Preparation method of nucleic acid fragment sorting magnetic beads and application thereof

Abstract

The present application relates to the technical field of biological separation materials, and in particular to a preparation method of magnetic beads for nucleic acid fragment sorting and application thereof. The present application is characterized in that a high-molecular polymer magnetic bead coated with a carboxyl group is prepared through one-step emulsion polymerization. The carboxyl magnetic microspheres synthesized through the emulsion polymerization method of the present application not only solve the problem of complicated process in the current preparation of carboxyl magnetic beads, but also solve the problem that the purity and recovery rate of the sorting or purification product are reduced due to the agglomeration of the magnetic beads after binding with nucleic acid during the sorting or purification of the carboxyl magnetic beads. The carboxyl magnetic microspheres prepared by the present application can efficiently realize the sorting or purification of nucleic acid fragments, significantly reduce the raw material cost of sequencing reagent (kit) developers, and promote the domestication of magnetic bead raw materials.

Description

Technical Field

[0001] This invention relates to the field of bio-separation materials technology, specifically to a method for preparing magnetic beads for nucleic acid fragment sorting and their application. Background Technology

[0002] Next-generation sequencing (NGS), also known as second-generation sequencing or high-throughput sequencing, represents a revolutionary advancement over first-generation sequencing technology. Its core characteristic is its ability to sequence hundreds of thousands to millions of DNA molecules simultaneously, achieving astonishingly high throughput. Since Roche introduced the 454 sequencing system in 2005, NGS technology has developed rapidly, drastically reducing sequencing costs. From the $3.8 billion cost of the Human Genome Project, the cost of sequencing a single genome in China today is only about $500, significantly advancing genomics research and biomedical applications.

[0003] In the NGS workflow, constructing high-quality sequencing libraries is crucial, and the accurate sorting and purification of nucleic acid fragments is one of the key steps. Traditional nucleic acid fragment sorting methods include solution precipitation and gel separation, but these methods are cumbersome and not conducive to automation. Currently, magnetic bead-based sorting technologies, especially solid-phase reversible fixation (SPRI) technology, can solve the above problems and have become the mainstream method. This method utilizes carboxyl-coated superparamagnetic beads to reversibly bind nucleic acids in the presence of polyethylene glycol (PEG) and salt. By adjusting the volume ratio of magnetic beads to samples, specific-sized nucleic acid fragments are selectively bound, thereby removing impurities such as primer dimers and unincorporated dNTPs, and recovering DNA fragments of the target length. Beckman AMPure XP Beads are considered the industry gold standard in commercial products. Although there are many types of magnetic bead products on the market, few meet the sequencing quality requirements. Therefore, imported magnetic beads still have a high market share in China, resulting in high reagent kit development costs, low profits, and weak core competitiveness for downstream companies.

[0004] There are various traditional methods for preparing magnetic beads. For example, CN 117861624 A discloses a method of coating iron oxide with silica to obtain core-shell magnetic beads, then introducing amino groups on the surface of the magnetic beads, and further modifying them with carboxyl groups to obtain the final product. CN 119775999A discloses a layer-by-layer self-assembly method, which uses silica as the core, coats iron oxide particles, and then coats them with silica before modifying the surface of the magnetic beads with carboxyl groups to obtain silicon carboxyl magnetic beads. These methods involve many steps and are cumbersome to operate, which is not conducive to controlling the stability of the process. Moreover, after the magnetic beads bind to nucleic acids, they are prone to agglomeration and clumping during the ethanol washing and elution stages, resulting in poor sorting purity and low loading capacity. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an emulsion polymerization method that can simplify the process of preparing magnetic beads for nucleic acid fragment separation, improve the aggregation problem of magnetic beads in downstream applications, and increase the purity and loading of the separation products.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for preparing nucleic acid fragment sorting magnetic beads involves a one-step emulsion polymerization reaction. The nucleic acid fragment sorting magnetic beads are high molecular polymer magnetic beads with carboxyl groups coated on their surface; The emulsion polymerization reaction includes: mixing 73%~98% V / V... 单体 Core monomer, 0.5%~10% V / V 单体 Emulsifier, 6%~17% V / V 单体 Functional monomer, 1%~11%V / V 单体 Crosslinking agent, 18%~23% W / V 单体 Fe3O4@OA / UA nanoparticles, 2.3%~3.3% W / V 单体 Initiator, 7.2%~23% V / V 单体 After solvent mixing, nucleic acid fragment sorting magnetic beads are obtained through ultrasonic emulsification polymerization, wherein V 单体 =V 核心单体 +V 功能单体 10V 交联剂 W / V 单体 It is in g / ml.

