CN115691994A - Preparation method and application of silicon hydroxyl magnetic beads - Google Patents
Preparation method and application of silicon hydroxyl magnetic beads Download PDFInfo
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Abstract
The invention discloses a preparation method of silicon hydroxyl magnetic beads, which comprises the following steps: providing magnetic particles; sequentially carrying out hydrophilic layer modification and silicon hydroxyl modification on the surface of the magnetic particle; and post-treating the magnetic particles modified by the silicon hydroxyl, wherein a post-treating reagent is inorganic strong acid or a mixture of the inorganic strong acid and a complexing agent. The invention also discloses a silicon hydroxyl magnetic bead. The invention also discloses application of the silicon hydroxyl magnetic beads in nucleic acid extraction.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, and in particular relates to a preparation method and application of silicon hydroxyl magnetic beads.
Background
Nucleic acids are a class of biological macromolecules formed by connecting nucleotides or deoxynucleotides through phosphodiester bonds, and include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). As a carrier of genetic information, nucleic acids are important as an object of research in the field of molecular biology. The quality of the nucleic acid sample, particularly the extraction effect thereof, has an important influence on the subsequent analysis process. The traditional nucleic acid extraction method comprises an alkaline lysis method, a boiling method, a column separation method and the like.
The magnetic microsphere method is a new extraction method developed in recent years, and the method is used for preparing superparamagnetic nano magnetic microspheres with some functional groups (such as hydroxyl and carboxyl) introduced on the surfaces. The magnetic microsphere can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface. Under the action of an external magnetic field, nucleic acid can be separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like. Compared with the traditional method, the method has the characteristics of no need of participation of highly toxic reagents, avoidance of multi-step high-rotation-speed centrifugation, and simplicity and feasibility. The silicon hydroxyl magnetic microspheres are common magnetic microspheres for nucleic acid extraction, but the traditional silicon hydroxyl magnetic beads have the defect of poor stability among batches, and even the detection stability of the silicon hydroxyl magnetic beads of the corresponding batches is unstable, the problem that a nucleic acid extraction signal cannot be detected exists, and the problem is particularly prominent in rapid RNA extraction based on magnetic beads.
Disclosure of Invention
Therefore, it is necessary to provide a preparation method and application of silicon hydroxyl magnetic beads for solving the problem of poor batch-to-batch stability of silicon hydroxyl magnetic beads obtained by the conventional preparation method.
A preparation method of silicon hydroxyl magnetic beads comprises the following steps:
providing magnetic particles;
sequentially carrying out hydrophilic layer modification and silicon hydroxyl modification on the surfaces of the magnetic particles; and
and (3) carrying out post-treatment on the magnetic particles modified by the silicon hydroxyl, wherein a post-treatment reagent is inorganic strong acid or a mixture of the inorganic strong acid and a complexing agent.
In one embodiment, the strong inorganic 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, chloropolylead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid.
In one embodiment, the complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, phenanthroline, 2' -bipyridine, 8-quinolinol, and nitrogen-based complexing agents.
In one embodiment, the concentration of the strong inorganic acid in the post-treatment reagent is 1 × 10 -4 mol/L～10mol/L。
In one embodiment, the concentration of the complexing agent in the post-treated reagent is 0.001mol/L to 1mol/L.
In one embodiment, the post-treatment step is to soak the magnetic particles modified by the silicon hydroxyl groups in the post-treatment reagent and to stand.
In one embodiment, the standing temperature is 20-70 ℃, and the standing time is 1-15 hours.
In one embodiment, the method comprises the following steps: and (3) washing the post-treated magnetic particles by using water, and then dispersing the magnetic particles into the water, wherein the pH of the obtained aqueous dispersion is adjusted to 5-7.
In one embodiment, the step of modifying the silicon hydroxyl group comprises:
dispersing the magnetic particles modified by the hydrophilic layer in anhydrous lower alcohol solution;
adding a monomer for endowing the magnetic particles with silicon hydroxyl functionalization for reaction.
In one embodiment, no water is added and no aqueous reagent other than a pH adjuster is used in the step of silicon hydroxyl modification.
In one embodiment, the magnetic particles have a particle size of 35nm to 80nm.
The invention also aims to provide the silicon hydroxyl magnetic bead prepared by the preparation method of the silicon hydroxyl magnetic bead.
The invention further aims to provide application of the silicon hydroxyl magnetic beads in nucleic acid extraction.
The invention also aims to provide the application of the silicon hydroxyl magnetic beads in RNA extraction.
