A magnetic bead for total RNA extraction and a preparation method thereof

Abstract

The application discloses a magnetic bead for total RNA extraction and a preparation method thereof, wherein the magnetic bead for total RNA extraction comprises, from inside to outside, a magnetic core, an organic small molecule polymer layer and an outer shell coating layer; the magnetic bead adopts a three-layer structure of the magnetic core-organic small molecule polymer layer-silica outer shell, the discrete ferroferric oxide magnetic core is gathered to form nanospheres through organic small molecule radical polymerization, the problems of weak magnetism, many discrete particles, difficult magnetic separation and easy residue of the existing magnetic bead are solved, the magnetic bead has strong magnetic field responsiveness, can realize rapid and efficient magnetic separation and is suitable for high-throughput automatic extraction; the dense silica outer shell can effectively isolate air, reduces the risk of oxidation of the magnetic core, enables the magnetic bead to be stored at room temperature for a long time, and solves the problem of poor stability of the existing magnetic bead.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, specifically a magnetic bead for total RNA extraction and its preparation method. Background Technology

[0002] Total RNA refers to the sum of all RNA molecules extracted from samples such as cells, tissues, whole blood, and plants, including various RNA types such as mRNA, rRNA, and tRNA. As an important carrier of genetic information in organisms, RNA (ribonucleic acid) typically participates in various biological processes such as protein synthesis and gene expression regulation. The quality of RNA, including its concentration, purity, and fragment integrity, directly determines the success or failure of downstream experiments such as qRT-PCR, next-generation sequencing, microarray analysis, in vitro translation, and molecular cloning.

[0003] Currently, the mainstream methods for total RNA extraction include the TRIZOL method, silica gel column centrifugation method, and magnetic bead method. Among these, the TRIZOL and silica gel column centrifugation methods typically require manual operation and may involve organic reagents, limiting RNA extraction throughput and potentially impacting the health of operators. Most commercially available kits utilize the magnetic bead method. Patents such as CN118459670A and CN121495923A describe methods for preparing magnetic beads that involve numerous steps and demanding synthesis conditions, hindering industrial-scale production.

[0004] Furthermore, the magnetic beads used for total RNA extraction mentioned above cannot simultaneously meet the requirements of high extraction capacity, purity, and intact 28S, 18S, and 5S fragments. Most magnetic beads have complex preparation processes and slightly weaker magnetism, making them prone to leaving residue, especially during high-throughput automated sample extraction, thus affecting nucleic acid yield. Therefore, it is necessary to design an improved magnetic bead for total RNA extraction and its preparation method to address these issues. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the present invention provides a magnetic bead for total RNA extraction and a method for preparing the same, which effectively solves the problems mentioned in the background.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a magnetic bead for total RNA extraction, comprising, from the inside out, a magnetic core, an organic small molecule polymer layer, and a shell coating layer; The magnetic core is composed of superparamagnetic iron oxide particles; The organic small molecule polymer layer is formed by free radical polymerization of organic small molecule monomers, wherein the organic small molecule monomers are selected from at least one of acrylic acid, methacrylic acid, and acrylates; The outer shell covering layer is a dense silicon dioxide layer, which is formed by the hydrolysis and polymerization of silane compounds, and the silane compounds are selected from at least one of tetramethyl orthosilicate, tetraethyl orthosilicate, and tetrapropyl orthosilicate.

[0007] Preferably, the magnetic core has a particle size of 50-200 nanometers, the organic small molecule polymer layer has a thickness of 20-50 nanometers, and the outer shell coating layer has a thickness of 30-80 nanometers.

[0008] Preferably, the total RNA is derived from at least one of cells, bacteria, fungi, tissues, whole blood, and plants.

[0009] A method for preparing magnetic beads for total RNA extraction includes the following steps: Step 1: Synthesizing the magnetic core Ferrous and ferrous salts are dissolved in an aqueous solution and stirred until homogeneous. An inert gas, selected from at least one of nitrogen and argon, is then introduced to remove oxygen for 0.5-1 hour. An alkaline regulator, selected from at least one of ammonia, sodium hydroxide, and potassium acetate, is then added to adjust the pH of the system to 11-12. The temperature is raised to 50-90°C, and the mixture is stirred at 100-300 rpm for 1-2 hours. After the reaction is complete, the mixture is cleaned and dried to obtain the magnetic core. The feeding ratio of water solvent, ferric salt, ferrous salt and alkaline regulator is (200-500) ml: (50-80) g: (20-50) g: (100-200) ml; the ferric salt is ferric chloride hexahydrate and the ferrous salt is ferrous sulfate heptahydrate.

