Preparation methods and applications of silanol magnetic beads

Abstract

This invention discloses a method for preparing silanol magnetic beads, comprising the following steps: providing magnetic microparticles; sequentially modifying the surface of the magnetic microparticles with a hydrophilic layer and then with silanol groups; and performing post-treatment on the silanol-modified magnetic microparticles, wherein the reagent for post-treatment is an inorganic strong acid or a mixture of an inorganic strong acid and a complexing agent. This invention also discloses a silanol magnetic bead. Furthermore, this invention discloses the application of silanol magnetic beads in nucleic acid extraction.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid extraction, specifically to a method for preparing and applying silanol magnetic beads. Background Technology

[0002] Nucleic acids are a class of biological macromolecules composed of nucleotides or deoxynucleotides linked by phosphodiester bonds, including ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). As carriers of genetic information, nucleic acids are an important research subject in molecular biology. The quality of nucleic acid samples, especially their extraction efficiency, has a significant impact on subsequent analytical processes. Traditional nucleic acid extraction methods include alkaline lysis, boiling, and column separation.

[0003] Magnetic microsphere extraction is a novel extraction method developed in recent years. This method involves preparing superparamagnetic nanospheres with functional groups (such as hydroxyl and carboxyl groups) introduced on their surface. These magnetic microspheres can specifically recognize and efficiently bind to nucleic acid molecules at the microscopic interface. Under the influence of an external magnetic field, nucleic acids can be separated from samples such as blood, animal tissues, food, and pathogenic microorganisms. Compared with traditional methods, it has the advantages of not requiring highly toxic reagents, avoiding multi-step high-speed centrifugation, and being simple and easy to implement. Silyl hydroxyl magnetic microspheres are a common type of magnetic microsphere used for nucleic acid extraction. However, traditional silyl hydroxyl magnetic beads suffer from poor batch-to-batch stability. Even within the same batch, the detection stability of silyl hydroxyl magnetic beads is unstable, leading to undetectable nucleic acid extraction signals, especially prominent in rapid RNA extraction based on magnetic beads. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing and applying silanol magnetic beads to address the problem of poor batch-to-batch stability of silanol magnetic beads prepared by traditional methods.

[0005] A method for preparing silanol magnetic beads includes the following steps:

[0006] Provide magnetic microparticles;

[0007] The surface of the magnetic microparticles was sequentially modified with a hydrophilic layer and then with silanol groups; and

[0008] The magnetic microparticles modified with silanol groups are post-treated, and the reagent for post-treatment is an inorganic strong acid or a mixture of an inorganic strong acid and a complexing agent.

[0009] In one embodiment, the inorganic strong acid is selected from any one or more of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, bromic acid, fluorosilicic acid, chlorolead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid.

[0010] In one embodiment, the complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, o-phenanthroline, 2,2'-bipyridine, 8-quinolinol, and nitrogen-based complexing agents.

[0011] In one embodiment, the concentration of the inorganic strong acid in the post-treatment reagent is 1 × 10⁻⁶. -4 mol / L~10mol / L.

[0012] In one embodiment, the concentration of the complexing agent in the post-treatment reagent is 0.001 mol / L to 1 mol / L.

[0013] In one embodiment, the post-processing step involves immersing the silanol-modified magnetic microparticles in the post-processing reagent and allowing them to stand.

[0014] In one embodiment, the settling temperature is 20°C to 70°C, and the settling time is 1 hour to 15 hours.

[0015] In one embodiment, the steps include: rinsing the post-treated magnetic microparticles with water and dispersing them in water, adjusting the pH of the resulting aqueous dispersion to 5-7.

[0016] In one embodiment, the silanol modification step includes:

[0017] Magnetic microparticles modified with a hydrophilic layer were dispersed in an anhydrous lower alcohol solution;

[0018] A monomer that functionalizes the silanol groups of the magnetic microparticles is added to initiate the reaction.

[0019] In one embodiment, no water is added and no aqueous reagents other than pH adjusters are used in the silanol modification step.

