WO2022135420A1 - Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof - Google Patents

Abstract

Disclosed is a method for preparing silicon hydroxyl magnetic microspheres, the method comprising: providing magnetic particles; carrying out a hydrophilic layer modification on the magnetic particles; and carrying out a functionalization treatment on the magnetic particles modified by a hydrophilic layer, or, carrying out a silicon hydroxyl modification on the magnetic particles modified by the hydrophilic layer and carrying out a post-treatment with a post-treatment reagent after the silicon hydroxyl modification. The functionalization treatment comprises: dispersing the modified magnetic particles in an anhydrous lower alcohol; and adding a silicon hydroxyl-functionalized monomer for reaction, wherein water is not added and no aqueous reagent other than a pH-adjusting agent is used during the step of functionalization treatment. The post-treatment reagent is selected from at least one of an organic strong acid, an inorganic strong acid, or a mixture of an inorganic strong acid and a complexing agent. The present invention further relates to the prepared silicon hydroxyl magnetic microspheres and the use thereof in nucleic acid extraction. The present invention improves the extraction effect of nucleic acid, especially RNA, and the inter-batch stability of detection.

Description

Preparation method, prepared product and use of magnetic microspheres for nucleic acid extraction

Cross References to Related Art

This application claims priority to Chinese patent application 202011537465.2 filed on December 23, 2020 and Chinese patent application 202110830842.X filed on July 22, 2021, the entire contents of which are incorporated herein by reference.

technical field

The present invention relates to the field of nucleic acid extraction, in particular to a preparation method of magnetic microspheres for nucleic acid extraction, and the prepared products and uses.

Background technique

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 the carrier of genetic information, nucleic acid is an important research object in the field of molecular biology. The quality of nucleic acid samples, especially the extraction effect, has an important impact on the subsequent analysis process. Traditional nucleic acid extraction methods include alkaline lysis method, boiling method, column separation method, etc.

The magnetic microsphere method is a new extraction method developed in recent years. This method is prepared into superparamagnetic nano-magnetic microspheres with some functional groups (such as hydroxyl and carboxyl groups) introduced on the surface, which can interact with the microscopic interface. Nucleic acid molecules specifically recognize and bind efficiently, so that nucleic acids can be isolated from blood, animal tissues, food, pathogenic microorganisms and other samples under the action of an external magnetic field. Compared with the traditional method, it has the characteristics of simple and easy operation without the participation of highly toxic reagents, avoiding multi-step high-speed centrifugation.

Silicon hydroxyl magnetic microspheres are a kind of common magnetic microspheres used for nucleic acid extraction. The magnetic microspheres have good effect in DNA extraction; however, in RNA extraction, there is a disadvantage that the signal is extremely weak and even cannot be extracted. In addition, the traditional silanol magnetic microspheres have the disadvantage of poor stability between batches. Even the detection stability of the corresponding batch of silanol magnetic microspheres is not stable, and there is a problem that the nucleic acid extraction signal cannot be detected. The problem is particularly prominent in the rapid extraction of RNA from magnetic microspheres.

Therefore, in the field of nucleic acid extraction, there is a strong need to improve the stability of silanol magnetic microspheres for assay batch-to-batch and RNA extraction.

SUMMARY OF THE INVENTION

Based on this, it is necessary to provide a preparation method of silanol magnetic microspheres for the problems of poor RNA extraction effect and poor stability between batches of silanol magnetic microspheres obtained by traditional preparation methods.

In order to solve the above problems, the inventors have studied the preparation process of silanol magnetic microspheres. The inventors found that one of the reasons for the above problems is: in the functionalization step in the preparation of magnetic microspheres, it is often necessary to add magnetic particles to aqueous ethanol, or to add water or aqueous reagents (such as dispersing agents after adding absolute ethanol) etc.), the use of water is aimed at promoting the hydrolysis rate and accelerating the silylation; however, it is surprising that the introduction of water in this step may lead to the difficulty of RNA extraction for the prepared magnetic microspheres. Accordingly, the first purpose of the present invention is to explore a functionalized treatment method using anhydrous solvent to prepare magnetic microspheres.

The invention also creatively finds that: by using strong acid to post-process the silanolated magnetic microparticles, the stability between detection batches of the silanolated magnetic microspheres can be significantly increased, which can be used for industrialized nucleic acid extraction, such as DNA extraction and RNA extraction. , DNA and RNA co-extraction, etc. In addition, the prepared silanol magnetic microspheres showed obvious advantages in the RNA quick extraction procedure, the extraction effect was significantly improved, and the sensitivity was high.

Accordingly, the first aspect of the present invention provides a method for preparing silanol magnetic microspheres, the gist of the involved method is: comprising the following steps:

S1. Provide magnetic particles;

S2, performing hydrophilic layer modification on the magnetic particles; and performing the following operations on the magnetic particles modified by the hydrophilic layer:

S3, performing functionalization treatment on the magnetic particles modified by the hydrophilic layer, and the functionalization treatment includes:

- Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

- adding silanol functional monomers to react,

wherein, no water is added or any water-containing reagents other than pH adjusters are not used in the functionalization step; or

S3', performing silanol modification on the magnetic particles modified by the hydrophilic layer; and post-processing the magnetic particles modified with silanol with a post-treatment reagent selected from organic strong acid, inorganic strong acid or inorganic strong acid and At least one of a mixture of complexing agents.

Preferably, in the above step S3', the step of modifying the silanol group comprises:

- Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

- adding silanol functional monomers to react,

Among them, it is preferable not to add any water or use any water-containing reagents other than pH adjusters in the step of silanol modification.

The lower alcohol in the above-mentioned step S3 or in the step of silanol modification is preferably an alcohol having 1 to 3 carbon atoms. The lower alcohol in the above step S3 or in the step of silanol modification is preferably at least one of methanol, ethanol, n-propanol and isopropanol.

