL centrifuge tube, dissolve it with ultrapure water, and clean it with oscillation and ultrasonication for 3 times; c. Use 160mL of ultrapure water to transfer the magnetic beads to a 1000mL round-bottom flask, 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 rack, and after magnetic absorption, discard the clarified liquid after clarification; 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 at clean room temperature to dry, and obtain magnetic beads coated with a silica layer on the surface, which are recorded as silicon-coated magnetic beads.

[0082] Then, amino groups are modified on the silica-coated magnetic beads to obtain amino magnetic beads, and the specific steps are as follows: a. Take 400 mg of silica-coated magnetic beads in a 50 mL centrifuge tube; b. Ultrasonic cleaning with anhydrous ethanol three times; c. Transfer the magnetic beads to a 250 mL round-bottom three-necked flask with 120 mL of anhydrous ethanol, with a total volume of 120 mL, and ultrasonicate again for 10 minutes; d. Fix the round-bottom three-necked flask on an overhead stirrer for stirring, slowly add 1.2 mL of 3-aminopropyltriethoxysilane, and stir overnight; e. Collect the amino magnetic bead reaction solution reacted overnight, ultrasonically clean it with anhydrous ethanol three times, then ultrasonically clean it with ultrapure water three times, rinse it with anhydrous ethanol once, and dry it to obtain amino magnetic beads, which are 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 with DMF and water twice, washed with ethanol once, 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 nanomagnetic beads with adjustable surface potential in this example is different from that in Example 1 only in that the ligand molecule used in this example for surface modification of amino magnetic beads is terpyridine-4-carboxylic acid, and the nanomagnetic beads with adjustable surface potential in this example are denoted as SD magnetic beads.

[0087] Example 3

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

[0089] Example 4

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

[0091] The structural formula of the ligand molecule used in Examples 1 to 4 is 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 in Examples 1 to 4 is adjustable at 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 produced 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 nanomagnetic beads in Examples 1 to 4 were incubated with metal ions respectively to make their surfaces positively charged. The specific process is as follows: a. The metal chloride compounds (FeCl3, AlCl3, ScCl3, CoCl2) were dissolved in water (concentration of 25 mmol / L), and the pH was adjusted to a suitable pH with sodium hydroxide solution to obtain different metal ion solutions with different pH values, specifically: FeCl3 solution with a pH of 2, FeCl3 solution with a pH of 3, FeCl3 solution with a pH of 4, AlCl3 solution with a pH of 5, AlCl3 solution with a pH of 6, and AlCl3 solution with a pH of 7. , ScCl3 solution with a pH of 4, ScCl3 solution with a pH of 5, ScCl3 solution with a pH of 6, CoCl3 solution with a pH of 4, and CoCl3 solution with a pH of 5; b. Take 10 mL of the above-mentioned metal ion solution with adjusted pH, add 100 mg of ligand magnetic beads, and incubate at room temperature for 24 hours to obtain a series of magnetic beads with different surface potentials, which are the nanomagnetic beads for nucleic acid extraction in this example, and then test the surface potential of the nanomagnetic beads for nucleic acid extraction in this example. For the convenience of comparison, the surface potential of the nanomagnetic beads in Examples 1 to 4 were tested as a control. The specific test results are as follows Figure 2 As shown by Figure 2 It can be seen that the surface potential-adjustable nanomagnetic beads in the present invention can be incubated with different metal ion solutions to obtain nanomagnetic beads with positive surface charge, and the surface potential of the nanomagnetic beads can be adjusted by adjusting the type of metal ions and the pH of the solution during incubation.

[0097] During the co-incubation process of the nanomagnetic beads with adjustable surface potential in the present invention with metal ions, the metal ions are loaded on the surface of the nanomagnetic beads with adjustable surface potential, so that the surface of the nanomagnetic beads with adjustable surface potential is positively charged, and then nucleic acids, microorganisms, etc. with negative charges on the surface in the environmental water sample are adsorbed on the surface of the nanomagnetic beads through the electrostatic adsorption of positive and negative charges. Subsequently, under the elution of the eluent, the metal ions adsorbed on the surface of the nanomagnetic beads are desorbed, and the surface of the nanomagnetic beads is negatively charged. Due to the repulsion between the negative charges, the nucleic acids, microorganisms, etc. are detached from the surface of the nanomagnetic beads, thereby realizing the extraction of nucleic acids and microorganisms. The specific principle diagram is shown in FIG. Figure 3 shown.