[0010] Step 2: Organic small molecules aggregate into magnetic cores to form spheres The magnetic core obtained in step 1 is dispersed in an aqueous solvent, and an emulsifier and an initiator are added. The emulsifier is selected from at least one of sodium dodecylbenzenesulfonate, maleate, and sodium dodecyl sulfate, and the initiator is selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate. The ultrasonic instrument is turned on, and the frequency is set to 40 kHz and the power to 200-400 W. The ultrasonic dispersion is carried out for 0.5-1 hour while stirring at 100-300 rpm. Then, while ultrasonicating, the temperature is raised to 40-60℃, and the small organic molecule monomer is slowly added dropwise. After the addition is completed, the reaction is stirred for 1-2 hours. After the reaction is completed, the core is washed and dried to obtain the magnetic core aggregated into spheres. The feeding ratio of water solvent, magnetic core, emulsifier, initiator and organic small molecule monomer is (200-500) ml: (10-20) g: (8-10) g: (2.5-5) g: (5-10) ml; the organic small molecule monomer is selected from at least one of acrylic acid, methacrylic acid and acrylates.

[0011] Step 3: Cover with outer shell The magnetic cores aggregated into spheres obtained in step 2 are dispersed in an aqueous-alcohol mixture, wherein the aqueous-alcohol mixture is composed of ethanol and water in a volume ratio of (8-9):1; an alkaline regulator is added to adjust the pH of the system to 11-12, wherein the alkaline regulator is selected from at least one of ammonia, sodium hydroxide, and potassium acetate; the mixture is stirred at room temperature at 100-400 rpm while slowly adding silane compounds, and the reaction is continued for 16-24 hours. After the reaction is completed, the mixture is washed and dispersed to obtain magnetic beads for total RNA extraction. The feeding ratio of the water-alcohol mixed solvent, the magnetic core aggregated into spheres, the silane compound, and the alkaline regulator is (1000-1500) ml: (10-40) g: (20-80) ml: (30-50) ml; the silane compound is selected from at least one of tetramethyl orthosilicate, tetraethyl orthosilicate, and tetrapropyl orthosilicate.

[0012] Preferably, in step 1, the cleaning is performed by washing with ultrapure water 3-5 times, and the supernatant is discarded after each cleaning by magnetic attraction; the drying temperature is 100-120℃, and the drying time is 2-3 hours.

[0013] Preferably, in step 2, the cleaning is performed by washing with ultrapure water 6-8 times, and the supernatant is discarded after each cleaning by magnetic attraction; the drying temperature is 100-120℃, and the drying time is 2-3 hours.

[0014] Preferably, in step 3, the washing involves washing with ultrapure water 6-8 times, and the supernatant is discarded after each washing by magnetic attraction; the dispersion involves dispersing the final product in ultrapure water, with the solid content controlled at 100-150 mg / mL.

[0015] Preferably, in step 1, the heating rate is 5-10°C / min, and the dropping rate of the alkaline regulator is 30-50 mL / min.

[0016] Preferably, in step 2, the dropping rate of the organic small molecule monomer is 1-2 ml / min, and the system temperature is kept below 30°C during ultrasonic dispersion.

[0017] Preferably, in step 3, the dropping rate of the silane compound is 2-5 mL / min, and the system is stirred at a constant speed during the stirring process at room temperature, without any localized excessive concentration.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. The magnetic beads of this invention adopt a three-layer structure of "magnetic core - organic small molecule polymer layer - silica shell". The discrete iron oxide magnetic core is aggregated into nanospheres through free radical polymerization of organic small molecules, which solves the problems of weak magnetism, many discrete particles, difficult magnetic separation and easy residue of existing magnetic beads. The magnetic beads have strong magnetic field responsiveness and can achieve rapid and efficient magnetic separation, which is suitable for high-throughput automated extraction. 2. The organic small molecule polymer layer of this invention provides sufficient active sites for the silica shell coating, significantly improving the silica coating efficiency and reducing the loss of uncoated iron oxide particles. At the same time, the active groups on the surface of the polymer layer can enhance the specific binding with RNA, improving the nucleic acid extraction load and purity. The dense silica shell can effectively isolate air, reduce the risk of the magnetic core being oxidized, and enable the magnetic beads to be stored stably at room temperature for a long time without agglomeration or failure, thus solving the problem of poor stability of existing magnetic beads.

