Magnetic microspheres, preparation method thereof, RNA extraction kit and RNA extraction method

Abstract

The disclosure provides a magnetic microsphere and a preparation method thereof, an RNA extraction kit and an RNA extraction method, which are applied to nucleic acid extraction and belong to the technical field of biological medicine, and aims to improve the extraction efficiency of RNA. The magnetic microsphere comprises a core and a shell wrapping the core. The core comprises magnetic nanoparticles, the shell comprises graphene oxide, and a target organic material is doped in the graphene oxide. The target organic material contains at least one of an amino functional group, a carboxyl functional group and a hydroxyl functional group.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, and in particular to a magnetic microsphere and its preparation method, an RNA extraction kit, and an RNA extraction method. Background Technology

[0002] Currently, the magnetic bead method is commonly used to extract RNA. However, during the traditional magnetic bead method for RNA extraction, the purity and yield of RNA are affected by the degradation of the RNA itself and the excessively long processing time of DNA. Summary of the Invention

[0003] Based on the background technology, this disclosure presents a magnetic microsphere and its preparation method, as well as an RNA extraction kit.

[0004] In a first aspect, this disclosure provides a magnetic microsphere for nucleic acid extraction, comprising a core and a shell encapsulating the core; wherein the core comprises magnetic nanoparticles, the shell comprises graphene oxide, and a target organic material doped within the graphene oxide, wherein the target organic material comprises at least one of an amino functional group, a carboxyl functional group, and a hydroxyl functional group.

[0005] Optionally, the target organic material includes at least one of amino coupling agents, carboxyl coupling agents, and hydroxyl coupling agents.

[0006] Optionally, the amino coupling agent includes γ-aminopropyltriethoxysilane, the carboxyl coupling agent includes mercaptopropionic acid, and the hydroxy coupling agent includes glycidyl methacrylate.

[0007] Optionally, the magnetic microspheres have a higher adsorption rate for RNA than they have for single-stranded DNA and proteins.

[0008] Optionally, the target organic material is doped into the graphene oxide at a ratio of 60%-75%.

[0009] Optionally, the magnetic microspheres further include a connecting layer located between the core and the shell;

[0010] The connecting layer includes at least one of silica, agarose gel, polyglycidyl methacrylate, and polyvinyl alcohol.

[0011] Optionally, the magnetic nanoparticles are magnetic nanoparticles, or...

[0012] The magnetic nanoparticles include organic polymer particles and a magnetic layer covering the organic polymer particles.

[0013] A second aspect of this disclosure provides a method for preparing magnetic microspheres, applicable to the preparation of magnetic microspheres as described in the first aspect, the method comprising:

[0014] Formation of magnetic nanoparticles;

[0015] The magnetic nanoparticles, graphene oxide, and target organic material are mixed in a mass ratio of 1:2-4:1.5-2.5 to obtain the magnetic microspheres; wherein the magnetic microspheres include a core and a shell enclosing the core, the core includes the magnetic nanoparticles, the shell includes graphene oxide, and the target organic material is doped inside the graphene oxide, the target organic material includes at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups.

[0016] Optionally, the magnetic nanoparticles, graphene oxide, and target organic material are mixed to obtain the magnetic microspheres, comprising:

[0017] Graphene oxide is activated using an activator.

[0018] The magnetic nanoparticles were dispersed in the buffer solution, and activated graphene oxide and the target organic material were added to obtain a first mixed solution.

[0019] The first mixed solution is placed at a first reaction temperature and stirred for a first time to obtain the magnetic microspheres.

[0020] Optionally, before mixing the magnetic nanoparticles, graphene oxide, and the target organic material, the method further includes:

[0021] The magnetic nanoparticles, monomers, and surfactants are added to water to obtain a second mixed solution;

[0022] After mixing the second mixed solution thoroughly, add the alkaline solution and stir.

[0023] The stirred second mixed solution is placed at a second reaction temperature for a second time to form a bonding layer on the surface of the magnetic nanoparticles.

[0024] A third aspect of this disclosure provides an RNA extraction kit comprising at least a magnetic bead suspension, said magnetic bead suspension comprising magnetic microspheres as described in the first aspect above, or magnetic microspheres prepared by the preparation method described in the second aspect above.

[0025] Optionally, the concentration of the magnetic bead suspension is 0.5-2 g / mL.

[0026] A fourth aspect of this disclosure provides a method for extracting RNA, using an RNA extraction kit as described in the third aspect above, the extraction method comprising:

[0027] Add lysis buffer and protease to the sample solution, and collect the supernatant after lysis by centrifugation;

[0028] A binding solution and a magnetic bead suspension were added to the supernatant to allow the magnetic beads to bind to RNA, and the RNA-bound magnetic beads were collected under an external magnetic field.

[0029] The magnetic beads that bind RNA were separated using an elution buffer, and the RNA was extracted.

[0030] The magnetic microspheres disclosed herein include a core and a shell enclosing the core; wherein the core comprises magnetic nanoparticles, the shell comprises graphene oxide, and a target organic material doped inside the graphene oxide, wherein the target organic material comprises at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups.

[0031] In the magnetic microspheres provided in this disclosure, at least one of the amino, carboxyl, and hydroxyl functional groups is doped into the framework structure of graphene oxide using a target organic material. This alters the binding properties of graphene oxide to RNA, allowing RNA to be directly eluted using conventional elution solutions, reducing elution time, and thereby improving RNA extraction efficiency.

[0032] The above description is merely an overview of the technical solution disclosed herein. In order to better understand the technical means of this disclosure and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this disclosure more apparent and understandable, specific embodiments of this disclosure are described below. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments or related technologies of this disclosure, the accompanying drawings used in the description of the embodiments or related technologies will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. It should be noted that the scale in the drawings is for illustration only and does not represent the actual scale.

[0034] Figure 1 A schematic diagram of the microstructure of the magnetic microspheres provided in an embodiment of this disclosure is shown;

[0035] Figure 2 A flowchart illustrating the steps of a method for preparing magnetic microspheres according to an embodiment of this disclosure is shown.