[0098] The LF magnetic beads in Example 1 were incubated with FeCl3 solution to make their surface positively charged. The specific process was as follows: a. FeCl3 was dissolved in water (concentration was 25 mmol / L), and pH=4, pH=5, and pH=6 were adjusted with sodium hydroxide solution to obtain FeCl3 solutions with different pH values. b. 10 mL of the FeCl3 solution with adjusted pH value was added to 100 mg of the LF magnetic beads in Example 1, and the mixture was incubated at room temperature for 24 hours to obtain Fe(III) coordinated 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 AlCl3 solution with adjusted pH 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 chloride 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 a 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, and 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 acid, and the specific steps are as follows:

[0103] Reference Figure 4 Schematic diagram of nucleic acid extraction process in. Taking surface water sample (lake water) as an example, 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 (in the eluent, the concentration of EDTA is 0.1mol / L, PO4 3- The concentration of is 0.1 mol / L, the mass percentage of sodium dodecyl sulfate is 2%, which is prepared by dissolving ethylenediaminetetraacetic acid, disodium hydrogen phosphate, and sodium dodecyl sulfate in 1×TE buffer, and the pH of the eluent is 7.5), and incubating in a water bath at 80°C for 30 minutes to elute the nucleic acid on the magnetic beads. The magnetic beads obtained after eluting the nucleic acid are recorded as Al(III) eluted QL magnetic beads. After the incubation is completed, the magnetic beads are separated from the eluent by magnetic attraction, and the eluent is collected in a new centrifuge tube. At this time, the nucleic acid is 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 elution solution 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 increased significantly, while after Al(III) elution, the Al content in the QL magnetic beads decreased significantly after elution with the elution solution, 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 the 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 carries a negative charge, which 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 QL magnetic beads coordinated by Al(III) 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 EDTA 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, and the pH of the eluent is 7.5), placed in a water bath and incubated at 80°C for 30 minutes, and then the DNA concentration in the eluent and the supernatant is tested respectively. The specific test results are as follows Fig. 9As shown. Fig. 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 (in the elution solution, the concentration of EDTA 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, and the pH of the eluent is 7.5), placed in a water bath and incubated at 80°C for 30 minutes, and then the DNA concentration in the eluent and the supernatant is tested respectively. The specific test results are as follows Fig.10 As shown. Fig.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, which further proves that better nucleic acid extraction effects can be obtained 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 optimizes the temperature, pH, chelating agent and anion type of the eluent during elution. The specific test method is: 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 ( Fig.11 a) 10-200mmol / L oxalate ( Fig.11 b) 10-200mmol / L fluoride ion ( Fig.11 c) 1-200mmol / L EDTA ( Fig.11 d) and 50-500mmol / L phosphate ( Fig.11 e), the specific test results are as follows Fig.11As shown in (ae), the elution solution 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 the components of the elution solution to elute the nucleic acids adsorbed on the nanomagnetic beads. The elution rate test results are shown in Fig.11 As shown in (f), Fig.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. Fig.11 (g) is shown by Fig.11 (g) It can be seen that under the elution condition of 80°C, the elution efficiency of the adsorbed nucleic acid is the highest. 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. Fig.11 As shown in (h), Fig.11 (h) It can be seen that under the elution condition of pH = 7, the elution efficiency of adsorbed nucleic acid 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: Fig.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 elution solution were detected by gel electrophoresis. The results are shown in Fig.12 (A) is shown. Then the SEM images of the Al(III)-coordinated QL magnetic beads after adsorbing viruses and bacteria are tested respectively. Fig.12 (B) and Fig.12 (C), and qPCR was used to detect the concentrations of bacteria and viruses in the original solution and the eluate, see Table 3. Fig.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, and 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 a large volume of liquid samples without adding any additional solvent, and are easy to elute, and can achieve 100% adsorption efficiency of nucleic acids ( Fig.10 ) and 90% elution efficiency ( Fig.11(h)), can achieve 100-1000 times nucleic acid enrichment (the Ct value of free nucleic acid in the eluate in Table 1 is about 9.5 lower than that in the initial solution, which means that the nucleic acid in the eluate is enriched about 700 times), and is suitable for different fragment lengths ( Fig.12 ), different forms of DNA extraction (Table 1, Fig.12 ), the operation process and elution solution composition are gentle and do not affect the properties of nucleic acids. The extracted nucleic acids can be directly used for subsequent routine analysis, 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 in 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 co-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 experiment, 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 with the Al(III)-coordinated QL magnetic beads in 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 detection. Every decrease of 1 in the Ct value means that the corresponding nucleic acid concentration has increased by 2 times. When the Ct value decreases by x, the concentration increases by x times of 2. Sd refers to the standard deviation of the results of multiple repeated tests.

