A stimulus-responsive polymer magnetic bead, its preparation method, and a method for targeted extraction of ribonucleic acid.

Abstract

This invention discloses a stimulus-responsive polymer magnetic bead, its preparation method, and a method for targeted extraction of ribonucleic acid (RNA), belonging to the fields of biomedical nanomaterials and molecular diagnostics. The polymer magnetic bead comprises a magnetic carrier and a stimulus-responsive polymer grafted onto its surface. This polymer contains thermosensitive N-isopropylacrylamide segments and functional monomer segments with phosphate group affinity. The preparation method includes surface bromination of the magnetic carrier, followed by grafting the copolymer via atom transfer radical polymerization. In RNA extraction applications, these magnetic beads utilize the stimulus-responsive properties of the polymer chains to achieve dynamic adsorption and static elution, enabling direct and efficient capture of RNA from tissue lysate. This invention significantly improves the concentration, purity, and integrity of extracted RNA, while simplifying the operation steps, shortening the extraction time, and greatly reducing the use of toxic reagents, offering outstanding advantages such as low cost, high efficiency, and environmental friendliness.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedical nanomaterials and molecular diagnostic technology, specifically relating to a stimulus-responsive polymer magnetic bead, its preparation method, and a method for targeted extraction of ribonucleic acid. Background Technology

[0002] Ribonucleic acid (RNA), as a key carrier of genetic information, plays a crucial role in gene expression regulation, disease diagnosis, and functional genomics research. Efficient and high-purity RNA extraction is a prerequisite for downstream molecular analysis of complex biological samples. Currently, the mainstream methods for RNA extraction mainly include the Trizol method and the commercial magnetic bead method.

[0003] The Trizol method, based on an acidic phenol-chloroform system, enriches RNA through phase separation. Its advantage lies in its high RNA yield, but it has significant disadvantages: the operation steps are cumbersome and time-consuming, and the use of toxic reagents such as phenol and chloroform poses risks to operators and the environment. At the same time, this method is susceptible to interference from impurities such as polysaccharides and polyphenols, resulting in insufficient RNA purity and affecting the reliability of subsequent experiments.

[0004] Commercial magnetic bead methods rely on functionalized magnetic particles to adsorb nucleic acids through electrostatic, hydrophobic, or hydrogen bonding interactions, offering advantages such as ease of operation and automation. However, traditional magnetic beads are typically modified with static functional groups such as silicon, amino, or carboxyl groups, resulting in a single adsorption mechanism, limited binding capacity and selectivity for RNA, and susceptibility to competitive adsorption interference from proteins and metabolites in complex samples, leading to decreased extraction efficiency and purity. Furthermore, commercial magnetic bead kits often rely on multiple specialized buffers, resulting in high costs, and some reagents still possess a degree of toxicity.

[0005] Therefore, developing a novel RNA extraction material that combines high adsorption capacity, excellent selectivity, low toxicity, and ease of operation has become an urgent goal in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention innovatively develops a stimulus-responsive polymer magnetic bead, its preparation method, and a method for targeted extraction of ribonucleic acid (RNA). This invention utilizes PNI... x -CO-ATBA 1-x By grafting onto the surface of magnetic beads, the extraction efficiency of traditional magnetic beads is improved, and a new generation of stimulus-responsive polymer magnetic beads is developed.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a stimulus-responsive polymer magnetic bead, the polymer magnetic bead comprising a magnetic carrier and a stimulus-responsive polymer grafted onto the surface of the magnetic carrier, the stimulus-responsive polymer comprising a stimulus-responsive N-isopropylacrylamide segment and a functional monomer segment having phosphate group affinity.

[0009] As a further optimization of the present invention, the polymer magnetic beads have at least one of the following features a1)-a3):

[0010] a1) The polymer magnetic beads have a size of 150-250 nm;

[0011] a2) The magnetic carrier is Fe3O4-SiO2 composite particles with amino groups on the surface;

[0012] a3) The functional monomer is 4-(3-acryloylthiourea)-benzoic acid or its derivative.

[0013] Secondly, the present invention provides a method for preparing the aforementioned stimulus-responsive polymer magnetic beads, comprising the following steps:

[0014] Obtain a magnetic carrier with a brominated surface;

[0015] N-isopropylacrylamide and functional monomers are polymerized in a solvent to form the stimulus-responsive polymer on the surface of a magnetic carrier, and the polymer magnetic beads are obtained after purification.

[0016] As a further optimization of the present invention, the method for obtaining a surface-brominated magnetic support includes the following steps:

[0017] Obtain a dispersion of magnetic nanoparticles;

[0018] In the presence of an alkaline catalyst, an orthosilicate compound is added to the dispersion to carry out a hydrolysis-condensation reaction, thereby coating the surface of the magnetic nanoparticles with a silica layer.

[0019] An aminosilane coupling agent was added to the reaction system to continue the reaction, thereby introducing amino groups onto the surface of the silica layer. After filtration, washing and drying, a magnetic carrier with amino groups on its surface was obtained.

[0020] The surface-modified amino magnetic carrier is reacted with an α-haloacyl halide compound in the presence of an organic base. After filtration, washing and drying, a surface-brominated magnetic carrier is obtained.

[0021] As a further optimization of the present invention, the method for obtaining a surface-brominated magnetic support has at least one of the following features b1)-b7):

[0022] b1) The magnetic nanoparticles are iron oxide nanoparticles;

[0023] b2) The alkaline catalyst is ammonia water;

[0024] b3) The orthosilicate compound is a tetraethoxysilane;

[0025] b4) The aminosilane coupling agent is 3-aminopropyltriethoxysilane;

[0026] b5) The α-haloacyl halide is 2-bromoisobutyryl bromide;

[0027] b6) The organic base is anhydrous pyridine;

[0028] b7) The process of reacting in the presence of an organic base includes: first, dispersing the surface-modified amino magnetic support in a mixture of organic base and aprotic polar solvent, then adding α-haloacyl halides dropwise while stirring under 0°C ice bath conditions and reacting in the dark for 0.5-1.5 hours, followed by transferring to room temperature and reacting in the dark for 8-15 hours.

[0029] As a further optimization of the present invention, the method for grafting the stimulus-responsive polymer onto the surface of a magnetic carrier includes the following steps:

[0030] A surface-brominated magnetic support, N-isopropylacrylamide, and functional monomers are dispersed in a solvent, and a copper salt catalyst and a polydentate amine ligand are added under an inert atmosphere. The mixture is then sealed to remove oxygen, and a polymerization reaction is carried out after deoxygenation, thereby grafting the stimulus-responsive polymer onto the surface of the magnetic support.