[0019] 3. The magnetic beads prepared by this invention are suitable for total RNA extraction from various samples such as cells, bacteria, fungi, tissues, whole blood, and plants. The extracted total RNA has high concentration and good purity, with complete 28S, 18S, and 5S fragments and a RIN value greater than 7, which is superior to existing control magnetic beads. It can meet the needs of various downstream molecular biology experiments and has broad market prospects and application value. Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0021] In the attached diagram: Figure 1 Electron micrograph of the magnetic beads used for total RNA extraction prepared in Example 1; Figure 2 Infrared spectrum of the total RNA extraction magnetic beads prepared in Example 1; Figure 3 The electrophoresis comparison image shows the total RNA extraction magnetic beads prepared in Examples 1 and 2 and the total RNA extracted from cells using the magnetic bead method tissue / cell / blood total RNA extraction kit at a mass of 500 ng. Lane 1 is the marker, lanes 2 and 3 are the total RNA extracted in Example 1, lanes 4 and 5 are the total RNA extracted in Example 2, and lanes 6 and 7 are the total RNA extracted from the control magnetic beads. Figure 4The electrophoresis comparison images are of total RNA extraction magnetic beads prepared in Examples 1 and 2 and total plant RNA extracted by the plant RNA extraction kit at a mass of 500 ng. Lane 1 is the marker, lanes 2 and 3 are the total RNA extracted in Example 1, lanes 4 and 5 are the total RNA extracted in Example 2, and lanes 6 and 7 are the total RNA extracted by the control magnetic beads. Figure 5 The total RNA was extracted from 5 × 10⁵ magnetic beads prepared in Example 1. 5 2100 gel images of Hacat cells; Figure 6 Image 2100 gel of 50 mg tomato leaf extract obtained from total RNA extraction beads prepared in Example 1; Detailed Implementation

[0022] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Depend on Figures 1-6 The present invention provides a magnetic bead for total RNA extraction, comprising, from the inside out, a magnetic core, an organic small molecule polymer layer, and a shell coating layer; The magnetic core consists of superparamagnetic iron oxide particles, which have strong magnetism and can achieve rapid magnetic separation, reducing magnetic bead residue. The organic small molecule polymer layer is formed by free radical polymerization of organic small molecule monomers. It can aggregate discrete magnetic cores into uniform nanospheres, while providing sufficient active sites for the formation of the outer shell coating layer, thus improving the coating efficiency. The outer shell is a dense silica layer, which can effectively isolate air, reduce the risk of the magnetic core being oxidized, improve the stability of the magnetic beads, prevent the magnetic beads from agglomerating, and enhance the adsorption capacity of the magnetic beads for total RNA, thereby improving the extraction load and purity.

[0024] The magnetic core has a particle size of 50-200 nanometers, which ensures that the magnetic core has good superparamagnetism and dispersibility; the organic small molecule polymer layer has a thickness of 20-50 nanometers, which can achieve effective aggregation of the magnetic core without affecting the overall magnetism of the magnetic bead; the outer shell coating layer has a thickness of 30-80 nanometers, which can ensure the compactness of the coating layer, while avoiding excessive thickness that would weaken the magnetism of the magnetic bead.

[0025] The organic small molecule monomers are selected from at least one of acrylic acid, methacrylic acid, and acrylates. These monomers have good polymerization activity, and the polymer layer formed after polymerization has a certain degree of hydrophilicity, which is conducive to the binding of magnetic beads and RNA. The silane compounds are selected from at least one of tetramethyl orthosilicate, tetraethyl orthosilicate, and tetrapropyl orthosilicate. These compounds can form a dense silica coating layer after hydrolysis and polymerization, and have strong stability.

[0026] A method for preparing magnetic beads for total RNA extraction includes the following steps: Step 1: Synthesizing the magnetic core Ferric salt (ferric chloride hexahydrate) and ferrous salt (ferrous sulfate heptahydrate) are dissolved in an aqueous solution. Stirring is started at 100-300 rpm to ensure complete dissolution and form a homogeneous solution. Then, an inert gas (nitrogen or argon) is introduced for deoxygenation for 0.5-1 hour to prevent oxygen from affecting the synthesis of the magnetic core. After deoxygenation, an alkaline regulator (ammonia, sodium hydroxide, or potassium acetate) is added to adjust the pH to 11-12. This pH range promotes the hydrolysis and crystallization of the ferric and ferrous salts, forming magnetite particles. Subsequently, [further steps are needed]. Heat the mixture to 50-90℃ at a rate of 5-10℃ / minute, and stir at 100-300 rpm for 1-2 hours to allow the iron oxide particles to grow fully and form a magnetic core. After the reaction, pour the reaction product into a container, place a magnet at the bottom of the container, and magnetize for 6-10 minutes. Then discard the supernatant and add ultrapure water to wash the product. Repeat the washing process 3-5 times, magnetizing and discarding the supernatant after each wash to remove unreacted raw materials and impurities. Finally, place the washed magnetic core in an oven and dry it at 100-120℃ for 2-3 hours. Weigh the dried core for later use.