According to the invention, the inorganic strong acid is adopted to carry out post-treatment on the silicon hydroxylated magnetic particles, so that the stability among batches of the silicon hydroxylated magnetic beads is obviously improved, and the method can be used for industrialized nucleic acid extraction, such as DNA extraction, RNA extraction, DNA/RNA co-extraction and the like. In addition, the silicon hydroxyl magnetic beads of the invention have obvious advantages in the RNA fast extraction process, the extraction effect is obviously improved, and the sensitivity is high.
Drawings
FIG. 1 is a diagram of pre-dispensing of reagents from a 96-well deep-well plate according to one embodiment of the present invention;
FIG. 2 is a diagram of a VSM of a magnetic bead in accordance with an embodiment of the present invention;
FIG. 3 is a CA diagram of a magnetic bead according to an embodiment of the present invention;
FIG. 4 is a TEM image of magnetic beads according to an embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully hereinafter with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, 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 terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Other than as shown in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients, physical and chemical properties, and so forth used in the specification and claims are to be understood as being modified in all instances 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 suitably varied by those skilled in the art in seeking to obtain the desired properties utilizing the teachings disclosed herein. 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 the like.
The embodiment of the invention provides a preparation method of silicon hydroxyl magnetic beads, which comprises the following steps:
providing magnetic particles;
performing hydrophilic layer modification on the surfaces of the magnetic particles;
carrying out silicon hydroxyl modification on the surface of the magnetic particle modified by the hydrophilic layer; and
and (2) carrying out post-treatment on the magnetic particles modified by the silicon hydroxyl, wherein a post-treatment reagent is inorganic strong acid or a mixture of the inorganic strong acid and a complexing agent.
According to the invention, the inorganic strong acid is adopted to carry out post-treatment on the silicon hydroxylated magnetic particles, so that the stability among batches of the silicon hydroxylated magnetic beads is obviously improved, and the method can be used for industrialized nucleic acid extraction, such as DNA extraction, RNA extraction, DNA/RNA co-extraction and the like. In addition, the silicon hydroxyl magnetic beads of the invention have obvious advantages in the RNA fast extraction process, the extraction effect is obviously improved, and the sensitivity is high.
As used herein, "magnetic microparticle" is used interchangeably with "magnetic matrix material" and refers to a magnetic particle having a superparamagnetic, intact and uniform crystalline form.
It will be appreciated from the context of the present invention that the magnetic particles of the present invention have not been functionally and/or hydrophilically modified. The magnetic fine particles (magnetic seed cores) may be, for example, fe 3 O 4 Form, or AFe 3 O 4 Forms wherein A is two or more of zinc, manganese, titanium, nickel, cobalt, zirconium, chromium.
The magnetic particles can be synthesized by methods well known in the art, such as chemical coprecipitation, hydrothermal, solvothermal, microemulsion, dc arc plasma, or pyrolysis. The synthesized magnetic particles have superparamagnetism and complete and uniform crystal form.
As used herein, "magnetic microspheres" are used interchangeably with "magnetic beads".
As used herein, "hydrophilic layer modification" refers to modification of magnetic microparticles with a hydrophilic substance. The hydrophilic layer modification is intended to improve the dispersibility of the particles.
In the present invention, the hydrophilic substance may be a citric acid (e.g., citrate), a polyethylene glycol, or a polyvinylpyrrolidone type hydrophilic substance.
Said Fe 2+ The soluble ferric salt solution is one or a mixture of at least two of ferrous chloride, ferrous sulfate and ferrous nitrate.
By selecting the concentration of the magnetic fluid of 2-14mg/mL in the step of silicon hydroxylation treatment, the invention further improves the effect of the prepared magnetic microspheres in the process of extracting nucleic acid.
In some embodiments, the step of modifying the silicon hydroxyl group comprises:
dispersing the magnetic particles modified by the hydrophilic layer in an anhydrous lower alcohol solution;
adding a monomer for endowing the magnetic particles with silicon hydroxyl functionalization for reaction.
It will be appreciated that the silicon hydroxylation step of the present invention is carried out with the introduction of water being avoided as much as possible; in other words, no water is added and no aqueous reagent other than the pH adjuster is used in the silicon hydroxylation modification step. Any water may be, for example: ultrapure water, distilled water, water left in or intentionally added to the reaction vessel, and the like; any aqueous agent may be, for example, a dispersant, an alcohol/water mixture, or the like. The removal of water can improve the efficiency of RNA extraction.