[0031] As a further optimization of the present invention, the method for grafting the stimulus-responsive polymer onto the surface of a magnetic carrier has at least one of the following features c1)-c5):

[0032] c1) The copper salt catalyst is cuprous bromide;

[0033] c2) The polydentate amine ligand is N,N,N′,N″,N″-pentamethyldiethylenetriamine;

[0034] c3) The sealing and deoxygenation process includes: filling the reaction system into a reaction bottle and sealing it, immersing it in liquid nitrogen to freeze it completely, then evacuating the reaction bottle, and repeating the freeze-thaw process multiple times after the reactants have fully thawed to completely remove the oxygen from the reaction system.

[0035] c4) The molar ratio between the N-isopropylacrylamide, the functional monomer, the cuprous bromide, the polydentate amine ligand, and the brominated group (i.e., the 2-bromoisobutyramide group) on the magnetic support is (3-5):1:(0.1-0.25):(0.3-0.8):(0.025-0.05).

[0036] c5) The conditions for the polymerization reaction include: reaction under light-protected conditions, reaction temperature of 50-70℃, and reaction time of 20-30 hours. 50-70℃ can ensure both the polymerization rate and maintain controllability.

[0037] Thirdly, the present invention provides a method for targeted extraction of ribonucleic acid, using the aforementioned stimulus-responsive polymer magnetic beads, the method comprising:

[0038] (1) The tissue sample was broken in water treated with diethyl pyrocarbonate, and the supernatant was collected after centrifugation;

[0039] (2) Add isopropanol and pretreated polymer magnetic bead suspension to the supernatant and mix to bind RNA to the magnetic beads;

[0040] (3) Adsorb the magnetic beads using a magnetic rack, discard the liquid, and wash the magnetic beads with ethanol;

[0041] (4) After drying the magnetic beads, add diethyl pyrocarbonate-treated water and shake to wash out the RNA. After separating the magnetic beads, obtain the RNA solution.

[0042] As a further optimization of the present invention, the pretreatment step of the polymer magnetic beads includes: adding the polymer magnetic beads to isopropanol for ultrasonic treatment, diluting and incubating to obtain a polymer magnetic bead suspension.

[0043] As a further optimization of the present invention, in step (1), guanidine hydrochloride is added to the water treated with diethyl pyrocarbonate.

[0044] More preferably, after adding guanidine hydrochloride to the water treated with diethyl pyrocarbonate, the concentration of guanidine hydrochloride is 0.3-0.8 mol / L.

[0045] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0046] 1. Traditional magnetic beads rely on static surface coatings (such as silicon-based or amino-based) to adsorb RNA through electrostatic or hydrophobic interactions, resulting in limited adsorption capacity and susceptibility to impurities. This invention grafts a copolymer of thermosensitive PNIPAAm segments and the phosphate-specific affinity monomer ATBA onto the magnetic bead surface. The multiple hydrogen bonds formed by thiourea and carboxyl groups in ATBA selectively bind RNA phosphate groups. Simultaneously, the PNIPAAm segments undergo conformational changes under external stimuli (such as temperature and solvent polarity), achieving dynamic adsorption and static elution. This design not only significantly increases the effective adsorption area but also exposes more binding sites through conformational regulation, significantly improving RNA binding capacity and selectivity. Experimental data show that compared to the Trizol method and commercial magnetic bead methods, this method increases RNA extraction concentration by 50–80% with simultaneous improvement in purity, while maintaining excellent extraction integrity in complex matrix samples such as white hyacinth bean, lotus seed, and honeysuckle.

[0047] 2. Traditional methods rely on multi-step phase separation or various specialized buffer systems, which are cumbersome and costly in terms of reagents. The magnetic beads of this invention can be directly added to the tissue lysis buffer. The polymer conformation and RNA binding can be controlled simply by adjusting the solvent composition (e.g., the isopropanol ratio), eliminating the need for multiple auxiliary reagents such as lysis buffer, binding buffer, and washing buffer. Extraction time is reduced from 1.5–2 hours in traditional methods to 40 minutes, increasing efficiency by over 55%. Simultaneously, it eliminates expensive or toxic reagents such as Trizol, chloroform, and mercaptoethanol, reducing the cost per hundred extractions by 78–93%, demonstrating significant economic advantages and ease of operation.

[0048] 3. The traditional Trizol method uses highly toxic organic reagents such as phenol and chloroform, posing hazards to operators and the environment; commercial magnetic bead methods also often involve guanidine salts and organic solvents. This invention achieves highly specific RNA capture and gentle elution through stimulus-responsive polymers, significantly reducing the types and amounts of toxic reagents used. Only low concentrations of isopropanol and ethanol are required for extraction and washing, making it green and environmentally friendly, and better meeting the safety and sustainable development requirements of modern laboratories.

[0049] 4. The polymer segments exhibit sensitive responses to external stimuli such as temperature and solvent polarity, enabling precise control of RNA adsorption and elution processes through simple physical condition adjustments, thus enhancing experimental reproducibility and automation potential. Furthermore, by adjusting the ratio of ATBA to PNIPAMAm segments (e.g., preferably 36% ATBA), the affinity and responsiveness balance can be further optimized to accommodate biological samples of varying origins and complexities (such as plant tissues and metabolite-rich samples). The introduction of auxiliary reagents such as guanidine hydrochloride further effectively inhibits RNase and dissolves RNA-impurity complexes, further improving extraction purity and integrity. Attached Figure Description

[0050] Figure 1Three different grafting ratios of PNI synthesized in Example 1 x -CO-ATBA 1-x The proton NMR spectrum of the polymer.

[0051] Figure 2 Three different grafting ratios of PNI synthesized in Example 1 x -CO-ATBA 1-x Infrared spectrum of the polymer.

[0052] Figure 3 PNI synthesized in Example 1 0.71 -CO-ATBA 0.29 XPS spectra of polymers.

[0053] Figure 4 PNI synthesized in Example 1 0.64 -CO-ATBA 0.36 XPS spectra of polymers.

[0054] Figure 5 PNI synthesized in Example 1 0.51 -CO-ATBA 0.49 XPS spectra of polymers.

[0055] Figure 6 PNI synthesized in Example 1 0.64 -CO-ATBA 0.36 Hydrodynamic radius distribution of polymers in aqueous solutions with different acetonitrile ratios.

[0056] Figure 7 Infrared spectra of polymer magnetic beads with different grafting ratios prepared in Examples 2-4.

[0057] Figure 8 PNI prepared in Example 2 0.71 -CO-ATBA 0.29 XPS spectra of polymer magnetic beads.

[0058] Figure 9 PNI prepared in Example 3 0.64 -CO-ATBA 0.36 XPS spectra of polymer magnetic beads.

[0059] Figure 10 PNI prepared in Example 4 0.51 -CO-ATBA 0.49 XPS spectra of polymer magnetic beads.

[0060] Figure 11 For PNI 0.64 -CO-ATBA 0.36Scanning electron microscope (SEM) image of polymer magnetic beads.

[0061] Figure 12 For PNI 0.64 -CO-ATBA 0.36 TEM-EDS energy spectrum mappings of polymer magnetic beads, where (a) is the TEM-EDS energy spectrum mapping of elements N and Fe, and (b) is the TEM-EDS energy spectrum mapping of elements S and Fe.