[0027] The feeding ratio of water solvent, ferric salt, ferrous salt and alkaline regulator is (200-500) ml: (50-80) g: (20-50) g: (100-200) ml, which can ensure that the ferric salt and ferrous salt react fully to form ferric oxide particles with strong magnetic properties, and avoid waste of raw materials or insufficient reaction.

[0028] Step 2: Organic small molecules aggregate into magnetic cores to form spheres Disperse the magnetic cores obtained in step 1 in an aqueous solvent and start stirring at 100-300 rpm to ensure uniform dispersion. Then add the emulsifier (sodium dodecylbenzenesulfonate, maleate, or sodium dodecyl sulfate) and initiator (ammonium persulfate, sodium persulfate, or potassium persulfate) and stir until homogeneous. Turn on the ultrasonic instrument, set the frequency to 40 kHz and the power to 200-400 W, and ultrasonically disperse for 0.5-1 hour. During ultrasonication, keep the system temperature below 30°C to avoid affecting the activity of the emulsifier and initiator. After ultrasonic dispersion, while ultrasonicating, raise the temperature to 40-60°C at a rate of 5-10°C / min, and then reduce the temperature at a rate of 1-2 ml / min. Slowly add small organic monomers (acrylic acid, methacrylic acid, or acrylates) dropwise. After the addition is complete, continue stirring at 100-300 rpm for 1-2 hours to allow the small organic monomers to undergo free radical polymerization, while simultaneously aggregating the discrete magnetic cores to form nanospheres. After the reaction is complete, pour the reaction product into a container, place a magnet at the bottom of the container, and magnetize for 10-15 minutes. Then discard the supernatant and add ultrapure water to wash the nanospheres. Repeat the washing process 6-8 times, magnetizing and discarding the supernatant after each wash to remove unreacted monomers, emulsifiers, and initiators. Finally, place the washed and aggregated magnetic cores into an oven and dry them at 100-120℃ for 2-3 hours. Weigh the dried nanospheres for later use.

[0029] The feeding ratio of water solvent, magnetic core, emulsifier, initiator and organic small molecule monomer is (200-500) ml: (10-20) g: (8-10) g: (2.5-5) g: (5-10) ml, which can ensure that the organic small molecule monomer is fully polymerized, realize the effective aggregation of magnetic core, and form nanospheres with uniform particle size.

[0030] Step 3: Cover with outer shell The magnetic cores aggregated into spheres obtained in step 2 are dispersed in an aqueous-alcohol mixture, wherein the aqueous-alcohol mixture is composed of ethanol and water at a volume ratio of (8-9):1. This mixture improves the dispersibility of the aggregated magnetic cores and promotes the hydrolysis of silane compounds. Stirring is started at 100-400 rpm to ensure uniform dispersion of the aggregated magnetic cores. Then, an alkaline regulator (ammonia, sodium hydroxide, or potassium acetate) is added to adjust the pH of the system to 11-12. This pH range promotes the hydrolytic polymerization of silane compounds. While maintaining stirring at room temperature, silane compounds (tetramethyl orthosilicate, tetramethyl orthosilicate, etc.) are slowly added at a rate of 2-5 mL / min. After adding tetraethyl or tetrapropyl orthosilicate, continue stirring at room temperature for 16-24 hours at 100-400 rpm to allow the silane compounds to fully hydrolyze and polymerize, forming a dense silica coating layer on the surface of the aggregated magnetic core. After the reaction, pour the reaction product into a container, place a magnet at the bottom of the container, and magnetically attract the magnet for 8-10 minutes. Then discard the supernatant, add ultrapure water to wash, and repeat the washing 6-8 times. After each washing, magnetically attract the magnet and discard the supernatant to remove unreacted silane compounds and impurities. Finally, disperse the washed product in ultrapure water and adjust the solid content to 100-150 mg / mL to obtain magnetic beads for total RNA extraction.

[0031] The ratio of water-alcohol mixed solvent, magnetic core aggregated into spheres, silane compound, and alkaline regulator is (1000-1500) ml: (10-40) g: (20-80) ml: (30-50) ml, which can ensure the density and uniformity of the silica coating layer and avoid insufficient coating or excessive coating layer.