[0007] Preferably, the preparation method of the Fe3O4@OA / UA nanoparticles (Fe3O4@OA / UA nanoparticles refer to oleic acid (OA) or undecenoic acid (UA) modified magnetite nanoparticles) is as follows: Weigh out FeCl3·6H2O and FeSO4·7H2O, and measure out water at the same time. Pour all of them into a flask, stir, heat, and pour out ammonia water to react. Add oleic acid to react. Continue to heat the reaction. Volatilize the ammonia water until sedimentation is observed with the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water. Finally, add n-hexane to resuspend to obtain Fe3O4@OA / UA nanoparticles.

[0008] Specifically, weigh 50±1 g FeCl3·6H2O and 30±1 g FeSO4·7H2O, and simultaneously measure 150 mL of water. Pour all of the mixture into a 1 L three-necked flask, stir at 500 rpm, and simultaneously introduce nitrogen gas. After the temperature rises to 70~75℃, quickly pour in 80~85 mL of ammonia water and react for 5 min. Add 12±0.5 mL of oleic acid and react for 1~1.5 h. Raise the temperature to 85℃ and continue the reaction for 1 h. Volatilize the ammonia water until precipitation is observed to the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water, 200~250 mL each time, for 4 washes. Finally, resuspend the solution in 200~250 mL of n-hexane to obtain Fe3O4@OA / UA nanoparticles.

[0009] The first aspect of this invention aims to provide a method for preparing carboxyl magnetic beads. The method specifically comprises: weighing 0.3-0.4 g of emulsifier into a reactor, adding purified water, and stirring to dissolve; sequentially adding 8-8.5 mL of core monomer, 0.8-2 mL of solvent, 0.6-1.5 mL of functional monomer, 0.1-1 mL of crosslinking agent, and 2 g of Fe3O4@OA / UA nanoparticles; ultrasonically emulsifying the mixture for 2-3 hours at a temperature not exceeding 20°C; after the reaction, raising the temperature to 75±5°C and adding 0.25-0.3 g of initiator to the reactor to initiate the reaction; after the reaction, washing, and finally resuspending in deionized water to obtain nucleic acid fragment sorting magnetic beads.

[0010] Specifically, the method is as follows: Weigh 0.3g of emulsifier and pour it into a 250mL three-necked flask, add 80mL of purified water, and stir to dissolve; add 8mL of core monomer, 0.8~2mL of solvent, 1mL of functional monomer, 0.1~1mL of crosslinking agent, and 2g of Fe3O4@OA / UA nanoparticles to the three-necked flask in sequence, and sonicate the mixture under nitrogen atmosphere for 2~3h at a stirring speed of 500±50rpm, with the reaction temperature not exceeding 20℃. After the reaction is completed, raise the reaction temperature to 75±5℃, add 0.25g of initiator to the three-necked flask to initiate the reaction for 18h at a stirring speed of 300±50rpm; after the reaction is completed, wash with 0.1% SDS solution, and finally resuspend in deionized water to obtain nucleic acid fragment sorting magnetic beads.

[0011] A second aspect of the present invention is to provide a carboxyl magnetic bead.

[0012] The third aspect of this invention aims to provide the application of carboxyl magnetic beads, as described in the second aspect of this invention, in DNA fragment sorting or DNA purification.

[0013] In a first aspect, the present invention provides a method for preparing magnetic beads, wherein high molecular polymer magnetic beads with carboxyl groups coated on their surface are prepared by one-step emulsion polymerization reaction.

[0014] According to an embodiment of the present invention, the emulsion polymerization reaction of the present invention includes: mixing a core monomer, an emulsifier, a functional monomer, a crosslinking agent, Fe3O4@OA / UA nanoparticles, an initiator, a solvent, etc., and then obtaining magnetic beads through an ultrasonic emulsification polymerization reaction. According to an embodiment of the present invention, the core monomer can be selected from monomers commonly used in the field of polymer microsphere synthesis, such as at least one selected from styrene and its derivatives, acrylates, etc. Preferably, the core monomer can be selected from styrene and acrylates.