[0020] In one embodiment, the magnetic microparticles have a particle size of 35 nm to 80 nm.

[0021] Another object of the present invention is to provide silanol magnetic beads prepared by the aforementioned method.

[0022] Another object of the present invention is to provide the application of the aforementioned silanol magnetic beads in nucleic acid extraction.

[0023] Another object of the present invention is to provide the application of the aforementioned silanol magnetic beads in RNA extraction.

[0024] This invention significantly increases the batch-to-batch stability of silanol magnetic beads by post-treating them with inorganic strong acids, making them suitable for industrial-scale nucleic acid extraction, such as DNA extraction, RNA extraction, and DNA / RNA co-extraction. Furthermore, the silanol magnetic beads of this invention exhibit significant advantages in rapid RNA extraction programs, demonstrating a marked improvement in extraction efficiency and high sensitivity. Attached Figure Description

[0025] Figure 1 This is a diagram of reagent pre-packaging for a 96-well deep well plate according to an embodiment of the present invention;

[0026] Figure 2 This is a VSM diagram of a magnetic bead according to an embodiment of the present invention;

[0027] Figure 3 This is a CA diagram of a magnetic bead according to an embodiment of the present invention;

[0028] Figure 4 This is a TEM image of a magnetic bead according to an embodiment of the present invention. Detailed Implementation

[0029] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0031] Unless otherwise shown or indicated in the operational embodiments, all figures used to represent the amounts, physicochemical properties, etc., of ingredients in the specification and claims are to be understood to be adjusted by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters listed in the foregoing specification and appended claims are approximations, and those skilled in the art can appropriately modify these approximations to obtain the desired characteristics by utilizing the teachings disclosed herein. The use of numerical ranges indicated by endpoints includes all numbers within that range and any range within that range; for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4, and 5, etc.

[0032] This invention provides a method for preparing siloxane magnetic beads, comprising the following steps:

[0033] Provide magnetic microparticles;

[0034] A hydrophilic layer is applied to the surface of the magnetic microparticles;

[0035] The surface of hydrophilic-modified magnetic microparticles was modified with silanol groups; and

[0036] The magnetic microparticles modified with silanol groups are post-treated, and the reagent for post-treatment is an inorganic strong acid or a mixture of an inorganic strong acid and a complexing agent.

[0037] This invention significantly increases the batch-to-batch stability of silanol magnetic beads by post-treating them with inorganic strong acids, making them suitable for industrial-scale nucleic acid extraction, such as DNA extraction, RNA extraction, and DNA / RNA co-extraction. Furthermore, the silanol magnetic beads of this invention exhibit significant advantages in rapid RNA extraction programs, demonstrating a marked improvement in extraction efficiency and high sensitivity.

[0038] As used in this article, "magnetic microparticles" can be used interchangeably with "magnetic matrix materials," referring to magnetic particles that are superparamagnetic and have a complete and uniform crystal shape.

[0039] As can be understood from the context of this invention, the magnetic microparticles in this invention have not undergone functionalization and / or hydrophilic modification. The magnetic microparticles (magnetic seed nuclei) may, for example, be in the form of Fe3O4 or AFe3O4, wherein A is two or more of zinc, manganese, titanium, nickel, cobalt, zirconium, and chromium.

[0040] Magnetic microparticles can be synthesized using methods well-known in the art, such as chemical coprecipitation, hydrothermal methods, solvothermal methods, microemulsion methods, DC arc plasma methods, or high-temperature pyrolysis methods. The synthesized magnetic microparticles exhibit superparamagnetism and have complete and uniform crystal morphology.

[0041] As used in this article, "magnetic microspheres" can be used interchangeably with "magnetic beads".

[0042] As used in this article, "hydrophilic layer modification" refers to the modification of magnetic microparticles with hydrophilic substances. The purpose of hydrophilic layer modification is to improve the dispersibility of the particles.

[0043] In this invention, the hydrophilic substance can be a citric acid (such as citrate), a polyethylene glycol, or a polyvinylpyrrolidone hydrophilic substance.