[0036] Figure 3 A flowchart illustrating the steps involved in forming magnetic microspheres in an embodiment of this disclosure is shown.

[0037] Figure 4 A flowchart illustrating the steps of the RNA extraction method provided in this embodiment is shown.

[0038] Figure 5 SEM images of the magnetic microspheres prepared in Example 1 are shown.

[0039] Figure 6 A TEM image of the magnetic microspheres prepared in Example 1 is shown;

[0040] Figure 7 The results of the 4200 assay for the product obtained using the RNA extraction kit with magnetic microspheres from Example 3 are shown.

[0041] Figure 8 The sequence diagrams of the probe primers designed for RNA and DNA in Experiment 2 are shown;

[0042] Figure 9 The figure shows the qRT-PCR experimental results of the product extracted using the RNA extraction kit with magnetic microspheres from Example 3. Detailed Implementation

[0043] To make the above-mentioned objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. Based on the embodiments of this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0044] RNA (ribonucleic acid), as a carrier of genetic information, is widely used in many aspects of tumor research and treatment. Before studying RNA, it is necessary to extract RNA from the sample. Conventional extraction methods utilize the adsorption properties of graphene oxide on RNA to achieve RNA extraction. However, the binding of graphene oxide and RNA usually requires the use of 8M urea solution for good separation, which increases RNA processing time and is not conducive to automated extraction.

[0045] In view of this, embodiments of the present disclosure provide a magnetic microsphere and its preparation method, an RNA extraction kit, and an RNA extraction method, wherein a target organic material containing at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups is doped into the backbone of graphene oxide to change the binding performance of graphene oxide and RNA, so that graphene oxide and RNA can be separated by conventional elution buffer, reducing RNA processing time and thereby improving RNA extraction efficiency.

[0046] Reference Figure 1 , Figure 1 A schematic diagram of the microstructure of the magnetic microspheres provided in an embodiment of this disclosure is shown, as follows. Figure 1 As shown, the magnetic microsphere includes a core A and a shell B enclosing the core A. The core A includes magnetic nanoparticles, the shell B includes graphene oxide, and a target organic material doped inside the graphene oxide. The target organic material includes at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups.

[0047] In this embodiment, the core A can be composed of organic nanoparticles and a magnetic layer encapsulating the organic nanoparticles, or it can be directly composed of magnetic nanoparticles. When the core A consists of organic nanoparticles and a magnetic layer encapsulating the organic nanoparticles, the organic nanoparticles can be organic polymers such as polystyrene, and the magnetic layer can be one or more of magnetic materials such as iron(II,III) oxide, iron(II,III) nitride, γ-iron(II,III) oxide, cobalt, and nickel oxide. When the core A is magnetic nanoparticles, the magnetic nanoparticles can be one or more of magnetic materials such as iron(II,III) oxide, iron(II,III) nitride, γ-iron(II,III) oxide, cobalt, and nickel oxide.

[0048] The shell B may include graphene oxide doped with amino materials, graphene oxide doped with carboxyl materials, graphene oxide doped with hydroxyl materials, or graphene oxide doped with both amino and carboxyl materials, or graphene oxide doped with amino, carboxyl, and hydroxyl materials.

[0049] In particular, doping at least one of the amino, carboxyl, and hydroxyl functional groups into the graphene oxide framework can alter the binding properties of graphene oxide to RNA, enabling separation using conventional elution solutions. Furthermore, modifying the surface of graphene oxide with amino, hydroxyl, or carboxyl groups can enhance its adsorption of RNA, thereby improving RNA extraction efficiency.

[0050] In one example, a connecting material can be used to connect the core A and the shell B. This connecting material provides more connection sites for the graphene oxide in the shell B, allowing the shell B to completely cover the core A and protect it from external demagnetization. Furthermore, a modification layer can be formed on the side of the shell B away from the core A. This modification layer can then modify the surface of the shell B with functional groups such as amino, hydroxyl, and carboxyl groups, improving the adsorption of RNA by the graphene oxide. Alternatively, a target organic material can be used to surface-modify the graphene oxide, adding functional groups such as amino, hydroxyl, and carboxyl groups to its surface.

[0051] The magnetic microspheres provided in this disclosure modify the binding properties of graphene oxide to RNA by doping the graphene oxide framework structure with at least one of amino, carboxyl, and hydroxyl functional groups. This allows graphene oxide and RNA to be separated by conventional elution solutions, reducing RNA processing time and thus improving RNA extraction efficiency.

[0052] In one embodiment, the target organic material includes at least one of an amino coupling agent, a carboxyl coupling agent, and a hydroxyl coupling agent.

[0053] In this embodiment, considering that the surface of graphene oxide includes carboxyl functional groups, which can react with amino and hydroxyl groups, making it difficult to directly dope organic materials containing amino, carboxyl, and hydroxyl groups, a coupling agent can be used to carry out the doping process. The carboxyl groups of graphene oxide are activated to form esters, and then the functional groups are doped into the framework structure of graphene oxide through the coupling agent, thereby achieving the doping of the target organic material into graphene oxide.

[0054] The amino coupling agent includes γ-aminopropyltriethoxysilane, the carboxyl coupling agent includes mercaptopropionic acid, and the hydroxyl coupling agent includes glycidyl methacrylate. Specifically, γ-aminopropyltriethoxysilane reacts with graphene oxide to dope amino functional groups into graphene oxide, mercaptopropionic acid can dope carboxyl functional groups into graphene oxide, and glycidyl methacrylate can dope hydroxyl functional groups into graphene oxide.

[0055] In one embodiment, the magnetic microspheres exhibit a higher adsorption rate for RNA than for single-stranded DNA (deoxyribonucleic acid) and protein.

[0056] In this embodiment, the concentration of amino groups in the magnetic microspheres can be controlled by adjusting the doping ratio of the target organic material, making the magnetic microspheres preferential for RNA, reducing the contamination of RNA by impurities such as single-stranded DNA and proteins, and improving the purity of the extracted RNA.

[0057] The target organic material has a doping ratio of 60%-75% in graphene oxide.