[0116] As can be seen from Table 1, the Al(III)-coordinated QL magnetic beads in the present invention have significant enrichment capabilities for nucleic acids, viruses and bacteria in different types of environmental water samples, and the enrichment degree can reach 100-1000 times. Compared with the traditional enrichment method of nucleic acids in environmental water (such as the membrane filter method, which has a low interception rate for free nucleic acids, is complex and time-consuming to operate, and requires 12 to 24 hours), the entire process is simpler and faster (1-2 hours), and does not require special equipment such as centrifuges and vacuum pumps. It can be used for nucleic acid extraction in the environment, and then plays a role in application scenarios such as environmental biodiversity surveys, endangered invasive species research, and daily monitoring of drug-resistant bacteria and ARGs.

[0117] In order to verify the feasibility of the nanomagnetic beads in the present invention in the background survey and daily monitoring of ARGs, we regularly collected environmental water samples for different application scenarios (surface rivers, sewage plant effluent, seawater, and mariculture wastewater), used the Al(III)-coordinated QL magnetic beads in the present invention to extract ARGs from them according to the method described above, and used qPCR for detection. The specific test results are shown in the figure. Fig.13 As shown, Fig.13 (A) is a diagram of daily monitoring of antibiotic resistance genes in community lakes; Fig.13 (B) is the daily monitoring map of antibiotic resistance genes in the effluent section of domestic sewage treatment plants; Fig.13 (C) is a monitoring chart of antibiotic resistance genes in seawater and mariculture wastewater. Fig.13 It can be seen that the nanomagnetic beads in the present invention are convenient and fast in the entire antibiotic resistance gene extraction and detection process, and meet the large-scale and high-frequency daily monitoring work of large-volume environmental water samples.

[0118] In addition, we used metagenomic technology to analyze the environmental nucleic acids enriched by the Al(III) coordinated QL magnetic beads in the present invention, and compared them with the integrated ARGs database to screen out the ARGs with the highest relative abundance and the most serious risks. The specific test results are as follows Fig.14 As shown by Fig.14 It can be seen that the nanomagnetic beads in the present invention are feasible in conducting background investigation and screening of ARGs and discovering potential ARGs.

[0119] Commonly used nucleic acid extraction techniques (precipitation method, column extraction method, traditional magnetic bead method, etc.) require the addition of a large amount of high-salt solvents, which are not suitable for the enrichment and extraction of nucleic acids in large-volume samples. Technologies such as the membrane filter method and the PEG precipitation method have limitations such as complex and time-consuming operations and reliance on special equipment. Based on the metal ion exchange strategy, the surface potential-adjustable nanomagnetic beads of the present invention are co-incubated with metal ions to obtain nanomagnetic beads with positive charges on the surface. Through electrostatic interactions, efficient adsorption of biological macromolecules and cells in large-volume water samples is achieved; by changing the surface potential of the nanomagnetic beads, efficient elution of the adsorbed molecules is achieved, and the nucleic acid recovery rate reaches 90%. The nanomagnetic beads in the present invention can also achieve efficient enrichment of nucleic acids in environmental water samples and urine samples, and are successfully applied to different scenarios in combination with PCR or sequencing technology, for example:

[0120] (1) Liquid biopsy

[0121] Urine is an easily accessible biological sample, and the nucleic acids in it show important potential in disease diagnosis, monitoring and research. The Al(III)-coordinated QL magnetic beads of the present invention were used to extract DNA of known sequences from environmental water samples and urine according to the above nucleic acid extraction and enrichment method. The specific test results are shown in Table 2 below:

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

[0123]

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

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

[0126] Studies have shown that monitoring pathogenic microorganisms in water environments can accurately reflect the prevalence of viral lineages and can detect emerging viral variants earlier than clinical samples. The Al(III)-coordinated QL magnetic beads of the present invention were used to extract pathogenic microorganisms and viruses from environmental water samples according to the above-mentioned nucleic acid extraction and enrichment method. The specific extraction results are shown in Table 3 below.