[0015] According to embodiments of the present invention, the emulsifier can be selected from emulsifiers commonly used in the field of polymer microsphere synthesis, such as anionic emulsifiers like sodium dodecyl sulfate and sodium dodecylbenzenesulfonate; cationic emulsifiers such as quaternary ammonium salts like hexadecyltrimethylammonium bromide (CTAB) and heterocyclic compounds like hexadecylpyridinium bromide (CPB); and nonionic emulsifiers such as at least one of Span 80 / 60, Tween 80 / 60, PVA, and cellulose. Preferably, the emulsifier can be selected from sodium dodecyl sulfate and sodium dodecylbenzene sulfate.

[0016] According to an embodiment of the present invention, the functional monomer may be selected from functional monomers commonly used in the field of polymer microsphere synthesis, such as at least one selected from acrylic acid, methacrylic acid, trans-butenedioic acid, succinic anhydride, hydroxypropyl acrylate, glycidyl methacrylate, hydroxyethyl methacrylate, acrylamide, N,N dimethylacrylamide, etc. Preferably, the functional monomer may be selected from at least one selected from acrylic acid, methacrylic acid, trans-butenedioic acid, etc.

[0017] According to embodiments of the present invention, the crosslinking agent can be selected from crosslinking agents commonly used in the field of polymer microsphere synthesis, such as aromatic hydrocarbons with polyene functional groups, such as p-divinylbenzene, m-divinylbenzene, o-divinylbenzene, 1,2,3-trivinylbenzene, 1,2,4-trivinylbenzene, 1,3,5-trivinylbenzene, etc.; aliphatic hydrocarbons with polyene functional groups, such as 1,3-butadiene, 1,4-pentadiene, 1,9-decadiene, etc.; and crosslinking agents containing crosslinkable functional groups, such as at least one of 1,3-diallylurea, allyl acrylate, allyl methacrylate, diallyl carbonate, triallylamine, ethylene glycol dimethacrylate, etc. Preferably, the crosslinking agent can be selected from at least one of divinylbenzene, tert-butyl polyacrylate-glycidyl methacrylate, and polymethyl methacrylate.

[0018] According to an embodiment of the present invention, the initiator can be selected from initiators commonly used in the field of polymer synthesis, such as oil-soluble initiators, such as azobisisobutyronitrile (AIBN) and benzoyl peroxide (BPO); or aqueous initiators, such as ammonium persulfate (APS) and potassium persulfate (KPS). Preferably, the initiator can be selected from at least one of ammonium persulfate and potassium persulfate.

[0019] According to embodiments of the present invention, the solvent may be selected from organic solvents known in the art, such as at least one selected from n-heptane, cyclohexane, n-hexane, toluene, xylene, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, ethylene glycol phenyl ether, diphenyl ether, sulfolane, diethylene glycol, benzyl alcohol, ethyl acetate, etc. Preferably, the organic solvent may be selected from at least one selected from toluene, ethyl acetate, n-heptane, and ethylene glycol dimethyl ether.

[0020] In a second aspect, the present invention provides carboxyl magnetic beads prepared by the preparation method of the first aspect of the present invention.

[0021] In some embodiments of the present invention, the particle size of the carboxyl magnetic beads is 50-300 nm.

[0022] In some preferred embodiments of the present invention, the particle size of the carboxyl magnetic beads is 100-200 nm.

[0023] In some embodiments of the present invention, the carboxyl magnetic beads contain 200 to 1000 nmol / mg of carboxyl groups.

[0024] In some preferred embodiments of the present invention, the carboxyl magnetic beads contain 600-900 nmol / mg of carboxyl groups.

[0025] In some embodiments of the present invention, the carboxyl magnetic beads dispersed in water have a zeta potential of -40 to -20 mV.

[0026] In some preferred embodiments of the present invention, the carboxyl magnetic beads dispersed in water have a zeta potential of -35 to -25 mV.

[0027] In a third aspect, the present invention provides the application of carboxyl magnetic beads of the second aspect of the present invention in DNA fragment sorting or DNA purification.

[0028] This method can also be used for immunodiagnostics, such as chemiluminescence detection and immunochromatographic detection.

[0029] The carboxyl magnetic beads prepared by this method can also be coupled with different CD series antibodies to prepare cell sorting magnetic beads for application in the field of cell sorting.

[0030] The individual units in this scheme can be further improved or replaced according to different application areas.