[0058] In this embodiment, the amino functional group can bind to RNA, while the carboxyl functional group can bind not only to RNA but also to single-stranded DNA. Therefore, to improve the purity of the extracted RNA, the preference of the magnetic microspheres for RNA can be controlled by adjusting the doping ratio of γ-aminopropyltriethoxysilane in graphene oxide.

[0059] The doping ratio of γ-aminopropyltriethoxysilane in graphene oxide affects the adsorption performance of graphene oxide for RNA. When the doping ratio of γ-aminopropyltriethoxysilane in graphene oxide is between 60% and 75%, the magnetic microspheres have a high preference for RNA, that is, the magnetic microspheres adsorb RNA better, which further improves the RNA extraction efficiency of the magnetic microspheres.

[0060] Specifically, the doping ratio of the target organic material in graphene oxide can be 60%, 65%, 70%, and 75%, etc.

[0061] In one embodiment, the magnetic microspheres further include a connecting layer located between the core and the shell;

[0062] The connecting layer includes at least one of silica, agarose gel, glycidyl methacrylate, and polyvinyl alcohol.

[0063] In this embodiment, a connecting layer including the core is first used on the surface of the magnetic microsphere, and then a shell including the core and the connecting layer is used. In this way, the connecting layer can provide more connection sites for the graphene oxide in the shell, so that the shell can well cover the surface of the core.

[0064] In one embodiment, the magnetic nanoparticles are magnetic nanoparticles, or the magnetic nanoparticles include organic polymer particles and a magnetic layer coating the organic polymer particles.

[0065] Specifically, magnetic nanoparticles can be used directly as the core, in which case they provide both a spherical framework and paramagnetism. These magnetic nanoparticles can be one or more of iron(III) oxide, iron nitride, γ-ferric oxide, and cobalt or nickel oxides. Alternatively, organic polymer particles and a magnetic layer coating the organic polymer particles can be used as the core, in which case the organic polymer particles provide a spherical framework, and the magnetic layer provides paramagnetism. The organic polymer particles can be organic polymer nanoparticles such as polyvinyl alcohol, and the magnetic layer can include one or more of iron(III) oxide, iron nitride, γ-ferric oxide, and cobalt or nickel oxides.

[0066] The magnetic microspheres provided in this disclosure are formed by doping graphene oxide with at least one of γ-aminopropyltriethoxysilane, mercaptopropionic acid, and glycidyl methacrylate to form the shell of the magnetic microspheres. On the one hand, the amino functional groups doped in the graphene oxide change the binding performance between the graphene oxide and RNA, reducing the RNA extraction time. On the other hand, the doping ratio of γ-aminopropyltriethoxysilane in the graphene oxide is between 60% and 75%, which makes the graphene oxide doped with amino functional groups have a preference for RNA, reducing the contamination of proteins and single-stranded DNA and improving the purity of the extracted RNA.

[0067] Based on the same inventive concept, this disclosure also provides a method for preparing magnetic microspheres, referring to... Figure 2 , Figure 2 A flowchart illustrating the steps of the method for preparing magnetic microspheres according to embodiments of this disclosure is shown, as follows: Figure 2 As shown, the preparation method specifically includes:

[0068] Step S1: Formation of magnetic nanoparticles.

[0069] In this embodiment, the magnetic nanoparticles can be magnetic nanoparticles or composed of organic polymer particles and a magnetic layer coating the organic polymer particles. Different magnetic nanoparticles are formed using different preparation methods. For example, when the magnetic nanoparticles are magnetic nanoparticles, they can be prepared directly. When the magnetic nanoparticles are composed of organic polymer particles and a magnetic layer coating the organic polymer particles, a precursor composite of the magnetic material and the monomer can be prepared first, and then the magnetic nanoparticles can be formed using the precursor.

[0070] Specifically, the magnetic nanoparticles can be one or more magnetic materials such as iron(II,III) oxide, iron(II) nitride, γ-iron(II) oxide, and cobalt and nickel oxides, or polyvinyl alcohol and iron(II,III) oxide, iron(II,III) nitride, γ-iron(II) oxide, and cobalt and nickel oxides encapsulating polyvinyl alcohol.

[0071] Step S2: Magnetic nanoparticles, graphene oxide, and target organic material are mixed in a mass ratio of 1:2-4:1.5-2.5 to obtain magnetic microspheres; wherein, the magnetic microspheres include a core and a shell including the core, the core including magnetic nanoparticles, the shell including graphene oxide, and the target organic material doped inside the graphene oxide, the target organic material including at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups.

[0072] In this embodiment, while forming graphene oxide on the surface of magnetic nanoparticles, organic materials with amino, carboxyl, or hydroxyl functional groups are doped into the graphene oxide, so that the graphene oxide in the shell of the magnetic microspheres is doped with amino, carboxyl, or hydroxyl functional groups, thereby changing the binding performance of graphene oxide to RNA and reducing the RNA processing time.

[0073] Specifically, the ratio of magnetic nanoparticles, graphene oxide, and the target organic material can be 1:2:1.5, 1:3:2, 1:4:2.5, 1:2:2.5, 1:3:2.5, and 1:4:2, etc. Before mixing the magnetic nanoparticles, graphene oxide, and the target organic material, the graphene oxide can be activated to facilitate the incorporation of the target organic material into the graphene oxide.

[0074] The method for preparing magnetic microspheres provided in this disclosure involves doping a target organic material containing amino and / or carboxyl functional groups into graphene oxide during shell formation. This alters the binding properties of graphene oxide to RNA, enabling the magnetic microspheres and RNA to be separated using conventional elution solutions, reducing RNA processing time, and thereby improving RNA extraction efficiency.

[0075] In one embodiment, reference is made to Figure 3 , Figure 3 A flowchart illustrating the steps of the method for forming magnetic microspheres in an embodiment of this disclosure is shown, as follows: Figure 3 As shown, step S2 specifically includes:

[0076] S201 uses an activator to activate graphene oxide.

[0077] In this embodiment, the activator can be EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide). The activator is used to activate the carboxyl functional group of graphene oxide, so that the carboxyl functional group forms a stable ester intermediate, thereby avoiding the direct reaction between amino and carboxyl groups during the reaction process, which makes it difficult for amino groups to be incorporated into graphene oxide.