[0062] Figure 13 For PNI 0.64 -CO-ATBA 0.36 Thermogravimetric analysis of polymer magnetic beads.

[0063] Figure 14 For Fe3O4 and PNI 0.64 -CO-ATBA 0.36 Magnetometer test diagram of polymer magnetic bead vibration sample.

[0064] Figure 15 For PNI 0.64 -CO-ATBA 0.36 Hydrodynamic radius distribution of polymer magnetic beads in aqueous solutions with different isopropanol ratios.

[0065] Figure 16 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A comparison chart of the concentrations of RNA extracted from white hyacinth bean using seven solution extraction methods.

[0066] Figure 17 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A comparison of A260 / 280 values ​​of RNA extracted from white hyacinth bean using seven solution extraction methods.

[0067] Figure 18 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36A comparison of A260 / 230 values ​​of RNA extracted from white hyacinth bean using seven solution extraction methods.

[0068] Figure 19 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 3D comparison of the RNA adsorption capacity of seven solution extraction methods on white hyacinth bean.

[0069] Figure 20 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 Figure showing the concentration of RNA extracted from lotus seeds using seven different solution extraction methods.

[0070] Figure 21 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A comparison of A260 / 280 values ​​of RNA extracted from lotus seeds using seven different solution extraction methods.

[0071] Figure 22 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A comparison of A260 / 230 of RNA extracted from lotus seeds using seven solution extraction methods.

[0072] Figure 23 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 3D comparison of the RNA adsorption capacity of seven solution extraction methods on lotus seeds.

[0073] Figure 24To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 Figure showing the concentration of RNA extracted from honeysuckle using seven different solution extraction methods.

[0074] Figure 25 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A260 / 280 comparison of RNA extracted from honeysuckle using seven different solution extraction methods.

[0075] Figure 26 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 A comparison of A260 / 230 values ​​of RNA extracted from honeysuckle using seven different solution extraction methods.

[0076] Figure 27 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 3D comparison of the RNA adsorption capacity of seven solution extraction methods on honeysuckle.

[0077] Figure 28 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 Gel electrophoresis images of RNA extracted from white hyacinth bean using seven solution extraction methods.

[0078] Figure 29 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 Gel electrophoresis images of RNA extracted from lotus seeds using seven solution extraction methods.

[0079] Figure 30 To employ the Trizol method, commercial magnetic bead method, polymer magnetic bead method, and polymer magnetic bead method 0.29 Solution extraction method, polymer magnetic beads 0.36 Solution extraction method, polymer magnetic beads 0.49 Solution extraction method, guanidine hydrochloride modified polymer magnetic beads 0.36 Gel electrophoresis images of RNA extracted from honeysuckle using seven different solution extraction methods. Detailed Implementation

[0080] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention are described below in conjunction with specific examples. However, these should not be construed as limiting the present invention and are merely examples.

[0081] Example 1

[0082] This embodiment provides a PNI x -CO-ATBA 1-x A method for preparing a polymer, comprising the following steps:

[0083] 1. Synthesis and purification of monomers

[0084] 1.1. Synthesis of ATBA Monomer

[0085] Step 1: First, weigh potassium thiocyanate (0.972 g, 10 mmol) into a 100 mL round-bottom flask and dry it under vacuum for 24 h. Then, add 20 mL of anhydrous acetone and stir thoroughly until the solid is completely dissolved. Use a pipette to add 0.976 mL (1.087 g, 12 mmol) of acryloyl chloride into a 25 mL centrifuge tube, and then use a syringe to add it dropwise into the round-bottom flask. Stir at room temperature for 12 h. Take the suspension after the reaction into a 10 mL centrifuge tube and centrifuge at 8000 rpm for 15 min. Concentrate the supernatant using a rotary evaporator to obtain an orange-red viscous liquid. Add 20 mL of anhydrous acetone to dissolve it completely, and then concentrate it again using a rotary evaporator. Repeat this process three times to wash away excess acryloyl chloride after the reaction.

[0086] Step 2: Dissolve the orange-red liquid obtained in Step 1 completely in 20 mL of anhydrous acetone, and place this solution in a separatory funnel; this is reaction solution A. Separately, weigh 1.372 g (10 mmol) of 4-aminobenzoic acid into a 100 mL round-bottom flask, add 20 mL of a mixture of acetone and water (volume ratio 1:1), and stir until the solid is completely dissolved; this is reaction solution B. While stirring, add reaction solution A dropwise to reaction solution B, and react at room temperature for 12 h. Remove the suspension after reaction and place it in a centrifuge tube; centrifuge at 8000 rpm for 15 min, discard the supernatant, and disperse the crude product in a mixture of 100 mL of acetone and 100 mL of water, then centrifuge at 8000 rpm for 15 min, discarding the supernatant. Repeat this process three times to wash away the remaining reactants (mainly 4-aminobenzoic acid). Dry the final precipitate under vacuum at 40 °C to obtain a pale yellow solid, pure ATBA product.

[0087] 1.2. Purification of N-isopropylacrylamide (NIPAAm)

[0088] Weigh 15g of NIPAAm into a 250mL flask, then add 100mL of n-hexane. Reflux at 65-75℃ for one hour. Filter the solution with qualitative filter paper to remove impurities insoluble in n-hexane. Collect the filtrate in a 250mL Erlenmeyer flask and allow it to crystallize fully at 4℃ to obtain a white needle-like solid. Filter the solution and solid mixture using a suction funnel, discard the solution, and dry the solid in the funnel under vacuum at 40℃. Repeat this process 2-3 times. Seal the resulting white needle-like solid for later use.

[0089] 1.3. Purification of Cuprous Bromide (CuBr)

[0090] Cuprous bromide (CuBr) is easily oxidized by oxygen in the air during long-term storage and is easily degraded by light. This deterioration can affect polymerization efficiency, therefore further purification is necessary. Take 1.0 g of CuBr in a 25 mL round-bottom flask, add 25 mL of glacial acetic acid, and stir for about 0.5-1 h under the protection of high-purity nitrogen (>99.999%). Then centrifuge at 8000 rpm for 5 min to remove the glacial acetic acid and wash three times with anhydrous ethanol. Then, repeat the same steps, washing three times with diethyl ether and drying with nitrogen to obtain a grayish-white solid powder. Purge with high-purity nitrogen, seal, and store in a light-proof, dry, low-temperature environment for later use.

[0091] 2. Three PNIs with different grafting ratios were prepared using ATRP (atom transfer radical polymerization) in solution. x -CO-ATBA 1-x polymer

[0092] 2.1. In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially in a molar ratio of 4:1. Simultaneously, 30 mL of DMF was added as a solvent. Nitrogen gas was purged while stirring. After the monomer dissolved, under nitrogen protection, the initiator 2-bromoisobutyric acid (4.2 mg, 0.025 mmol), the catalyst CuBr (0.016 g, 0.11 mmol), and N,N,N',N'',N''-pentamethyldiethylenetriamine PMDETA (0.08 mL, 0.38 mmol) were added. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction temperature was controlled at approximately 60 °C, and the reaction was stirred for 24 h. After the reaction was completed, the mixture was dialyzed with a large amount of ethanol to remove excess monomer and DMF, and then rotary evaporated to obtain PNI. 0.71 -CO-ATBA 0.29 The polymers contain 71.5% NIPAAm and 29.5% ATBA.