[0031] The beneficial effects of this invention are as follows: ① Compared to traditional methods that use multiple reactions to synthesize magnetic beads, this invention uses emulsion polymerization to obtain magnetic beads in a single reaction, significantly simplifying the synthesis steps. It is rapid, simple, highly productive, and the process is controllable, making it easy to scale up production. The carboxyl magnetic microspheres (i.e., nucleic acid fragment sorting magnetic beads) rapidly synthesized via emulsion polymerization solve the problem of cumbersome procedures in current carboxyl magnetic bead preparation methods.

[0032] ② The core component of this invention for DNA sorting and purification, the nucleic acid fragment sorting magnetic beads, has a particle size at the nanoscale (50-300 nm). By increasing the ultrasonic power or extending the homogenization time, Fe3O4@OA / UA nanoparticles are dispersed into fine droplets, and the emulsifier concentration is increased to better cover the surface of the newly formed fine oily droplets, effectively reducing and stabilizing the particle size. Compared with the 0.5-2.8 μm large-particle-size magnetic beads reported in current literature and commercial products, the magnetic beads prepared by this invention can provide a larger specific surface area, reduce the sedimentation coefficient, increase the DNA capture capacity, and reduce nonspecificity. This solves the problem of carboxyl magnetic beads agglomerating after binding nucleic acids during nucleic acid fragment sorting or purification, which reduces the purity and recovery rate of the sorted or purified products.

[0033] ③ The nucleic acid fragment sorting magnetic beads prepared in this invention can effectively purify and sort DNA fragments, with sorting results close to those of commercially available products. They offer high cost-effectiveness and fast magnetic response (within the same time frame, the magnetic beads of this invention can be completely magnetically attracted, resulting in a clear and transparent supernatant, while some commercially available products are not completely magnetically attracted, leaving a cloudy and opaque supernatant). They are suitable for automated equipment, meeting the high-throughput requirements of NGS library establishment. The prepared carboxyl magnetic microspheres can efficiently achieve nucleic acid fragment sorting or purification, significantly reducing raw material costs for sequencing reagent (kit) developers and promoting the domestic production of magnetic bead raw materials. Attached Figure Description

[0034] Figure 1 Scanning electron microscope images of Preparation Example 1 and Comparative Example 1; Figure 2 Image of magnetic beads during the first round of 80% ethanol cleaning in Example 1; Figure 3 Graph of magnetic beads during the first elution stage of Example 1; Figure 4 Image of magnetic beads during the second round of 80% ethanol cleaning in Example 1; Figure 5 Graph of magnetic beads during the second elution stage of Example 1; Figure 6Electrophoresis diagram showing the nucleic acid fragment sorting results of the control magnetic beads; Figure 7 Electrophoresis diagram of nucleic acid fragment sorting results of the magnetic beads obtained in Example 1 of this invention; Figure 8 Example 1: DL5000 marker image preparation; Figure 9 The image shows the magnetic beads during the first round of 80% ethanol cleaning in Comparative Example 1. Figure 10 This is a diagram of the magnetic beads during the first elution stage of Comparative Example 1. Figure 11 The image shows the magnetic beads during the second round of 80% ethanol cleaning in Comparative Example 1. Figure 12 This is a diagram of the magnetic beads during the second elution stage of Comparative Example 1. Figure 13 This is a diagram showing the nucleic acid fragment sorting results of Comparative Example 1. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but is not limited thereto.

[0036] The instruments used in the following embodiments are as follows: Particle size potential detector: Potential particle and molecular measurement system, ELSZ-2000, Otsuka, Japan; SEM: Super-high resolution field emission scanning electron microscope, Hitachi Regulus 8100, Japan; Saturation magnetic intensity (VSM) measurement equipment: Vibrating sample magnetometer, model 7404 / 8604, LakeShore Corporation, USA; Electrical conductivity titration equipment (for determining surface carboxyl content): conductivity meter, FE38, Mettler Toledo; Magnetic response time measurement equipment: Sepmag® System, A200mL.