In a specific embodiment, "lower alcohol" refers to an alcohol having a carbon chain length of 3 or less.
In exemplary embodiments, the lower alcohol may be selected from one or more of methanol, ethanol, n-propanol, and isopropanol.
In the functionalization treatment step, after the modified magnetic particles are dispersed in the lower alcohol solution, a certain concentration of magnetic fluid is formed. The magnetic fluid 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 300mg of the modified magnetic particles were dispersed in 150mL of a lower alcohol, the magnetofluid concentration was 2mg/mL.
The present invention is not particularly limited with respect to the length of the sub-step of the treatment with a lower alcohol, as long as the treated magnetic particles can be sufficiently dispersed.
As used herein, "silicon hydroxyl functionalized monomer" is used interchangeably with "donor of silicon hydroxyl" and refers to a substance that imparts silicon hydroxyl functionalization to the modified magnetic microparticles.
Specifically, the "silicon hydroxyl-functionalized monomer" may be selected from one or more of the group consisting of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, butyl orthosilicate, alkyltrimethoxysilane, alkyltriethoxysilane, alkyltriethylsilane, dialkyldimethoxysilane, dialkyldiethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, aminopropyltrimethoxysilane, and epoxypropyltrimethoxysilane.
Before or while the "silicon hydroxyl-functionalized monomer" is added for the reaction, the method of the present invention may further comprise a step of adjusting the reaction solution to a suitable reaction condition. Suitable pH conditions may for example be 8-13; the adjustment of the pH can be effected, for example, by adding a weakly basic pH regulator, such as 1-2mL of aqueous ammonia. Suitable reaction temperatures may be, for example, from 50 to 90 ℃.
Inorganic strong acids, which are defined as "an acid unsaturated full dissociation" (i.e., an acid that can be completely ionized by itself, such as 1mol HCl to 1mol H in water) + And also another 1mol Cl - ). Acids meeting this condition are generally inorganic acids. The acid that is completely ionized in solution is a strong acid, and the strong acid is ionized using an equal sign, such as: HCl = H + +Cl - . The standard for strong acids is their ionization constant in aqueous solution, and those with pKa (acidity coefficient, negative logarithm of ionization constant) of less than 1 are strong acids (pKa =1 to 4 is medium strong acid, and more than 4 is weak acid), and some acids with pKa values of a few tenths may also be considered as strong acids.
In some embodiments, the strong inorganic 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, chloropolybdic acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid.
In certain embodiments, a strong organic acid may be used in place of or in combination with a strong inorganic 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.
In some embodiments, the complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, phenanthroline, 2' -bipyridine, 8-quinolinol, and nitrogen-based complexing agents.
In some embodiments, the concentration of strong inorganic acid in the post-treated reagent is 1 × 10 -4 mol/L to 10mol/L. Specifically, it may 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 and 10mol/L. The research result of the invention shows that the inorganic strong acid has obvious difference with the common acidic solution or buffer solution for the effect of the application. The concentration of the strong inorganic acid is correlated with the effect of the post-treatment. Preferably, the concentration of the strong inorganic acid is 10 -3 mol/L～1mol/L。
The post-treatmentThe reagents of (a) may or may not contain a complexing agent. In some embodiments, the concentration of the complexing agent in the post-treated reagent is between 0.001mol/L and 1mol/L. Specifically, it may 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、1mol/L。
In some embodiments, the post-treatment step is to soak the magnetic microparticles modified with a silicon hydroxyl group in the post-treatment reagent and to stand.
In some embodiments, the temperature of the standing is 20 ℃ to 70 ℃, and the time of the standing is 1 hour to 15 hours. The specific standing temperature can be 20 deg.C, 30 deg.C at normal temperature, or 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C at high temperature. The time of standing may 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.
In some embodiments, the method comprises the steps of: and (3) washing the post-treated magnetic particles by using water, and then dispersing the magnetic particles into the water, wherein the pH of the obtained aqueous dispersion is adjusted to 5-7. Specifically, 5, 5.5, 6, 6.5, 7 may be used.
In some embodiments, the magnetic particles of the present invention have a particle size of no greater than 80nm, for example, from 35nm to 50nm.
The invention further aims to provide application of the silicon hydroxyl magnetic beads in any embodiment in nucleic acid extraction.
The silicon hydroxyl magnetic beads can be used for DNA extraction or RNA extraction or DNA and RNA co-extraction.
