Hydroxyl nano magnetic bead with uniform particle size as well as preparation method and application of hydroxyl nano magnetic bead

Abstract

The invention provides a hydroxyl nano magnetic bead with uniform particle size and a preparation method and application thereof, the preparation method comprises the following steps: in a nitrogen atmosphere, mixing ferric acetylacetonate, 2-pyrrolidone and a dendritic silicon ball template to obtain an iron precursor mixture, and then performing high-temperature thermal decomposition reaction to form a ferroferric oxide / dendritic silicon ball magnetic compound; and dispersing the ferroferric oxide / dendritic silicon sphere magnetic compound in an ethanol / water mixed solution, ultrasonically mixing uniformly, adding ammonia water and tetraethoxysilane, and mechanically stirring at room temperature to obtain the ferroferric oxide / dendritic silicon sphere / silicon dioxide hydroxyl magnetic bead. The particle size of the prepared hydroxyl magnetic beads is 400-500nm, and the hydroxyl magnetic beads have the advantages of large specific surface area, good suspension property and the like, and are uniform in particle size and uniform in distribution; as the surface of the magnetic bead contains abundant silicon hydroxyl, nucleic acid can be reversibly captured and released in an acidic / alkaline environment.

Description

Technical Field

[0001] The invention relates to the technical field of magnetic composite nanomaterials, and in particular to hydroxy nano magnetic beads with uniform particle size, and a preparation method and application thereof. Background Art

[0002] At present, early screening of tumors usually adopts the method of liquid biopsy. Compared with traditional tissue biopsy, liquid biopsy has the characteristics of non-invasiveness, sensitivity, and dynamics. Liquid biopsy technology is usually based on deep gene sequencing of tumor free DNA (cfDNA) shed by tumor cells into the blood. cfDNA refers to partially degraded endogenous DNA of the body that is free from cells in circulating blood. Under normal physiological conditions, cfDNA mainly comes from the degradation of genomic DNA of aging apoptotic cells. When the body is diseased, such as trauma, tissue and organ failure, organ transplant rejection, malignant tumors, trauma and infection of major diseases, abnormal necrotic cells will release a large amount of DNA into the blood circulation. The first step in using cfDNA for testing is to extract and purify cfDNA in human peripheral blood. The quality of the extracted product directly affects the accuracy and reliability of subsequent test results. However, the content of cfDNA is very low and is extremely easy to degrade. The extraction method of ultra-trace tumor cfDNA at the source greatly affects the efficiency of the detection platform.

[0003] Traditional cfDNA extraction methods include phenol-chloroform method, Trizol method, and adsorption column method. They use toxic phenol and chloroform reagents and adopt precipitation and centrifugation processes. The extraction process requires a large amount of samples, and the sample processing process is cumbersome and complicated. The recovery efficiency of cfDNA is low, and it is difficult to achieve high-throughput and automated operations. At present, the mainstream extraction method of cfDNA is the magnetic bead method. The principle of magnetic bead method for extracting nucleic acids is: using magnetic beads as carriers, the magnetic beads can adsorb nucleic acids under high salt and low pH, and separate from nucleic acids under low salt and high pH, ​​and then move the magnetic beads or transfer liquids to achieve the entire extraction and purification process of nucleic acids.

[0004] Most of the magnetic beads used for nucleic acid extraction in the prior art are prepared by the swelling method. The preparation process generally includes preparing monodisperse macroporous polystyrene microspheres by seed polymerization technology, and then sulfonating the inside and outside of the microsphere pores (-SO 3 ) or nitrification (-NO 2 ) to make it have a hydrophilic interface, and then immerse the microspheres in an iron salt aqueous solution, and under appropriate reaction conditions, superparamagnetic Fe 3 O 4 or γ-Fe 3 O 4The microspheres are generated in the pores, and finally the monomers containing active functional groups are used to swell, polymerize, and coat the microspheres, so that the microsphere pores are closed and the surface is functionalized. The magnetic beads prepared by the swelling method are micron-sized, with a particle size of generally 1 to 4 μm. They have poor suspension in the solution and are easy to settle, which affects the interaction with cfDNA in the sample. In particular, the micron-sized magnetic beads have a small specific surface area and fewer binding sites for DNA. The amount of magnetic beads needs to be increased to achieve the purpose of effectively separating DNA from the sample, which increases the cost. Summary of the invention

[0005] In view of this, in order to solve the above problems, the present invention provides a hydroxyl nano-magnetic bead with uniform particle size and a preparation method thereof, aiming to solve the technical problems of large particle size, small specific surface area and poor suspension of magnetic beads in the prior art.