[0127] Table 3 Extraction effect of pathogens and viruses

[0128]

[0129] The nanomagnetic beads of the present invention can be applied to the rapid extraction of nucleic acids related to pathogenic microorganisms in environmental water samples and used in routine epidemiological investigations.

[0130] (3) Ecological survey

[0131] Environmental DNA refers to the sum of nucleic acid fragments directly extracted from environmental samples (such as water, soil, and air). It is widely used in the fields of biodiversity research, invasive alien species monitoring, and endangered species monitoring. The nanomagnetic beads in the present invention can achieve rapid enrichment and extraction of water environmental DNA, helping relevant personnel to conduct ecological surveys and research, including biodiversity surveys, endangered / invasive species monitoring, etc. Fig.15 shown.

[0132] (4) Antibiotic resistance gene screening and routine monitoring

[0133] The environment is an important medium for the spread of ARGs. Wastewater discharged from hospitals, breeding farms, sewage treatment plants and other facilities is an important source of ARGs entering the environment. Comprehensive investigation and daily monitoring of ARGs in relevant wastewater and surface water is a prerequisite for formulating management policies and controlling the spread of ARGs. The nanomagnetic beads of the present invention can be used to identify and screen drug-resistant genes in surface water and sewage treatment plant effluent, specifically Fig.16 The nanomagnetic beads of the present invention can be used for screening and daily monitoring of antibiotic resistance genes in surface water, as shown in FIG. Fig.17As shown, the nanomagnetic beads in the present invention are easy to operate when performing drug resistance gene identification and screening and antibiotic resistance gene screening, and can achieve rapid in-situ extraction of nucleic acids in large volumes of environmental water, meeting the needs of large-scale and high-frequency sample processing in daily ARGs monitoring. In previous studies, we enriched and extracted ARGs in different water bodies, and achieved screening and daily monitoring of major ARGs pollutants through sequencing and qPCR technology.

[0134] In summary, the nanomagnetic beads in 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 in different volumes (10 μL to 10 L) and different types of liquid samples (including blood, urine, wastewater, seawater, etc.).

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

[0138] The nucleic acid extraction method based on traditional magnetic beads often requires the addition of a large amount of high-concentration salt solution to help the nucleic acids in the liquid sample aggregate and adsorb on the magnetic beads. Therefore, it is difficult to apply to large-volume, low-concentration samples, such as urine samples or environmental water samples. The nanomagnetic beads in the present invention can enrich and extract nucleic acid molecules in liquid samples without adding any solvent. Experiments have shown that it only takes 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.

[0139] (3) Quick and easy operation

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

[0141] (4) Mild elution conditions

[0142] The nucleic acid elution conditions in the nucleic acid extraction process of the present invention are mild and do not affect the properties of the nucleic acid. At the same time, the components of the elution liquid have been systematically optimized so that it 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 the basic requirement of high nucleic acid recovery efficiency, and the cost is greatly reduced.

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

[0146] The currently commonly used amino magnetic bead method also uses electrostatic interaction to adsorb nucleic acid molecules in liquid samples, which is similar to the principle of the present invention. However, its adsorption and elution efficiency are not as good as the nanomagnetic beads with adjustable surface potential used in the present invention. The present invention uses 1.25μg magnetic beads / μL Al(III) coordinated QL magnetic beads to adsorb nucleic acids in an initial solution with a nucleic acid concentration of 49.67ng / μL, and then tests the nucleic acid concentration in the supernatant and the eluate after magnetic bead adsorption. The adsorption capacity of magnetic beads for nucleic acids is calculated according to the following formula: The calculation formula for the adsorption capacity of magnetic beads for nucleic acids is: (nucleic acid concentration in the initial solution-nucleic acid concentration in the supernatant after magnetic bead adsorption) / magnetic bead dosage. The specific test results are shown in Table 4 below.