[0044] The Fe 2+ The soluble iron salt solution is one or a mixture of at least two of ferrous chloride, ferrous sulfate and ferrous nitrate.

[0045] By selecting a magnetic fluid concentration of 2-14 mg / mL in the silanization process, this invention further improves the effectiveness of the prepared magnetic microspheres in nucleic acid extraction.

[0046] In some embodiments, the silanol modification step includes:

[0047] Magnetic microparticles modified with a hydrophilic layer were dispersed in an anhydrous lower alcohol solution;

[0048] A monomer that functionalizes the silanol groups of the magnetic microparticles is added to initiate the reaction.

[0049] It should be understood that the silanization step of the present invention is performed with minimal or no water introduction; in other words, no water is added and no aqueous reagents other than pH adjusters are used in the silanization modification step. Any water may be, for example, ultrapure water, distilled water, water retained in or intentionally added to the reaction vessel, etc.; any aqueous reagent may be, for example, a dispersant, an alcohol / water mixture, etc. Water removal can improve the efficiency of RNA extraction.

[0050] In specific implementations, "lower alcohols" refer to alcohols with a carbon chain length of 3 or less.

[0051] In an exemplary embodiment, the lower alcohol may be selected from one or more of methanol, ethanol, n-propanol, and isopropanol.

[0052] In the functionalization process, after dispersing the modified magnetic microparticles in a lower alcohol solution, a magnetic fluid of a certain concentration is formed. The magnetic fluid concentration is defined as the ratio of the mass of the modified magnetic microparticles used to the volume of the lower alcohol solution. For example, when 300 mg of modified magnetic microparticles is dispersed in 150 ml of lower alcohol, the magnetic fluid concentration is 2 mg / mL.

[0053] The present invention does not have any particular limitation on the duration of the sub-step of treatment with lower alcohols, as long as the treated magnetic particles are sufficiently dispersed.

[0054] As used in this article, "silanol-functionalized monomer" can be used interchangeably with "silanol donor" and refers to a substance that functionalizes modified magnetic microparticles with silanol.

[0055] Specifically, the "silanol-functionalized monomer" may be selected from one or more of the group consisting of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, butyl orthosilicate, alkyltrimethoxysilane, alkyltriethoxysilane, alkyltriethylpropylsilane, dialkyldimethoxysilane, dialkyldiethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, aminopropyltrimethoxysilane, and glycidyltrimethoxysilane.

[0056] Before or during the reaction with the "silanol-functionalized monomer", the method of the present invention may further include a step of adjusting the reaction solution to suitable reaction conditions. Suitable pH conditions may be, for example, 8-13; for example, pH adjustment can be achieved by adding a weakly alkaline pH adjuster (such as 1-2 mL of ammonia). Suitable reaction temperature may be, for example, 50-90°C.

[0057] Inorganic strong acids are defined as "an acid that undergoes fully dissociation" (i.e., an acid that can spontaneously and completely ionize, such as 1 mol of HCl reacting with water to produce 1 mol of H+). + There is another 1 mol Cl - Acids that meet this condition are generally inorganic acids. Acids that completely ionize in solution are strong acids; the ionization of strong acids uses an equal sign, such as: HCl = H₂. + +Cl - The current standard for judging strong acids is their ionization constant in aqueous solution. Acids with a pKa (acidity coefficient, the negative logarithm of the ionization constant) less than 1 are strong acids (pKa = 1 to 4 are moderately strong acids, and greater than 4 are weak acids). Some acids with pKa values ​​of a few tenths of a percent can also be considered strong acids.

[0058] In some embodiments, the inorganic strong acid of the present invention is selected from any one or more of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, bromic acid, fluorosilicic acid, chlorolead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid.

[0059] In some embodiments, an organic strong acid may be used instead of the inorganic strong acid, or the organic strong acid may be used in combination with the inorganic strong acid. The organic strong acid is selected from one or more of trifluoroacetic acid (TFA), trichloroacetic acid, methanesulfonic acid, benzenesulfonic acid, KMD acid (cyclohexanethiol sulfonic acid), and 2-chloroethanethiol.