[0006] In order to achieve the above object, the present invention provides a method for preparing hydroxyl nano magnetic beads, comprising the following steps:

[0007] S1. Preparation of an iron precursor mixture: Under a nitrogen atmosphere, ferric acetylacetonate, 2-pyrrolidone and a dendritic silicon sphere template were mixed and stirred to obtain a uniformly mixed mixture of the iron precursor;

[0008] S2. Preparing a ferroferric oxide / dendritic silicon sphere magnetic composite: subjecting the iron precursor mixture to a high-temperature thermal decomposition reaction, and then magnetically separating and washing the mixture to obtain the ferroferric oxide / dendritic silicon sphere magnetic composite; wherein, in the ferroferric oxide / dendritic silicon sphere magnetic composite, the ferroferric oxide particles are loaded inside the dendritic silicon sphere template to form an inner core layer structure;

[0009] S3. Preparation of ferroferric oxide / dendritic silica spheres / silica hydroxyl magnetic beads: disperse the ferroferric oxide / dendritic silica sphere magnetic complex in an ethanol / water mixture, mix evenly by ultrasonication, add ammonia water, and after secondary ultrasonication, add tetraethyl orthosilicate, and mix evenly by mechanical stirring at room temperature to form ferroferric oxide / dendritic silica spheres / silica hydroxyl magnetic beads; the hydroxyl silica obtained after the reaction of tetraethyl orthosilicate is coated on the surface of the dendritic silica spheres to form microspherical hydroxyl nanomagnetic beads with smooth surface and regular shape.

[0010] S4. Preparation of hydroxy nanomagnetic beads: magnetically separating the ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads, and then washing them to obtain the ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads.

[0011] Preferably, in S1, the mass ratio of ferric acetylacetonate, 2-pyrrolidone and dendritic silica sphere template is (0.1-1.0):(11-33):(0.01-1.0); and the stirring time is 15-30 min.

[0012] Preferably, in S2, the high temperature thermal decomposition reaction comprises a temperature of 200 to 245° C. and a reaction time of 10 to 60 minutes under stirring conditions.

[0013] Preferably, in S3, the mass / volume ratio of the ferroferric oxide / dendritic silicon sphere magnetic composite to the ethanol / water mixture is 5-10 mg: 3-10 mL.

[0014] Preferably, the mass / volume ratio of the ferroferric oxide / dendritic silicon sphere magnetic composite to ammonia water is 50-100 mg: 0.1-10 mL.

[0015] Preferably, the mass / volume ratio of the ferrosoferric oxide / dendritic silicon sphere magnetic composite to tetraethyl orthosilicate is 50-100 mg: 0.1-5 mL.

[0016] Preferably, the preparation method of the dendritic silicon spheres comprises: dissolving triethanolamine in deionized water and stirring to form a triethanolamine solution;

[0017] Add hexadecyltrimethylammonium bromide, sodium salicylate and ethyl orthosilicate to the triethanolamine solution in sequence, and after the reaction is completed, cool to room temperature and collect precipitate A by centrifugation;

[0018] The precipitate A is dispersed in a mixed solution of hydrochloric acid and ethanol to react. After the reaction is completed, the precipitate B is collected by centrifugation to obtain the dendritic silicon sphere.

[0019] Preferably, the mass volume concentration of the triethanolamine solution is 0.1-10 mg / mL.

[0020] Preferably, in the mixed solution of hydrochloric acid and ethanol, the volume ratio of hydrochloric acid to ethanol is (1-5):(25-100).

[0021] Preferably, the entire process of preparing the dendritic silicon spheres is carried out under stirring conditions, and the reaction temperature is 60-80°C.

[0022] Preferably, the mass ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is (0.1-10):(1-10):(1-10).

[0023] Preferably, the mass / volume ratio of triethanolamine to hydrochloric acid is (0.01-1.0) g: (1-5) mL.

[0024] To achieve another purpose, the present invention also provides the above-mentioned hydroxyl nano magnetic beads with uniform particle size, including: a core-shell structure with double-layer coating composed of ferroferric oxide nanoparticles, a dendritic silicon sphere template and a silicon dioxide layer; the core-shell structure includes an inner core and an outer shell; wherein the ferroferric oxide nanoparticles are loaded inside the dendritic silicon sphere template to form the inner core; and the silicon dioxide layer is coated on the surface of the dendritic silicon sphere template to form the outer shell.