[0037] Nucleic acid concentration measurement equipment: One Drop TM Spectrophotometer, OD1000+, Nanjing Zihanmu Scientific Instruments Co., Ltd. Example 1:

[0038] Preparation Example 1: Magnetic beads 1 (methacrylic acid) were prepared using the emulsion polymerization method of the present invention. (1) Preparation of Fe3O4@OA / UA nanoparticles Weigh 50 g FeCl3·6H2O and 30 g FeSO4·7H2O, and simultaneously measure 150 mL of water. Pour all of the mixture into a 1 L three-necked flask, stir at 500 rpm, and simultaneously introduce nitrogen gas. After the temperature rises to 70℃, quickly pour in 80 mL of ammonia water and react for 5 min. Add 12 mL of oleic acid and react for 1 h. Raise the temperature to 85℃ and continue the reaction for 1 h. Volatilize the ammonia water until precipitation is observed to the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water, 200 mL each time, for 4 washes. Finally, resuspend the solution in 200 mL of n-hexane to obtain Fe3O4@OA / UA nanoparticles (98.0 mg / mL).

[0039] (2) Preparation of carboxyl magnetic microspheres Weigh 0.3 g of sodium dodecyl sulfate (SDS) and pour it into a 250 mL three-necked flask. Add 80 mL of purified water and stir to dissolve. Add 8 mL of styrene, 0.8 mL of n-heptane, 1 mL of methacrylic acid, 1 mL of divinylbenzene, and 2 g of Fe3O4@OA / UA nanoparticles (converted to liquid form containing n-hexane) to the three-necked flask in sequence. Under nitrogen atmosphere, sonicate the mixture for 2 h with a stirring speed of 500 rpm and a reaction temperature not exceeding 20 °C. After the reaction is complete, raise the reaction temperature to 80 °C and add 0.25 g of potassium persulfate to the three-necked flask to initiate the reaction for 18 h with a stirring speed of 300 rpm. After the reaction is complete, wash with 0.1% SDS solution and finally resuspend in deionized water to obtain carboxyl magnetic microspheres. Example 2:

[0040] Preparation Example 2: Magnetic beads 2 (acrylic acid) were prepared using the emulsion polymerization method of the present invention. (1) Preparation of Fe3O4@OA / UA nanoparticles Weigh 50 g FeCl3·6H2O and 30 g FeSO4·7H2O, and simultaneously measure 150 mL of water. Pour all of the mixture into a 1 L three-necked flask, stir at 500 rpm, and simultaneously introduce nitrogen gas. After the temperature rises to 70℃, quickly pour in 80 mL of ammonia water and react for 5 min. Add 12 mL of oleic acid and react for 1 h. Raise the temperature to 85 ℃ and continue the reaction for 1 h. Volatilize the ammonia water until precipitation is observed to the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water, 200 mL each time, for 4 washes. Finally, resuspend the solution in 200 mL of n-hexane to obtain Fe3O4@OA / UA nanoparticles (99.2 mg / mL).

[0041] (2) Preparation of carboxyl magnetic microspheres Weigh 0.3 g of sodium dodecyl sulfate (SDS) and pour it into a 250 mL three-necked flask. Add 80 mL of purified water and stir to dissolve. Add 8 mL of styrene, 0.8 mL of n-heptane, 1 mL of acrylic acid, 0.2 mL of polymethyl methacrylate, and 2 g of Fe3O4@OA / UA nanoparticles (converted to liquid form containing n-hexane) to the three-necked flask in sequence. Under nitrogen atmosphere, sonicate the mixture for 2 h with a stirring speed of 500 rpm and a reaction temperature not exceeding 20 °C. After the reaction is complete, raise the reaction temperature to 80 °C and add 0.25 g of potassium persulfate to the three-necked flask to initiate the reaction for 18 h with a stirring speed of 300 rpm. After the reaction is complete, wash with 0.1% SDS solution and finally resuspend in deionized water to obtain carboxyl magnetic microspheres. Example 3:

[0042] Preparation Example 3: Magnetic beads 3 (fumaric acid) were prepared using the emulsion polymerization method of the present invention. (1) Preparation of Fe3O4@OA / UA nanoparticles Weigh 50 g FeCl3·6H2O and 30 g FeSO4·7H2O, and simultaneously measure 150 mL of water. Pour all of the mixture into a 1 L three-necked flask, stir at 500 rpm, and purge with nitrogen gas. After the temperature rises to 70℃, quickly pour in 80 mL of ammonia water and react for 5 min. Add 12 mL of oleic acid and react for 1 h. Raise the temperature to 85℃ and continue the reaction for 1 h. Volatilize the ammonia water until precipitation is observed to the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water, 200 mL each time, for 4 washes. Finally, resuspend the solution in 200 mL of n-hexane to obtain Fe3O4@OA / UA nanoparticles (98.5 mg / mL).