[0093] 2.2. In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially in a molar ratio of 4:1. Simultaneously, 30 mL of DMF was added as a solvent. Nitrogen gas was purged while stirring. After the monomer dissolved, 2-bromoisobutyric acid (4.2 mg, 0.025 mmol), CuBr (0.032 g, 0.22 mmol), and PMDETA (0.16 mL, 0.76 mmol) were added under nitrogen protection as an initiator. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction temperature was controlled at approximately 60 °C, and the reaction was stirred for 24 h. After the reaction was completed, the mixture was dialyzed with a large amount of ethanol to remove excess monomer and DMF, and then rotary evaporated to obtain PNI. 0.64 -CO-ATBA 0.36 The polymers contain 64.41% NIPAAm and 35.59% ATBA.

[0094] 2.3. In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially in a molar ratio of 4:1. Simultaneously, 30 mL of DMF was added as a solvent. Nitrogen gas was introduced while stirring. After the monomer dissolved, 2-bromoisobutyric acid (8.4 mg, 0.05 mmol), CuBr (0.032 g, 0.22 mmol), and PMDETA (0.16 mL, 0.76 mmol) were added under nitrogen protection as an initiator. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction temperature was controlled at approximately 60 °C, and the reaction was stirred for 24 h. After the reaction was completed, the mixture was dialyzed with a large amount of ethanol to remove excess monomer and DMF, and then rotary evaporated to obtain PNI. 0.51 -CO-ATBA 0.49 The polymers, in which NIPAAm was grafted at a rate of 51.46% and ATBA at a rate of 48.54%.

[0095] Example 2

[0096] This embodiment provides a method for preparing stimulus-responsive polymer magnetic beads, comprising the following steps:

[0097] 1. Preparation of brominated NH2@Fe3O4-SiO2 particles

[0098] 1.1. 200 mL of ethanol and 20 mL of ultrapure water were mixed, and 2 g of magnetite particles were added. The resulting dispersion was sonicated for 1 hour. Subsequently, 5 mL of ammonia and 6 mL of tetraethoxysilane (TEOS) were added to the reaction solution. The dispersion was continuously mechanically stirred for 3 hours at room temperature. Next, to prepare NH2-modified magnetic silica particles, 4 mL of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was stirred for 12 hours. The NH2-modified magnetic silica particles were collected by vacuum filtration, washed with water and ethanol, and dried overnight in an oven at 60 °C.

[0099] 1.2. Weigh 2g of amino magnetic beads (hereinafter referred to as NH2@Fe3O4-SiO2) and place them in a 100mL round-bottom flask. Then add 50mL of pre-dried dichloromethane and 2mL of anhydrous pyridine. Sonicate the mixture for a period of time to ensure the silica beads are fully dispersed. Then, mechanically stir the mixture under 0℃ ice bath conditions while adding 2mL of 2-bromoisobutyryl bromide dropwise. Continue stirring under 0℃ ice bath conditions in the dark for 1 hour, then transfer the mixture to room temperature and stir for 10 hours in the dark. Separate the solution and magnetic beads by filtration using a Buchner funnel. Wash the surface of the magnetic beads with 200mL of dichloromethane to remove excess 2-bromoisobutyryl bromide. Then transfer the silica gel to a vacuum drying oven at 60℃ and seal it for later use at room temperature.

[0100] 2. Grafting copolymer PNI onto brominated NH2@Fe3O4-SiO2 x -CO-ATBA 1-x (x=0.71)

[0101] In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially, with a molar ratio of 4:1. Simultaneously, 30 mL of LDM was added as a solvent to ensure complete dissolution of the reactants. 0.5 g of brominated magnetic beads prepared in step (1) was added, and the mixture was sonicated for 10 min to ensure complete dispersion. Nitrogen gas was introduced during stirring. After the monomers dissolved, PMDETA (0.08 mL, 0.38 mmol) and cuprous bromide (0.016 g, 0.11 mmol) were added under nitrogen protection. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction was maintained in a 60 °C oil bath and stirred for 24 h under light-protected conditions. After the reaction was complete, the reaction solution, along with the magnetic beads, was transferred to a centrifuge tube and centrifuged at 5000 rpm for 5 min. The supernatant was discarded. Then, 60 mL of LDM was added, and the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was discarded. The polymer magnetic beads were washed with 100 mL of ethanol using the same method. Finally, the mixture was vacuum dried at 40 °C for 24 h to obtain PNI. 0.71 -CO-ATBA 0.29 Polymer magnetic beads should be sealed and stored for later use.

[0102] Example 3

[0103] This embodiment provides a method for preparing stimulus-responsive polymer magnetic beads, comprising the following steps:

[0104] 1. The steps for preparing brominated NH2@Fe3O4-SiO2 particles are the same as in Example 2.

[0105] 2. Grafting copolymer PNI onto brominated NH2@Fe3O4-SiO2 x -CO-ATBA 1-x (x=0.64)

[0106] In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially, with a molar ratio of 4:1. Simultaneously, 30 mL of LDM was added as a solvent to ensure complete dissolution of the reactants. 0.5 g of brominated magnetic beads prepared in step (1) was added, and the mixture was sonicated for 10 min to ensure complete dispersion. Nitrogen gas was introduced during stirring. After the monomers dissolved, PMDETA (0.16 mL, 0.76 mmol) and cuprous bromide (0.032 g, 0.22 mmol) were added under nitrogen protection. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction was then maintained in a 60 °C oil bath and stirred for 24 h under light-protected conditions. After the reaction was complete, the reaction solution, along with the magnetic beads, was transferred to a centrifuge tube and centrifuged at 5000 rpm for 5 min. The supernatant was discarded. Then, 60 mL of LDM was added, and the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was discarded. The polymer magnetic beads were washed with 100 mL of ethanol using the same method. Finally, the mixture was vacuum dried at 40 °C for 24 h to obtain PNI. 0.64 -CO-ATBA 0.36 Polymer magnetic beads should be sealed and stored for later use.

[0107] Example 4

[0108] This embodiment provides a method for preparing stimulus-responsive polymer magnetic beads, comprising the following steps:

[0109] 1. The steps for preparing brominated NH2@Fe3O4-SiO2 particles are the same as in Example 2.