[0025] Preferably, the average particle size of the hydroxyl nanomagnetic beads is 400nm to 500nm.

[0026] Preferably, the hydroxy nanomagnetic beads have uniform particle sizes and are in the shape of regular microspheres.

[0027] The hydroxyl nanomagnetic beads prepared based on the above technical solution can be used for nucleic acid capture or nucleic acid release, wherein the nucleic acid includes DNA ladder, cfDNA in plasma or urine.

[0028] Preferably, the method for nucleic acid capture comprises the following steps:

[0029] (1) dispersing the hydroxy nano magnetic beads according to claim 1 or 2 into deionized water to obtain a magnetic bead suspension;

[0030] (2) The nucleic acid, the acidic buffer and the magnetic bead suspension are mixed and incubated at room temperature under shaking conditions until the adsorption of the nucleic acid by the magnetic beads reaches equilibrium, and then the product in the mixed solution obtained by the incubation is collected by a magnet. The collected product is then washed with an acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.

[0031] Preferably, the method for releasing nucleic acid comprises the following steps:

[0032] The magnetic bead-nucleic acid complex is dispersed in an alkaline buffer and eluted at room temperature under shaking conditions until the desorption of the nucleic acid by the magnetic beads reaches equilibrium, so that the nucleic acid is separated from the magnetic beads, that is, the release of the nucleic acid is completed, and then the magnetic beads in the eluted mixed solution are collected with a magnet.

[0033] Preferably, the concentration of the magnetic bead suspension is 10-50 mg / mL.

[0034] Preferably, the acidic buffer comprises 5-6 mol / L guanidine thiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, and 40%-60% (v / v) isopropanol.

[0035] Preferably, the alkaline buffer comprises 50 mM TE buffer, pH=10.0.

[0036] Preferably, the amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.

[0037] Beneficial technical effects obtained by the present invention:

[0038] 1. The hydroxyl nano magnetic beads provided by the technical solution of the present invention have uniform particle size, a regular spherical shape, and an average particle size of 400-500nm. Compared with the prior art, they have the characteristics of small particle size and large specific surface area. In particular, they have good dispersibility and suspension properties in water or solvents.

[0039] 2. The hydroxyl nanomagnetic beads provided by the technical solution of the present invention have a core-shell structure, and can load iron-containing compounds in the dendritic silica sphere template to form a core structure, and the silica layer is coated on the surface of the dendritic silica sphere template to form an outer shell. The surface of the silica layer of the outer shell has abundant hydroxyl groups, which can make the composite particles easy to further biofunctionalize, and can effectively adsorb nucleic acids to achieve the capture and release of nucleic acids, especially cfDNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a transmission electron microscope image of the dendritic silicon sphere provided in Example 1 of the present invention.

[0041] Figure 2 This is a scanning electron microscope image of the ferroferric oxide / dendritic silicon sphere magnetic composite provided in Example 1 of the present invention;

[0042] Figure 3 A scanning electron microscope image of ferroferric oxide / dendritic silicon spheres / silicon dioxide hydroxyl magnetic beads provided in Example 1 of the present invention;

[0043] Figure 4 This is a transmission electron microscope image of ferrosoferric oxide nanoparticles provided in Comparative Example 1 of the present invention;

[0044] Figure 5 This is a transmission electron microscope image of ferroferric oxide / silicon dioxide hydroxyl magnetic beads provided in Comparative Example 1 of the present invention.

[0045] Figure 6a and Figure 6b They are respectively the transmission electron microscope picture and the magnified electron microscope picture provided for comparative example 2 of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments.

[0047] The present invention provides a method for preparing hydroxyl nano magnetic beads with uniform particle size, comprising the following steps:

[0048] (1) under a nitrogen atmosphere, mixing and stirring iron acetylacetonate, 2-pyrrolidone and a dendritic silicon sphere template to obtain an iron precursor mixture;

[0049] (2) subjecting the iron precursor mixture to a high temperature thermal decomposition reaction to form a ferroferric oxide / dendritic silicon sphere magnetic composite;

[0050] (3) magnetically separating the ferroferric oxide / dendritic silicon sphere magnetic composite and cleaning it;

[0051] (4) dispersing the ferroferric oxide / dendritic silica ball magnetic composite in an ethanol / water mixture, ultrasonically mixing, adding ammonia water, and then ultrasonically mixing, adding tetraethyl orthosilicate, and mechanically stirring at room temperature to form ferroferric oxide / dendritic silica ball / silicon dioxide hydroxy magnetic beads; the tetraethyl orthosilicate forms a silica sol after reaction, which is coated on the surface of the dendritic silica ball;

[0052] (5) Magnetic separation of ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads and cleaning thereof.