[0043] (2) Preparation of carboxyl magnetic microspheres Weigh 0.3g sodium dodecyl sulfate (SDS) and 1g fumaric acid (converted to 0.61mL based on density) into a 250mL three-necked flask, add 80mL purified water, and stir to dissolve. Add 8mL styrene, 0.1mL tert-butyl polyacrylate-glycidyl methacrylate, 0.8mL n-heptane, and 2g Fe3O4@OA / UA nanoparticles (converted to liquid form containing n-hexane) sequentially to the three-necked flask. Under nitrogen atmosphere, sonicate the mixture for 2 hours at a stirring speed of 500rpm, keeping the reaction temperature below 20℃. After the reaction, raise the temperature to 80℃ and add 0.25g potassium persulfate to the three-necked flask to initiate the reaction for 18 hours at a stirring speed of 300rpm. After the reaction, wash with 0.1% SDS solution and finally resuspend in deionized water to obtain carboxyl magnetic microspheres.

[0044] Comparative Example 1: Preparation of magnetic beads by layer-by-layer self-assembly using traditional methods 4 (1) Preparation of Fe3O4@OA / UA nanoparticles Weigh 50 g FeCl3·6H2O and 30 g FeSO4·7H2O, and simultaneously measure 150 mL of water. Pour all of the mixture into a 1 L three-necked flask, stir at 500 rpm, and simultaneously introduce nitrogen gas. After the temperature rises to 70℃, quickly pour in 80 mL of ammonia water and react for 5 min. Add 12 mL of oleic acid and react for 1 h. Raise the temperature to 85℃ and continue the reaction for 1 h. Volatilize the ammonia water until precipitation is observed to the naked eye. After the reaction solution cools to room temperature, wash it alternately with ethanol and water, 200 mL each time, for 4 washes. Finally, resuspend the solution in 200 mL of n-hexane to obtain Fe3O4@OA / UA nanoparticles (98.0 mg / mL).

[0045] (2) Preparation of 400nm silica microsphere core 73.8 mL of ethanol, 10.8 mL of deionized water, 9.8 mL of ammonia, and 5.6 mL of tetraethyl orthosilicate were measured and poured into a 250 mL three-necked flask. The mixture was reacted for 12 h with stirring at 500 rpm and 30°C. The product was washed alternately with ethanol and ultrapure water. Silica microspheres with a particle size of approximately 400 nm were obtained.

[0046] (3) Preparation of aqueous magnetic particles Measure 320 mL of acetone, 220 mL of n-hexane, and weigh 2.5 g of dimercaptosuccinic acid. Pour all the contents into a 1 L three-necked flask, stir to dissolve, then add 100 mL of Fe3O4@OA / UA nanoparticles. Stir the reaction at 60 °C / 500 rpm for 4 hours. After cooling, wash the product with ultrapure water until the supernatant is clear. Disperse the product in 1 L of ultrapure water, adjust the pH to 10, sonicate for 30 minutes to stabilize the pH at 10, adjust the pH to neutral with dilute hydrochloric acid solution, sonicate for another 30 minutes, and filter through a 0.22 μm filter membrane to obtain aqueous particles.

[0047] (4) Preparation of magnetic microspheres by layer-by-layer self-assembly: Measure 300 mL of polyethyleneimine (PEI) solution (5 mg / mL) and 25 mL of 3 M NaCl solution and add them to a 1 L three-necked flask. Stir mechanically at 500 rpm. Add 10 g of 400 nm silica microsphere solution dropwise to the PEI solution. Sonicate for 45 minutes, centrifuge at 8000 rpm for 5 minutes, and then wash several times with purified water under the same centrifugation conditions to obtain PEI-coated microspheres. Add 200 mL of aqueous magnetic particles (10 mg / mL), 25 mL of NaCl, and 200 mL of PEI-coated microspheres to a 1 L three-necked flask. Sonicate for 45 minutes at 500 rpm, centrifuge at 8000 rpm for 5 minutes, and then wash several times with purified water under the same centrifugation conditions until the supernatant is colorless and transparent to obtain a layer of Fe3O4-coated microspheres. The above PEI coating and Fe3O4 coating steps are repeated alternately until microspheres coated with 5 layers of Fe3O4 are obtained. These microspheres are then dispersed in ultrapure water to obtain an aqueous solution of SiO2@Fe3O4 microspheres.