In the above-mentioned step S3 and/or the step of silanol modification, the modified magnetic particles are preferably dispersed in anhydrous lower alcohol so that the magnetic fluid concentration is 1-15 mg/mL. In the above-mentioned step S3 and/or in the step of silanol modification, the modified magnetic particles are preferably dispersed in anhydrous lower alcohol so that the magnetic fluid concentration is 2-14 mg/mL.

Moreover, the second aspect of the present invention provides a silanol magnetic microsphere, and the gist of the silanol magnetic microsphere involved in the present invention is that it is prepared by the method provided in the first aspect of the present invention.

Moreover, the third aspect of the present invention provides an application of the silanol magnetic microspheres in nucleic acid extraction.

Preferably, the nucleic acid is DNA, RNA, or DNA and RNA, in particular RNA such as from SARS-CoV-2.

Invention effect

In the present invention, by not adding any water or using any water-containing reagents except pH regulators in the functionalization step, the prepared silanol magnetic microspheres have significantly improved effects when used for nucleic acid, especially RNA extraction, and the sensitivity is improved. high, and improved repeatability and detection rate.

Moreover, the method of the present invention significantly increases the batch-to-batch stability of the silicon hydroxyl magnetic beads, and can be used for industrialized nucleic acid extraction, such as DNA extraction, RNA extraction, DNA/RNA co-extraction, and the like.

In addition, the silanol magnetic microspheres of the present invention have obvious advantages in the RNA quick extraction procedure, the extraction effect is significantly improved, and the sensitivity is high.

Description of drawings

Figure 1 shows the preparation process route of the silanol magnetic microspheres of Examples 1-9.

Figure 2 shows the SEM image of the magnetic particles prepared in Example 1;

FIG. 3 shows the VSM test results of the magnetic particles prepared in Example 1.

Fig. 4 is the reagent pre-packing diagram of the 96-well deep-well plate according to the embodiment of the present invention;

Fig. 5 is the VSM diagram of the magnetic bead of Example 15 of the present invention;

6 is a CA diagram of the magnetic bead of Example 15 of the present invention;

FIG. 7 is a TEM image of magnetic beads in Example 15 of the present invention.

Detailed ways

Hereinafter, the present invention will be described in detail.

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 belongs. The terms used herein in the description of the present invention are for the purpose of describing the embodiments only, and are not intended to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Except as shown in the working examples or otherwise indicated, all numbers used in the specification and claims indicating amounts, physicochemical properties, etc. of ingredients are understood to be adjusted in all cases by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the foregoing specification and attached claims are approximations that can be modified by those skilled in the art to obtain the desired properties sought to be obtained by the teachings disclosed herein. approximation. Unless otherwise defined, the use of numerical ranges 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 and so on.

As used herein, the terms "having", "comprising" or "including" are used in a non-exclusive manner. Accordingly, these terms may refer to both situations in which no additional features are present in the entity described in this context other than the features introduced by the terms, and situations in which one or more additional features are present. As an example, the expressions "A has B," "A includes B," and "A includes B" can both refer to situations where no additional elements other than B are present in A (ie, situations where B alone and exclusively consists of B). ), and may refer to situations where in addition to B there are one or more additional elements in entity A such as element C, elements C and D, or even other elements.

The term "about" in the context of a particular value or ratio of the present invention means +/- 10% of the stated value or ratio, or in one embodiment +/- 5% of a given value or ratio.

Herein, the term "magnetic particle" can be used interchangeably with "magnetic matrix material" and "magnetic seed core", and refers to magnetic particles with superparamagnetic properties and a complete and uniform crystal form. It can be understood from the context of the present invention that the magnetic microparticles of the present invention have not been functionalized and/or hydrophilically modified.

Herein, the term "magnetic microspheres" is used interchangeably with "magnetic beads".

A first aspect of the present invention provides a method for preparing silanol magnetic microspheres, comprising the following steps:

S1. Provide magnetic particles;

S2, performing hydrophilic layer modification on the magnetic particles; and performing the following operations on the magnetic particles modified by the hydrophilic layer:

S3, performing functionalization treatment on the magnetic particles modified by the hydrophilic layer, and the functionalization treatment includes:

- Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

- adding silanol functional monomers to react,

wherein, no water is added or any water-containing reagents other than pH adjusters are not used in the functionalization step; or

S3', performing silanol modification on the magnetic particles modified by the hydrophilic layer; and post-processing the magnetic particles modified with silanol with a post-treatment reagent selected from organic strong acid, inorganic strong acid or inorganic strong acid and At least one of a mixture of complexing agents.

Herein, the "silyl group modification" refers to the modification of magnetic microparticles with a silyl group functional monomer. According to the present invention, "silyl group functional monomer" may be used interchangeably with "silyl group donor" and refers to a substance that imparts silyl group functionality to the magnetic particles. Specifically, the "silicon hydroxyl functional monomer" can be selected from methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, butyl orthosilicate, alkyl trimethoxysilane, alkyl triethoxy Silane, Alkyltriethylpropylsilane, Dialkyldimethoxysilane, Dialkyldiethoxysilane, Phenyltrimethoxysilane, Phenyltriethoxysilane, Aminopropyltrimethoxysilane At least one of silane and glycidyltrimethoxysilane.

According to the present invention, the step of silanol modification preferably includes:

- Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

- adding silanol functional monomers to react,

Among them, it is preferable not to add any water or use any water-containing reagents other than pH adjusters in the step of silanol modification.