[0053] Preferably, the preparation method of dendritic silicon spheres comprises: dissolving triethanolamine in deionized water, stirring and mixing, then adding hexadecyltrimethylammonium bromide and sodium salicylate, and continuing stirring; then adding ethyl orthosilicate, heating and stirring to react; cooling the product to room temperature, collecting the precipitate by centrifugation, and washing with ethanol several times; dispersing the product in concentrated hydrochloric acid and ethanol, and after reflux reaction, collecting the precipitate by centrifugation to obtain dendritic silicon spheres.

[0054] As a preferred solution, in step (1), the mass of ferric acetylacetonate is 0.1-1.0 g, the volume of 2-pyrrolidone is 10-30 mL, and the mass of the dendritic silicon spheres is 0.01-1.0 g.

[0055] As a preferred solution, the high temperature thermal decomposition reaction temperature in step (2) is 200-245°C, and the reaction time is 10-60 minutes.

[0056] As a preferred solution, in step (4), the volume ratio of ethanol to water in the ethanol / water mixture is 10-50:10-50, the volume of ammonia water is 0.01-10 mL, and the volume of tetraethyl orthosilicate is 0.1-10 mL.

[0057] As a preferred solution, after the ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads are dispersed in ethanol, the particle size thereof is 400 nm to 500 nm.

[0058] In some specific embodiments, the preparation method of the ferroferric oxide / dendritic silicon sphere magnetic composite comprises: in a nitrogen atmosphere, 0.1-1.0 g of ferric acetylacetonate, 10-30 mL of 2-pyrrolidone and 0.01-1.0 g of a dendritic silicon sphere template are stirred for 15-30 min to mix evenly, the mixed solution is stirred and reacted at 200-245° C. for 10-60 min, and when the temperature drops to room temperature, the product is collected by magnetic separation, and washed several times with ethanol to obtain a ferroferric oxide / dendritic silicon sphere magnetic composite.

[0059] In some specific embodiments, the preparation method of ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads comprises:

[0060] Disperse 50-100 mg of ferroferric oxide / dendritic silica sphere magnetic complex in 30-100 mL of ethanol / water mixture, mix by ultrasonication, add 0.1-10 mL of ammonia water, continue ultrasonication for 30 min, add 0.1-5 mL of ethyl orthosilicate, stir mechanically at room temperature for 5-10 h, collect the product by magnetic separation, wash it with ethanol several times, and obtain ferroferric oxide / dendritic silica sphere / silicon dioxide hydroxy magnetic beads.

[0061] Further preferably, the preparation method of dendritic silicon spheres comprises the following steps: dissolving triethanolamine in deionized water, stirring and mixing, then adding hexadecyltrimethylammonium bromide and sodium salicylate, and continuing stirring; then adding ethyl orthosilicate, heating and stirring to react; cooling the product to room temperature, collecting the precipitate by centrifugation, and washing with ethanol several times; dispersing the product in concentrated hydrochloric acid and ethanol, and after reflux reaction, collecting the precipitate by centrifugation to obtain dendritic silicon spheres.

[0062] In some specific embodiments, the method for preparing dendritic silicon spheres comprises the following steps:

[0063] 1) Dissolve 0.01-1.0 g triethanolamine in 10-100 mL deionized water and stir magnetically at 60-80 °C for 15-30 min;

[0064] 2) Add 0.1-1.0 g of hexadecyltrimethylammonium bromide and 0.1-1.0 g of sodium salicylate, and stir magnetically at 60-80° C. for 15-30 min;

[0065] 3) Add 1-10 mL of ethyl orthosilicate and stir magnetically at 60-80°C for 1-10 hours;

[0066] 4) The product was cooled to room temperature, the precipitate was collected by centrifugation, and washed several times with ethanol;

[0067] 5) The product is dispersed in 1-5 mL of concentrated hydrochloric acid and 25-100 mL of ethanol and refluxed at 60-80°C for 12-24 h;

[0068] 6) Collect the precipitate by centrifugation to obtain dendritic silicon spheres.

[0069] The hydroxy nanomagnetic beads prepared by the above method are used in nucleic acid capture or nucleic acid release, and the nucleic acid is DNA ladder, cfDNA in plasma or urine.