In particular embodiments, the sample may be selected from the group consisting of blood, pharyngeal swab, sputum, alveolar lavage, tissue, food, and environmental samples, among others.
In an exemplary embodiment, the RNA is an RNA derived from a virus, such as the RNA of a novel coronavirus (SARS-CoV-2).
The following are specific examples.
Example 1: synthesis process of magnetic microspheres
The method comprises the following steps: magnetic seed core
Taking 5.5g of ferrous sulfate heptahydrate and 10.5g of ferric chloride hexahydrate in 200mL of water, setting the stirring speed at 200rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, then adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano-magnetic particles with the particle size of about 5-15 nm.
Step two: magnetic particle hydrophilic layer modification
And dispersing the prepared magnetic seed core in 200mL of 0.003M PVP aqueous solution, and keeping the temperature at 90 ℃ for 1h to obtain the surface-modified magnetic particles.
Step three: modification of silicon hydroxyl groups
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the second step in 150mL of absolute ethanol solution, adding 1 mL of ammonia water after 30min, heating to 77 ℃, adding 5 mL of TEOS after the temperature is kept constant, and stopping the reaction after 2 h. The product is directly dispersed in water to obtain the silicon hydroxyl magnetic beads.
Example 2: the silicon hydroxyl magnetic bead treatment method for improving the magnetic responsiveness aims at: it is verified whether or not the improvement of the magnetic responsiveness can directly improve the extraction performance.
The magnetic beads with silica groups obtained in example 1 were directly dispersed in a 0.01% (w/v) sodium chloride solution. The experiment shows that: the magnetic response performance of the product can be obviously improved after the sodium chloride solution is added.
And (3) detection of extraction performance:
a) Instrument for measuring the position of a moving object
Ausheng Auto-Pure32A nucleic acid extractor, mike N32 nucleic acid extractor
b) Reagent components and amounts
| Component name | Component amounts/test |
| Proteinase K | 100-400ug |
| Lysis solution | 500-700ul |
| Magnetic bead | 200- |
| Cleaning solution | |
| 1 | 600- |
| Washing solution | |
| 2 | 600-800ul |
| Eluent | 40-80ul |
c) The reagent pre-dispensing of 96-well deep-well plates is shown in FIG. 1. Wherein, each hole of the 1 st column and the 7 th column contains magnetic beads; the 2 nd column and the 8 th column are effective working hole sites, and each hole contains a lysis solution; the 3 rd and 9 th rows each contain washing solution 1; the 4 th and 10 th rows each contain washing solution 2; column 6 and 12 each contain an eluent.
d) The instrument operation fast extraction program comprises the following steps:
e) The instrument runs the slow extraction program (in this patent, this program is used only for the HCV kit) as:
after nucleic acid extraction is carried out on a sample to be detected, a novel coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) (Mike biology) is adopted for detection, and the test results are shown in a table 1:
TABLE 1 evaluation results of magnetic beads in Rapid extraction procedure
In the table, "/" indicates undetected values.
And (4) conclusion: table 1 shows the evaluation results of the magnetic beads in the new crown fast extraction program. From the table, it can be seen that: the products of example 1 and example 2 were not detectable in the fast extraction procedure, indicating that this type of magnetic beads is not suitable for the fast extraction procedure.
Example 3: method for processing silicon hydroxyl magnetic beads
20g (wet weight) of the silicon hydroxyl magnetic beads obtained in example 1 was dispersed in 100mL of 1X 10 -4 The standing treatment time is 1 to 24 hours in the hydrochloric acid water solution with mol/L to 10mol/L. And then washing with water for several times, and dispersing in purified water to obtain the post-processed magnetic silicon hydroxyl magnetic beads.
TABLE 2 evaluation results of post-treatment beads in the Rapid extraction procedure
And (4) conclusion: table 2 shows the results of the assay performed by the novel coronavirus 2019-nCoV nucleic acid assay kit (fluorescence PCR method) (michael biosome) after the magnetic beads were subjected to nucleic acid extraction in the new corona fast extraction procedure. From the table, it can be seen that:
1) The magnetic beads synthesized in example 1 were not detectable in the fast extraction procedure, indicating that the magnetic beads are not suitable for the fast extraction procedure.
2) In example 3, the detection rate of the magnetic beads in the sensitivity sample was 100%, and the CT value of the magnetic beads in the precision sample was advanced by 0.3CT to 1.1CT from that of the magnetic beads in example 1.