It should be understood that the functionalization step and the preferred silanol modification step of the present invention are performed while avoiding the introduction of water as much as possible; in other words, the functionalization step and the preferred silanol modification step are performed without adding any water or using any aqueous reagents other than pH adjusters (or without introducing water in the functionalization step and the preferred silanol modification step in any way other than pH adjusters) . Any water can be, for example, ultrapure water, distilled water, water retained or intentionally added to the reaction vessel, or any aqueous reagent, etc.; any aqueous reagent can be, for example, a dispersant, an aqueous alcohol (alcohol/water mixture), and the like. Therefore, it can also be understood that, in the functionalization treatment step and the preferred silanol modification step, there may be a step of adjusting pH as required, for example, before or during the reaction by adding the "silanol functional monomer", adjusting pH to conditions suitable for the reaction. The pH adjusting step is achieved by adding a pH adjusting agent, so that the pH adjusting agent may contain a very small amount of water, but this very small amount of water has little effect on the functionalization treatment step. According to a specific embodiment of the present invention, the water brought into the functionalization treatment step and the preferred silanol group modification step system by the pH adjuster does not exceed 5% of the total mass of the system, such as 0-5%, 0.1% -5%, 0.5%-5% or 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5 and any value in between. In some embodiments, after dispersing the modified magnetic microparticles in anhydrous lower alcohol and before or at the time of adding the silanol functional monomer for the reaction, the reaction solution is adjusted to a suitable reaction pH by adding a pH adjuster condition.

According to the present invention, in step S1, the magnetic particles (magnetic seed cores) can be magnetic particles commonly used in the field, for example, can be in the form of Fe ₃ O ₄ , or in the form of AFe ₃ O ₄ , wherein A is zinc, manganese, titanium, nickel, Two or more of cobalt, zirconium, and chromium. The magnetic particles can be commercially available products or synthesized by methods well known in the art, such as chemical co-precipitation, hydrothermal, solvothermal, microemulsion, DC arc plasma or pyrolysis. Wait. The morphology, crystal structure and particle size of the obtained magnetic particles can be confirmed by SEM photographs of the magnetic particles. Magnetic results can be confirmed by VSM results. The synthesized magnetic particles are superparamagnetic and have a complete and uniform crystal form. According to some specific embodiments, the particle size of the magnetic particles is 5-20 nm, such as 5 nm, 10 nm, 15 nm, 20 nm and any value therebetween.

According to some preferred embodiments of the present invention, the magnetic particles are heated (eg, 80°C-110°C) after reacting the Fe ²⁺ soluble iron salt solution with an alkaline solution such as ammonia water at a certain temperature (eg, 20°C-40°C). ℃) made by aging. Specific examples of the Fe ²⁺ soluble iron salt solution include, but are not limited to, one or a mixture of at least two of ferrous chloride, ferrous sulfate and ferrous nitrate.

According to the present invention, in step S2, the "hydrophilic layer modification" refers to modifying the magnetic particles with a hydrophilic substance. The hydrophilic layer modification is aimed at improving the dispersibility of the particles. The step of modifying the hydrophilic layer may specifically include dispersing the magnetic particles in the hydrophilic substance solution (eg, an aqueous solution) for a certain period of time. The temperature and time of the holding can be selected in a wide range, and in some preferred embodiments, the temperature is 60-80°C. In some preferred embodiments, the time is 12-30 h.

In the present invention, the hydrophilic substance is preferably citric acid (such as citrate), polyethylene glycol, and polyvinylpyrrolidone, such as sodium citrate. In an exemplary embodiment, in the case of using sodium citrate as the hydrophilic substance, 0.01M sodium citrate can be added and kept at 90° C. for 1 h, thereby obtaining surface-modified magnetic particles. In the case of using PVP as the hydrophilic substance, 0.003 MPVP aqueous solution can be added and kept at 90° C. for 1 h to obtain surface-modified magnetic particles.

According to some embodiments of the present invention, in step S3 and/or in the step of silanol modification, the "lower alcohol" refers to an alcohol with 3 or less carbon atoms, such as methanol, ethanol, n-propanol and At least one of isopropanol. In some preferred embodiments, the anhydrous lower alcohol is anhydrous ethanol and/or isopropanol, and from the perspective of further improving the RNA extraction effect, the anhydrous lower alcohol is most preferably anhydrous ethanol.

In the functionalization treatment step S3 and the preferred silanol modification step, after the modified magnetic particles are dispersed in the lower alcohol, a certain concentration of magnetic fluid will be formed. The ferrofluid concentration is defined as the ratio of the mass of the modified magnetic particles used to the volume of the lower alcohol solution. For example, when 300 mg of the modified magnetic particles are dispersed in 150 ml of lower alcohol, the magnetic fluid concentration is 2 mg/mL.

In the present invention, the ferrofluid concentration may be 1-15 mg/mL. For example, about 1 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, or about 15 mg/mL.

In a preferred embodiment, the ferrofluid concentration of the present invention is 2-14 mg/mL. According to the research of the present invention, by selecting the magnetic fluid concentration of 2-14 mg/mL, the effect of the prepared magnetic microspheres in extracting nucleic acid is further improved.

According to the present invention, the temperature and time for treating the modified magnetic particles with lower alcohol in step S3 and/or in the silanol modification step are not particularly limited, as long as the treated magnetic particles can be sufficiently dispersed. For example, in some embodiments, the temperature is 0-40°C, and is preferably performed under closed conditions. In some embodiments, the time is 1-96 h.

According to the present invention, in step S3 and/or in the step of silanol modification, before or when the "silicon hydroxyl functional monomer" is added for the reaction, the method of the present invention may further comprise adjusting the solution to be reacted to a suitable Steps for reaction conditions. The conditions suitable for the reaction are, for example, pH conditions suitable for the reaction. In the present invention, a suitable pH condition can be, for example, 8-13; for example, pH adjustment can be achieved by adding a weakly alkaline pH adjusting agent (eg, 1-2 mL of ammonia water). The suitable reaction conditions are, for example, temperature conditions suitable for the reaction. In the present invention, the suitable reaction temperature may be, for example, 50-90°C. In the present invention, the time selection range for adding silanol functional monomers for the reaction is wide, so as to fully react to realize the silanol functionalization of magnetic particles. In some specific embodiments, the adding silanol functionalization The sub-steps of the reaction of the monomers can last for 1-6h, for example.