[0048] Silica encapsulation and carboxyl modification: Measure 200 mL of ethanol and 20 mL of SiO2@Fe3O4 microsphere aqueous solution into a three-necked flask, respectively. Stir at 300 rpm for 15 minutes under sonication, add 5 mL of ammonia, and continue sonicating for 15 minutes. Add 10 mL of tetraethyl orthosilicate (TEOS), and react for 2 hours in a 30°C water bath with stirring at 300 rpm. Magnetic separation of the product: wash 4 times each with anhydrous ethanol and water, and disperse in anhydrous ethanol to obtain silica-coated magnetic beads. Add 20 mL of silica-coated magnetic beads, 1 mL of polyacrylic acid (PAA), and 80 mL of water to a 500 mL three-necked flask, stir for 30 minutes, wash with water, wash once with MES buffer, resuspend in 80 mL of MES, sonicate for 3 minutes, add 20 mL of MES solution containing 100 mg EDC, react at 37°C for 2 hours, add 15 mL of ethanolamine solution for blocking for 30 minutes. Wash with water and magnetic separation, adjust pH to 7, wash once with water, and obtain the final product, silicon carboxyl magnetic microspheres.

[0049] SEM characterization images of Preparation Example 1 and Comparative Example 1 are shown in Figure 1. Figure 1 The performance data of the magnetic beads prepared in Examples 1-3 and Comparative Example 1 are shown in Table 1 below.

[0050] Table 1 Summary of Performance Data Note: The surface carboxyl group density of the magnetic beads was determined using a conductivity titration method.

[0051] Nucleic acid fragment sorting performance test of carboxyl magnetic beads (Preparation Example 1) 1) Preparation of binding buffer: Weigh 180g PEG8000, 100g sodium chloride, and 25mL Tris (1M) into a beaker, add an appropriate amount of purified water and stir to dissolve, adjust the pH to 7-8, and finally bring the volume to 1L.

[0052] 2) Preparation of samples to be sorted: Add an equal volume of purified water to the DL5000 marker solution (catalog number: BR0013-01) produced by our company, mix repeatedly, and set aside.

[0053] 3) Vortex or thoroughly invert the magnetic beads to ensure uniform mixing. Add different volumes of the mixture containing carboxyl magnetic beads and the sample to be sorted according to Table 2. Mix well and incubate at room temperature for 5-10 minutes.

[0054] 4) After incubation, place the centrifuge tube on a magnetic separator. Once the solution has clarified, carefully transfer the supernatant to a clean centrifuge tube as the sample to be sorted in the second round. At the same time, add 50 μl of purified water to the magnetic beads, mix well, and let stand at room temperature for 5 min. Place the centrifuge tube on the magnetic separator and transfer the supernatant to another clean centrifuge tube as the elution product for the first round.

[0055] 5) Add different volumes of mixed liquid beads containing carboxyl magnetic beads to the supernatant solution collected in step 4) according to Table 2, mix well, and incubate at room temperature for 5-10 minutes.

[0056] 6) After incubation, place the centrifuge tubes on a magnetic separator and carefully aspirate any remaining liquid with a pipette.

[0057] 7) Add 200 μl of 80% ethanol solution, vortex to mix, place the centrifuge tube on a magnetic separator, and carefully remove the remaining liquid with a pipette.

[0058] 8) Repeat step 6) once. Carefully aspirate any remaining liquid with a pipette and allow to air dry at room temperature for about 5 minutes.

[0059] 9) Add 50 μl of purified water, mix well, and let stand at room temperature for 5 min. Place the centrifuge tube on a magnetic separator and transfer the supernatant to another clean centrifuge tube as the elution product for the second round.

[0060] Table 2. Quantity Ratio of Magnetic Beads and Samples Note: "×" indicates the DNA sample volume. For example, if the length of the inserted fragment in the library is 250 bp and the sample volume is 100 μl, then the first round of sorting magnetic beads will be 0.7 × 100 μl = 70 μl; the second round of sorting magnetic beads will be 0.2 × 100 μl = 20 μl.