[0032] Example 1 A method for preparing magnetic beads for total RNA extraction, comprising the following steps: Step 1: Synthesizing the magnetic core (1) Set up the reaction vessel apparatus and add 300 ml of ultrapure water into the reaction vessel; (2) Weigh 60g of ferric chloride hexahydrate powder and 30g of ferrous sulfate heptahydrate powder respectively, and slowly add them into the reaction vessel. During the operation, pay attention to avoid spilling the powder and ensure that all the raw materials are added. (3) Turn on the mechanical stirring device and adjust the stirring speed to 240-250 rpm to fully dissolve the two powders in the ultrapure water; (4) Open the nitrogen cylinder switch, adjust the nitrogen pressure reducing valve to control the gas pressure, and observe that continuous bubbles emerge from the exhaust port of the reactor. Then start timing for deoxygenation. The deoxygenation time is 0.5 hours. (5) Start the heating device. After the temperature of the reaction system rises to 82°C, add 150 ml of ammonia water drop by drop at a non-continuous droplet flow rate. The droplet process lasts for about 4 minutes. Start timing after the droplet is completed and keep the reaction for 1 hour.

[0033] (6) After the reaction is complete, turn off the nitrogen pressure reducing valve, nitrogen cylinder switch and mechanical stirring device in sequence; (7) Pour the magnetic core product in the reactor into a beaker, place a magnet at the bottom of the beaker, let it stand for 6-7 minutes until the magnetic core is fully attracted, then discard the supernatant and remove the magnet at the bottom. (8) Pour 1000 ml of ultrapure water into the beaker and stir with a glass rod for 1-2 minutes to fully disperse the magnetic core. Place the beaker on the magnet again to attract the magnet. After 6-7 minutes, discard the supernatant and remove the magnet. (9) Repeat step (8) twice to ensure that the impurities on the surface of the magnetic core are thoroughly cleaned; (10) When discarding the supernatant liquid for the last time, try to discard as little supernatant water as possible. Put the beaker together with the magnetic core into the oven and dry it at 120°C for 2 hours. After drying, weigh the solid mass and collect it for later use.

[0034] Step 2: Organic small molecules aggregate into magnetic cores to form spheres (1) Reassemble the reactor apparatus and add 300 ml of ultrapure water into the reactor; (2) Weigh 10g of the magnetic core powder prepared above, 8g of sodium dodecyl sulfate and 2.5g of potassium persulfate respectively, and add them to the reaction vessel. Avoid spilling the powder during the operation. (3) Turn on the ultrasonic instrument and set the ultrasonic frequency to 40 kHz and the ultrasonic power to 300 watts; (4) Turn on the mechanical stirrer, adjust the speed to 240-250 rpm, stir while sonicating and heating, and continue ultrasonic dispersion for 0.5 hours; (5) When the temperature of the reaction system rises to 50°C, slowly add 5 ml of acrylic acid to the reaction vessel. After the addition is complete, start the timer and continue stirring the reaction for 1 hour. (6) After the reaction is complete, pour the magnetic core that has gathered into a ball in the reactor into a beaker, place a magnet at the bottom of the beaker, let it stand for 14-15 minutes to attract the magnet, then discard the supernatant and remove the magnet. (7) Pour 1000 ml of ultrapure water into the beaker, stir with a glass rod for 4-5 minutes, place the beaker on a magnet to attract the magnet, discard the supernatant after 14-15 minutes, and remove the magnet. (8) Repeat step (7) 7 times to completely remove unreacted monomers, emulsifiers and initiators; (9) After the last pouring of the supernatant, remove as much water as possible, put the beaker together with the magnetic core that has agglomerated into a ball into the oven, dry at 120°C for 2 hours, weigh the solid mass and collect it for later use.

[0035] Step 3: Cover with outer shell (1) Set up a reaction vessel, measure 100 ml of ultrapure water and 180 ml of anhydrous ethanol respectively, add them to the reaction vessel, and mix them evenly to form a water-ethanol mixture system; (2) Weigh 4 grams of the magnetic core powder that has been aggregated into spheres and add it to the reaction vessel. Avoid spilling the powder during operation and ensure that all the raw materials are added. (3) Turn on the mechanical stirrer and adjust the speed to 240-250 rpm to make the magnetic cores that have aggregated into balls evenly dispersed in the water-alcohol mixture; (4) Measure 10 ml of ammonia water and add it to the reaction vessel to adjust the pH value of the reaction system; (5) While stirring, slowly add 8 ml of tetraethyl orthosilicate (TEOS). Start timing after the addition is complete and continue stirring for 20 hours. (6) After the reaction is complete, pour the total RNA extraction magnetic beads in the reaction vessel into a beaker, place a magnet at the bottom of the beaker, let it stand for 9-10 minutes to attract the magnet, then discard the supernatant and remove the magnet. (7) Pour 1000 ml of ultrapure water into the beaker, stir with a glass rod for 1-2 minutes, place the beaker on a magnet to attract the magnet, discard the supernatant after 9-10 minutes, and remove the magnet. (8) Repeat step (7) 7 times to thoroughly remove unreacted tetraethyl orthosilicate and other impurities; (9) After discarding the supernatant for the last time, remove as much water as possible. Place the beaker and magnetic beads in an oven and dry at 120°C for 2 hours. After drying, weigh the powder and disperse it in ultrapure water. Adjust the final solid content to 120 mg / mL to obtain total RNA extraction magnetic beads.