[0060] In some embodiments, the complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, o-phenanthroline, 2,2'-bipyridine, 8-quinolinol, and nitrogen-based complexing agents.

[0061] In some embodiments, the concentration of the inorganic strong acid in the post-treatment reagent is 1 × 10⁻⁶. -4 mol / L to 10 mol / L. Specifically, it can be 1×10⁻⁶ mol / L. -4 mol / L, 5×10 -4 mol / L, 1×10 -3 mol / L, 5×10 -3 mol / L, 1×10 -2 mol / L, 5×10 -2 mol / L, 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L, 10 mol / L. The research results of this invention show that inorganic strong acids and ordinary acidic solutions or buffer solutions have a significant difference in the effect of this application. The concentration of the inorganic strong acid is related to the effect of post-treatment. Preferably, the concentration of the inorganic strong acid is 10 mol / L. -3 mol / L to 1mol / L.

[0062] The post-treatment reagent may or may not contain a complexing agent. In some embodiments, the concentration of the complexing agent in the post-treatment reagent is 0.001 mol / L to 1 mol / L. Specifically, it can be 1 × 10⁻⁶ mol / L. -5 mol / L, 5×10 -5 mol / L, 1×10 -4 mol / L, 5×10 -4 mol / L, 1×10 -3 mol / L, 5×10 -3 mol / L, 1×10 -2 mol / L, 5×10 -2 mol / L, 1mol / L.

[0063] In some embodiments, the post-treatment step involves immersing the silanol-modified magnetic microparticles in the post-treatment reagent and allowing them to stand.

[0064] In some embodiments, the settling temperature is 20°C to 70°C, and the settling time is 1 hour to 15 hours. Specifically, the settling temperature can be room temperature (20°C, 30°C) or high temperature (40°C, 50°C, 60°C, 70°C). The settling time can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours.

[0065] In some embodiments, the method includes the step of rinsing the post-treated magnetic particles with water and then dispersing them in water, adjusting the pH of the resulting aqueous dispersion to 5-7. Specifically, it can be 5, 5.5, 6, 6.5, or 7.

[0066] In some embodiments, the particle size of the magnetic microparticles of the present invention is not greater than 80 nm, for example, 35 nm to 50 nm.

[0067] Another object of the present invention is to provide the application of silanol magnetic beads of any of the above embodiments in nucleic acid extraction.

[0068] The silanol magnetic beads of the present invention can be used for DNA extraction, RNA extraction, or co-extraction of DNA and RNA.

[0069] In specific implementations, samples can be selected from blood, throat swabs, sputum, bronchoalveolar lavage fluid, tissue, food, and environmental samples, etc.

[0070] In an exemplary embodiment, the RNA is viral RNA, such as RNA from the novel coronavirus (SARS-CoV-2).

[0071] The following are specific examples.

[0072] Example 1: Synthesis process of magnetic microspheres

[0073] Step 1: Magnetic Seeding

[0074] Take 5.5g of ferrous sulfate heptahydrate and 10.5g of ferric chloride hexahydrate in 200mL of water, set the stirring speed to 200rpm, stir for 30min to mix the solution thoroughly, heat to 40℃, then add 50mL of ammonia water, react for 30min, and heat again to 90℃ for 1h to obtain superparamagnetic nanoparticles with a particle size of about 5-15nm.

[0075] Step 2: Modification of the hydrophilic layer with magnetic particles

[0076] The magnetic seed nuclei prepared above were dispersed in 200 mL of 0.003 M PVP aqueous solution and kept at 90 °C for 1 h to obtain surface-modified magnetic particles.

[0077] Step 3: Silanol modification

[0078] Silyl group modification: 1 g of the magnetic particles obtained in step two were dispersed in 150 mL of anhydrous ethanol solution. After 30 min, 1 mL of ammonia water was added and the temperature was raised to 77 °C. After maintaining a constant temperature, 5 mL of TEOS was added, and the reaction was stopped after 2 h. The product was directly dispersed in water to obtain silyl group magnetic beads.