According to the present invention, in step S3 and/or the sub-step of adding a silanol functional monomer to react in the step S3 and/or the silanol modification step, the amount of the silanol functional monomer added can be adjusted, for example, in some preferred In the embodiment of the present invention, the mass ratio of the amount of the added silanol functional monomer to the magnetic particles modified by the hydrophilic layer is 1:1-1:20.

As mentioned above, the silanol magnetic microspheres prepared by the method of adding water in the functionalization step have the problem that the signal is weak or even cannot be extracted when used for RNA extraction, which greatly restricts the silanol magnetic microspheres Applications in nucleic acid extraction. In the present invention, anhydrous lower alcohol solvent is combined with other steps to prepare silanol magnetic microspheres for nucleic acid extraction, such as DNA extraction, RNA extraction, or DNA/RNA co-extraction, in the functionalization process, especially for RNA extraction. Significantly improved performance, sensitivity, and improved repeatability and detection rates.

Strong acids are defined in this article as "an acid undergoes fully dissociation" (that is, an acid that can fully ionize autonomously, such as 1 mol of HCl in water to generate 1 mol of H ⁺ , and another 1 mol of Cl ^- ). Acids that meet this condition are generally inorganic acids. An acid that is completely ionized in solution is a strong acid, and the ionization of a strong acid uses the equal sign, such as: HCl=H ⁺ +Cl ^- . The current judgment standard of strong acid is its ionization constant in aqueous solution, pKa (acidity coefficient, negative logarithm of ionization constant) is less than 1 is strong acid (pKa=1～4 is medium strong acid, greater than 4 is weak acid), some pKa values A fraction of an acid can also be considered a strong acid.

According to some embodiments of the present invention, the inorganic strong acid of the present invention is selected from sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, bromic acid, fluorosilicic acid, lead chloride Any one or more of acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid and metaperiodic acid.

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

According to some embodiments of the present invention, the complexing agent is selected from the group consisting of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, phenanthroline, 2, Any one or more of 2'-bipyridine, 8-quinolinol, and nitrogen-based complexing agents.

According to some preferred embodiments of the present invention, the concentration of the strong acid (eg, inorganic strong acid) in the post-treatment reagent is 1×10 ⁻⁴ mol/L˜10 mol/L. Specifically, it can be 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, 2mol/L, 3mol/L, 4mol/L, 5mol/L, 6mol/L, 7mol/L, 8mol/L, 9mol/L, 10mol/L. The research results of the present invention show that, for the effect of the present application, the inorganic strong acid is significantly different from the common acidic solution or buffer, and the concentration of the inorganic strong acid is related to the effect of the post-treatment. Preferably, the concentration of the inorganic strong acid is 10 ^-3 mol/L～1mol/L.

In the present invention, the post-treatment reagent may or may not contain a complexing agent. According to some preferred embodiments of the present invention, 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 ^-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, 1 mol/L. It is found through research that the use of strong acid, especially inorganic strong acid, in the present invention to post-process the silanolated magnetic particles can significantly increase the batch-to-batch stability of the silanolated magnetic beads, which can be used for industrial nucleic acid extraction, such as DNA extraction, RNA extraction, etc. Extraction, DNA/RNA co-extraction, etc. In addition, the silicon hydroxyl magnetic beads of the present invention have obvious advantages in the RNA quick extraction procedure, the extraction effect is significantly improved, and the sensitivity is high.

According to the present invention, the post-treatment includes soaking the silanol-modified magnetic particles in the post-treatment reagent. According to some embodiments of the present invention, the post-processing step includes soaking the silanol-modified magnetic particles in the post-processing reagent and standing. In some embodiments, the standing temperature is 20-70°C. In some embodiments, the standing time is 1-15 h. The specific standing temperature can be 20°C, 30°C at normal temperature, or 40°C, 50°C, 60°C, and 70°C at high temperature. The specific standing 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, 15 hours.

According to some specific embodiments of the present invention, step S3' of the method of the present invention further includes the steps of washing, dispersing and adjusting pH of the magnetic particles after post-treatment with the post-treatment reagent. Specifically, for example, the post-treated magnetic microparticles are washed with water and then dispersed in water, and the pH of the resulting aqueous dispersion is adjusted to 5-7 (specifically, 5, 5.5, 6, 6.5, and 7).

According to the present invention, in addition to the above-mentioned steps, the method of the present invention may also include general steps for preparing magnetic microspheres in the art, or as required, within the scope of not hindering the nucleic acid extraction effect and batch stability of the magnetic microspheres of the present invention, additional steps to take. For example, the steps of magnetic separation, washing, and drying are performed after the functionalization treatment step or the strong acid post-treatment step, and the relevant steps can be performed by using conventional or known methods in the art, which will not be repeated here.

According to some embodiments of the present invention, the particle size of the magnetic microspheres of the present invention is not greater than 80 nm, preferably 5-80 nm, preferably 35-80 nm, more preferably 35-50 nm.

The second aspect of the present invention provides a silanol magnetic microsphere, which is prepared by the method for preparing a silanol magnetic microsphere according to the first aspect of the present invention, preferably the particle size of the magnetic microsphere is not greater than 80 nm, preferably 5 to 80 nm, preferably 35 to 80 nm, more preferably 35 to 50 nm.

The third aspect of the present invention provides the application of the silanol magnetic microspheres prepared by the method described in the first aspect of the present invention or the silanol magnetic microspheres described in the second aspect of the present invention in nucleic acid extraction.

According to the present invention, the silicon hydroxyl magnetic microspheres prepared by the present invention can be used for DNA extraction or RNA extraction or co-extraction of DNA and RNA.

According to the present invention, the samples to which the silicon hydroxyl magnetic microspheres prepared by the present invention are applied are not specifically limited, as long as they contain/have nucleic acid. Specific examples of samples include but are not limited to blood, throat swabs, sputum, alveoli lavage fluid, tissue, food and environmental samples, etc.