[0036] Example 2 A method for preparing magnetic beads for total RNA extraction, comprising the following steps: Step 1: Synthesizing the magnetic core (1) Set up the reaction vessel apparatus and add 300 ml of ultrapure water into the reaction vessel; (2) Weigh 60g of ferric chloride hexahydrate powder and 30g of ferrous sulfate heptahydrate powder respectively, and add them to the reaction vessel. Take care to avoid spilling the powder during the operation. (3) Turn on the mechanical stirrer and set the speed to 240-250 rpm; (4) Open the nitrogen cylinder switch, adjust the gas pressure of the nitrogen pressure reducing valve, observe that there are continuous bubbles coming out of the exhaust port, and time the deoxygenation for 0.5 hours; (5) Start heating. After the temperature reaches 82°C, start adding 150 ml of ammonia water dropwise at a non-continuous droplet flow rate for about 4 minutes. After adding the ammonia water, start timing the reaction for 1 hour.

[0037] (6) After the reaction is complete, close the nitrogen pressure reducing valve and the gas cylinder switch, and turn off the stirring. (7) Pour the magnetic core of the reactor into the beaker, place a magnet at the bottom of the beaker, wait 6-7 minutes for the magnet to be attracted, then discard the supernatant and remove the bottom magnet; (8) Pour in 1000 ml of ultrapure water, stir with a glass rod for 1-2 minutes, place the beaker on a magnet to attract the magnet, wait 6-7 minutes to attract the magnet, then discard the supernatant liquid and remove the bottom magnet; (9) Repeat step (8) twice; (10) When discarding the supernatant for the last time, try to remove as much water as possible from the supernatant. Then, put the beaker together with the magnetic core into an oven and dry it at 120°C for 2 hours. Weigh the solid and collect it for later use.

[0038] Step 2: Organic small molecules aggregate into magnetic cores to form spheres (1) Set up the reaction vessel apparatus and add 300 ml of ultrapure water into the reaction vessel; (2) Weigh 10g of magnetic core powder, 8g of sodium dodecylbenzenesulfonate and 2.5g of potassium persulfate and add them to the reaction vessel. Take care to avoid spilling the powder during the operation. (3) Turn on the mechanical stirrer and set the speed to 240-250 rpm, stirring and heating at the same time; (4) When the temperature rises to 50°C, slowly add 5 ml of methacrylic acid to the reaction vessel. After the addition is complete, start the reaction time for 1 hour. (5) Pour the magnetic core that has been agglomerated into a ball from the reactor into a beaker. Place a magnet at the bottom of the beaker. After 14-15 minutes, the magnet will be attracted. Then, discard the supernatant and remove the magnet from the bottom. (6) Pour in 1000 ml of ultrapure water, stir with a glass rod for 4-5 minutes, place the beaker on a magnet to attract the magnet, wait 14-15 minutes to attract the magnet, then discard the supernatant liquid and remove the bottom magnet. (7) Repeat step (6) 7 times; (8) When discarding the supernatant for the last time, try to remove as much water as possible from the supernatant. Then, put the beaker and the magnetic core into an oven and dry it at 120°C for 2 hours. Weigh the solid and collect it for later use.

[0039] Step 3: Cover with outer shell (1) Set up the reaction vessel apparatus, and measure 100 ml of ultrapure water and 180 ml of anhydrous ethanol and add them to the reaction vessel respectively; (2) Weigh 4 grams of magnetic core powder aggregated into spheres and add it to the reaction vessel. Take care to avoid spilling the powder during the operation. (3) Turn on the mechanical stirrer and set the speed to 240-250 rpm; (4) Measure 10 ml of ammonia water and add it to the reaction vessel; (5) Then, while stirring, slowly add 8 ml of tetraethyl orthosilicate (TEOS) dropwise. After the addition is complete, start the reaction time for 20 hours. (6) Pour the total RNA extraction magnetic beads from the reactor into a beaker, place a magnet at the bottom of the beaker, wait 9-10 minutes for the magnet to be attracted, then discard the supernatant and remove the bottom magnet; (7) Pour in 1000 ml of ultrapure water, stir with a glass rod for 1-2 minutes, place the beaker on a magnet to attract the magnet, wait 9-10 minutes to attract the magnet, then discard the supernatant liquid and remove the bottom magnet; (8) Repeat step (7) 7 times; (9) When discarding the supernatant liquid for the last time, try to remove as much water as possible from the supernatant. Then put the beaker together with the magnetic beads into the oven and dry at 120°C for 2 hours. Weigh the powder and disperse it in ultrapure water. The final solid content is 120 mg / mL, and the total RNA extraction magnetic beads are obtained.