[0079] Example 2: Method for improving magnetic responsiveness of silanol magnetic beads. Objective: To verify whether improving magnetic responsiveness can directly enhance extraction performance.

[0080] The silanol magnetic beads obtained in Example 1 were directly dispersed in a 0.01% (w / v) sodium chloride solution. Experiments showed that the addition of sodium chloride solution significantly improved the magnetic response performance of the product.

[0081] Extraction performance testing:

[0082] a) Instruments

[0083] Ausen Auto-Pure32A Nucleic Acid Extractor, Mike N32 Nucleic Acid Extractor

[0084] b) Reagent components and dosage

[0085] Component Name Component dosage / testing Proteinase K 100-400ug pyrolysis solution 500-700ul Magnetic beads 200-300ug Washing liquid 1 600-800ul Washing liquid 2 600-800ul Elution solution 40-80ul

[0086] c) 96-well deep-well plate reagent pre-dispensing, such as... Figure 1 As shown. Among them, each well in columns 1 and 7 contains magnetic beads; columns 2 and 8 are effective working wells, each containing lysis buffer; each well in columns 3 and 9 contains washing buffer 1; each well in columns 4 and 10 contains washing buffer 2; and each well in columns 6 and 12 contains elution buffer.

[0087] d) The instrument's quick-run procedure is as follows:

[0088]

[0089] e) The instrument's slow-release program (in this patent, this program is only used for HCV kits) is as follows:

[0090]

[0091] After nucleic acid extraction from the samples, the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) (Maccura Biotechnology) was used for detection. The test results are shown in Table 1:

[0092] Table 1 Evaluation results of magnetic beads in the fast extraction program

[0093]

[0094] In the table, " / " indicates values ​​that were not detected.

[0095] Conclusion: Table 1 shows the evaluation results of the magnetic beads in the COVID-19 rapid detection procedure. The table shows that the products from Examples 1 and 2 were undetectable in the rapid detection procedure, indicating that these magnetic beads are not suitable for the rapid detection procedure.

[0096] Example 3: Method for processing silanol magnetic beads

[0097] Take 20g (wet weight) of the silanol magnetic beads obtained in Example 1 and disperse them in 100mL of a solution with a concentration of 1×10⁻⁶. -4 The magnetic silica hydroxyl beads are treated by standing in an aqueous solution of hydrochloric acid ranging from mol / L to 10 mol / L for 1 to 24 hours. After washing several times with water, they are then dispersed in purified water to obtain the post-treated magnetic silica hydroxyl beads.

[0098] Table 2 Evaluation results of post-processing magnetic beads in the fast extraction process.

[0099]

[0100]

[0101] Conclusion: Table 2 shows the evaluation results of the novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) (MacMed Biotechnology) after nucleic acid extraction using magnetic beads in the rapid COVID-19 extraction procedure. From the table, we can see that:

[0102] 1) The magnetic beads synthesized in Example 1 could not be detected in the fast extraction process, indicating that the magnetic beads are not suitable for the fast extraction process.

[0103] 2) In Example 3, the detection rate of the magnetic beads in the sensitivity sample was 100%, and the CT value in the precision sample was 0.3CT to 1.1CT earlier than that of the magnetic beads in Example 1.

[0104] 3) Compared with commercial products, the magnetic beads of Example 1 are less effective than commercial products because they cannot be detected in sensitive samples. Although the detection rate of both Example 3 and commercial products is 100% for sensitive samples, the CT value of Example 3 is earlier than that of commercial products. Therefore, the magnetic beads of Example 3 are better than commercial products.

[0105] Example 4: Method for processing siloxane magnetic beads

[0106] Take 20g (wet weight) of the silanol magnetic beads obtained in Example 1 and disperse them in 100mL of medium with a concentration of 1×10⁻⁶. -4 The mixture was prepared in a solution of 0.01 mol / L sulfuric acid and 0.01 mol / L EDTA and allowed to stand at room temperature for 1 hour. The solid particles were then washed with water and magnetically separated. The collected magnetic solid particles were treated with an aqueous solution of sodium citrate. After repeated washing several times, the product was finally dispersed in water. The pH of the resulting product was adjusted to 7 using an aqueous solution of sodium hydroxide to obtain post-treated silanol magnetic beads.