[0110] 2. Grafting copolymer PNI onto brominated NH2@Fe3O4-SiO2 x -CO-ATBA 1-x (x=0.51)

[0111] In a 50 mL pear-shaped reaction flask with a side arm, 0.452 g of recrystallized N-isopropylacrylamide (NIPAAm, 4 mmol) and 0.250 g of prepared and purified ATBA (1.0 mmol) functional monomer were added sequentially, with a molar ratio of 4:1. Simultaneously, 30 mL of LDM was added as a solvent to ensure complete dissolution of the reactants. 1 g of brominated magnetic beads prepared in step (1) was added, and the mixture was sonicated for 10 min to ensure complete dispersion. Nitrogen gas was introduced during stirring. After the monomers dissolved, PMDETA (0.16 mL, 0.76 mmol) and 0.032 g of cuprous bromide (0.032 g, 0.22 mmol) were added under nitrogen protection. The reaction system was then sealed and subjected to three cycles of liquid nitrogen freezing-vacuuming-nitrogen purging to remove oxygen. The reaction was then maintained in a 60 °C oil bath and stirred for 24 h under light-protected conditions. After the reaction was complete, the reaction solution, along with the magnetic beads, was transferred to a centrifuge tube and centrifuged at 5000 rpm for 5 min. The supernatant was discarded. Then, 60 mL of LDM was added, and the mixture was centrifuged at 5000 rpm for 5 min. The supernatant was discarded. The polymer magnetic beads were washed with 100 mL of ethanol using the same method. Finally, the mixture was vacuum dried at 40 °C for 24 h to obtain PNI. 0.51 -CO-ATBA 0.49 Polymer magnetic beads should be sealed and stored for later use.

[0112] Examples 2-4 can be carried out with reference to the following reaction formulas:

[0113]

[0114] Example 5

[0115] This embodiment provides a method for the targeted extraction of RNA from white hyacinth bean, lotus seed, and honeysuckle, respectively. This method uses the stimulus-responsive polymer magnetic beads prepared in Example 2, i.e., polymer magnetic beads. 0.29 Solution extraction method includes the following steps:

[0116] (1) Weigh 7.5 mg of PNI 0.71 -CO-ATBA 0.29 After adding 1 ml of isopropanol to polymer magnetic beads and sonicating for 10 min, take 10 μl of the suspension, add 90 μl of isopropanol to dilute 10 times, and incubate for 30 min.

[0117] (2) Take 0.2g of sample tissue and add 2ml of diethyl pyrocarbonate-treated water, and disrupt the cells in an ice bath for 5-10min in a cell disruptor;

[0118] (3) Centrifuge at 12000 rpm and 4℃ for 5 minutes; take 300 μL of the supernatant, add 750 μL of isopropanol, and then add 10 μL of the polymer magnetic bead suspension in step (1), and rotate to mix for 20 minutes.

[0119] (4) Magnetic rack adsorption, carefully pour out the liquid;

[0120] (5) Add 1000 μL of anhydrous ethanol, shake and wash for 2 minutes; use a magnetic rack to adsorb the liquid, and carefully pour it off.

[0121] (6) Place in a clean bench for 5-10 minutes, dry, add 20 μL of diethyl pyrocarbonate to treat the water, shake for 2 minutes; use a handheld centrifuge for 1 minute, use a magnetic rack to remove the magnetic beads, and collect the RNA solution.

[0122] Example 6

[0123] This embodiment provides a method for the targeted extraction of RNA from white hyacinth bean, lotus seed, and honeysuckle, respectively. This method uses the stimulus-responsive polymer magnetic beads prepared in Example 3, i.e., polymer magnetic beads. 0.36 Solution extraction method.

[0124] The difference between this embodiment and embodiment 5 is that the PNI in step (1) is changed. 0.71 -CO-ATBA 0.29 Polymer magnetic beads replaced with PNI 0.64 -CO-ATBA 0.36 Polymer magnetic beads, otherwise the same as in Example 5.

[0125] Example 7

[0126] This embodiment provides a method for the targeted extraction of RNA from white hyacinth bean, lotus seed, and honeysuckle, respectively. This method uses the stimulus-responsive polymer magnetic beads prepared in Example 4, i.e., polymer magnetic beads. 0.49 Solution extraction method.

[0127] The difference between this embodiment and embodiment 5 is that the PNI in step (1) is changed. 0.71 -CO-ATBA 0.29 Polymer magnetic beads replaced with PNI 0.51 -CO-ATBA 0.49 Polymer magnetic beads, otherwise the same as in Example 5.

[0128] Example 8

[0129] This embodiment provides a guanidine hydrochloride-modified polymer magnetic bead for the targeted extraction of RNA from white hyacinth bean RNA, lotus seed RNA, and honeysuckle RNA, respectively. 0.36The solution extraction method differs from that in Example 6 in that 0.1 g of guanidine hydrochloride (concentration of 0.5 mol / L) is added to the 2 ml of diethyl pyrocarbonate treated water in step (2), and the rest is the same as in Example 6.

[0130] Comparative Example 1

[0131] In this comparative example, RNA was extracted from white hyacinth bean, lotus seed, and honeysuckle using the Trizol method. The extraction methods are as follows:

[0132] (1) Take 200 μL of sample liquid, add 800 μL of Trizol, shake vigorously until homogeneous, and let stand at room temperature for 5 min;

[0133] (2) Add 200 μL of chloroform, shake vigorously for 15 seconds to mix thoroughly, and let stand for 5 minutes;

[0134] (3) Centrifuge at 12000 rpm for 5 min at 4℃;

[0135] (4) Transfer 600 μL of supernatant to a new centrifuge tube, add an equal volume of isopropanol, and mix by inverting.

[0136] (5) Place at -20℃ for 20 minutes;

[0137] (6) Centrifuge at 12000 rpm for 20 min at 4℃. The white precipitate at the bottom of the tube is RNA.

[0138] (7) Gently discard the liquid (to avoid suspending the RNA), add 1 ml of 100% anhydrous ethanol, invert the container, and wash the precipitate.

[0139] (8) Centrifuge at 12000 rpm for 10 min at 4℃;

[0140] (9) Gently discard the liquid (avoid suspending the RNA); centrifuge again at 12000 rpm for 10 min, carefully aspirate the supernatant, avoiding aspirating the precipitate, and place the centrifuge tube on a clean bench to air dry, generally for 5-10 min;

[0141] (10) Add 20 μL of diethyl pyrocarbonate to treat the water to dissolve the RNA. Dissolve at room temperature, and repeatedly tap the bottom of the tube and briefly remove the tube from the centrifuge until the RNA is completely dissolved.

[0142] Comparative Example 2

[0143] In this comparative example, RNA was extracted from white hyacinth bean, lotus seed, and honeysuckle using the traditional commercial magnetic bead method. The extraction methods are as follows:

[0144] (1) Take 300 μL of the decoction of the pretreated sample or 0.2 g of the pretreated medicinal material tissue respectively;

[0145] (2) Add 600 μL of lysis buffer and 50 μL of β-mercaptoethanol, shake to mix, and lyse for 10 min; centrifuge at 12000 rpm for 5 min.