[0078] S202, magnetic nanoparticles are dispersed in water, and activated graphene oxide and the target organic material are added to obtain a first mixed solution.

[0079] The dosage of magnetic nanoparticles is 0.5-2 g / 100 mL, the dosage of graphene oxide is 1-4 g / 100 mL, and the dosage of the target organic material is 0.5-4 g / 100 mL.

[0080] S203, the first mixed solution is placed at the first reaction temperature and stirred for a first time to obtain magnetic microspheres.

[0081] The first reaction temperature is 10-30℃, and the first reaction time is 4-8h.

[0082] For example, the first mixed solution can be stirred at a reaction temperature of 10°C for 8 hours to obtain magnetic microspheres; the first mixed solution can also be stirred at a reaction temperature of 20°C for 6 hours to obtain magnetic microspheres; or the first mixed solution can be stirred at a reaction temperature of 30°C for 4 hours to obtain magnetic microspheres.

[0083] In one embodiment, before step S2, a connecting layer may be formed first. Specifically, the process may be as follows: First, magnetic nanoparticles, monomers and surfactants are added to water to obtain a second mixed solution; then, the second mixed solution is mixed and an alkaline solution is added and stirred; then, the stirred second mixed solution is placed at a reaction temperature of 20-40°C for 4-8 hours to form a connecting layer on the surface of the magnetic nanoparticles.

[0084] The monomer material is determined based on the linker material. If the linker material is silicon dioxide, the monomer is tetraethyl orthosilicate. If the linker material is agarose gel, the monomer is agarose. If the linker material is polyglycidyl methacrylate, the monomer is glycidyl methacrylate. If the linker material is polyvinyl alcohol, the monomer can be vinyl acetate.

[0085] In this disclosure, the magnetic nanoparticles can be magnetic nanoparticles or can be composed of organic polymer particles and a magnetic layer encapsulating the organic polymer particles, and different methods can be used to prepare different nanoparticles.

[0086] In one example, the magnetic nanoparticles are magnetic nanoparticles. In this case, the preparation method of the magnetic nanoparticles includes: first, mixing ferric chloride, ferric chloride, surfactant, alkaline solution and water to obtain a first reaction solution; then, placing the first reaction solution at a reaction temperature of 70-100℃ for 2-8 hours, and performing magnetic separation, washing and drying on the first reaction solution after the reaction to obtain magnetic nanoparticles.

[0087] In another example, the magnetic nanoparticles consist of organic polymer particles and a magnetic layer encapsulating the organic polymer particles. The preparation method for the magnetic nanoparticles includes: first, under a nitrogen atmosphere, adding ferric chloride hexahydrate and ferric chloride tetrahydrate to distilled water and stirring until completely dissolved to obtain a third mixed solution; next, heating the third mixed solution to 60-100℃ and adding a second monomer and a 25% ammonia solution to the third mixed solution to obtain a fourth mixed solution; after the fourth mixed solution is uniformly mixed, reacting it at a reaction temperature of 60-100℃ for 2-6 hours to obtain a precursor; then, ultrasonically dispersing the precursor in a solvent, adding an initiator and distilled water, and stirring for 10-30 minutes to obtain a second reaction solution; finally, reacting the second reaction solution at a reaction temperature of 60-100℃ for 2-6 hours, and washing, drying, and separating the reacted second reaction solution to obtain magnetic nanoparticles.

[0088] The method for preparing magnetic microspheres provided in this disclosure involves doping a target organic material containing at least one of amino, carboxyl, and hydroxyl functional groups into the framework structure of graphene oxide. This alters the binding properties between graphene oxide and RNA, allowing the magnetic microspheres and RNA to be separated using conventional elution solutions. This reduces processing time during RNA extraction, thereby improving RNA extraction efficiency. Furthermore, the method uses a mixture of magnetic nanoparticles, graphene oxide, and the target organic material in a mass ratio of 1:2-4-1.5-2.5 to ensure the magnetic microspheres' preference for RNA and improve the purity of RNA extraction.

[0089] Based on the same inventive concept, this disclosure also provides an RNA extraction kit, including a magnetic bead suspension; wherein the magnetic bead suspension includes magnetic microspheres as described in any of the above embodiments or magnetic microspheres prepared by the method described in any of the above embodiments.

[0090] In this embodiment, the magnetic bead suspension is obtained by adding the magnetic microspheres provided in the above embodiment to a Tris-HCl solution.

[0091] The concentration of the magnetic bead suspension is 0.5-2 g / mL.

[0092] Specifically, the concentration of the magnetic bead suspension can be 0.5 g / mL, 1 g / mL, 1.5 g / mL, and 2 g / mL, etc. Among them, when the concentration of the magnetic bead suspension is 1 g / mL, the RNA extraction kit has a better RNA extraction effect.

[0093] It is understandable that the RNA extraction process also requires steps such as lysis, washing, and elution. Therefore, the RNA extraction kit also includes lysis buffer, binding buffer, washing buffer, and elution buffer. The binding buffer may include a first binding buffer and a second binding buffer, and the washing buffer may include a first washing buffer and a second washing buffer.

[0094] The lysis buffer comprises 2-6 M guanidine isothiocyanate, 1-3 mM Tris-HCl, and 0.01%-0.1% Tween 20, with a pH of 7.0-8.0. For example, the lysis buffer comprises 3 M guanidine isothiocyanate, 1 mM Tris-HCl, and 0.05% Tween 20, with a pH of 7.2.

[0095] The first binding solution comprises 10-20% PEG8000, 0.5-1M NaCl, 10-30mM Tris-HCl, 10-30mM EDTA, and 0.01%-0.1% Tween 20, with a pH of 7-8; the second binding solution comprises isopropanol. For example, the first binding solution comprises 15% PEG8000, 1M NaCl, 10mM Tris-HCl, 10mM EDTA, and 0.05% Tween 20, with a pH of 7.5; the second binding solution comprises isopropanol.