3) Compared with the commercial product, the magnetic bead of the example 1 has poor performance compared with the commercial product because the magnetic bead of the example 1 has a phenomenon that can not be detected in a sensitivity sample; although the detection rate of the sensitivity sample is 100% for example 3 and the commercial products, the CT value of example 3 is earlier than that of the commercial products, so the magnetic bead performance of example 3 is better than that of the commercial products.
Example 4: method for processing silicon hydroxyl magnetic beads
20g (wet weight) of the silica-based magnetic beads obtained in example 1 were dispersed in 100mL of a 1X 10 solution -4 A mixed solution of a sulfuric acid aqueous solution of mol/L and an EDTA solution of 0.01mol/L, and the system is allowed to stand at room temperature for 1 hour. 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. And (4) repeatedly washing with water for several times, finally dispersing the product in water, and adjusting the pH of the obtained product to 7 by using a sodium hydroxide aqueous solution to obtain the post-treated silicon hydroxyl magnetic beads.
TABLE 3 evaluation of magnetic beads in New crown (2019-nCoV) Rapid extraction procedure
Table 4 evaluation results of magnetic beads in HCV slow extraction program
And (4) conclusion:
a) 2019-nCoV project test extraction program: the method is rapid; HCV project test extraction procedure: and (4) slow speed. The fast extraction procedure was the procedure of example 2 d); the slow extraction procedure was the procedure of example 2 e).
b) 2019-nCoV, the CT value obtained by evaluation of the example 4 is earlier than that of a commercial product on the whole, so that the product of the example 4 is better than the commercial product on the whole.
c) In the HCV project, the CT value obtained by evaluation in example 4 is earlier than that of the commercial product, regardless of the sensitivity sample or the precision sample, so that the product performance of example 4 is better than that of the commercial product.
Example 5: treatment of silicon hydroxyl magnetic beads by acid with different concentrations
20g (wet weight) of the silica-based magnetic beads obtained in example 1 were collected, and the particle size of the beads was 50nm. Respectively dispersing in 100mL of 10mol/L, 0.1mol/L, 1 × 10 -3 mol/L、1×10 -5 mol/L hydrochloric acid aqueous solution. Standing at normal temperature for 1-24 h, washing with water, and magnetically separating to collect solid particles. The collected magnetic solid particles were dispersed in purified water.
TABLE 5 evaluation results of magnetic beads in New Guankuaiji procedure
And (4) conclusion: from table 5, it can be seen that:
when the concentration of hydrochloric acid is 10mol/L, 0.1mol/L, 1 × 10 -3 At mol/L, the CT value of the R2 sample is not obviously different, and the S3 sample can be completely detected.
When the concentration of hydrochloric acid is 1 x 10 -5 At mol/L, no detectable phenomenon was observed in the low concentration sample (S3 sample).
In combination with the above conclusions, it can be preliminarily concluded that for Si-OH magnetic beads with a particle size of less than 100nm, the acid concentration in the post-treatment stage needs to be higher than 1 × 10 to ensure that the magnetic beads are completely detected in the S3 sample -5 mol/L。
Comparative example 1: the three batches of experiment of example 1 were repeated to verify the stability between batches.
TABLE 6 evaluation results of magnetic beads in New Guanguan fast extraction program
And (4) conclusion: as can be seen from table 6:
the product of example 4 was superior in new crown evaluation performance compared to the commercial product.
Comparative examples 1-1, comparative examples 1-2, and comparative examples 1-3 are three batches of experiments of example 1, and it can be seen from the table that three batches can be detected in precision samples, but the difference between the CT values of ORF1ab channel is as high as 1.5CT, which indicates that the magnetic beads synthesized by the three batches of experiments have the disadvantage of large batch-to-batch difference.
Sensitivity sample: comparative example 1 three batches of magnetic beads all had the phenomenon that the sensitivity sample could not be detected, which indicates that they were not qualified in the new crown fast extraction.
Comparative example 2: the three batches of experiment of example 3 were repeated to verify the stability between batches.
TABLE 7 evaluation results of magnetic beads in New Guanguan fast extraction program
And (4) conclusion: comparative examples 2-1, comparative examples 2-2, and comparative examples 2-3 were obtained by post-processing the three batches of the experiment of comparative example 1, and it can be seen from the table that all three batches were detectable in precision samples, and the four-channel CT values were not significantly different, indicating that the post-processing means can reduce the batch-to-batch difference.
Example 6
The post-treated magnetic silicon hydroxyl magnetic beads prepared in example 3 were subjected to VSM test and the results are shown in fig. 2 and table 8.