In an exemplary embodiment, the RNA is RNA derived from a virus, such as RNA from a novel coronavirus (SARS-CoV-2).

Example

Hereinafter, the present invention will be described in detail using examples. The preparation process route of the silanol magnetic microspheres of Examples 1-9 of the present invention is shown in FIG. 1 .

The magnetic bead preparation method of the commercial product in the embodiment of the present invention is as follows: contacting 0.1 wt% Fe ₃ O ₄ magnetic seed core cyclohexane dispersion with an aqueous solution containing 0.01 wt % surfactant SDS to form a monodisperse oil drop suspension. The cyclohexane in the oil droplets was evaporated under reduced pressure, so that the oil droplets containing Fe ₃ O ₄ shrunk into magnetic nanoparticle assemblies. In a 500mL round-bottomed flask, 0.25g of the prepared magnetic nanoparticle assembly, 200g of deionized water, 250g of absolute ethanol, 5mL of 25wt% concentrated ammonia and 2.5mL of tetraethyl orthosilicate were sequentially added, and the mixture was fully stirred for 30 minutes. The reaction was carried out at room temperature for 12 hours, and then the obtained product was washed and dried to finally obtain silicon oxide magnetic microspheres with an average particle size of about 500 nm, and the silicon oxide coating thickness was about 10 nm.

The mass percentage concentration of ammonia water used in the embodiments of the present invention is 25%-28% (ie, ammonia water containing 25%-28% ammonia).

Example 1

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

The obtained magnetic seed nuclei were observed under SEM, and the results are shown in Figure 2 (under the scale of 5 nm and 10 nm, respectively); the SEM results showed that the magnetic seed nuclei were basically spherical and had a good crystal structure with a particle size of about 10 nm. At the same time, the obtained magnetic seed nuclei were measured by magnetic experiments using VSM, and the results are shown in Figure 3; the VSM results show that the magnetic seed nuclei have superparamagnetic properties.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silanol modification: Disperse 1 g of the magnetic particles obtained in step 2 in 150 mL of absolute ethanol, add 1 mL of ammonia water after 30 min, and raise the temperature to 80 °C. After keeping the temperature constant, add 5 mL of TEOS, and stop the reaction after 2 h to obtain silanol magnetic microspheres. The particle size is about 35-50nm.

Example 2

Using the silicon hydroxyl magnetic microspheres obtained in Example 1, using Aosheng Auto Pure32A extractor, extract sample 1 (SARS-CoV-2 high concentration sample 5 × 103 copies/mL) and sample 2 (SARS-CoV-2 low concentration sample) Nucleic acids in sample 400copies/mL), wherein sample 1 is a pseudovirus sample containing SARS-CoV-2 gene at high concentration (5×103copies/mL), and sample 2 is a low concentration (400copies/mL) containing SARS-CoV-2 - Pseudovirus samples of the CoV-2 gene.

The RNA extracted from sample 1 and sample 2 was amplified by reverse transcription using Hongshi SLAN96P amplifier. The detection results are shown in Table 1.

Table 1

*Channels 1 to 4 represent the detection results of four different genes of SARS-CoV-2, the same below;

*Detection rate: when there is no Ct value or the Ct value is greater than 38, it is regarded as not detected, the same below.

It can be seen from Table 1 that the experimental data of the high-concentration sample 1 shows that the magnetic microspheres prepared in Example 1 have better extraction effect and good repeatability.

According to the results of the low concentration sample 2, the detection rate of the magnetic microspheres prepared in Example 1 was 100%, and the repeatability was good.

Comparative Example 1

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse 1 g of the magnetic particles obtained in step 2 in 150 mL of ethanol/water solution (the volume ratio of ethanol and water is 5:1), add 1 mL of ammonia water after 30 min and heat up to 77 ° C, add 5 mL of TEOS after the temperature is kept constant, The dilution ratio of TEOS and ethanol was 1:1 to 1:10, and the reaction was stopped after 2 h. Silanol magnetic microspheres were obtained.

Comparative Example 2

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse 1 g of the magnetic particles obtained in step 2 in 120 mL of absolute ethanol, add 2 mL of ammonia water after 30 min, and heat up to 80 ° C. After the temperature is kept constant, add 5 mL of TEOS, wherein the dilution ratio of TEOS and ethanol is 1:1～ 1:10, after the reaction for 2 hours, 30 mL of ultrapure water was added, the reaction was continued for 2 hours, the heating was stopped, and the mixture was stirred overnight. Silanol magnetic microspheres were obtained.

Comparative Example 3

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse 1 g of the magnetic particles obtained in step 2 in 30 mL of water, stir for 30 min to disperse, and then add 120 mL of anhydrous ethanol and continue to stir for 30 min. Add 2 mL of ammonia water and raise the temperature to 80°C. After keeping the temperature constant, add 5 mL of TEOS, wherein the dilution ratio of TEOS and ethanol is 1:1 to 1:10. After the reaction for 2 hours, add 30 mL of ultrapure water, continue the reaction for 2 hours, and stop heating. Stir overnight. Silanol magnetic microspheres were obtained.

Comparative Example 4

Samples 1 and 2 were tested according to the extraction method and detection method in Example 2 using the silanol magnetic microspheres prepared in Comparative Examples 1-3 respectively, and the results are shown in Table 2 below.

Table 2

In the methods of Comparative Examples 1-3, water was added in the functionalization treatment step in a different order. The results in Table 2 show that the magnetic microspheres synthesized by these three methods have almost no effect on RNA extraction, and only the part that can be detected in channel 2, and the rest of the channels cannot be detected. It shows that the silanol magnetic microspheres synthesized by alcohol/water reaction system in the functionalization step cannot be used for RNA extraction.