[0107] Table 3 Evaluation results of magnetic beads in the COVID-19 (2019-nCoV) rapid extraction program.

[0108]

[0109]

[0110] Table 4 Evaluation results of magnetic beads in HCV slow extraction procedure

[0111]

[0112] in conclusion:

[0113] a) 2019-nCoV project testing extraction procedure: rapid; HCV project testing extraction procedure: slow. The rapid extraction procedure is procedure d) of Example 2; the slow extraction procedure is procedure e) of Example 2.

[0114] b) In the 2019-nCoV project, the CT value obtained in Example 4 was generally earlier than the CT value of the commercial product, so the product of Example 4 was generally superior to the commercial product.

[0115] c) In the HCV project, whether it is the sensitivity sample or the precision sample, the CT value obtained by Example 4 is earlier than the CT value of the commercial product. Therefore, the product performance of Example 4 is better than that of the commercial product.

[0116] Example 5: Treatment of silanol magnetic beads with different concentrations of acid

[0117] Take 20g (wet weight) of the silanol magnetic beads obtained in Example 1, with a particle size of 50nm. Disperse them in 100mL of solutions with concentrations of 10mol / L, 0.1mol / L, and 1×10⁻⁶ m³ / dL, respectively. -3 mol / L, 1×10 -5 Dissolve in mol / L hydrochloric acid aqueous solution. Allow to stand at room temperature for 1–24 h, then wash with water and collect solid particles by magnetic separation. Disperse the collected magnetic solid particles in purified water.

[0118] Table 5 Evaluation results of magnetic beads in the COVID-19 rapid response program.

[0119]

[0120]

[0121] Conclusion: As shown in Table 5:

[0122] When the hydrochloric acid concentration is 10 mol / L, 0.1 mol / L, 1×10 -3 At mol / L, there was no significant difference in CT values ​​for R2 samples, while S3 samples could be completely detected.

[0123] When the hydrochloric acid concentration is 1×10 -5 At mol / L, the low-concentration sample (S3 sample) was undetectable.

[0124] Based on the above conclusions, it can be preliminarily inferred that, for silanol magnetic beads with a particle size of less than 100 nm, to ensure complete detection of the magnetic beads in the S3 sample, the acid concentration in the post-processing stage needs to be higher than 1 × 10⁻⁶. -5 mol / L.

[0125] Comparative Example 1: Repeat the experiment of Example 1 for three batches to verify its batch-to-batch stability.

[0126] Table 6 Evaluation results of magnetic beads in the COVID-19 rapid response program.

[0127]

[0128]

[0129]

[0130] Conclusion: As can be seen from Table 6:

[0131] Compared to commercial products, the product in Example 4 exhibits superior performance in COVID-19 testing.

[0132] Comparative Examples 1-1, 1-2, and 1-3 represent three batches of experiments in Example 1. As shown in the table, all three batches were detectable in the precision samples, but the difference between the CT values ​​of the ORF1ab channel was as high as 1.5 CT, indicating that the magnetic beads synthesized in these three batches had the drawback of large batch-to-batch variation.

[0133] Sensitivity Samples: In Comparative Example 1, all three batches of magnetic beads exhibited undetectable sensitivity samples, indicating that they were substandard in the COVID-19 rapid testing process.

[0134] Comparative Example 2: Repeat the experiment of Example 3 for three batches to verify its batch-to-batch stability.

[0135] Table 7 Evaluation results of magnetic beads in the COVID-19 rapid response program.

[0136]

[0137]

[0138] Conclusion: Comparative Examples 2-1, 2-2, and 2-3 are the results of post-processing of three batches of Comparative Example 1. As can be seen from the table, all three batches can be detected in the precision samples, and there is no significant difference in the four-channel CT values, indicating that this post-processing method can reduce the inter-batch difference.