[0070] In some specific embodiments, the steps of nucleic acid adsorption are as follows:

[0071] (1) dispersing hydroxy nano magnetic beads in deionized water to prepare a suspension having a hydroxy nano magnetic bead concentration of 10 to 50 mg / mL;

[0072] (2) Mix the nucleic acid, acidic buffer and magnetic bead suspension, incubate at room temperature under shaking conditions until the adsorption of the magnetic beads to the nucleic acid reaches equilibrium, then use a magnet to collect the product in the mixed solution obtained by incubation, and then wash the collected product with acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.

[0073] In some specific embodiments, the steps of nucleic acid release are as follows: dispersing the magnetic bead-nucleic acid complex in an alkaline buffer, eluting at room temperature under shaking conditions until the desorption of nucleic acid by the magnetic beads reaches equilibrium, separating the nucleic acid from the magnetic beads, i.e., completing the release of the nucleic acid, and then collecting the magnetic beads in the eluted mixed solution with a magnet; the amount of the alkaline buffer used is at least enough to submerge the magnetic bead-nucleic acid complex.

[0074] In some specific embodiments, the concentration of the magnetic bead suspension is 10-50 mg / mL.

[0075] In some specific embodiments, the acidic buffer comprises: 5-6 mol / L guanidine thiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, 40%-60% (volume percentage, v / v) isopropanol, and the rest is water.

[0076] In some specific embodiments, the alkaline buffer comprises: 50 mM TE buffer, pH=10.0.

[0077] In some specific embodiments, the amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.

[0078] It should be noted that, unless otherwise specified, the raw materials and reagents involved in the present invention are all commercially available.

[0079] The technical solution of the present invention is described in detail below through specific embodiments.

[0080] Example 1

[0081] The preparation method of hydroxy magnetic beads provided in this embodiment specifically includes:

[0082] 1. Provide dendritic silicon spheres, the preparation method comprising:

[0083] 1) 68 mg of triethanolamine was dissolved in 25 mL of deionized water and stirred at 80 °C for 15 min;

[0084] 2) Add 380 mg of hexadecyltrimethylammonium bromide and 218 mg of sodium salicylate, and stir magnetically at 80°C for 15 min;

[0085] 3) Add 4 mL of ethyl orthosilicate and stir under magnetic stirring at 80°C for 3 h;

[0086] 4) The product was cooled to room temperature, the precipitate was collected by centrifugation, and washed several times with ethanol;

[0087] 5) The product was dispersed in 3 mL of concentrated hydrochloric acid (37%) and 50 mL of ethanol and refluxed at 60°C for 24 h;

[0088] 6) Collect the precipitate by centrifugation to obtain dendritic silicon spheres.

[0089] 2. Provide a ferroferric oxide / dendritic silicon sphere magnetic composite, the preparation method comprising:

[0090] Under nitrogen atmosphere, 0.7063 g of ferric acetylacetonate, 20 mL of 2-pyrrolidone and 0.05 g of dendritic silica sphere template were stirred for 30 min to mix evenly, and the mixture was stirred and reacted at 245° C. for 30 min. After the temperature dropped to room temperature, a magnetic stand was used for magnetic separation and the product was collected, and washed several times with ethanol to obtain a ferroferric oxide / dendritic silica sphere magnetic composite.

[0091] 3. Provide ferroferric oxide / dendritic silicon spheres / silicon dioxide hydroxy magnetic beads, the preparation method of which includes:

[0092] 60 mg of ferroferric oxide / dendritic silica sphere magnetic complex was dispersed in 37 mL of ethanol / water mixture (ethanol: water = 25: 12), ultrasonically mixed, 0.6 mL of ammonia water was added, and ultrasonication was continued for 30 min. 0.4 mL of tetraethyl orthosilicate was added, and mechanical stirring was performed at room temperature for 5 h. The product was magnetically separated and collected using a magnetic rack, and washed several times with ethanol to obtain ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads.

[0093] See also Figure 1, is a transmission electron microscope picture of the dendritic silicon spheres prepared in the above step 1. It can be seen from the figure that the dendritic silicon spheres obtained by the technical solution of the present invention have uniform particle size and are evenly distributed without agglomeration. The surface of the silicon spheres has a large number of pores and a regular shape.