[0146] (3) Take 300 μL of supernatant, add 300 μL of binding solution, add 480 μL of isopropanol, and add 10 μL of magnetic bead suspension diluted 10 times; rotate and mix for 20 minutes.

[0147] (4) Magnetic rack adsorption, carefully pour out the liquid;

[0148] (5) Add 500 μL of RNA pre-wash buffer, shake and wash once; use a magnetic rack to adsorb the liquid, then carefully pour it out;

[0149] (6) Add 500 μL of RNA washing buffer, shake and wash once; use a magnetic rack to absorb the liquid, then carefully pour it off; repeat the above washing steps once.

[0150] (7) Add 500 μL of anhydrous ethanol, shake and wash for 2 minutes; use a magnetic rack to adsorb, then carefully pour off the liquid;

[0151] (8) Place in a clean bench for 5-10 minutes, dry, add 20 μL of diethyl pyrocarbonate to treat the water, shake for 2 minutes; use a handheld centrifuge for 1 minute, use a magnetic rack to remove the magnetic beads, and collect the RNA solution.

[0152] Comparative Example 3

[0153] In this comparative example, RNA was extracted from white hyacinth bean, lotus seed, and honeysuckle using polymer magnetic beads. The difference between this extraction method and that of Comparative Example 2 is that in step (3), traditional magnetic beads were replaced with PNI. 0.64 -CO-ATBA 0.36 Polymer magnetic beads, the rest are the same as comparative example 2.

[0154] Experimental methods:

[0155] 1. Experimental Measurement of Hydrodynamic Radius Distribution of Polymers and Polymer Magnetic Beads

[0156] 1.1 Hydrodynamic radius distribution measurement experiment of polymer in aqueous solutions containing different proportions of acetonitrile

[0157] Take 1 ml of each aqueous solution containing 50%, 33.3%, 25%, and 0% acetonitrile, respectively, add 7.5 mg of polymer, and sonicate for 10 min. Then, use a dynamic light scattering instrument to measure the hydrodynamic radius distribution of the polymer in the aqueous solution containing different proportions of acetonitrile in a liquid phase environment.

[0158] 1.2 Hydrodynamic radius distribution measurement experiment of polymer magnetic beads in aqueous solutions containing different proportions of isopropanol

[0159] Take 1 ml of aqueous solutions containing 100%, 70%, and 0% isopropanol respectively, add 0.75 mg of polymer magnetic beads, and sonicate for 10 min. Then, use a dynamic light scattering instrument to measure the hydrodynamic radius distribution of polymer magnetic beads in aqueous solutions containing different proportions of isopropanol in a liquid phase environment.

[0160] The following will combine Figures 1-30 Tables 1-5 illustrate the synthesis results and performance characterization of each embodiment and comparative example of the present invention. Table 1 shows the PNI prepared in Example 1. x -CO-ATBA 1-x The organic element content of the polymer. Table 2 is a summary of the production cost analysis of the Trizol method in Example 8 and Comparative Example 1 of the present invention. Table 3 is a summary of the extraction process analysis of the Trizol method in Example 8 and Comparative Example 1 of the present invention. Table 4 is a summary of the production cost analysis of the commercial magnetic bead method in Example 8 and Comparative Example 2 of the present invention. Table 5 is a summary of the extraction process analysis of the commercial magnetic bead method in Example 8 and Comparative Example 2 of the present invention.

[0161] Table 1

[0162]

[0163] Table 2

[0164]

[0165] Table 3

[0166]

[0167] Table 4

[0168]

[0169] Table 5

[0170]

[0171] Combination Figures 1-6 And Table 1, analyze Example 1, from Figure 1 The 1H NMR spectrum clearly shows the disappearance of the characteristic peaks of olefins at 6.02 (C=CH), 6.42 (C=CH), and 6.61-6.66 (C=CH), and the appearance of the proton peak unique to NIPAAm at 0.84 (C(CH3)2); simultaneously, Figure 2 Further verification using infrared spectroscopy confirmed the successful synthesis of three polymers with different ratios. Figures 3-5 The presence of the characteristic peak of the S2p orbital indicates the presence of the S element, which is unique to the ATBA monomer, indicating that the polymers in three different proportions were successfully synthesized. Figure 6This indicates that as solvent polarity decreases, the copolymer chains that move freely in the solution phase exhibit a more extended state; conversely, as solvent polarity increases, the polymer chains become more compacted. Based on the proportions of C, S, O, and N in the polymers analyzed by organic elements in Table 1, the grafting ratios of ATBA in the three polymers with different ratios were calculated to be 29.5%, 35.59%, and 48.54%, respectively.

[0172] Combination Figures 7-15 Examples 2-4 were analyzed. Figure 7 Peak of characteristic groups: 564 cm⁻¹ -1 (Fe-O), 1101cm -1 (Si-OC), carboxyl group: 2972cm -1 amide tape: 1650cm -1 and 1530cm -1 Thiourea 776cm -1 Methyl group: 1388 cm -1 Benzene ring 856cm -1 The emergence indicates the successful synthesis of polymer magnetic beads in three different proportions. Figures 8-10 The appearance of peaks for the characteristic elements Fe, Si, O, C, N, and S further verifies the synthesis of polymer magnetic beads in three different proportions. Figure 11 Scanning electron microscopy images show that the polymer magnetic beads are about 200-300 nm in size. Figure 12 Since S and N elements are unique to polymers, the TEM-EDS energy dispersive spectroscopy map indicates that the polymer magnetic beads were successfully prepared. Figure 13 This indicates that the polymer grafting rate of the polymer magnetic beads is 4.5%. Figure 14 This indicates that the magnetization of the polymer magnetic beads is lower than that of Fe3O4, proving that Fe3O4 is encapsulated by the polymer. Figure 15 This indicates that as the solvent polarity decreases, the copolymer chains of the magnetic beads that move freely in the solution phase exhibit a more extended state; conversely, as the solvent polarity increases, the polymer chains become more compacted.

[0173] Combination Figures 16-30 Tables 2-5 analyze Examples 5-8 and Comparative Examples 1-3:

[0174] Combination Figures 16-19It can be seen that (1) the polymer magnetic bead method (Comparative Example 3) is superior to the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration and purity of white hyacinth bean RNA, indicating that the polymer magnetic beads prepared in this invention are superior to the traditional commercial magnetic beads in terms of the effective content of white hyacinth bean RNA extraction. (2) Among the basic polymer magnetic bead solution extraction methods (Examples 5-7), the ATBA grafting ratio of 36% (Example 6) is the best, indicating that the affinity group and the stimulus response chain achieve a better synergistic effect at this ratio. (3) The basic polymer magnetic bead solution extraction methods (Examples 5-7) are superior to the Trizol method (Comparative Example 1) and the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration of white hyacinth bean RNA, indicating that the polymer magnetic bead extraction method proposed in this invention is superior to the Trizol method and the traditional commercial magnetic bead method in terms of the effective content of white hyacinth bean RNA extraction. (4) Guanidine hydrochloride improves polymer magnetic beads 0.36 Solution extraction (Example 8) significantly improves upon the low purity of RNA extracted from white hyacinth bean using the basic polymer magnetic bead solution extraction method (Example 6), achieving a level comparable to or better than the Trizol method and commercial magnetic bead method. Simultaneously, guanidine hydrochloride improves the polymer magnetic beads... 0.36 Solution extraction also yields better RNA concentrations from white hyacinth bean than the Trizol method and commercial magnetic bead method.