[0096] The first cleaning solution comprises 1-2 M guanidine isothiocyanate, 1-2 mM Tris-HCl, 0.01%-0.1% Tween 20, 0.5-1 M NaCl, and 40%-60% isopropanol, with a pH of 7.0-8.0; the second cleaning solution comprises a 60%-80% ethanol solution. For example, the first cleaning solution comprises 1.5 M guanidine isothiocyanate, 1 mM Tris-HCl, 0.05% Tween 20, 1 M NaCl, and 50% isopropanol, with a pH of 7.6; the second cleaning solution is an 80% ethanol solution.

[0097] The eluent comprises an enzyme-free aqueous solution of 0-20 mmol / L Tris-EDTA, for example, the eluent is an enzyme-free aqueous solution of 10 mmol / L Tris-EDTA.

[0098] The RNA extraction kit provided in this embodiment of the invention has a good adsorption effect on RNA by magnetic microspheres, which eliminates the need to use DNase and DNA reaction mix to process the extracted single-stranded DNA, thereby improving the RNA extraction efficiency and ensuring the purity of the extracted RNA.

[0099] Based on the same inventive concept, this disclosure also provides an RNA extraction method, which uses the RNA extraction kit described in the above embodiments for extraction, referring to... Figure 4 , Figure 4 A flowchart illustrating the steps of the RNA extraction method provided in this disclosure embodiment is shown, as follows: Figure 4 As shown, the method specifically includes:

[0100] S301, add lysis buffer and protease to the sample solution, and collect the supernatant after lysis by centrifugation.

[0101] S302: Binding solution and magnetic bead suspension are added sequentially to the supernatant to bind the magnetic microspheres to RNA, and the RNA-bound magnetic microspheres are collected under an external magnetic field.

[0102] S303 uses an elution buffer to separate the magnetic microspheres that have bound RNA, thereby extracting the RNA.

[0103] Specifically, the sample solution can be a cell suspension. By adding a lysis buffer to the sample solution to disrupt the cell membrane, nuclear membrane, and other biological membrane structures, the cell contents are released. Proteinase K degrades proteins, including histones that bind to nucleic acids, allowing RNA to be released from the protein-nucleic acid complex, facilitating RNA extraction. Afterward, the sample solution can be centrifuged, and the supernatant is collected to remove impurities. Next, a binding buffer can be used to provide an environment for the magnetic microspheres to bind to RNA, enabling the magnetic microspheres to specifically bind to RNA. Impurities are thus retained in the solution; by collecting the magnetic microspheres, RNA and impurities can be separated.

[0104] Before separating the magnetic microspheres, the RNA-bound microspheres can be washed to remove impurities such as single-stranded DNA and proteins that may be present on the surface of the microspheres, ensuring the purity of the RNA. After washing, the magnetic microspheres can be separated using an elution buffer to extract the RNA.

[0105] The specific components of the lysis buffer, binding buffer, washing buffer, and elution buffer can be referred to the components shown in the above-mentioned RNA extraction kit examples, and will not be repeated here.

[0106] The RNA extraction method provided in this embodiment of the invention includes graphene oxide doped with amino, carboxyl, and hydroxyl groups in the magnetic microspheres, which alters the binding properties between graphene oxide and RNA. This eliminates the need for an additional urea solution elution step in the RNA extraction process, reducing the RNA extraction time and thus improving the RNA extraction efficiency.

[0107] To enable those skilled in the art to better understand the magnetic microspheres and their preparation methods, RNA extraction kits, and RNA extraction methods provided in the embodiments of this disclosure, the performance of the magnetic microspheres and the RNA extraction methods will be tested through some exemplary embodiments below:

[0108] Example 1

[0109] In a 500 mL three-necked flask, 300 mL of distilled water was added, and nitrogen gas was continuously bubbled through the flask for 30 min, maintaining the nitrogen atmosphere thereafter. Then, 5 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were added, and stirring continued until completely dissolved. The temperature was then raised to 80 °C, 100 mg of oleic acid was added, followed by 20 mL of 25% ammonia solution. After thorough mixing, the mixture was reacted at 80 °C for 4 h to obtain the Fe3O4-OA precursor.

[0110] 0.5 g of Fe3O4-OA precursor was ultrasonically dispersed in 5 mL of styrene, and then 0.5 g of benzoyl peroxide, 0.2 g of cetyl alcohol and 100 mL of ultrapure water were added. After stirring for 0.5 h, the temperature was raised to 80 °C and stirred continuously for 4 h. After washing and drying, the PS@Fe3O4 nanoparticles were obtained by separation with a magnet.

[0111] Place 1g of cleaned PS@Fe3O4 nanoparticles into a test tube, then add 10ml of TEOS, 0.2g of Triton X-100 and 100ml of deionized water in sequence. After sonication for 10min, add 20mL of 25% ammonia water and react in a sealed container at 80℃ for 4h to obtain PS@Fe3O4@SiO2 nanoparticles.

[0112] 765 mg EDC and 115 mg NHS were weighed and prepared into 10 mL solutions respectively. These solutions were then added together to 2 g of graphene oxide for activation. 1 g of the prepared PS@Fe3O4@SiO2 nanoparticles were added to 100 mL of enzyme-free water and ultrasonically dispersed. Then, 2 g of activated graphene oxide and 0.5 g APTES were added, and the mixture was stirred and reacted at 30 °C overnight or for 12 h. After magnetic separation, washing, and drying, PS@Fe3O4@SiO2@GO-NH2 nanoparticles were obtained. The structural diagram of the obtained PS@Fe3O4@SiO2@GO-NH2 nanoparticles is shown below. Figure 1 As shown, the SEM and TEM images of the nanoparticles are as follows: Figure 5 , Figure 6 As shown.

[0113] Example 2

[0114] A reaction solution was prepared by mixing 3g ferric chloride, 1g ferric chloride, 0.3g surfactant, 4mL alkaline solution and 100mL water. The reaction solution was reacted at 70-100℃ for 2-8h, and then subjected to magnetic separation, washing and drying to obtain magnetic Fe3O4 nanoparticles.