[0094] See also Figure 2 , is a scanning electron microscope image of the ferroferric oxide / dendritic silicon sphere magnetic composite prepared in the above step 2. It can be seen from the figure that in the ferroferric oxide / dendritic silicon sphere magnetic composite prepared in this embodiment, the ferroferric oxide particles are loaded inside the dendritic silicon sphere, and the pores of the dendritic silicon sphere are almost completely filled to form a microsphere shape with a dense structure, and the size of the particles has obvious uniformity, which can provide a core structure of uniform size for the preparation of hydroxyl magnetic beads. In particular, it can be clearly observed that the pores on the surface of the composite have been loaded and filled with ferroferric oxide, indicating that the technical solution of this embodiment can successfully load ferroferric oxide into the interior of the dendritic silicon sphere.

[0095] See also Figure 3 , is a scanning electron microscope image of the hydroxy magnetic beads prepared in the above step 3. It can be seen from the figure that the hydroxy nano magnetic beads prepared in this embodiment have a regular microsphere structure and a smooth surface without a hole structure, which is different from the microspheres of the ferroferric oxide / dendritic silicon ball magnetic composite having a large number of holes obtained in step 2, indicating that silicon dioxide is successfully wrapped on the surface of the dendritic silicon ball, and the size of the magnetic bead microspheres is uniform and evenly distributed.

[0096] The hydroxy nanomagnetic beads prepared by the technical solution of the present invention can reversibly capture and release nucleic acids in an acidic / alkaline environment, thereby enabling nucleic acid extraction from DNA ladders, cfDNA in plasma or urine.

[0097] Example 2

[0098] The hydroxy nanomagnetic beads prepared based on the technical solution of Example 1 are applied to the extraction of cfDNA from human plasma, specifically including DNA capture and DNA release, and the specific steps include:

[0099] (1) Take 2 mL of plasma into a 10 / 15 mL centrifuge tube, add 2.5 mL of acidic buffer (5 mol / L guanidine thiocyanate, 0.5 mmol / L polyoxyethylene ether Brij58, 50 mmol / L pH=5.0 NaAc-HAc buffer, 40% isopropanol (v / v), and the rest is water) and 30 μL of mixed magnetic beads (hydroxy magnetic beads prepared in Example 1, with a magnetic bead concentration of 40 mg / mL), vortex mix at room temperature for 10 min; then centrifuge at 1000 rpm for 1 min; then place on a magnetic rack for 5 min or until the solution is clear, and remove the clear solution by aspiration.

[0100] (2) Remove the centrifuge tube from the magnetic rack, add 1 mL of 80% ethanol, shake for 30 seconds, and centrifuge. Place the centrifuge tube on the magnetic rack for 2 minutes or until the solution is clear; use a 200 μL pipette tip to remove the clear solution as thoroughly as possible, and repeat once; open the centrifuge tube cap and air-dry the magnetic beads for 2 to 5 minutes.

[0101] (3) Remove the centrifuge tube from the magnetic rack, add 15 μL of alkaline buffer, and mix by pipetting back and forth; shake for 5 minutes and then briefly centrifuge. Place the centrifuge tube on the magnetic rack for 2 minutes or until the liquid is clear, and then use a pipette to pipette the clear liquid into a new 1.5 mL centrifuge tube to obtain cfDNA. Repeat the extraction three times. The cfDNA quantification results are shown in Table 1.

[0102] Table 1 cfDNA quantification results of Example 2

[0103] cfDNA mass in 2mL plasma (ng) 1 22.65 2 20.25 3 19.7

[0104] Comparative Example 1

[0105] This comparative example provides a method for preparing hydroxy magnetic beads, in which no dendritic mesoporous silica is added during the preparation process, and specifically includes:

[0106] 1. Provide a method for preparing ferroferric oxide nanoparticles, the preparation method comprising:

[0107] Under a nitrogen atmosphere, 0.7063 g of ferric acetylacetonate and 20 mL of 2-pyrrolidone were stirred for 20 min to mix evenly, and the mixture was stirred and reacted at 245° C. for 30 min. When the temperature dropped to room temperature, a magnetic stand was used for magnetic separation and the product was collected, and washed several times with ethanol to obtain ferrosoferric oxide nanoparticles.

[0108] 2. Disperse 60 mg of ferroferric oxide in 37 mL of ethanol / water mixture (ethanol: water = 25: 12), mix by ultrasonication, add 0.6 mL of aqueous ammonia, continue ultrasonication for 30 min, add 0.4 mL of tetraethyl orthosilicate, stir mechanically at room temperature for 5 h, use a magnetic stand for magnetic separation and collect the product, wash it several times with ethanol, and obtain ferroferric oxide / silicon dioxide hydroxy magnetic beads.