[0040] Performance verification To verify the extraction performance of the magnetic beads of the present invention, the magnetic beads prepared in Examples 1 and 2 were compared with the control magnetic beads in commercially available magnetic bead-based tissue / cell / blood total RNA extraction kits (brand: Tiangen Biotech (Beijing) Co., Ltd., catalog number: DP761) and plant RNA extraction kits (brand: Suzhou Beaver Biomedical Engineering Co., Ltd., catalog number: 70420-20). The details are as follows: 1. Experimental sample: Hacat cells (5×10⁻⁶) 5 Tomato leaves (50 mg / serving); 2. Extraction method: Refer to the instructions of the control kit, replace the control magnetic beads with the magnetic beads of Examples 1 and 2 of this invention, and perform manual extraction; 3. Detection indicators: The electrophoresis effect of total RNA was detected using a gel imaging system, the fragment integrity (RIN value) was detected using a 2100 bioanalyzer, and the concentration and purity of total RNA (A260 / 280, A260 / 230) were detected using a TGem Pro spectrophotometer.

[0041] The experimental results are as follows: (1) Figure 1 This is an electron micrograph of the total RNA extraction magnetic beads prepared in Example 1. As can be seen from the electron micrograph, the total RNA extraction magnetic beads prepared in Example 1 have a relatively uniform particle size and good dispersibility. Figure 2This is the infrared spectrum of the total RNA extracted magnetic beads prepared in Example 1. As can be seen from the image, at 1650 cm⁻¹... -1 The peak at 1080 cm⁻¹ is the -COOH vibration peak. -1 The presence of the Si-O-Si vibration peak at this point confirms the successful preparation of total RNA extraction magnetic beads. (2) Electrophoresis results: The total RNA extracted from the magnetic beads prepared in Examples 1 and 2 showed clear bands at 28s, 18s, and 5s with no obvious tailing; the total RNA extracted from the control magnetic beads showed weaker bands and some showed tailing. (3) Fragment integrity: The total RNA extracted from the magnetic beads prepared in Examples 1 and 2 had RIN values ​​greater than 7, indicating complete fragments; the total RNA extracted from the control magnetic beads had RIN values ​​of 6.5-7.0, indicating slightly poor fragment integrity. (4) Concentration and purity: The total RNA concentration of Hacat cells extracted by magnetic beads in Example 1 was 184.3-189.5 ng / µL, A260 / 280 was 2.05-2.08, and A260 / 230 was 1.85-1.92; the total RNA concentration of tomato leaves extracted by magnetic beads in Example 1 was 246.4-256.9 ng / µL, A260 / 280 was 2.03, and A260 / 230 was 1.84-1.89; the total RNA concentration of Hacat cells extracted by control magnetic beads was 141.4-149.6 ng / µL, and the total RNA concentration of tomato leaves was 179.6-181.4 ng / µL. The purity index was slightly lower than that of the magnetic beads of this invention.

[0042] The above experimental results show that the total RNA extracted by the magnetic beads prepared in this invention has better loading capacity, purity and fragment integrity than the existing control magnetic beads, and the extraction effect is excellent and can meet the needs of downstream experiments.

[0043] Table 1: Raw data on total RNA concentration and purity extracted from cells by magnetic beads prepared in Examples 1 and 2 and control magnetic beads.

[0044] Table 2: Original data on the concentration and purity of total plant RNA extracted from magnetic beads prepared in Examples 1 and 2 and control magnetic beads.

[0045] 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 process, method, article, or apparatus.

[0046] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A magnetic bead for total RNA extraction, characterized in that, From the inside out, it consists of a magnetic core, an organic small molecule polymer layer, and an outer shell coating layer; The magnetic core is composed of superparamagnetic iron oxide particles; The organic small molecule polymer layer is formed by free radical polymerization of organic small molecule monomers, wherein the organic small molecule monomers are selected from at least one of acrylic acid, methacrylic acid, and acrylates; The outer shell covering layer is a dense silicon dioxide layer, which is formed by the hydrolysis and polymerization of silane compounds, and the silane compounds are selected from at least one of tetramethyl orthosilicate, tetraethyl orthosilicate, and tetrapropyl orthosilicate.