[0061] The agglomeration of magnetic beads prepared in Example 1 of this invention during the sorting process is shown in the figure. Figures 2-5 The magnetic beads showed no aggregation. The sorted nucleic acid fragments were then subjected to agarose gel electrophoresis. Figures 6-8 As shown in the figure, compared with Beckman AMPure XP, which is the industry gold standard, the sorted bands are clearly visible, and the size of the sorted fragments shows the expected gradient. The aggregation of magnetic beads prepared in Comparative Example 1 of this invention is shown in the figure. Figures 9-12 The magnetic beads showed obvious aggregation and clumping. The sorted nucleic acid fragments were then subjected to agarose gel electrophoresis. Figure 13 As shown in the figure, although the sorted bands also exhibit a gradient, it is not regular. Significant aggregation occurs in both the ethanol washing and elution stages, leading to reduced purity and elution efficiency of the eluted product. In summary, the magnetic beads of this invention are suitable for sorting fragments of specific read lengths in NGS.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for preparing nucleic acid fragment sorting magnetic beads, characterized in that, Nucleic acid fragment sorting magnetic beads were prepared by one-step emulsion polymerization. The nucleic acid fragment sorting magnetic beads are high molecular polymer magnetic beads with carboxyl groups coated on their surface; The emulsion polymerization reaction includes: mixing 73%~98% V / V... 单体 Core monomer, 0.5%~10% V / V 单体 Emulsifier, 6%~17% V / V 单体 Functional monomer, 1%~11%V / V 单体 Crosslinking agent, 18%~23% W / V 单体 Fe3O4@OA / UA nanoparticles, 2.3%~3.3% W / V 单体 Initiator, 7.2%~23% V / V 单体 After solvent mixing, nucleic acid fragment sorting magnetic beads are obtained through ultrasonic emulsification polymerization, wherein V 单体 =V 核心单体 +V 功能单体 10V 交联剂 W / V 单体 It is in g / ml.

2. The preparation method according to claim 1, characterized in that, The core monomer is selected from at least one of styrene and its derivatives, acrylates and their derivatives.

3. The preparation method according to claim 1, characterized in that, The emulsifier is selected from anionic emulsifiers, cationic emulsifiers, or nonionic emulsifiers.

4. The preparation method according to claim 3, characterized in that, The anionic emulsifier is selected from sodium dodecyl sulfate or sodium dodecylbenzene sulfonate; the cationic emulsifier is selected from hexadecyltrimethylammonium bromide or hexadecylpyridinium bromide; and the nonionic emulsifier is selected from at least one of Span 80 / 60, Tween 80 / 60, PVA, and cellulose.

5. The preparation method according to claim 1, characterized in that, The functional monomer is selected from at least one of acrylic acid, methacrylic acid, trans-butenedioic acid, succinic anhydride, hydroxypropyl acrylate, glycidyl methacrylate, hydroxyethyl methacrylate, acrylamide, and N,N-dimethylacrylamide.

6. The preparation method according to claim 1, characterized in that, The crosslinking agent is selected from at least one of p-divinylbenzene, m-divinylbenzene, o-divinylbenzene, 1,2,3-trivinylbenzene, 1,2,4-trivinylbenzene, 1,3,5-trivinylbenzene, 1,3-butadiene, 1,4-pentadiene, 1,9-decadiene, 1,3-diallylurea, allyl acrylate, allyl methacrylate, diallyl carbonate, triallylamine, and ethylene glycol dimethacrylate.

7. The preparation method according to claim 1, characterized in that, The initiator is selected from at least one of azobisisobutyronitrile, benzoyl peroxide, ammonium persulfate, and potassium persulfate.

8. The preparation method according to claim 1, characterized in that, The solvent is selected from at least one of n-heptane, cyclohexane, n-hexane, toluene, xylene, ethylene glycol monomethyl ether, ethylene glycol dimethyl ether, diethylene glycol monomethyl ether, diethylene glycol dimethyl ether, ethylene glycol phenyl ether, diphenyl ether, sulfolane, diethylene glycol, benzyl alcohol, and ethyl acetate.

9. The preparation method according to claim 1, characterized in that, The method is as follows: Weigh the emulsifier into the reactor, add purified water, and stir to dissolve; add the core monomer, solvent, functional monomer, crosslinking agent, and Fe3O4@OA / UA nanoparticles in sequence, perform ultrasonic emulsification reaction, and keep the reaction temperature below 20°C. After the reaction is completed, raise the reaction temperature to 75±5°C, add an initiator to the reactor to initiate the reaction; after the reaction is completed, wash, and finally add deionized water to resuspend to obtain nucleic acid fragment sorting magnetic beads.

10. The application of nucleic acid fragment sorting magnetic beads prepared by any one of claims 1-9 in DNA fragment sorting or DNA purification.

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