[0115] 1g of magnetic Fe3O4 nanoparticles, 10mLTEOS, and 0.1g of surfactant were added to 100mL of water, mixed well, and then 10mL of alkaline solution was added. The mixture was stirred and reacted at 20-40℃ for 4-8h. After magnetic separation, washing, and drying, Fe3O4@SiO2 nanoparticles were obtained.

[0116] Graphene oxide was activated using EDC / NHS solution. 1g of Fe3O4@SiO2 nanoparticles were dispersed in 100mL of water, and 2g of activated graphene oxide and 2g of APTES were added. The mixture was stirred and reacted at 10-30℃ for 4-8h to allow APTES to be doped into the graphene oxide and modified onto the silica layer. After magnetic separation, washing and drying, Fe3O4@SiO2@GO-NH2 nanoparticles were obtained.

[0117] Example 3

[0118] In a 500 mL three-necked flask, 300 mL of distilled water was added, and nitrogen was continuously bubbled through the flask for 30 min, maintaining the nitrogen atmosphere thereafter. Then, 5 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were added, and stirring continued until completely dissolved. The temperature was then raised to 80 °C, 100 mg of oleic acid was added, followed by 20 mL of 25% ammonia solution. After thorough mixing, the mixture was reacted at 80 °C for 4 h to obtain the Fe3O4-OA precursor.

[0119] 0.5 g of Fe3O4-OA precursor was ultrasonically dispersed in 5 mL of styrene, and then 0.5 g of benzoyl peroxide, 0.2 g of cetyl alcohol and 100 mL of ultrapure water were added. After stirring for 0.5 h, the temperature was raised to 80 °C and stirred continuously for 4 h. After washing and drying, the PS@Fe3O4 nanoparticles were obtained by separation with a magnet.

[0120] Place 1g of cleaned PS@Fe3O4 nanoparticles into a test tube, then add 10ml of TEOS, 0.2g of Triton X-100 and 100ml of deionized water in sequence. After sonication for 10min, add 20mL of 25% ammonia water and react in a sealed container at 80℃ for 4h to obtain PS@Fe3O4@SiO2 nanoparticles.

[0121] Weigh 765 mg EDC and 115 mg NHS, prepare 10 mL solutions respectively, and then pour them together into 2 g of graphene oxide for activation. Add 1 g of the prepared PS@Fe3O4@SiO2 nanoparticles to 100 mL of enzyme-free water, disperse by ultrasonication, add 2 g of activated graphene oxide and 1 g of APTES, stir, and react at 30 °C overnight or for 12 h. After magnetic separation, washing, and drying, PS@Fe3O4@SiO2@GO-NH2 nanoparticles are obtained.

[0122] Example 4

[0123] In a 500 mL three-necked flask, 300 mL of distilled water was added, and nitrogen was continuously bubbled through the flask for 30 min, maintaining the nitrogen atmosphere thereafter. Then, 5 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were added, and stirring continued until completely dissolved. The temperature was then raised to 80 °C, 100 mg of oleic acid was added, followed by 20 mL of 25% ammonia solution. After thorough mixing, the mixture was reacted at 80 °C for 4 h to obtain the Fe3O4-OA precursor.

[0124] 0.5 g of Fe3O4-OA precursor was ultrasonically dispersed in 5 mL of styrene, and then 0.5 g of benzoyl peroxide, 0.2 g of cetyl alcohol and 100 mL of ultrapure water were added. After stirring for 0.5 h, the temperature was raised to 80 °C and stirred continuously for 4 h. After washing and drying, the PS@Fe3O4 nanoparticles were obtained by separation with a magnet.

[0125] Place 1g of cleaned PS@Fe3O4 nanoparticles into a test tube, then add 10ml of TEOS, 0.2g of Triton X-100 and 100ml of deionized water in sequence. After sonication for 10min, add 20mL of 25% ammonia water and react in a sealed container at 80℃ for 4h to obtain PS@Fe3O4@SiO2 nanoparticles.

[0126] Weigh 765 mg EDC and 115 mg NHS, prepare 10 mL solutions respectively, and then pour them together into 2 g of graphene oxide for activation. Add 1 g of the prepared PS@Fe3O4@SiO2 nanoparticles to 100 mL of enzyme-free water, disperse by ultrasonication, add 2 g of activated graphene oxide and 1.5 g APTES, stir, and react at 30 °C overnight or for 12 h. After magnetic separation, washing, and drying, PS@Fe3O4@SiO2@GO-NH2 nanoparticles are obtained.

[0127] Example 5

[0128] In a 500 mL three-necked flask, 300 mL of distilled water was added, and nitrogen was continuously bubbled through the flask for 30 min, maintaining the nitrogen atmosphere thereafter. Then, 5 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were added, and stirring continued until completely dissolved. The temperature was then raised to 80 °C, 100 mg of oleic acid was added, followed by 20 mL of 25% ammonia solution. After thorough mixing, the mixture was reacted at 80 °C for 4 h to obtain the Fe3O4-OA precursor.

[0129] 0.5 g of Fe3O4-OA precursor was ultrasonically dispersed in 5 mL of styrene, and then 0.5 g of benzoyl peroxide, 0.2 g of cetyl alcohol and 100 mL of ultrapure water were added. After stirring for 0.5 h, the temperature was raised to 80 °C and stirred continuously for 4 h. After washing and drying, the PS@Fe3O4 nanoparticles were obtained by separation with a magnet.

[0130] Place 1g of cleaned PS@Fe3O4 nanoparticles into a test tube, then add 10ml of TEOS, 0.2g of Triton X-100 and 100ml of deionized water in sequence. After sonication for 10min, add 20mL of 25% ammonia water and react in a sealed container at 80℃ for 4h to obtain PS@Fe3O4@SiO2 nanoparticles.

[0131] Weigh 765 mg EDC and 115 mg NHS, prepare 10 mL solutions respectively, and then pour them together into 2 g of graphene oxide for activation. Add 1 g of the prepared PS@Fe3O4@SiO2 nanoparticles to 100 mL of enzyme-free water, disperse by ultrasonication, add 2 g of activated graphene oxide and 2 g of APTES, stir, and react at 30 °C overnight or for 12 h. After magnetic separation, washing, and drying, PS@Fe3O4@SiO2@GO-NH2 nanoparticles are obtained.