Table 8 magnetic beads VSM data
And (4) conclusion: the following conclusions are drawn from fig. 2 and table 8:
all the magnetic beads are superparamagnetic.
The specific saturation magnetic strength is more than 70emu/g.
Four batches of coercive force is 7.4 multiplied by 10 -4 ～7.7×10 -4 The coercivity is negligible.
The post-treated magnetic silicon hydroxyl magnetic beads prepared in example 3 were subjected to a contact angle test, and the results are shown in fig. 3. As can be seen from fig. 3: the contact angle between the magnetic beads and water is 20.2 degrees, which primarily shows that the hydrophilicity of the magnetic beads is better. According to the standard test method T/FSI-049-2020 for the content of silicon hydroxyl on the surface of fumed silica, the amount of silanol on the surface of the magnetic bead before post-treatment is 0.526%, the amount of silanol on the surface after post-treatment is 0.527%, and the amount of silanol on the surface of the magnetic bead before post-treatment is not obviously changed, so that the performance improvement is realized by the post-treatment method without increasing the amount of silanol on the surface of the magnetic bead.
The post-treated magnetic silicon hydroxyl magnetic beads prepared in example 3 were subjected to particle size detection. As shown in fig. 4. As can be seen from FIG. 4, the magnetic bead particle size is less than 50nm.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the patent protection scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims, and the description can be used to interpret the contents of the claims.
Claims (10)
1. A preparation method of silicon hydroxyl magnetic beads is characterized by comprising the following steps:
providing magnetic particles;
sequentially carrying out hydrophilic layer modification and silicon hydroxyl modification on the surface of the magnetic particle; and
and (3) carrying out post-treatment on the magnetic particles modified by the silicon hydroxyl, wherein a post-treatment reagent is inorganic strong acid or a mixture of the inorganic strong acid and a complexing agent.
2. The method of claim 1, wherein the strong inorganic acid is selected from the group consisting of sulfuric acid, nitric acid, perchloric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, perbromic acid, chloric acid, bromic acid, fluorosilicic acid, chloropolylead acid, metaphosphoric acid, permanganic acid, selenic acid, ferric acid, fluoroboric acid, fluorosulfonic acid, and metaperiodic acid; and/or the presence of a gas in the atmosphere,
the complexing agent is selected from any one or more of EDTA, citrate, thiocyanate, 2-mercaptoethanol, dithioglycerol, dithiotrimethylolpropane, o-phenanthroline, 2' -bipyridine, 8-quinolinol and nitrogen-based complexing agents.
3. The method of claim 1, wherein the concentration of the strong inorganic acid in the post-treatment reagent is 1X 10 -4 mol/L-10 mol/L; and/or the presence of a gas in the gas,
the concentration of the complexing agent in the post-treatment reagent is 0.001-1 mol/L.
4. The method of any one of claims 1 to 3, wherein the post-treatment step comprises immersing the silicon hydroxyl-modified magnetic particles in the post-treatment reagent and standing, preferably at a temperature of 20 ℃ to 70 ℃ for 1 hour to 15 hours.
5. The method for preparing silicon hydroxyl magnetic beads according to any one of claims 1 to 3, comprising the steps of: and (3) washing the post-treated magnetic particles by using water, and then dispersing the magnetic particles into the water, wherein the pH of the obtained aqueous dispersion is adjusted to 5-7.
6. The method for preparing silicon hydroxyl magnetic beads according to any one of claims 1 to 3, wherein the step of modifying the silicon hydroxyl groups comprises the steps of:
dispersing the magnetic particles modified by the hydrophilic layer in an anhydrous lower alcohol solution;
adding a monomer for endowing the magnetic particles with silicon hydroxyl functionalization for reaction.
7. The method of claim 6, wherein no water is added and no aqueous reagent other than a pH adjusting agent is used in the step of modifying the silicon hydroxyl groups.
8. The method of manufacturing a silicon hydroxyl magnetic bead according to any one of claims 1 to 3 and 7, wherein the magnetic fine particles have a particle size of 35nm to 80nm.
9. The silicon hydroxyl magnetic bead prepared by the method according to any one of claims 1 to 8.
10. Use of the silicon hydroxyl magnetic beads according to claim 9 in nucleic acid extraction.
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| PCT/CN2021/140204 WO2022135420A1 (en) | 2020-12-23 | 2021-12-21 | Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof |
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| CN (1) | CN115691994A (en) |
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2021
- 2021-07-22 CN CN202110830842.XA patent/CN115691994A/en active Pending
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