[0175] Combination Figures 20-23 It can be seen that (1) the polymer magnetic bead method (Comparative Example 3) is superior to the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration of lotus seed RNA, and is basically equivalent to the traditional commercial magnetic bead method in terms of extraction purity, indicating that the polymer magnetic beads prepared in this invention are superior to the traditional commercial magnetic beads in terms of the effective content of lotus seed RNA extraction. (2) Among the basic polymer magnetic bead solution extraction methods (Examples 5-7), the ATBA grafting ratio of 36% (Example 6) is the best, indicating that the affinity group and the stimulus response chain achieve a better synergistic effect at this ratio. (3) The basic polymer magnetic bead solution extraction methods (Examples 5-7) are all superior to the Trizol method (Comparative Example 1) and the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration of lotus seed RNA, indicating that the polymer magnetic bead extraction method proposed in this invention is superior to the Trizol method and the traditional commercial magnetic bead method in terms of the effective content of lotus seed RNA extraction. (4) Guanidine hydrochloride improves polymer magnetic beads 0.36 Solution extraction (Example 8) can significantly improve the low purity of lotus seed RNA extraction by the basic polymer magnetic bead solution extraction method (Example 6), achieving a level comparable to or better than the Trizol method and commercial magnetic bead method; simultaneously, guanidine hydrochloride improves the polymer magnetic beads. 0.36 Solution extraction also yields better RNA concentrations from lotus seeds than the Trizol method and commercial magnetic bead method.

[0176] Combination Figures 24-27It can be seen that (1) the polymer magnetic bead method (Comparative Example 3) is superior to the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration of honeysuckle RNA, and is basically equivalent to the traditional commercial magnetic bead method in terms of extraction purity, indicating that the polymer magnetic beads prepared in this invention are superior to the traditional commercial magnetic beads in terms of the effective content of honeysuckle RNA extraction. (2) Among the basic polymer magnetic bead solution extraction methods (Examples 5-7), the ATBA grafting ratio of 36% (Example 6) is the best, indicating that the affinity group and the stimulus response segment achieve a better synergistic effect at this ratio. (3) The basic polymer magnetic bead solution extraction methods (Examples 5-7) are all superior to the Trizol method (Comparative Example 1) and the traditional commercial magnetic bead method (Comparative Example 2) in terms of the extraction concentration of honeysuckle RNA, indicating that the polymer magnetic bead extraction method proposed in this invention is superior to the Trizol method and the traditional commercial magnetic bead method in terms of the effective content of honeysuckle RNA extraction. (4) Guanidine hydrochloride improves polymer magnetic beads 0.36 Solution extraction (Example 8) can improve upon the low purity of honeysuckle RNA extraction using the basic polymer magnetic bead solution extraction method (Example 6), and is superior to the Trizol method and commercial magnetic bead method; simultaneously, guanidine hydrochloride improves the polymer magnetic beads. 0.36 Solution extraction also yields better RNA concentrations from honeysuckle than the Trizol method and commercial magnetic bead method.

[0177] Figure 28 Agarose gel electrophoresis images of RNA extracted from white hyacinth bean using seven methods are shown. White hyacinth bean often contains a large amount of interfering substances such as polysaccharides and polyphenols. Traditional methods (Trizol method and commercial magnetic bead method): The bands are blurry and have obvious tailing, indicating that the RNA has been partially degraded, especially in the high molecular weight region where the band strength is insufficient. This indicates that the traditional method failed to fully inhibit RNase activity or caused mechanical damage during the extraction process. Basic polymer magnetic bead solution extraction methods (Examples 5-7): With the change of ATBA grafting ratio, the band quality improved significantly; among them, the magnetic beads with an ATBA grafting ratio of 36% (Example 6) produced the clearest and most complete bands without obvious tailing, indicating that the polymer chain extension and affinity site exposure were optimally balanced at this ratio. Guanidine hydrochloride improved polymer magnetic beads 0.36 Solution extraction method (Example 8): Among all methods, the bands were the clearest, sharpest, and most complete, with almost no signs of degradation. The bands were bright and well-proportioned. This indicates that the introduction of guanidine hydrochloride effectively inhibited RNase activity and enhanced the specific binding of RNA to ATBA on the magnetic bead surface, significantly improving the integrity and purity of RNA extraction.

[0178] Figure 29Seven methods for extracting RNA from lotus seeds were presented using gel electrophoresis. Lotus seed samples typically contain high levels of starch and fiber, making extraction challenging. Traditional methods: Both the Trizol method and commercial magnetic bead methods showed significant degradation bands, especially severe tailing of small RNA fragments, indicating that RNA is easily damaged during lysis and washing. Basic polymer magnetic bead solution extraction methods (Examples 5-7): With adjustments to the ATBA grafting ratio, band quality gradually improved. Magnetic beads with a 36% ATBA grafting ratio (Example 6) again demonstrated optimal band integrity and clarity. Guanidine hydrochloride-modified polymer magnetic beads... 0.36 Solution extraction method (Example 8): The bands remained highly intact with almost no degradation and uniform brightness, indicating that the method has strong adaptability to complex matrix samples such as lotus seeds. Guanidine hydrochloride not only inhibits RNase, but may also improve RNA accessibility by destroying starch-RNA complexes.

[0179] Figure 30 Electrophoresis images of seven methods for extracting RNA from honeysuckle are shown. Honeysuckle, as a medicinal plant, often contains numerous metabolites (such as flavonoids and organic acids), which can easily interfere with RNA extraction. Traditional methods: The Trizol method and the commercial magnetic bead method produce blurred bands with slightly dark backgrounds, suggesting possible polysaccharide or polyphenol co-precipitation, affecting RNA purity and integrity. Basic polymer magnetic bead solution extraction methods (Examples 5-7): The bands of magnetic beads with a 36% ATBA grafting ratio (Example 6) show significant improvement compared to the traditional method. Guanidine hydrochloride-modified polymer magnetic beads... 0.36 Solution extraction method (Example 8): The bands were clear, sharp, and the background was clean with no visible degradation. This indicates that the introduction of guanidine hydrochloride can effectively denature proteins and dissolve some metabolite impurities, thereby reducing competitive interference with RNA binding and improving extraction specificity.