[0147] Table 4 Test data of the adsorption capacity of nucleic acids in water by Al(III) coordinated QL magnetic beads

[0148]

[0149]

[0150] As shown in Table 4, the Al(III)-coordinated QL magnetic beads used in the present invention have an adsorption capacity of 18 ng nucleic acid / μg magnetic beads for nucleic acid (including DNA and RNA) in water, which is much higher than the currently commonly used amino magnetic beads (1-2 ng nucleic acid / μg magnetic beads). The nucleic acid elution efficiency after adsorption exceeds 90% (by Fig.11 It can be seen that the nucleic acid elution efficiency is better than that of the general amino magnetic bead method (<85%).

[0151] (7) Recoverable nucleic acids in different forms

[0152] Nucleic acids exist in complex forms in liquid samples, including free nucleic acids, intracellular nucleic acids, and adsorbed nucleic acids. The nucleic acid extraction method of the present invention realizes the efficient extraction of DNA and RNA of different fragment sizes, intracellular nucleic acids, viral nucleic acids, and free nucleic acids. In addition, the method has excellent extraction efficiency for nucleic acids in free form, solving the current problem of difficulty in recovering free nucleic acids in large-volume water samples.

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

[0154] At present, the main methods for extracting "soluble nucleic acids" (nucleic acids that can pass through a 0.22μm filter membrane) from large-volume samples are the PEG method and the coagulation precipitation method. Both methods will produce harmful waste liquid (PEG waste liquid or metal ion waste liquid) equivalent to the volume of the liquid sample, which requires additional treatment. However, the present invention does not require the addition of any chemical reagents to the liquid sample, which is green and environmentally friendly.

[0155] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without 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; and the ligand unit can be coordinated and connected with metal ions.

2. The nanomagnetic bead according to claim 1, characterized in that: The ligand unit is selected from At least one of; or, The ligand unit is selected from A polymer material having at least one functional group.

3. The nanomagnetic bead according to claim 1, characterized in that: The nanomagnetic beads also contain metal ions, and coordination bonds are formed between the metal ions and the ligand units.

4. The nanomagnetic bead according to claim 3, characterized in that: 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 .

5. The method for preparing the nanomagnetic beads according to any one of claims 3 to 4, characterized in that: The following steps are involved: S1: Mix and react the amino-containing magnetic silica beads, ligand molecules, 1,3-diisopropylcarbodiimide and N,N-diisopropylethylamine to obtain coordinated nano magnetic beads; S2: mixing the coordinated nanomagnetic beads with a solution containing metal ions to obtain the nanomagnetic beads.

6. The method for preparing nano magnetic beads according to claim 5, characterized in that: The pH of the solution containing metal ions is 2-7.

7. A method for extracting negatively charged biomacromolecules, characterized in that: The following steps are involved: (1) mixing the nanomagnetic beads according to any one of claims 3 to 4 with a test sample, so that negatively charged biomacromolecules in the test sample are adsorbed on the surface of the nanomagnetic beads, and separating the adsorbed nanomagnetic beads; (2) mixing the adsorbed nanomagnetic beads with an eluent for elution, so that the negatively charged biomacromolecules are desorbed from the surface of the nanomagnetic beads; The eluent contains anions.

8. The method for extracting negatively charged biomacromolecules according to claim 7, characterized in that: The negatively charged biomacromolecules include at least one of nucleic acids, proteins, phospholipid membranes, cells, bacteria, viruses, and vesicles.

9. The method for extracting negatively charged biomacromolecules according to claim 7, characterized in that: 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; taking 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.

10. The method for extracting negatively charged biomacromolecules according to claim 7, characterized in that: The mixed elution temperature is 60-100°C; And / or, the mixed elution time is 20 to 60 minutes.

11. Use of the nanomagnetic beads according to any one of claims 1 to 4 and / or the method for extracting negatively charged biomacromolecules according to any one of claims 7 to 10 in the field of biological detection.

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