[0109] See also Figure 4 , is a transmission electron microscope picture of the ferroferric oxide prepared in the above step 1. It can be seen from the figure that the ferroferric oxide obtained in Comparative Example 1 is agglomerated.

[0110] See also Figure 5 , is a scanning electron microscope picture of the hydroxyl magnetic beads prepared in the above step 2. It can be seen from the figure that the hydroxyl nanomagnetic beads prepared in this comparative example have irregular morphology and non-uniform particle size.

[0111] The ferroferric oxide / silicon dioxide hydroxy magnetic beads prepared in this comparative example were used as a substitute for the cfDNA quantitative test. The test method was the same as that in Example 2, and the extraction was repeated 3 times. The cfDNA quantitative results are shown in Table 2. Comparing the results of Table 1 and Table 2, it can be seen that the nucleic acid extraction efficiency of the hydroxy magnetic beads prepared in Example 1 is significantly higher than that of Comparative Example 1.

[0112] Table 2 Quantitative results of cfDNA extraction using hydroxy magnetic beads prepared in Comparative Example 1

[0113] cfDNA mass in 2mL plasma (ng) 1 7.5 2 6.75 3 6.05

[0114] Comparative Example 2

[0115] This comparative example provides a step-by-step method for preparing a ferroferric oxide / dendritic silicon sphere magnetic composite, which specifically includes:

[0116] 1. Under nitrogen atmosphere, 0.7063 g of ferric acetylacetonate and 20 mL of 2-pyrrolidone were stirred for 20 min to mix evenly. The mixture was stirred and reacted at 245 ° C for 30 min. When the temperature dropped to room temperature, a magnetic stand was used for magnetic separation and the product was collected. It was washed several times with ethanol to obtain ferrosoferric oxide nanoparticles.

[0117] 2. 60 mg of ferrosoferric oxide and 0.05 g of dendritic silica sphere template were evenly dispersed in 30 mL of deionized water, mechanically stirred at room temperature for 12 h, magnetically separated and collected using a magnetic stand, washed several times with ethanol, and the product was dispersed in ethanol.

[0118] See also Figure 6a and Figure 6b , is a transmission electron microscope picture of the product obtained in the above step 2. From the picture, the structure of ferroferric oxide of about 10 nm can be seen, and the structure of dendritic mesoporous silicon is not seen, which means that the magnetic composite of ferroferric oxide / dendritic silicon ball cannot be obtained in Comparative Example 2.

[0119] As can be seen from the above, the particle size of the dendritic mesoporous silicon prepared by the technical solution of Example 1 is uniform, thereby ensuring the uniform particle size of the composite, while there is no dendritic mesoporous silicon in Comparative Example 1, and it is obvious that the ferroferric oxide is seriously agglomerated and the particle size distribution is not uniform. The comparison results of Example 1 and Comparative Example 1 show that adding mesoporous silicon in the preparation process can achieve uniform particle size of the final product.

[0120] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or capable of achieving the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A method for preparing hydroxyl nanoparticles with uniform particle size, characterized in that: The following steps are involved: S1. Preparation of an iron precursor mixture: Under a nitrogen atmosphere, ferric acetylacetonate, 2-pyrrolidone and a dendritic silicon sphere template were mixed and stirred to obtain a uniformly mixed mixture of the iron precursor; S2. Preparation of ferroferric oxide / dendritic silicon ball magnetic composite: After the iron precursor mixture is subjected to a high temperature thermal decomposition reaction, magnetic separation and washing are performed to obtain the ferroferric oxide / dendritic silicon ball magnetic composite; S3. Preparation of ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxyl magnetic beads: dispersing the ferroferric oxide / dendritic silica sphere magnetic composite in an ethanol / water mixture, ultrasonically mixing, adding ammonia water, ultrasonicating for a second time, adding tetraethyl orthosilicate, and mechanically stirring and mixing at room temperature to form ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxyl magnetic beads; S4. Preparation of hydroxy nanomagnetic beads: magnetically separating the ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads, and then washing them to obtain the ferroferric oxide / dendritic silica spheres / silicon dioxide hydroxy magnetic beads.