[0139] Example 6

[0140] The post-treated magnetic silanol beads prepared in Example 3 were subjected to VSM testing, and the results are as follows: Figure 2 See Table 8.

[0141] Table 8. VSM Data for Magnet Beads

[0142]

[0143]

[0144] Conclusion: From Figure 2 Based on Table 8, the following conclusions can be drawn:

[0145] All the magnetic beads are superparamagnetic.

[0146] It has a saturation magnetic strength greater than 70 emu / g.

[0147] The coercivity of the four batches was 7.4 × 10⁻⁶. -4 ~7.7×10 -4 Its coercivity is negligible.

[0148] The contact angle of the post-treated magnetic silica-hydroxyl beads prepared in Example 3 was tested, and the results are as follows: Figure 3 .from Figure 3 It can be seen that the contact angle between the magnetic beads and water is 20.2°, which initially indicates that the magnetic beads have good hydrophilicity. According to the standard "Test Method for Silicon Hydroxyl Content on Fumed Silica Surface T / FSI-049-2020", the amount of silanol groups on the surface of the magnetic beads before post-treatment is 0.526%, and the amount of silanol groups after treatment is 0.527%. There is no significant change in the amount of silanol groups on the surface of the magnetic beads before and after post-treatment, which indicates that the post-treatment method does not improve performance by increasing the number of silanol groups on the surface of the magnetic beads.

[0149] The particle size of the post-treated magnetic silanol beads prepared in Example 3 was measured. Figure 4 .from Figure 4 It can be seen that the particle size of the magnetic beads is less than 50nm.

[0150] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0151] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of patent protection. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a silicon hydroxyl magnetic bead, characterized by, Includes the following steps: Provide magnetic microparticles; The surface of the magnetic microparticles is sequentially modified with a hydrophilic layer and then with silanol groups. as well as The magnetic microparticles modified with silanol groups are post-treated, wherein the reagent for post-treatment is an inorganic strong acid or a mixture of an inorganic strong acid and a complexing agent; The post-treatment step involves immersing the silanol-modified magnetic microparticles in the post-treatment reagent and allowing them to stand at a temperature of 20°C to 70°C for 1 hour to 15 hours.

2. The method for preparing silanol magnetic beads according to claim 1, characterized in that, The inorganic strong acid is selected from any one or more of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, bromic acid, fluorosilicic acid, chlorolead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid; and / or, The complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, o-phenanthroline, 2,2'-bipyridine, 8-quinolinol, and nitrogen-based complexing agents.

3. The method for preparing silanol magnetic beads according to claim 1, characterized in that, The concentration of the inorganic strong acid in the reagent for the post-treatment is 1 x 10 -4 mol / L~10 mol / L; and / or, The concentration of the complexing agent in the post-treatment reagent is 0.001 mol / L to 1 mol / L.

4. The method for preparing silanol magnetic beads according to any one of claims 1 to 3, characterized in that, The process includes the following steps: rinsing the post-treated magnetic microparticles with water and then dispersing them in water, adjusting the pH of the resulting aqueous dispersion to 5-7.

5. The method for preparing silanol magnetic beads according to any one of claims 1 to 3, characterized in that, The silanol modification step includes: Magnetic microparticles modified with a hydrophilic layer were dispersed in an anhydrous lower alcohol solution; A monomer that functionalizes the silanol groups of the magnetic microparticles is added to initiate the reaction.

6. The method for preparing silanol magnetic beads according to claim 5, characterized in that, No water is added and no aqueous reagents other than pH adjusters are used in the silanol modification step.

7. The method for preparing silanol magnetic beads according to any one of claims 1 to 3 and 6, characterized in that, The magnetic microparticles have a particle size of 35nm to 80nm.

8. Silicon hydroxyl magnetic beads prepared by the method according to any one of claims 1 to 7.

9. The application of the silanol magnetic beads as described in claim 8 in nucleic acid extraction.

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