Example 3

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic particles prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse the magnetic particles obtained in step 2 in 150 mL of absolute ethanol to make the magnetic fluid concentration 2 mg/mL. After 30 min, add 2 mL of ammonia water and raise the temperature to 80 °C. After the temperature is kept constant, add 5 mL of TEOS. The reaction is stopped after 2 h. Heat and stir overnight. Silanol magnetic microspheres were obtained.

Example 4

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse the magnetic particles obtained in step 2 in 150 mL of absolute ethanol to make the magnetic fluid concentration 14 mg/mL. After 30 min, add 2 mL of ammonia water and raise the temperature to 80 °C. After the temperature is kept constant, add 5 mL of TEOS, and the reaction is stopped after 2 h. Heat and stir overnight. Silanol magnetic microspheres were obtained.

Example 5

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse the magnetic particles obtained in step 2 in 150 mL of absolute ethanol to make the magnetic fluid concentration 1 mg/mL respectively. After 30 min, add 2 mL of ammonia water and raise the temperature to 80 °C. After the temperature is kept constant, add 5 mL of TEOS, and react for 2 h. Heating was stopped and stirred overnight. Silanol magnetic microspheres were obtained.

Example 6

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse the magnetic particles obtained in step 2 in 150 mL of absolute ethanol to make the magnetic fluid concentration 15 mg/mL. After 30 min, add 2 mL of ammonia water and raise the temperature to 80 °C. After the temperature is kept constant, add 5 mL of TEOS. The reaction is stopped after 2 h. Heat and stir overnight. Silanol magnetic microspheres were obtained.

Example 7

The silanol magnetic microspheres prepared in Examples 3-4 and 5-6 were used respectively, and samples 1 and 2 were tested according to the extraction method and detection method in Example 2. The results are shown in Table 3 below. .

It can be seen from Table 3 that there is no significant difference in the extraction effect of the magnetic microspheres obtained in Example 3 and Example 4 in Sample 1; in the extraction of Sample 2, the Ct value of Example 3 in the first channel is about 1.5 ahead of Example 4, In the third channel, the effect of the Ct value of Example 4 is about 0.6 higher than that of Example 3; and the detection rates of the magnetic microspheres prepared by these two examples are both 100%. When the concentration of the magnetic fluid is 1 mg/mL (ie Example 5), the number of extraction cycles of sample 1 lags behind that of Example 3 by 1.5-3 Ct; the number of extraction cycles of sample 2 lags by 0.5-3.5 Ct, and the detection rate is low . When the ferrofluid concentration is 15 mg/mL (ie Example 6), the number of extraction cycles of sample 1 lags behind that of Example 3 by 0-4 Ct on average; the number of extraction cycles of sample 2 lags by 0.8-2.5 Ct, and there is no detection phenomenon . It shows that the optimal concentration of magnetic fluid is 2～14g/mL.

Example 8

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

Take 4.0g of ferrous sulfate·heptahydrate and 7.0g of ferric chloride·hexahydrate in 150mL of water, set the stirring speed to 300rpm, stir for 30min to fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Hydrophilic layer modification of magnetic particles

Disperse the magnetic seed core prepared above in 200 mL of 0.01 M sodium citrate aqueous solution, and keep at 90 °C for 1 h to obtain surface-modified magnetic particles.

Step 3: Functionalization

Silicon hydroxyl group modification: Disperse 1 g of the magnetic particles obtained in step 2 in 150 mL of anhydrous isopropanol, add 2 mL of ammonia water after 30 min, and raise the temperature to 80 °C. After the temperature is kept constant, add 5 mL of TEOS, stop heating after 2 h of reaction, and stir overnight. Silanol magnetic microspheres were obtained.

Example 9

Using the silanol magnetic microspheres prepared in Example 8, according to the extraction method and detection method in Example 2, samples 1 and 2 were tested, and the results are shown in Table 4 below.

Table 4

It can be seen from Table 4 that the silanol magnetic microspheres prepared in Example 8 using isopropanol as anhydrous lower alcohol also achieved good results in nucleic acid extraction.

Example 10

Step 1: Synthesis of Magnetic Particles (Magnetic Seed Nuclei)

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 fully mix the solution, heat up to 40°C, then add 50mL of ammonia water, react for 30min, and then again The temperature is raised to 90° C. and matured for 1 h to obtain superparamagnetic nanomagnetic particles with a particle size of about 5-15 nm.

Step 2: Modification of the hydrophilic layer of magnetic particles

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

Step 3: Silanol Modification

Silicon hydroxyl group modification: Disperse 1 g of the magnetic particles obtained in step 2 in 150 mL of absolute ethanol, add 1 mL of ammonia water after 30 min, and raise the temperature to 77 °C, add 5 mL of TEOS after keeping the temperature constant, and stop the reaction after 2 h. The product is directly dispersed in water to obtain silanol magnetic beads.

Example 11: Treatment method for improving magnetic responsiveness of silicon hydroxyl magnetic beads, purpose: to verify whether improving the magnetic responsiveness can directly improve the extraction performance.

The silicon hydroxyl magnetic beads obtained in Example 10 were directly dispersed in 0.01% (w/v) sodium chloride solution. Experiments show that the magnetic response performance of the product can be significantly improved after adding sodium chloride solution.

Extraction performance check:

a) Instruments

Aosheng Auto-Pure32A Nucleic Acid Extractor

b) Reagent components and dosage (using the nucleic acid extraction reagent in Mike Bio’s new coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method))

component name	Component dosage/test
Proteinase K	100-400ug
Lysate	500-700ul
Magnetic beads	200-300ug
washing liquid 1	600-800ul
washing liquid 2	600-800ul
eluent	40-80ul

c) 96-well deep-well plate reagent pre-packing is shown in Figure 1. Among them, the 1st and 7th columns contain magnetic beads in each well; the 2nd and 8th columns contain effective working wells, and each well contains lysis buffer; the 3rd and 9th columns each well contains washing solution 1; Column and 10th column each well contains washing solution 2; 6th column and 12th column each well contains eluate.

d) The instrument operation quick-lift procedure is:

e) The slow lifting procedure of the instrument operation (in this patent, this procedure is only used for the HCV kit) is:

After nucleic acid extraction from the sample to be tested, the new coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) (Mike Bio) was used for detection. The test results are shown in Table 5:

Table 5 Evaluation results of magnetic beads in the quick-lift procedure

"/" in the table is the undetected value.