[0132] Example 6

[0133] In a 500 mL three-necked flask, 300 mL of distilled water was added, and nitrogen was continuously bubbled through the flask for 30 min, maintaining the nitrogen atmosphere thereafter. Then, 5 g of FeCl3·6H2O and 2 g of FeCl2·4H2O were added, and stirring continued until completely dissolved. The temperature was then raised to 80 °C, 100 mg of oleic acid was added, followed by 20 mL of 25% ammonia solution. After thorough mixing, the mixture was reacted at 80 °C for 4 h to obtain the Fe3O4-OA precursor.

[0134] 0.5 g of Fe3O4-OA precursor was ultrasonically dispersed in 5 mL of styrene, and then 0.5 g of benzoyl peroxide, 0.2 g of cetyl alcohol and 100 mL of ultrapure water were added. After stirring for 0.5 h, the temperature was raised to 80 °C and stirred continuously for 4 h. After washing and drying, the PS@Fe3O4 nanoparticles were obtained by separation with a magnet.

[0135] Place 1g of cleaned PS@Fe3O4 nanoparticles into a test tube, then add 10ml of TEOS, 0.2g of Triton X-100 and 100ml of deionized water in sequence. After sonication for 10min, add 20mL of 25% ammonia water and react in a sealed container at 80℃ for 4h to obtain PS@Fe3O4@SiO2 nanoparticles.

[0136] Weigh 765 mg EDC and 115 mg NHS, prepare 10 mL solutions respectively, and then pour them together into 2 g of graphene oxide for activation. Add 1 g of the prepared PS@Fe3O4@SiO2 nanoparticles to 100 mL of enzyme-free water, disperse by ultrasonication, add 2 g of activated graphene oxide and 2.5 g APTES, stir, and react at 30 °C overnight or for 12 h. After magnetic separation, washing, and drying, PS@Fe3O4@SiO2@GO-NH2 nanoparticles are obtained.

[0137] The magnetic beads obtained in Examples 1 and 3-6 were respectively prepared into magnetic bead suspensions of 1 g / mL to obtain RNA extraction kits numbered 1-5. The other solution components in RNA extraction kits numbered 1-5 are the same, specifically including: lysis buffer: 3M guanidine isothiocyanate, 1mM Tris-HCl and 0.05% Tween 20, pH value 7.2.

[0138] First binding solution: 15% PEG8000, 1M NaCl, 10mM Tris-HCl, 10mM EDTA and 0.05% Tween 20, pH 7.5.

[0139] Second binding agent: isopropanol;

[0140] First cleaning solution: 1.5M guanidine isothiocyanate, 1mM Tris-HCl, 0.05% Tween 20, 1M NaCl and 50% isopropanol, pH 7.6.

[0141] Second cleaning solution: 80% ethanol solution;

[0142] Elution buffer: 10 mmol / L Tris-EDTA in an enzyme-free aqueous solution.

[0143] Experiment 1

[0144] Take 200 μL of sample solution and put it into a 1.5 mL centrifuge tube. Then add 300 μL of lysis buffer and 20 μL of proteinase K. Vortex to mix and incubate at 60 °C for 10 min. After centrifugation, transfer the supernatant to a new 1.5 mL centrifuge tube.

[0145] Add 270 μL of the first binding solution to a centrifuge tube containing the supernatant, vortex to mix, then add 30 μL of the magnetic bead suspension, vortex to mix, then add 300 μL of the second binding solution, vortex to mix, and let stand for 5 minutes. Finally, centrifuge at low speed, place the centrifuge tube on a magnetic rack, let stand for two minutes, and then remove the supernatant.

[0146] Add 600 μL of the first cleaning solution to the centrifuge tube, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant; then add 400 μL of the second cleaning solution, vortex to mix, let stand for two minutes, centrifuge at low speed, place on a magnetic rack, let stand for two minutes, and then remove the supernatant.

[0147] Next, add 80 μL of elution buffer to the centrifuge tube, vortex to mix, let stand for 2 minutes, centrifuge at low speed, place on a magnetic rack, aspirate the supernatant and store it, which is the RNA to be extracted.

[0148] Experiment 1 was performed using RNA extraction kits numbered 1-5, and the results are shown in Table 1 below. The extracted RNA was analyzed using a fully automated nucleic acid analyzer at 4200 nm, and the results are as follows: Figure 7 As shown, Figure 7 The horizontal axis represents the size of the nucleic acid fragment, and the vertical axis represents the concentration value. Figure 7 It can be seen that the product has signals at 5s, 18s and 28s, that is, the 5s, 18s and 28s bands of RNA are clear and complete, and its RIN value is 9.2, indicating good integrity.

[0149] Table 1 shows the results of 4200, Qubit, and Nanodrop assays on the extracted nucleic acid products.

[0150] serial number Average RNA concentration (ng / mL) Average DNA concentration (ng / mL) A260 / 280 1 80.4 29.2 1.76 2 90.2 20.6 1.82 3 98.4 15.4 1.87 4 118.2 5.68 1.96 5 102.5 9.26 1.91

[0151] As shown in Table 1, when the amount of γ-aminopropyltriethoxysilane added is between 1.5g and 2.5g, the magnetic microspheres have a good extraction effect on RNA. When the content of γ-aminopropyltriethoxysilane is 2g, the RNA concentration is the highest, the residual DNA concentration is the lowest, and the RNA purity is also the highest.

[0152] Experiment 2

[0153] Primers and probes for DNA and RNA were designed separately. The designed probes and primers are as follows: Figure 8 As shown, D1 represents DNA probe primer and R1 represents RNA probe primer. Based on the results of Experiment 1, the extract of magnetic beads with an APTES addition of 2g was selected, divided into two portions, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiment was performed according to the following protocol.

[0154] RNA reverse transcription: Reverse transcription was performed using the Quant cDNA First Strand Synthesis Kit (KR103) from Tiangen (Beijing) Biotechnology Co., Ltd., following the instructions for use.