[0180] Combining Tables 2 and 3, compared with the traditional Trizol method: (1) Production cost comparison: The method of the present invention (Example 8) saves RMB 87.108 per 100 extractions compared with the Trizol method, a reduction of 78%. This shows that the present invention significantly reduces extraction costs by replacing expensive special reagents (Trizol, chloroform) and simplifying the operation process. (2) Extraction process and efficiency comparison: The extraction time is shortened from 1.5–2 hours to 40 minutes, and the efficiency is increased by 55.5–66.7%; the extraction concentration is increased from 200–500 ng / μl to 300–1300 ng / μl, an increase of 50–80%; the extraction purity is increased from ≥60% to ≥70%, a purity increase of 16.67%; the types of toxic reagents are reduced from more than four to one, and the amount of toxic reagents used is reduced by more than 80%. This shows that the present invention is significantly better than the traditional Trizol method in terms of time, yield, purity, and safety.

[0181] Combining Tables 4 and 5, compared with the traditional commercial magnetic bead method: (1) Production cost comparison: The total cost of the method of the present invention (Example 8) is 317.0833 yuan less per 100 extractions compared with the commercial magnetic bead method, a reduction of up to 92.8%. This shows that the present invention has achieved a great reduction in cost by eliminating a variety of special buffer solutions and auxiliary reagents and adopting a one-step extraction method. (2) Comparison of extraction process and efficiency: The extraction time is shortened from 1.5 hours to 40 minutes, and the efficiency is increased by 55.5%; the extraction concentration is increased from 50-400 ng / μl to 300-1300 ng / μl, an increase of 50-80%; the extraction purity is maintained at a high purity level of ≥70%; the types of toxic reagents are reduced from two to one, and the amount of toxic reagents used is reduced by 50%. This shows that the present invention has greatly improved the extraction speed and yield while maintaining high purity, and further reduced the use of toxic reagents.

[0182] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for the targeted extraction of ribonucleic acids, characterized in that, The use of stimulus-responsive polymer magnetic beads, the polymer magnetic beads comprising a magnetic carrier and a stimulus-responsive polymer grafted onto the surface of the magnetic carrier, the stimulus-responsive polymer comprising a stimulus-responsive N-isopropylacrylamide segment and a 4-(3-acryloylthiourea)-benzoic acid segment having phosphate group affinity. The method includes: (1) The tissue sample was broken in water treated with diethyl pyrocarbonate, and the supernatant was collected after centrifugation; (2) Add isopropanol and pretreated polymer magnetic bead suspension to the supernatant and mix to bind RNA to the magnetic beads; (3) Adsorb the magnetic beads using a magnetic rack, discard the liquid, and wash the magnetic beads with ethanol; (4) After drying the magnetic beads, add water treated with diethyl pyrocarbonate and shake to wash out the RNA. After separating the magnetic beads, obtain the RNA solution.

2. The method of claim 1, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, and a combination thereof. The polymer magnetic beads have at least one of the following characteristics a1)-a2): a1) The polymer magnetic beads have a size of 150-250 nm; a2) The magnetic carrier is Fe3O4-SiO2 composite particles with amino groups on the surface.

3. The method of claim 1, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, and snRNA. The method for preparing the stimulus-responsive polymer magnetic beads includes the following steps: Obtain a magnetic carrier with a brominated surface; N-isopropylacrylamide and functional monomers are polymerized in a solvent to form the stimulus-responsive polymer on the surface of a magnetic carrier, and the polymer magnetic beads are obtained after purification.

4. The method of claim 3, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, and a combination thereof. The method for obtaining a surface-brominated magnetic support includes the following steps: Obtain a dispersion of magnetic nanoparticles; In the presence of an alkaline catalyst, an orthosilicate compound is added to the dispersion to carry out a hydrolysis-condensation reaction, thereby coating the surface of the magnetic nanoparticles with a silica layer. An aminosilane coupling agent was added to the reaction system to continue the reaction, thereby introducing amino groups onto the surface of the silica layer. After filtration, washing and drying, a magnetic carrier with amino groups on its surface was obtained. The surface-modified amino magnetic carrier is reacted with an α-haloacyl halide compound in the presence of an organic base. After filtration, washing and drying, a surface-brominated magnetic carrier is obtained.

5. The method of claim 4, wherein the ribonucleic acid is selected from the group consisting of: The method for obtaining a surface-brominated magnetic carrier has at least one of the following features b1)-b7): ​ b1) The magnetic nanoparticles are iron oxide nanoparticles; b2) The alkaline catalyst is ammonia water; b3) The orthosilicate compound is a tetraethoxysilane; b4) The aminosilane coupling agent is 3-aminopropyltriethoxysilane; b5) The α-haloacyl halide is 2-bromoisobutyryl bromide; b6) The organic base is anhydrous pyridine; b7) The process of reacting in the presence of an organic base includes: first, dispersing the surface-modified amino magnetic support in a mixture of organic base and aprotic polar solvent, then adding α-haloacyl halides dropwise while stirring under 0°C ice bath conditions and reacting in the dark for 0.5-1.5 hours, followed by transferring to room temperature and reacting in the dark for 8-15 hours.

6. The method of claim 4, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, and snRNA. The method for grafting the stimulus-responsive polymer onto the surface of a magnetic carrier includes the following steps: A surface-brominated magnetic support, N-isopropylacrylamide, and functional monomers are dispersed in a solvent, and a copper salt catalyst and a polydentate amine ligand are added under an inert atmosphere. The mixture is then sealed to remove oxygen, and a polymerization reaction is carried out after deoxygenation, thereby grafting the stimulus-responsive polymer onto the surface of the magnetic support.

7. The method of claim 6, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, and snRNA. The method for grafting the stimulus-responsive polymer onto the surface of a magnetic carrier has at least one of the following features c1)-c5): ​ c1) The copper salt catalyst is cuprous bromide; c2) The polydentate amine ligand is N,N,N′,N″,N″-pentamethyldiethylenetriamine; c3) The sealing and deoxygenation process includes: filling the reaction system into a reaction bottle and sealing it, immersing it in liquid nitrogen to freeze it completely, then evacuating the reaction bottle, and repeating the freeze-thaw process multiple times after the reactants have fully thawed to completely remove the oxygen from the reaction system. c4) The molar ratio between the N-isopropylacrylamide, the functional monomer, the cuprous bromide, the polydentate amine ligand, and the brominated group on the magnetic support is (3-5):1:(0.1-0.25):(0.3-0.8):(0.025-0.05). c5) The conditions for the polymerization reaction include: reaction under light-protected conditions, reaction temperature of 50-70℃, and reaction time of 20-30 hours.

8. The method of claim 1, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, siRNA, miRNA, and a combination thereof. The pretreatment steps of the polymer magnetic beads include: adding the polymer magnetic beads to isopropanol for ultrasonic treatment, diluting and incubating to obtain a polymer magnetic bead suspension.

9. The method of claim 1, wherein the RNA is selected from the group consisting of mRNA, tRNA, rRNA, snRNA, siRNA, miRNA, and a combination thereof. In step (1), guanidine hydrochloride is added to the water treated with diethyl pyrocarbonate.

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