Conclusion: Table 5 shows the evaluation results of magnetic beads in the new crown quick-lift procedure. It can be seen from the table that the products of Example 10 and Example 11 cannot be detected in the quick-lift procedure, indicating that the magnetic beads are not suitable for the quick-lift procedure.

Example 12: Processing method of silanol magnetic beads

Take 20 g (wet weight) of silicon hydroxyl magnetic beads obtained in Example 10, disperse in 100 mL of aqueous hydrochloric acid with a concentration of 1 × 10 ^-4 mol/L to 10 mol/L, and stand for 1 to 24 hours. After that, it is washed several times with water, and then dispersed in purified water to obtain post-treated magnetic silanol magnetic microspheres.

Conclusion: Table 6 shows the evaluation results of the new coronavirus 2019-nCoV nucleic acid detection kit (fluorescent PCR method) (Mike Bio) after nucleic acid extraction by magnetic beads in the new crown quick extraction procedure. It can be seen from the table that:

1) The magnetic beads synthesized in Example 10 cannot be detected in the quick-lift procedure, indicating that this type of magnetic beads is not suitable for the quick-lift procedure.

2) In Example 12, the detection rate of magnetic beads in the sensitivity sample was 100%, and the CT value of the magnetic beads in the precision sample was 0.3 CT to 1.1 CT earlier than that of the magnetic beads in Example 10.

3) Compared with the commercial product, since the magnetic beads of Example 10 cannot be detected in the sensitivity samples, the magnetic beads of Example 10 have poorer performance than the commercial products; The detection rate of the sensitivity sample was 100%, but the CT value of Example 12 was earlier than that of the commercial product, so the performance of the magnetic beads of Example 12 was better than that of the commercial product.

Example 13: Processing method of silanol magnetic beads

Get 20g (wet weight) of silicon hydroxyl magnetic beads obtained in Example 10, disperse in 100mL of the mixed solution of 1 × ^10-4 mol/L sulfuric acid aqueous solution and 0.01mol/L EDTA solution, and mix the system. Let stand for 1 h at room temperature. The solid particles were then collected by water washing and magnetic separation. The collected magnetic solid particles were treated with an aqueous solution of sodium citrate. After repeated washing with water for several times, the product is finally dispersed in water, and the pH of the obtained product is adjusted to 7 using an aqueous sodium hydroxide solution to obtain post-treated silanol magnetic beads.

Table 7 Evaluation results of magnetic beads in the new crown (2019-nCoV) quick pick-up procedure

Table 8 Evaluation results of magnetic beads in HCV slow lifting procedure

in conclusion:

a) 2019-nCoV project test extraction procedure: fast; HCV project test extraction procedure: slow. The fast extraction procedure is the d) procedure of Example 11; the slow extraction procedure is the e) procedure of Example 11.

b) In the 2019-nCoV project, the CT value obtained by the evaluation of Example 13 is generally ahead of the CT value of the commercial product, so the product of Example 13 is overall better than the commercial product.

c) In the HCV project, whether it is a sensitivity sample or a precision sample, the CT value obtained by the evaluation in Example 13 is earlier than that of the commercial product, so the product performance of Example 13 is better than the commercial product.

Example 14: Treatment of Silanol Magnetic Beads with Different Concentrations of Acid

Take 20 g (wet weight) of silanol magnetic beads obtained in Example 10, and the particle size of the magnetic beads is about 5-50 nm. Disperse in 100 mL of aqueous hydrochloric acid solutions with concentrations of 10 mol/L, 0.1 mol/L, 1×10 ^-3 mol/L and 1×10 ^-5 mol/L, respectively. The solid particles were collected by standing at room temperature for 1 to 24 hours, and then washed with water and magnetically separated. The collected magnetic solid particles were dispersed in purified water.

Table 9 Evaluation results of magnetic beads in the new crown quick-lift procedure

Conclusion: It can be seen from Table 9 that:

When the concentration of hydrochloric acid was 10mol/L, 0.1mol/L and 1×10 ^-3 mol/L, there was no significant difference in CT values of R2 samples, but S3 samples could be detected completely.

When the concentration of hydrochloric acid was 1×10 ^-5 mol/L, the low concentration sample (S3 sample) could not be detected.

Based on the above conclusions, it can be preliminarily inferred that for silanol magnetic beads with a particle size of less than 100 nm, in order to ensure that the magnetic beads are completely detected in the S3 sample, the acid concentration in the post-processing stage should be higher than 1×10 ^-5 mol/L.

Comparative Example 5: Repeat the experiment of Example 10 for three batches to verify the stability between batches.

Table 10 Evaluation results of magnetic beads in the new crown quick pick-up procedure

Conclusion: From Table 10 it can be seen that:

Compared with the commercial product, the product of Example 13 has better performance in the new crown evaluation.

Comparative Example 5-1, Comparative Example 5-2, and Comparative Example 5-3 are three batches of experiments in Example 10. It can be seen from the table that all three batches can be detected in the precision samples, but the ORF1ab channel CT value The difference between them is as high as 1.5CT, indicating that the magnetic beads synthesized in the three batches of experiments have the disadvantage of large batch-to-batch differences.

Sensitivity samples: The three batches of magnetic beads in Comparative Example 5 could not be detected by the sensitivity samples, indicating that they were unqualified in the new crown quick extraction.

Comparative Example 6: Repeat the experiment of Example 12 for three batches to verify the stability between batches.