[0155] The qPCR experiments used the PCR reaction solution (Taq Pro U+MultipleProbe qPCR Mix) from Nanjing Novizan Biotechnology Co., Ltd. qPCR experiments were performed on both reverse-transcribed and non-reverse-transcribed samples according to the instructions.

[0156] Get as Figure 9 The results are shown, where curve 1 represents the fluorescence signal response curve of RNA, and curve 2 represents the fluorescence signal response curve of DNA. According to... Figure 9 The RNA extracted using the magnetic microsphere kit provided in Example 2 did not show a fluorescent signal of gDNA, but a fluorescent signal of cDNA was detected. This indicates that the DNA content in the extracted product is very low and will not affect the detection of RNA signal.

[0157] Based on the results of Experiments 1 and 2, it can be seen that the magnetic microspheres provided in this embodiment of the present disclosure are used for RNA extraction with high extraction efficiency and high RNA purity.

[0158] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0159] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0160] The foregoing has provided a detailed description of the magnetic microspheres and their preparation method, RNA extraction kit, and RNA extraction method provided in this disclosure. Specific examples have been used to illustrate the principles and implementation methods of this disclosure. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this disclosure. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this disclosure. Therefore, the content of this specification should not be construed as a limitation of this disclosure.

[0161] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the following claims.

[0162] It should be understood that this disclosure is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The scope of this disclosure is limited only by the appended claims.

[0163] The terms "an embodiment," "embodiment," or "one or more embodiments" as used herein mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of this disclosure. Furthermore, please note that the examples of the phrase "in one embodiment" do not necessarily all refer to the same embodiment.

[0164] Numerous specific details are set forth in the specification provided herein. However, it will be understood that embodiments of this disclosure may be practiced without these specific details. In some instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0165] In the claims, any reference signs placed between parentheses should not be construed as limiting the claims. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. This disclosure can be implemented by means of hardware comprising a plurality of different elements and by means of a suitably programmed computer. In a unit claim enumerating a plurality of means, several of these means may be embodied by the same item of hardware. The use of the words first, second, and third, etc., does not indicate any order. These words may be interpreted as names.

[0166] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this disclosure.

Claims

1. A magnetic microsphere, characterized in that, An application for nucleic acid extraction includes a core and a shell encapsulating the core; wherein the core comprises magnetic nanoparticles, the shell comprises graphene oxide, and a target organic material doped within the graphene oxide, wherein the target organic material comprises at least one of an amino functional group, a carboxyl functional group, and a hydroxyl functional group.

2. The magnetic microspheres according to claim 1, characterized in that, The target organic material includes at least one of amino coupling agents, carboxyl coupling agents, and hydroxyl coupling agents.

3. The magnetic microspheres according to claim 2, characterized in that, The amino coupling agent includes γ-aminopropyltriethoxysilane, the carboxyl coupling agent includes mercaptopropionic acid, and the hydroxy coupling agent includes glycidyl methacrylate.

4. The magnetic microspheres according to claim 1, characterized in that, The magnetic microspheres exhibit a higher adsorption rate for RNA than they do for single-stranded DNA and proteins.

5. The magnetic microspheres according to claim 4, characterized in that, The target organic material is doped into the graphene oxide at a ratio of 60%-75%.

6. The magnetic microspheres according to claim 1, characterized in that, The magnetic microspheres also include a connecting layer located between the core and the shell; The connecting layer includes at least one of silica, agarose gel, polyglycidyl methacrylate, and polyvinyl alcohol.

7. The magnetic microspheres according to claim 1, characterized in that, The magnetic nanoparticles are magnetic nanoparticles, or... The magnetic nanoparticles include organic polymer particles and a magnetic layer covering the organic polymer particles.

8. A method for preparing magnetic microspheres, characterized in that, The method is applied to the preparation of magnetic microspheres as described in any one of claims 1-7, and comprises: Formation of magnetic nanoparticles; The magnetic nanoparticles, graphene oxide, and target organic material are mixed in a mass ratio of 1:2-4:1.5-2.5 to obtain the magnetic microspheres; wherein the magnetic microspheres include a core and a shell enclosing the core, the core includes the magnetic nanoparticles, the shell includes graphene oxide, and the target organic material is doped inside the graphene oxide, the target organic material includes at least one of amino functional groups, carboxyl functional groups, and hydroxyl functional groups.

9. The method for preparing magnetic microspheres according to claim 8, characterized in that, The magnetic nanoparticles, graphene oxide, and target organic material are mixed to obtain the magnetic microspheres, comprising: Graphene oxide is activated using an activator. The magnetic nanoparticles were dispersed in the water, and activated graphene oxide and the target organic material were added to obtain a first mixed solution. The first mixed solution is placed at a first reaction temperature and stirred for a first time to obtain the magnetic microspheres.

10. The method for preparing magnetic microspheres according to claim 8, characterized in that, Before mixing the magnetic nanoparticles, graphene oxide, and the target organic material, the method further includes: The magnetic nanoparticles, monomers, and surfactants were added to water to obtain a second mixed solution; After mixing the second mixed solution thoroughly, add the alkaline solution and stir. The stirred second mixed solution is placed at a second reaction temperature for a second time to form a bonding layer on the surface of the magnetic nanoparticles.

11. An RNA extraction kit, characterized in that, It includes at least a magnetic bead suspension, wherein the magnetic bead suspension comprises magnetic microspheres as described in any one of claims 1-7, or magnetic microspheres prepared by the preparation method as described in any one of claims 8-10.

12. The RNA extraction kit according to claim 11, characterized in that, The concentration of the magnetic bead suspension is 0.5-2 g / mL.

13. A method for RNA extraction, characterized in that, RNA extraction is performed using the RNA extraction kit as described in any one of claims 11-12, wherein the extraction method comprises: Add lysis buffer and protease to the sample solution, and collect the supernatant after lysis by centrifugation; A binding solution and a magnetic bead suspension were added to the supernatant to bind the magnetic microspheres to RNA, and the RNA-bound magnetic microspheres were collected under an external magnetic field. The magnetic microspheres bound to RNA were separated using an elution buffer, and the RNA was extracted.

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