2. The method for preparing hydroxy nanoparticles with uniform particle size according to claim 1, characterized in that: In S1, the mass ratio of ferric acetylacetonate, 2-pyrrolidone and dendritic silica sphere template is 0.1-1.0:11-33:0.01-1.0; the stirring time is 15-30 min; In S2, the high temperature thermal decomposition reaction includes a temperature of 200 to 245° C. and a reaction time of 10 to 60 minutes under stirring conditions; In S3, based on the mass of the ferroferric oxide / dendritic silicon sphere magnetic composite, the volume of the ethanol / water mixture is 0.3-2 mL / mg; The volume of the added ammonia water is 1-200 mL / g based on the mass of the ferroferric oxide / dendritic silicon sphere magnetic composite. Based on the mass of the ferrosoferric oxide / dendritic silicon sphere magnetic composite, the added volume of ethyl orthosilicate is 1-100 mL / g.

3. The method for preparing hydroxy nanoparticles with uniform particle size according to claim 1, characterized in that: The preparation method of the dendritic silicon sphere comprises: dissolving triethanolamine in deionized water and stirring to form a triethanolamine solution; Add hexadecyltrimethylammonium bromide, sodium salicylate and ethyl orthosilicate to the triethanolamine solution in sequence, and after the reaction is completed, cool to room temperature and collect precipitate A by centrifugation; The precipitate A is dispersed in a mixed solution of hydrochloric acid and ethanol to react. After the reaction is completed, the precipitate B is collected by centrifugation to obtain the dendritic silicon sphere.

4. The method for preparing hydroxy nano-magnetic beads with uniform particle size according to claim 3, characterized in that: The mass volume concentration of triethanolamine solution is 0.1-10 mg / mL; And / or, in the mixed solution of hydrochloric acid and ethanol, the volume ratio of hydrochloric acid to ethanol is 1-5:25-100; the concentration of hydrochloric acid is 36%-38%; And / or, the entire process of preparing the dendritic silicon spheres is carried out under stirring conditions, and the reaction temperature is 60-80° C.; And / or, the mass ratio of triethanolamine, hexadecyltrimethylammonium bromide and sodium salicylate is 0.1-10:1-10:1-10.

5. A hydroxy nano-magnetic bead prepared by the preparation method according to any one of claims 1 to 4, comprising: A double-layer core-shell structure consisting of ferroferric oxide nanoparticles, dendritic silicon sphere templates and silicon dioxide layers; the core-shell structure comprises a core and an outer shell; wherein, Ferroferric oxide nanoparticles are loaded inside the dendritic silicon sphere template to form the core; The silicon dioxide layer covers the surface of the dendritic silicon sphere template to form the shell.

6. The hydroxy nano-magnetic beads with uniform particle size according to claim 5, characterized in that: The average particle size of the hydroxy nano magnetic beads is 400nm to 500nm; The hydroxy nano magnetic beads have uniform particle sizes and are in the shape of microspheres.

7. The use of the hydroxy nanomagnetic beads as claimed in claim 5 or 6 in nucleic acid capture or nucleic acid release can achieve reversible capture and release of nucleic acids in an acidic / alkaline environment; wherein, The nucleic acid includes DNA ladder, cfDNA in plasma or urine.

8. The use according to claim 7, wherein the method for nucleic acid capture comprises the following steps: (1) dispersing the hydroxy nano magnetic beads in deionized water to obtain a magnetic bead suspension; (2) The nucleic acid, the acidic buffer and the magnetic bead suspension are mixed and incubated at room temperature under shaking conditions until the adsorption of the nucleic acid by the magnetic beads reaches equilibrium, and then the product in the mixed solution obtained by the incubation is collected by a magnet. The collected product is then washed with an acidic buffer and ethanol respectively to remove protein and salt, thereby obtaining a magnetic bead-nucleic acid complex and completing the capture of the nucleic acid.

9. The use according to claim 8, wherein the method for releasing nucleic acid comprises the following steps: The magnetic bead-nucleic acid complex is dispersed in an alkaline buffer and eluted at room temperature under shaking conditions until the desorption of the nucleic acid by the magnetic beads reaches equilibrium, so that the nucleic acid is separated from the magnetic beads, that is, the release of the nucleic acid is completed, and then the magnetic beads in the eluted mixed solution are collected with a magnet.

10. The use according to claim 9, characterized in that The concentration of the magnetic bead suspension is 10 to 50 mg / mL; The acidic buffer comprises 5-6 mol / L guanidine isothiocyanate, 0.1-1 mmol / L polyoxyethylene ether Brij58, 50-100 mmol / L pH=5.0 NaAc-HAc buffer, and 40%-60% (v / v) isopropanol; The alkaline buffer comprises: 50 mM TE buffer, pH=10.0; The amount of the alkaline buffer is at least enough to submerge the magnetic bead-nucleic acid complex.

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