Table 11 Evaluation results of magnetic beads in the COVID-19 quick pick-up procedure

Conclusion: Comparative Example 6-1, Comparative Example 6-2, and Comparative Example 6-3 are obtained from three batches of experiments in Comparative Example 6 after post-processing. It can be seen from the table that all three batches can be detected in the precision samples. , and there was no significant difference in the four-channel CT value, indicating that the post-processing method can reduce the batch difference.

Example 15

The VSM test was performed on the post-treated magnetic silanol magnetic beads prepared in Example 12, and the results are shown in Figure 5 and Table 12.

Table 12 Magnetic Bead VSM Data

Conclusion: From Figure 5 and Table 12 the following conclusions are drawn:

The magnetic beads are superparamagnetic.

Than the saturation magnetism is greater than 70emu/g.

The coercivity of the four batches was 7.4×10 ^-4 to 7.7×10 ^-4 , and the coercivity was negligible.

The contact angle test was performed on the post-treated magnetic silanol magnetic beads prepared in Example 12, and the results are shown in Figure 6. It can be seen from Figure 6 that the contact angle between the magnetic beads and water is 20.2°, which preliminarily shows that the hydrophilicity of the magnetic beads is relatively good. According to the standard "Test Method for Silanol Content on the Surface of Fumed Silica 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 on the surface after treatment is 0.527%. The amount of silanol groups on the surface of the magnetic beads did not change significantly, indicating that the post-treatment method did not improve the performance by increasing the number of silanol groups on the surface of the magnetic beads.

The particle size detection was carried out on the post-treated magnetic silanol magnetic beads prepared in Example 12. Figure 7. It can be seen from Fig. 7 that the particle size of the magnetic beads is about 5-50 nm, which is not much changed compared with the particle size of the magnetic beads before the acid treatment.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present invention, so as to facilitate a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as a limitation on the scope of the invention patent protection. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be based on the appended claims, and the description can be used to interpret the content of the claims.

Claims (10)

1. A method for preparing silanol magnetic microspheres, comprising:

   S1. Provide magnetic particles;

   S2, performing hydrophilic layer modification on the magnetic particles; and performing the following operations on the magnetic particles modified by the hydrophilic layer:

   S3, performing functionalization treatment on the magnetic particles modified by the hydrophilic layer, and the functionalization treatment includes:

   - Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

   - adding silanol functional monomers to react,

   wherein, no water is added or any water-containing reagents other than pH adjusters are not used in the functionalization step; or

   S3', performing silanol modification on the magnetic particles modified by the hydrophilic layer; and post-processing the magnetic particles modified with silanol with a post-treatment reagent selected from organic strong acid, inorganic strong acid or inorganic strong acid and At least one of a mixture of complexing agents.
2. The method according to claim 1, wherein the step of silanol modification comprises:

   - Disperse the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol;

   - adding silanol functional monomers to react,

   Among them, it is preferable not to add any water or use any water-containing reagents other than pH adjusters in the step of silanol modification.
3. The method according to claim 1 or 2, wherein the lower alcohol is an alcohol having 1 to 3 carbon atoms, preferably at least one selected from methanol, ethanol, n-propanol and isopropanol;

   And/or the silanol functional monomer is selected from methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, butyl orthosilicate, alkyl trimethoxysilane, alkyl triethoxy Silane, Alkyltriethylpropylsilane, Dialkyldimethoxysilane, Dialkyldiethoxysilane, Phenyltrimethoxysilane, Phenyltriethoxysilane, Aminopropyltrimethoxysilane and at least one of glycidyltrimethoxysilane.
4. The method according to claim 2 or 3, wherein in step S3 and/or in the step of silanol modification, the magnetic particles modified by the hydrophilic layer are dispersed in anhydrous lower alcohol, so that the formed The ferrofluid concentration is 1-15 mg/mL, preferably 2-14 mg/mL.
5. The method according to any one of claims 2-4, characterized in that, in step S3 and/or in the step of silanol modification, it further comprises before or during the reaction by adding silanol functional monomers, The reaction solution is adjusted to a suitable reaction pH by adding a pH adjuster, preferably pH 8-13.
6. The method according to any one of claims 1-5, wherein the inorganic strong acid is selected from sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, At least one of bromic acid, fluorosilicic acid, chlorolead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid and metaperiodic acid; and/or,

   The organic strong acid is selected from at least one of trifluoroacetic acid, trichloroacetic acid, methanesulfonic acid, benzenesulfonic acid, cyclohexanethiolsulfonic acid and 2-chloroethanethiol, and/or,

   The complexing agent is selected from EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, o-phenanthroline, 2,2'-bipyridine, 8- At least one of a quinolinol and a nitrogen-based complexing agent.
7. The method according to any one of claims 1-6, wherein the concentration of the strong acid in the post-treatment reagent is 1 × 10 ^-4 mol/L to 10 mol/L, preferably 10 ^-3 mol/L ~1 mol/L; and/or,

   The concentration of the complexing agent in the post-treatment reagent is 0.001 mol/L to 1 mol/L.
8. The method according to any one of claims 1-7, wherein the post-treatment comprises immersing the silanol-modified magnetic particles in the post-treatment reagent for standing, preferably, the The temperature for standing is 20°C to 70°C, and/or the time for standing is 1 hour to 15 hours;

   More preferably, the method step S3' further comprises washing the post-treated magnetic particles with water and then dispersing them in water, and adjusting the pH of the resulting aqueous dispersion to 5-7.
9. Silicon hydroxyl magnetic microspheres prepared by the method of any one of claims 1-8, preferably the particle size of the silicon hydroxyl magnetic microspheres is not greater than 80 nm, preferably 5-80 nm, more preferably 35-80 nm, More preferably, it is 35 to 50 nm.
10. Application of the silanol magnetic microspheres prepared by the method according to any one of claims 1-8 or the silanol magnetic microspheres as claimed in claim 9 in nucleic acid extraction, preferably the nucleic acid is RNA, DNA Or DNA and RNA, such as RNA from SARS-CoV-2.

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