2. The magnetic beads for total RNA extraction according to claim 1, characterized in that, The magnetic core has a particle size of 50-200 nanometers, the organic small molecule polymer layer has a thickness of 20-50 nanometers, and the outer shell coating layer has a thickness of 30-80 nanometers.

3. The magnetic beads for total RNA extraction according to claim 1, characterized in that, The total RNA is derived from at least one of the following: cells, bacteria, fungi, tissues, whole blood, and plants.

4. A method for preparing magnetic beads for total RNA extraction, characterized in that, Includes the following steps: Step 1: Synthesizing the magnetic core Ferrous and ferrous salts are dissolved in an aqueous solution and stirred until homogeneous. An inert gas, selected from at least one of nitrogen and argon, is then introduced to remove oxygen for 0.5-1 hour. An alkaline regulator, selected from at least one of ammonia, sodium hydroxide, and potassium acetate, is then added to adjust the pH of the system to 11-12. The temperature is raised to 50-90°C, and the mixture is stirred at 100-300 rpm for 1-2 hours. After the reaction is complete, the mixture is cleaned and dried to obtain the magnetic core. The feeding ratio of water solvent, ferric salt, ferrous salt, and alkaline regulator is (200-500) ml: (50-80) g: (20-50) g: (100-200) ml; the ferric salt is ferric chloride hexahydrate, and the ferrous salt is ferrous sulfate heptahydrate; Step 2: Organic small molecules aggregate into magnetic cores to form spheres The magnetic core obtained in step 1 is dispersed in an aqueous solvent, and an emulsifier and an initiator are added. The emulsifier is selected from at least one of sodium dodecylbenzenesulfonate, maleate, and sodium dodecyl sulfate, and the initiator is selected from at least one of ammonium persulfate, sodium persulfate, and potassium persulfate. The ultrasonic instrument is turned on, and the frequency is set to 40 kHz and the power to 200-400 W. The ultrasonic dispersion is carried out for 0.5-1 hour while stirring at 100-300 rpm. Then, while ultrasonicating, the temperature is raised to 40-60℃, and the small organic molecule monomer is slowly added dropwise. After the addition is completed, the reaction is stirred for 1-2 hours. After the reaction is completed, the core is washed and dried to obtain the magnetic core aggregated into spheres. The feeding ratio of water solvent, magnetic core, emulsifier, initiator, and organic small molecule monomer is (200-500) ml: (10-20) g: (8-10) g: (2.5-5) g: (5-10) ml; the organic small molecule monomer is selected from at least one of acrylic acid, methacrylic acid, and acrylates. Step 3: Cover with outer shell The magnetic cores aggregated into spheres obtained in step 2 are dispersed in an aqueous-alcohol mixture, wherein the aqueous-alcohol mixture is composed of ethanol and water in a volume ratio of (8-9):1; an alkaline regulator is added to adjust the pH of the system to 11-12, wherein the alkaline regulator is selected from at least one of ammonia, sodium hydroxide, and potassium acetate; the mixture is stirred at room temperature at 100-400 rpm while slowly adding silane compounds, and the reaction is continued for 16-24 hours. After the reaction is completed, the mixture is washed and dispersed to obtain magnetic beads for total RNA extraction. The feeding ratio of the water-alcohol mixed solvent, the magnetic core aggregated into spheres, the silane compound, and the alkaline regulator is (1000-1500) ml: (10-40) g: (20-80) ml: (30-50) ml; the silane compound is selected from at least one of tetramethyl orthosilicate, tetraethyl orthosilicate, and tetrapropyl orthosilicate.

5. The method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 1, the cleaning is performed by washing with ultrapure water 3-5 times, and the supernatant is discarded after each cleaning by magnetic attraction; the drying temperature is 100-120℃, and the drying time is 2-3 hours.

6. The method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 2, the cleaning is performed by washing with ultrapure water 6-8 times, and the supernatant is discarded after each cleaning by magnetic attraction; the drying temperature is 100-120℃, and the drying time is 2-3 hours.

7. The method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 3, the washing involves washing with ultrapure water 6-8 times, and discarding the supernatant after each washing by magnetic attraction; the dispersion involves dispersing the final product in ultrapure water, with the solid content controlled at 100-150 mg / mL.

8. The method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 1, the heating rate is 5-10℃ / min, and the dropping rate of the alkaline regulator is 30-50 ml / min.

9. The method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 2, the dropping rate of the organic small molecule monomer is 1-2 ml / min, and the system temperature is kept below 30℃ during ultrasonic dispersion.

10. A method for preparing magnetic beads for total RNA extraction according to claim 4, characterized in that, In step 3, the dropping rate of the silane compound is 2-5 mL / min, and the system is stirred at a constant speed during the stirring process at room temperature, without any localized excessive concentration.

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